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British Journal of Industrial Medicine 1982;39:306-307 Progression of vinyl chloride induced hepatic fibrosis to angiosarcoma of the liver D B JONES AND P M SMITH From the Department of Gastroenterology, Llandough Hospital, Penarth, S Glamorgan, UK 1 abstract Two vinyl chloride monomer (VCM) workers, who developed non-cirrhotic portal fibrosis and portal hypertension, died from angiosarcoma of the liver five and ten years later respectively, despite withdrawal from occupational exposure. We suggest that non-cirrhotic por tal fibrosis caused by exposure to VCM is potentially prcmalignant and that those workers who already have the condition should be carefully monitored. Since the original communication by Creech and results showed a normal serum bilirubin with a Johnson1 there have been several further case raised alkaline phosphatase (201 IU/1) and reports of vinyl chloride monomer (VCM) induced y-glutnmyi transpeptidase (83 IU/1). Radioisotope angiosarcoma of the liver. A commoner hepatic liver scan showed a small liver with no filling defects lesion is non-cirrhotic portal fibrosis2 leading to por and needle liver biopsy showed a well-pronounced tal hypertension. It has been suggested that this micronodular cirrhosis with no evidence to tumour. lesion may be a precursor to angiosarcoma He responded to protein restriction and lactulose formation,*"* but there has been only one report of a and was discharged. Within a week of discharge he patient with documented hepatic fibrosis developing was readmitted with an endoscopicaily confirmed angiosarcoma at a later date.4 We describe two bleeding duodenal ulcer. After this, he lapsed into further cases. hepatic coma and died. Necropsy showed multiple malignant tumours in the liver (wt 1130 g), one of Case reports which was haemorrhagic. Histologically, a great variety of liver cell lesions were present, some resem CASE 1 bling angiosarcomas, some hepatocardnomas, and A 60-year old white man initially presented in 1969 some adenomas. There were also atypical sinusoidal to the dermatology department with a skin eruption. cells and fibrosis in the non-tumorous parts of the Examination at that time showed anaemia, thrombo liver. cytopenia, hepatospienomegaly, and occult faecal blood loss. He had an occupational history of expos CASH 2 ure to VCM at high concentration for seven years A 49-ycar-oid man with a 12-year history of expos while working as a polycleaner and a blowdown ure to vinyl chloride monomer while working as a recovery operator. After two haematemeses, barium spray drier bagger, premix operator, and paste studies and splenic venography confirmed portal charging operator was, during a factory survey of hypertension and oesophageal varices, and he process workers in 1974, found to have thrombo underwent an end-to-side portocaval shunt. At cytopenia. This was later shown to be due to laparotomy the liver looked nodular, but operative hypcrsplcnism and presinusoidal portal hyperten liver biopsy showed non-cirrhotic portal fibrosis sion. He was otherwise well and drank five pints of only. Over the next four years the patient developed beer a night. Liver function test results were normal chronic portosystemic encephalopathy and maturity apart from a raised y-glutamyl transpeptidase level onset diabetes mellitus that was treated with oral of 82 IU/1. Varices were shown by barium studies hypoglycaemic agents. In May 1980 he presented and endoscopy, and liver biopsy showed fatty with worsening mental deterioration, hepatic foetor, change and slight non-drrhotic portal fibrosis. Two asterixis, and an enlarging liver. Liver function test years later the patient developed insulin dependent diabetes mellitus, but otherwise remained well until Received 5 October 1981 Accepted 20 November 1981 October 1979 when he presented with a large haematemesis. Endoscopy confirmed oesophageal 306 AP00024374 Progression of vinyl chloride induced hepatic fibrosis to angiosarcoma of the liver 307 varices to be the source of haemorrhage, and these were treated by injection sclerotherapy. The serum alkaline phosphatase and y-glutamyl transpeptidase levels rose over the next three months to 334IU/1 and 106 IU/1 respectively. Radioisotope liver scan showed multiple filling defects consistent with tumour deposits. Ascites and ankle oedema developed, and he died suddenly in January 1980 from an intraperitoneal bleed after a liver biopsy. Necropsy showed that a large haemorrhagic tumour, occupying most of the right lobe of the liver, had ruptured into the peritoneum. Several smaller haemorrhagic tumours were found elsewhere in the liver. An enlarged spleen (wt 760 g) and thrombosed, oesphageal varices were also noted. Histology showed the tumours to be angiosarcomas, with areas of sinusoidal dilatation and portal fibrosis in unaf fected areas of the liver. absence of portal hypertension, and may then be difficult to detect. It does not lead to a disturbance of liver function tests and may even be missed on needle biopsy of the liver.1 Greyscale ultrasonogra phy is a useful diagnostic aid," but it has yet to be used in a large industrial survey, and there are prob ably several unrecognised affected VCM workers. Stringent measures to control levels of exposure should lead to eventual disappearance of non cirrhotic fibrosis. Those patients who already have the condition, however, are still at risk of developing hepatic angiosarcoma in the future. A problem highlighted by the second case is that angiosarcoma is, by definition, a vascular lesion, and there is a risk of uncontrolled haemorrhage after blind liver biopsy. We believe, therefore, that the diagnosis should be sought either by laparoscopic biopsy or by hepatic angiography. Discussion Vinyl chloride monomer is transformed by hepatic microsomal enzymes to toxic metabolites that coval ently bind to DNA.1 After exposure to VCM hepatocytic proliferation, sinusoidal lining cell pro liferation, and focal sinusoidal dilatation occur* and angiosarcoma may later develop from the sinusoidal lining cells. Enlarged lipocytes may be seen in the space of Disse*; these cells are fibroblast precursors and can lay down collagen.' Hepatic fibrosis results and may be associated with presinusoidal portal hypertension.10 It is commoner than angiosarcoma.1 Although fibrosis was found in tumour-free portions of the liver tissue from VCM workers dying of angiosarcoma,1 transition of hepatic fibrosis to angiosarcoma, although postulated,1 has not been observed. The two patients described here were originally included in a series of seven VCM workers with por tal hypertension.1 They were followed for five and 10 years respectively from the time of diagnosis of portal hypertension to death from angiosarcoma. During this period they were not further exposed to VCM. Their case histories indicate that hepatic fibrosis in a VCM worker may be a precursor of future malignant change and life-long follow-up is necessary. We know of only one other similar patient who died seven years after a portacaval shunt from an angiosarcoma.* Non-cirrhotic fibrosis can also occur in the We thank Dr D M D Evans and Professor Peter Scheuer for their help in interpreting the biopsy material. References 1 Creech JL, Johnson MN. Angiosarcoma of the liver in the manu facture of polyvinyl chloride. JOM 1974;16:150-1. 1 Smith PM, Crossley IR, William* DMJ. Pottal hypettearion to vinyl chloride monomer worker*. Lancet 1976:11:602-4. 1 Popper H, Maltoni C, Selikoff U. Vinyl chloride-induced hepatic lesions in man and rodents. A companion. Liver 1981;1:7-20. 4 Tamburro CH. The hepatic role in carcinogenesis and its early detection--the vinyi chloride model. Yale J Bid Melt 1978;51:67-80. * Popper H, Thomas LB, Telles NC, Falk H, Selikoff U. Develop ment of hepatic angiosarcoma in man induced by vinyl chloride, thorotrast and arsenic Am J Pathol 1978;92:549-69. ' Makk L, Delmore F, Creech SL,etal. Clinical and morphological patterns of hepatic angiosarcoma in vinyl chloride workers. Cancer 1976;37:149-63. ' Ostermann-Golkar S, Hultmatk D. Segetback D, et el. Alkyla tion of DNA and protein in mice exposed to vinyl chloride. Biochem Biophys Ret Commun 1977;76:259-66. * Schaffner F, Popper H, Selikoff U. Initial features of vinyl chloride (VC) hepatic injury. Gastroenterology 1976;72:A3S. Kent G, Gay S. Inouye T, Bahu R, Minick OT, Popper H. Vita min A containing lipocytes and formation of type III collagen in liver injury. Proc Nad Acad Sci USA 1976;73:3719-22. ' Blendis LM, Smith PM, Laurie BW. Stephens MR. Evans WD. Portal hypertension in vinyi chloride monomer workers. A hemodynamic study. Gastroenterology 1978;75:206-11. ' Williams DMJ, Smith PM, Taylor KJW. Guadey IR. Duck BW. Monitoring liver disorders in vinyl chloride moooeser workers using greyscale ultrasonography. Br J Ini Med 1976^3:1327. AP00024375 Animal Medals Angiosarcoma o( Rats and Mice Induced by Vtayi Chloride Catrtbated by: C. B. Hong, OVM, PhD, J. M. Winston, PhO, and C. C. Lae. PhD, Midwest Research Institute, Kansas City, Missouri. Vinyl chloride (VC, CH* " CHCL) is a monomer used mainly to make polyvinyl chloride (PVC), an essential material to our daily life. In 1074, Creech and Johnson reported hepatic angiosarcoma in workers associated with the manufacture of PVC The first case of hepatic angiosarcoma in VC workers was identified in 196L* This finding was later substantiated by experimental inhalation study in laboratory animals.** Although the definite mechanism has not been elucidated, recent ani mal studies have suggested that VC, like many other carcinogens, is not carcinogenic without metabolic activation.* Study in rodents revealed that VC was activated by cytochrome P-450 into epoxide (chloroetfaylene oxide), which could alkylate the macromolecules of cells and lead to neo plastic transformation. This explanation is strengthened by die findings that the exposide is a strong mutagen.' Mice exposed to 50 to 1000 ppm of VC in the air for 6 hr/day, 5 days/ wk. develop hepatic angiosarcoma after 6-12 months.4 The incidence of this tumor is related to the VC level and the length of exposure. Angiosar coma also developed in spleen, mesentery, and subcutaneous tissues. Rats exposed to the same levels of VC require a longer time to develop tumors and do not have the toxic effects seen in some mice, such as acute toxic hepatitis, tubular nephrosis, and early death in groups given higher doses. Exposure to VC followed fay a 1-year recovery period did not diminish the incidence of development of these tumors.7 Vinyl chloride-induced hepatic angiosarcomas in mice or rats usually PuhllreHnn iporaored by the Registry of CorapmUv Pathology of tb Araad Fotew InaUtuf oi htholocy wd wpported by Public Kaaltfi Secvioa Cnnt RK-00301 fra Hm DtvWna of Snirl 1Usooreet, US Department of Health, Education tad Walfue, aoder lb* awptas of UafvonttiM Aaeeiated for Reaoaidi and Education in Pktfaologr, lac. Addraa reprint nquortt to Dr. C B. Hoag IM. riocfc DbooaeDtasncetic Center, UnfvonttyofKantucbr,I41B Newtown Men. lariastBa.ET40BH. 0002-0440/80/1200-0737S01.00 VMMnGM MtOC*(DOn Of PHimOOWI 737 AP00024376 or fi - * !* u V HONQCTAL i :;: >' i are multioentxic, with no spedaTprevalence in any lo^eof.the liver. Grossly, the tumors are soft, friable, and red in color The large one* have often ruptured and formed a hiood dot or filled the abdominal cavity with a blood-tinged fluid. Microscopically, die earliest recognizable neoplastic foci consist of erythrocytes admixed with a cluster of immature endo thelial cells. These foci are usually located in dilated intralobular sinusoi dal spaces (Figure 1). The well-developed angiosarcomas are composed of aggregates of irregular vascular channels that are lined by neoplastic endothelial cells and filled with erythrocytes (Figure 2). The neoplastic mass expands and progressively replaces die liver cords. The neoplastic cells can be divided into two types: 1) large spindle cells that resemble endothelial cells and are arranged in capillary networks and 2) large, round, or polygonal cells with no special pattern of arrangement These cells may fuse to form giant cells. Mitotic figures are not an uncommon finding. Feliosis, extramedullary hematopoiesis, and liver necrosis are commonly associated with this tumor. The clinical signs are not detected until late in the course of the disease. The animals with advanced angiosarcoma have anemia with generalized edema, apparently due to rupture of the tumor or leaking of blood from the neoplastic mass. The anemia could also be due to abnormal vascula ture, which induces a microangiopathic type of hemolytic anemia. Comparison With Human Dheets Microscopic features and tumorigenesis of hepatic angiosarcoma from rats and mice induced by VC resembles closely that which develops in man exposed to VC, thorotrust, and arsenic.* In rodents, primary angiosar comas can also be found in spleen, mesentery, subcutaneous tissue, and other sites. In man, the liver is the only organ affected. Furthermore, VC induces a variety of tumors other than hepatic angiosarcoma in rodents, including hepatocellular carcinoma, skin tumors, and osteochondroma,7* bronchioloalveolar and mammary gland tumors 4,7 in mice, and malignant lymphomas4,7 in mice and rats. In man, hepatocellular carcinoma is die only tumor besides hepatic angiosarcoma suspected to be associated with VC exposure.10 Vinyl chloride is able to induce hepatic angiosarcoma in many spedes of laboratory animals. The model can be used to set up guidelines for (lie tolerance level of VC for workers in PVC plants. The model also provides an opportunity to study the possible advene effects on die hepafocytes, including tumor induction, caused by die dysfunction of the other com- AP00024377 AP00024378 'T' V 1 i. ) i / 3 D E 0 L Creech JL Jr, Johnson MN: Ac^ioarcoma of fryer in the manufacture of polyvinyl chloride. J Occup Med 1874.16:150-151 1 Heath CW Jr. Falk H, Creech JLJn Cfcamcteriatfcf ofcamaofangioaeicQniaofthe liver among vinyl chloride workers in the United States. Ann NY Acad Set 1875, 246:231-236 3. Msltooi C, Lefemfoa CT: Carctaogenicity bioaaays of vinyl chloride: Current re sults. Ann NY Acad Sd 1975. 46:186-218 4. Lee CC, Bhandari JC. Winston JM, House WB, Dixon RL, Woods JS: Carcinogenicity of vinyl chloride and vinylidaoe chloride. J Toxiool Environ Health 1078,4:15-00 5. AntweilerH: Studiermi tlnmetabolismnirinyl AUA. EnvironHealth Penpact 1876,17:217-218 6. Malavellle C. Bartsdi H. Baihin A. Camus AM, Mooteaano R. Croisy A, Jaoquignon P: Mutangenicity of vinyl chloride, chloroethyieoeoside, chloraoetaldehyde and chloroethanoL Biochem Blophys Res Cnsnmnn 1875,83J63-370 7. Hong CB, Winston JM, Thornburg LP, Lee CC, Diinn RL, Woods JS: Follow-up study on the carcinogenicity of vinyl chloride. J Toxicol Environ Health (To be sub mitted) 8. Popper H, Thomas LB, TeUee NC, Falk H, SelikofE IJ: Development of hepatic angiosarcoma in man induced by vinyl chloride, thoratsmt, and arsenic. Am J Pathol 1978,92:349-376 8. Viola PL, Bigotti A, Caputo A: Oncogenic response of rat ridn, hmg. andbooea to vinyl chloride. Cancer Rea 1871,31516-552. 10. Chen KTK, Bolles JC, Cilber EF: Angtoaaicama of dm spleen. Arch Pathol Lab Med 1879,103:122-124 AP00024379 i Patterns of Finger Capillary Abnormalities in Connective | Tissue Disease by "Wide-Field" Microscopy Hildegard Rand Maricq and E. Carwile LeRoy The fingers of 75 patients with connective tissue disorders were examined by "wide-field" capillary microscopy. Four diagnostic groups were in cluded in this study: rheumatoid arthritis--28, scleroderma--22, dermatomyositis--8, and systemic lupus erythematosus--17. On the basis of different patterns of elementary microvascular abnormalities and their distribution, 3 distinct groups were recognized among these patients: 1) increased visibility of nailfold subpapiilary plexus in rheumatoid arthri tis, 2) massive capillary dilatation in scleroderma-dermatomyositis, and 3) focal loss of capillaries and prominence of subpapiilary vessels with "punched-out" lesions in systemic lupus erythematosus. . Skin lesions arc prominent features of rheu* italic or connective tissue diseases. A variety of | riinirally recognizable cutaneous ch.-mgcs have httn described, most of which include tilieriiliuni at the mierorireulaiory level (1.2). Many investigators have observed these ehanges mitrasropitally in t'ivn (3-19). liy ibis method oT intravital miemsedpy several microvascular anomalies have been described: viz... a) dilata tion of capillary loops; l>) dilatation til subpjpillary venules; t) distortion of the normal xchilcnurc of microvesscls in the form of exces sive elongation, tortuosity, or sacculatioo; ti) Vim of capillaries; and f) extravasation of retl i tdls. Such elementary microvascular changes j ire not specific, having (icon reported in sev. cral different connective tissue diseases, as well ^ is other disorders. The small field of observation has made it dilliruh to demonstrate characteristic patients From (hr Veterans Administration Hospital, Lynns, New Jersey, Rssex County llospiwil Center, (ledar (Jnvve. N<r Jersey. un<l Columbia University College of IMiysituns and .Surgeons, New Vork, New York. Reprint requests should J>c addressed to: |)r. II R. Mjricq. Veterans Administration Hospital. Lyons. N| 0*939. Submitted for publication Jan 19, 1973; accepted June X 1973. of these elementary lesions. To compare one disease group to another or to normal controls, it has been necessary to count and/or to mea sure the anomalous microvesscls in one or sev eral (iclds of observation (19). In the present study 4 groups of patients with connective tissue disorders have been examined by the "wide-field" method (20) in an attempt to define patterns of microvascular pathology in these four disease categories. Three separable patterns of microvascular abnormalities can lie recognized. MATERIALS AND METHODS Subjects Seventy-live patients attending the Edward Daniels Faulkner Arthritis Clinic of the Frrsbytemn Hospital with the following connective tissue disorders were studied1 rheumatoid anhritis--2d. scleroderma -22. tlerinaiunivusitis--ti, and systemic lupus erythematosus--17. They ranged in age from 5 io 73 and included f3 women (2s KA. 16 SD. 6 DM. It, SLK) and 12 men (3 KA. 6 SD, 2 DM, and I Sl.K). All patients with KA routd lie classified as classical or definite hy the revised American Khcumaiisnt Association Criteria (21). The diagnosis of.SD was made by the definite presence of hideltound skin, often with biopsy confirmation. The diagnosis of DM was made by the presente of myositis, requiring muscle weakness, elevated serum levels of muscle* Arthritis and Rheumatism, Vol. 16. No. 5 (September-October 1973) ppfo | ^ ^ q j AP00024380 MARICQ U LEROl Table 1. Selected Clinical Parameter* of Patient* Studied / Age, mean, years (range) Sex. temale/male Duration, years from symptom onset from diagnosis Raynaud's phenomenon Therapy Aspirin Gold Steroids Immunosuppressive* Vasoactivet Rheumatoid arthritis (N = 28) ' 48 (15-72) 25/3 12.5 11.0 -- 21 17 3 1 0 Scleroderma (N = 22) 46 (16-62) 16/6 5.3 2.3 19/22 5 0 4 0 9 Dermatomyositis (N = 8) Systemic lupus erythematosus (N = 17) 34 (5-73) 6/2 31 (9-68) 16/1 1.3 1.1 1 0/8 7.1 4.6 -- 04 00 8 14 2 3 00 * Aiathiopnne (3), cyclophosphamide (1). or methotrexate (2). fGuanethidine flNC t i- 1 derived enzymes and abnormal electromyographic findings, and the characteristic dermatitis of the hands or face. All patients with Sl.E fulfilled at least 4 of the M criteria re* ccmly suggested for that diagnosis (22). Patients with features nf more than one rheumatic syn drome ("mixed'* or "overlap'* syndromes) were excluded from the study. Because of the clinical variability of the syn drome. patients with polymyositis were also excluded from the present study. Patients with wcll-estahlished diagnoses were examined at all stagrsnfilieirdisca.se without selection. Selected clini cal features of these patients are shown in Table l. Methods score (PVS) of the individual. No attempt was made t*Mw sure individual capillary loops. Although the capillaries are not exactly alike in ant pm urea of the skin, the field of olwcrvation under a mtcroMiyr is rather uniform in normal subjects (Figures 1A andH. With the wide field of observation. it was possibletnolnem patterns of dilated or deformed mirmvesscls and to inntr* them with surrounding normal capillaries, the appe.tu** of which was known from previous experience. Eurzt* tation of red blood cells was also recorded. Observations were supplemented by photographs uln with a single-lens rellcx ramera fitted wiih a m.Kr*-|A tographir lens and Ixdluws using the light of an eletto*, flash, as previously drsrrifxxi (20). I I The nailfolri area of all 10 linger! was examined in e.nrh patient under a widc-field stereo microscope (magnification X 12) after placing a drop of immersion oil on the skin to fa cilitate visualization of the vessels. The light source for ob servations was a low-voltage lamp which delivered no ap preciable heat to the skin area under study. Other pans of the fingers (linger pads, dorsum of lingers from distal to proximal phalanges) were then inspected for any visible vascular changes which were examined as above. `The ex tern of the visible suhpaplllary plexus in the nailfolds was measured according to a previously described scoring sys tem (20), which scores each finger from 0 to 4 based on the size of the area where the subpapillary plexus can lie seen. The total score for 10 fingers is the plexus visualization RESULTS Rheumatoid Arthritis < A large number of patients with rheum.umt i arthritis (RA) had an extensive visible sut. ) papillary plexus in the nailfold (Figure IBi ! Twenty-two of 28 patients (79%) had a fV'i of more than 10 points, a value observed it ; approximately 3% to 6% of healthy nduht 3 (23,24). The lingers of rheumatoid jo tients were also unusually pale, requirmt ! : to 4 times less photographic exposure that t f 620 Arthritis and Rheumatism, Vol. 16, No. 5 (September-October Wt Arthri Si AP00024381 Z8tZ000dV MARICQ t LEROY Fig 2. (A) Representative area of surface microvessels on the dorsum of the middle phalanx in a healthy subiect. (B) Prominent subpapillary plexus and scarcity of capillary loops on the dorsum ol the middle phalanx of a patient with SLE. (C) A "window" lesion on the dorsal Finger shin of a patient with chronic SLE showing clinically visible erythematous lesions on the finger. (D) A cluster of large dilated capillaries on the side ol the proximal phalanx in a patient with scleroderma. those of normal subjects. Capillary hemor rhages were noted in 6 patients. No grossly vis ible vascular alterations were observed clini cally on other pans of (he fingers in RA subjects; these areas were therefore not system- aticallv studied under the microscope. The ex amination was dilliculi in many patients due in hand deformities. Prominent visible venules on the palmar side of ihc lingers were noted in 5 patients; palmar erythema, in 2 patients. Thus 622 Arthritis and Rheumatism, Vol. 16, No. 5 (September-October 1973) AP00024383 < t ' -------- ------ - , ,,i - - WiGER CAPILLARY ABNORMALITIES Fig 3. (Left) Finger pad of a patient with scleroderma. Note the cluster of dilated capillaries contrasting with the normal capillaries visible along the finger ridges. (Right) Finger pad of a patient with SLE. Note the absence ol capillaries and prominence of the subpapillary plexus compared to usual ridge pattern visible in lower right corner. iht predominant pattern in rheumatoid subjects i was an extensive visible suhpapillarv plexus of I the nailfold. ciated with obviously atrophic skin. Subpapillary plexus w;is oec asionallv also observed in proximal linger skin where irlangirc tatit* spot is were visible m t lie naked eye. Capillary Scleroderma and Dermatomyosilis hemorrhages were frequent in Ixith categories These two diagnostic categories presented many similarities anil no major differences in ihcir vascular patterns, anil the results are therefore dose ribnl together. In Ixith gfoups the most striking finding was the presence of enormously dilated capillaries runt rasting with the surrounding eapillary of patients and were especially numerous in the 2 patients with acute dcrmatomvusitis wlm died a few clays alter examination. There were only 3 patients in the sclero derma (SD) group who had not reported having Raynaud's attacks. M icTovascular patterns in these patients were similar to those of oilier loops..These giant capillaries were especially patients with SD and all .3 had large dilated $ well seen in the nailfold (figures 1(1 and .ID), eapillary loops in their nailfolds. None* of the K t but clusters of dilated capillary loops occ urred patients with dermatomyosilis (DM) had expe also on more proximal parts of the lingers (fig rienced Raynaud's phenomenon. ure 2D) and on linger pads (figure 3, I .eft). The dilated capillaries were purple and fre Systemic Lupus Erythematosus i quently seen on a background of pale* skin. This group or patients had the widest variety (iroups of moderately dilated capillaries were of microvascular anomalies and the greatest also seen, but these were not characteristic* and variation from patient to patient. This could he i could also lie observed in tlie systemic lupus ery due In the fact that, whereas patients with SI) i thematosus group. Small avascular scarred or DM all had clinically recognizable in areas surrounded by a circle of dilated capil volvement of the fingers, only 6 patients with t laries were noted in the knuckles. The sub- systemic* lupus erythematosus (Sl.f.) had obvi i papillary plexus in the nailfold was seen less ous lesions on the lingers, lrt these 6 patients, frequently here than in the rheumatoid group areas of loss of capillaries with prominent sub- and was of a different character, being asso papillary plexus were the major findings (fig- ; }; } Arthritis and Rheumatism, Vol. 16, No. 5 (September-October 1973) ! 623 11 ; i AP00024384 MARICQ & LEftOt .. >vsv m if 'm<x* r I; ' `V1 : _ V*V: * <'.> w '_. ' ^Vii. v'. f , Fig 4. (A) Finger of a patient with scleroderma. Some capillaries can be seen by the naked eye. (The skin Is slightly oily from preceding microscopic examination and makes such observa. tions easier). (B) Nailfold vascular pattern of the same finger with the wide-field technioue. (Area within frame of Figure 4A at higher magnification.) (C) A portion of the same nailfold. (Area within frame of Figure 4B at higher magnification using leitz microscope with 6.5> Ultropak objective). ures 2B, 3Right, 5A, and 5B). In 1 acute case these lesions were diffuse and atvnmpnnicd l>y extravasation of red cells and a yellowish exu date (Figure 211). The more chronic lesions gave the impression of "punched out windows" in the skin thrmich which deeper vascular layers could lie seen. The center of the lesion was almost avascular, with Arthritis and Rheumatism, Vol. 16. No. 5 (September-October 19731 AP00024385 98frZ000dV MARICQ 2. LEROl UNGER Table 2. Microvascular Observations Rheumatoid Systemic tupui arthritis Scleroderma Dermatomyositis erythematovm (N = 28) (N = 22) <N = 8) (N = Pi trnetal1 graphs In III has alb llmnnn Nailfold III! "in V Large dilated capillaries 0/28* 17/22 7/8 1/17 mu rose PVS > 10 (visible subpapillary plexus) 22/28 6/18 .1/7 7/15 \.M u\,\'. i Other parts of finger 1*.' v Ousters ol dilated capillaries Generalized visible subpapillary plexus 0/28 -- 12/16 0/14 6/8 0/7 0/17 4/14 iml l*\ "Window" lesions -- 0/22 0/8' 5/17 <.m r;i" Capillary hemorrhages 6/28 16/22 6/8 14/17 irrns (o munui Number of subjects with given abnormality in the numerator, total subiects examined for that feature in the denominator. The denominator varies for certain observations due to pigmentation in dark-skinnee subjects which interfered with visualization of capillaries to a varying degree at eachanatomic site. vinuen widc-ln nliscrva Ml. 5C rare subpapillary vessels (Figure 2C). Even in patients whose hands showed no clinical lesions a subpapillary plexus was frequently seen (if not in the nailfold then on the proximal finger skin). There were also groups of small, sharplydefined capillaries--darker than the surround ing capillaries--that gave the skin a mottled microscopic appearance. It was difficult to esti mate capillary dilatation here because only the tips of vessels were visible. Occasionally, dus ters of definite enlarged capillaries were seen in addition to the mottling. Large nailfold loops nailfold skin or mention gross lesions when re porting capillary microscopy. Other invcstigaiors have studied indivirlii.il skin lesions in various areas of the body and compared these with the skin of health people (3, 5-7. 0, 10, 13). Because of the .mull field of observation, however, only a few ele mentary microvascular lesions tire visible in previous illustrations. (For example, giant cap illaries have been reported in .SI) and DM In many previous workers who have scanned dir nailfold area, but their illustrations include<>nh me the drmnns parts nl lion in rquipni parts ol Tire show o sinus in of ihc p ml the ni/cib were observed in only one patient, but unlike scleroderma, ihc giant loops were nssociaicd with an adjacent "punched out'' lesion (Figure 5A). Capillary hemorrhages were frequently observed in patients with SLE, with or without obvious skin involvement. A tabulation of ele mentary lesions seen in the three groups is shown in Tabic 2. DISCUSSION Most investigations of capillary microscopy of human skin have been performed on the nail fold because it is technically the easiest area to examine. Although skin lesions in connective tissue disease may involve the nailfold areas, few authors describe the gross appearance of a very limited number of capillaries.) Althinnh in 1953 Giljc advocated capillary microscopy ,i> a "biopsy" of living tissue and proposed thr technique as an aid in differential diagnosis, ii has not found broad clinical application. I'min the present work it is proposed that disease en tities arc characterized by specific patterns nl elementary lesions rather than any specifiriiy nf the elementary lesions themselves; moreover, widc-ficld microscopy is essential for the olivrr. vat ion of patterns of elementary lesions rather than the individual lesions separately. (Elemen tary lesions seen in such patterns arc similar in and in agreement with thoisc reported earlier In smaller field techniques.) Although use of loupes and low-power microscopes has Ix-rn recommended earlier, capillary microscopy has 1. '1 plexus nr dclm istic ol rilic foi lions rheum: and sch 2. 1. fold an were mmyiw value. 3. ii finding ncncc 626 Arthritis and Rheumatism, Vol. 16, No. S (September-October 1573) Arthriti: AP00024387 1 JWGER CAPILLARY ABNORMALITIES pticrally liccn restricted to small Odd phumjraphs and drawings. In the presem study the wide-licld method Lis allowed observations of patients ol vascular abnormalities and comparisons with summiid> irg ''ntirmal" skin, allowing one to relate the Btcniscopic examination to clinically visible uvular lesions. Skin areas with erylhemaintts where the subpapillary plexus is normally not visible. Flits phenomenon was most pro nounced in discoid lesions but was also present to a lesser degree where only ill-deiinetl lesions were seen. The circle of horizontally-oriented capillaries surrounding a small avascular area found es|x`- quits barely detet'tabic grossly can lie chat aclerutil bv wide-licld microscopy and smaller areas un easily he scleeietl from the widc-lidd paltents for study at higher magnification. The scqucncc of photographs from the .same skin area ihinen in Figure 4 illustrates this point. The widc-lidd technique also facilitates sequential observations of niierovtiseulaf lesions (Figures 5B. SCI, and SI)) been use ol the e;tse ill rdoeat- eially on knuckles in .SI). DM. and sometimes in SI.F. apparently represents a phase in wound healing (25). Since this study was designed to explore and define patterns of micmvascular lesions in vari ous connective tissue disorders, the specificity or the proposed characteristic patterns should lie verified by another study in which microvascular patterns arc examined "blindly"' from i inq the skin ;icca and lesion, 'litis work also demonstrates that the examination of other photographs. ! parts of the linger adds mierov.iscular infonna- ACKNOWLEDGMENTS 1 tion in these disorders without the additional equipment needed for examination of other parts of the Ixtdy.' The results of this investigation (Table 2) thmv overlap of elementary nticmvascidar 1c- The authors would like to thank Miss Kdnn Farrington, nurse of the Falwarrl Faulkner Arthritis < Uinir. for invaluable assistance in scheduling patients and reviewing the rlimcal data, anti Miss Agnes Knr/u anti Mrs. Karen Month for valuable technical assistance jttins til all -I groups ol disorders. On tin- basis of (he pattern and distribution of these elements tin the linger, .1 distinct patterns can be rcrognired:1 REFERENCES 1. Gopctnan WSO: Textl>ook of the Rheumatic Diseases. Third edition Edinburgh. Liv ingstone. 1964 1. The presence of visible subpapillary plexus in the nailfold without gross dilatation or deformation of capillary loops was chnraetcrisltc of the R4 group. This pattern is not s|)e- 2. Rook A, Wilkinson I)S. i'.bUrn^ FJ(!: Textbook of Dermatology. Oxford. Blackwell. I9(>8 3. Mtiller C): Die Kapillaren dor mensohlirhen KorpcroherfUichc in gesunden und kranken lo gon. Stuttgart, Ferdinand Knkc. 1922 rilir for R.A and can be found in other condi 4. Brown GE, O'Leary PA: Skin capillaries in tions such as rheumatic Tever, chronic ' rheumatic heart disettse, artcritiselerosis (12), | and sdii/ophreitia (24). 2. I.arge dilated capillary loops in the ttails fold and on finger pads or proximal phalanges f were characteristic of sclcmdcrma-dcrma| tomyosilis patients and may have diagnostic value. i. In the Sl.E group, (he most characteristic [ finding was the loss of capillaries and promi- scleroderma. Arch Intern Med 30:73-88, 1926 5. Giljc O: Capillary microscopy in the diHcmuial diagnosis of skin diseases. Acta Dcrmatovcnci 33:303-317.1953 6. Giljc O, Kierland R, Knlrics EJ: Capillary nticroscopy in the diagnosis of dermatologic dis eases. J Invest Dermatol 22; 199-206, 195-1 7. Davis MJ and Lawler JO: The capillary circu lation of the skin. Arch Dermatol (Chicago) 77:690-703, 1958 8. Illig L: Die terminate Strohmbahn. Gapillarbett * < nence of subpapillary vessels in linger skin und Mikrnzirkulation. Berlin-Gottingen- > j Arthritis and Rheumatism, Vol, 16, No. 5 (September-October 1973) 627 AP00024388 MARICQ L LERO Heidelberg. Springer-Yerlag. Idol 9. Zimmer JG. Demis DJ: Studies on the micro circulation or the skin in disease. J Invest Der matol 39:501-509, 1902 10. Fukushiro R: Capillary microscopic exam ination in various skin diseases. Jap J Dermatol (Series B) 75:486-502. 1965 11. Jablonskn S: Scleroderma and Pscudoscleroderma. Warsaw, Polish Medical Publishers, 1965 12. Davis F., Landau J: Clinical Capillary Micros copy. Springfield, Thomas, 1966 13. Kawashima V: Capillary microscopic exam ination in collagen disease. Jap J Dermatol (Se ries B)76:24-31. I960 14. Lcighcb G. Visctti M. Abcrar.zi F: II quadro capillaroscopiro della dernialnmiosite. Minerva Dermatol 41:199-201. 1966 15. Buchanan IS. Ilumpston DJ: Nail-fold capil laries in connective tissue disorders. Lancet 1:845-847. 1908 16. Cospite M. Palazzolo F. Bruno S, Hallo M: Alteraciones dr los capilarrs cn la enfermedad reumatoitlc. Angiologia 21:11-15. 1969 17. Redisch W. Messina F,J. IlughesG. NlcEwan C: Capillaroscopic observations in rheumatic diseases. Ann Rheum Dis 29:244-253. I9"M 18. Maricq HR. HI time RS. LeRov EC: Wide-fide study of nailfold capillary lied in disorders i/ connective tissue. 6th European Cnnferenrr Microcirculation. Aalborg. 1970. p 116 19. Rouen LR, Terry EN. Dofl BH, et al: Cl.id> cation and measurement of surface mirruveswt' in man. Microvasc Res 4:285-292, 1972 20. Maricq HR: `Wide-ficld' photography of ns:' fold capillary bed and a scale of plexus visual ization scores (PVS). Mirrovasc Res 2:335-31" 1970 y 21. Ropes MW, Bennett GA. Cobb S, et al: Km sion of diagnostic criteria for rheumatoid anhr. tis. Bull Rheum Dis 9:175-176, 1958 22. Cohen AS, and Canoso JJ: Criteria for il classification of systemic lupus erythematosus ' status 1972. Arthritis Rheum 15:540-543. I"".' 23. Maricq HR: Nailfold capillaries in normal thi1 dren. J Ncrv Mem Dis 141:19~-2fl3. I9(>5 24. Maricq HR: Capillary morphology and lhr course of illness in schizophrenic patients | Nerv Ment Dis 142:63-71. |9(>o 25. Zimmer JG. Demis DJ: Burns and other`L: lesiops: mierocirrular responses in- man durtnt healing. Science 140:994-996. 1963 628 Arthritis and Rheumatism, Vol. 16, No. 5 (September-October 197JI FenoprofenC A Controlled Dou James F. Fries Fenoprofen Cf gesic and antiitold arthritis a study compari with high-dose fective and sul equal to aspiri safety of the d these short-tei likely that Fen tional agent wi Toxicity limits the trmic 'medication cm management of I'hcr Even when a tnedii.o f.utnrilv controlling tinns of the disease. I the patient has often particularly of the "a new agents proposed mic h as cvclophospliar Irr hope of altering tldisease i lowcver. it si of these drugs will bo use in an\ hut the patients. Clearly then From the I trparlmeul "I vtiiMil of Medicine. Staid"' JAMES f. FRIES. MD: Assn jurtment of Medicine. Nt.ii I.-T- Mr.LVIN C- BRITTON, ' 4 Medicine. Department ol SshiKil of Medicine, and a ill Clinic. Reprint requests should fries. Department of Mo t niirrsity .School of Mcdn sotimitted for puhiicaiii'i 2.1973. Arthritis and Rheumatis AP00024389 & i V* it * TSmuAiA/if V- Capillary Abnormalities in Polyvinyl Chloride Production Workers Examination by In Vivo Microscopy Hiidegard R. Maricq, MD; Maurice N. Johnson, MD; Charles L. Whetstone, MD; E. Carwile LeRoy, MD ' Examination by wlde-field capillary microscopy of the hands of 152 workers In vinyl chloride (VC) polymerization plants demonstrated scat tered, scleroderma-like microvascular abnormalities in 21 workers and iso lated capillary abnormalities In 27, as compared with only three Isolated ab normalities In 50 manual workers not exposed to vinyl chloride. Thirteen of' 17 VC workers with objective evidence of VC-assoclated abnormalities (an giosarcoma or tlbrosls ot liver, acroosteolysls, or scleroderma-llke skin le sions) were observed to have microvascular abnormalities. If prospective studies confirm the implications of this study, capillary mi croscopy may become a useful mass-screening procedure in the early de tection and prevention of VC-assoclated disease. (JAMA 236:1368-1371, 1976) INITIAL reports of human disease in which vinyl chloride (VC) has been implicated as pathogenetic were lim ited to descriptions of such peripheral changes as distal phalangeal resorp tion, ie, acroosteolysis (AOL).'-' Re cently, a systemic effect of VC has been suggested by an unusual pat tern of hepatic portal, intralobular, and capsular fibrosis; angiosarcoma of the liver; and pulmonary functional abnormalities suggesting early pul monary fibrosis, all of which occur more frequently among workers em ployed in polyvinyl chloride produc tion plants than among the general population.4-11' Furthermore, animal From the Division of Rheumatology and !m. munology. Department ot Medicine, Medical University ol South Carolina. Charleston (Drs Maricq and LeRoy); The B. F. Goodrich Com pany. Akron, Ohio (Dr Johnson); and Continen tal Oil Company, Ponca City, Okla (Dr Whet stone). Reprint requests to Division ol Rheumatology and Immunology, Department of Medicine, Med ical University of South Carolina, SO Barra St, Charleston, SC 29401 (Dr Maricq). experiments have demonstrated that VC can produce a variety of tumors in rodents."'* There are several similarities be tween VC disease and scleroderma (systemic sclerosis): Raynaud phe nomenon is prominent among the symptoms associated with AOL,,*-*,,,"; localized scleroderma-like lesions have been reported in some patients with AOL5"*; and, in both VC-associ ated disease and scleroderma, smallblood-vessel lesions and interstitial fibrosis are thought to be important in pathogenesis. In scleroderma, changes in small blood vessels of the skin have been observed in vivo by capillary micros-. copy.1*-1* Using the technique of widefield capillary microscopy, distinctive abnormal capillary patterns in the fingers of patients with Raynaud syn drome and scleroderma have been demonstrated.1* Furthermore, a posi tive correlation is noted between the severity of capillary pattern abnor malities and multisystem involve ment in these connective-tissue dis orders." The possibility that VCassociated disease might be charac terized by microvascular abnormal ities similar to those observed in scleroderma led to the present study of VC workers, using techniques of wide-field capillary microscopy. The specific questions of this study were the following; (1) Do symptomatic VC workers have finger-skin-capillary abnormalities? (2) Are the abnormal ities similar to or different from those seen in patients with idiopathic scleroderma and Raynaud syndrome? (3) Are microvascular abnormalities related to the type of VC-associated abnormality (peripheral vs systemic), especially liver abnormalities? (4) Are microvascular abnormalities related to the nature and duration of VC-exposure? (5) Are microvascular abnor malities found in a control group of manual workers not exposed to VC? SUBJECTS VC Workers Selected by Symptoms (n* 44).-The 44 subjects in this group gave a history of symptoms and signs possibly related to VC exposure. They were 31- to 57-year-old men (mean age, 42 years) who had worked in the polyvinyl chloride industry for 5 to 28 years (mean, 15 years). A history of past reactor-cleaning experience (ranging from 0.2 to 8 years; mean, 2.5 years) was present in 35 of 44 workers. Table 1 shows the com plaints and the pathologic findings in group 1. VC Workers Selected by Question- 1368 JAMA, Sept 20, 1976-Vol 236, No. 12 Capillary Abnormalities--Maricq et al AP00024390 Two of the seven patients with ma jor bleeding episodes in the inter rin resistance and embolism. mci. mittent therapy group were switched from intermittent to continuous COMMENT It is difficult to indict the concomi tant use of warfarin and heparin in a treatment. These two patients were In this study, patients receiving in bleeding diathesis since the majority selected because of ease of observing termittent heparin therapy had sig of patients in this study were given bleeding and because of need for fur nificantly more major bleeding epi both drugs simultaneously at some ther treatment with anticoagulants sodes (33%) than patients receiving time, but only two patients bled while (both had pulmonary emboli). One pa continuous therapy (0%). This agrees receiving both drugs Basil et al* sug tient had gross hematuria and one with the findings of Salzman et al' gests that keeping the aPTT greater had extensive surgical wound bleed who reported 1% major occurrences of than lli times the control will prevent ing. Both bleeding episodes were al bleeding in patients on continuous pulmonary emboli in patients on con lowed tp stop, with the return of the heparin treatment vs 8% in the inter tinuous therapy. Five of the 162 pa aPTT to normal prior to instituting mittent group. However, Mant et a!5 tients in that study had a clinical i. continuous treatment, and neither found no difference in bleeding epi ly evident recurrence, generally when patient was given a bolus of heparin sodes between continuous and inter- the aPTT was less than lid times the prior to continuous heparin therapy. mitte.ntly treated patients. In their control for more than two to five Neither patient demonstrated any re study, continuously treated patients days. We could not define a lower currence of bleeding while on contin received a significantly greater daily limit of heparin dosage to prevent re uous treatment. Two patients from heparin dose than the intermittent current thromboembolism since only I!1 { the continuous therapy group were group, whereas in the present study one patient had proved pulmonary changed to intermittent therapy; one and in the one by Salzman et al,' in emboli while on treatment, nor could because of difficulty maintaining an termittently treated patients re we define an upper limit for contin intravenous line and one because the ceived significantly higher doses. The uous treatment since no patients in attending physician thought that discrepancy between the results of the group bled excessively. continuous therapy was inadvisable. these studies may be explainable on Because the question arises of The latter patient had no evidence of this basis: Mant et al* had 12.5% ma whether patients who are bleeding bleeding in the seven day3 on contin jor hemorrhages in tbeir continuous while receiving intermittent treat uous therapy, but four days after therapy group receiving 34,151 units ment can continue to receive anti starting intermittent therapy, heavy of heparin daily compared to no ma coagulants, two patients with severe surgical wound bleeding ensued and jor hemorrhages in our patients re bleeding episodes on intermittent treatment had to be stopped. For sta ceiving 24,000 units of heparin daily. treatment were switched to contin tistical purposes, this patient is It seems probable that the tran uous treatment and in neither case counted as a nonbleeder on contin siently high blood levels of heparin did bleeding resume. This indicates uous treatment. achieved with intermittent therapy that some patients who bleed while predisposes these patients to hemor receiving intermittent treatment Pulmonary Emboli rhage. need not have their anticoagulant Minor bleeding episodes in the pres permanently discontinued if the Three patients were suspected of ent study were also more frequent, heparin can be given by continuous having pulmonary emboli while on but not significantly so, in the inter infusion. heparin therapy because of symptoms mittent therapy group (48%) of pa of either acute dyspnea and chest pain, pleuritic chest pain, or he tients than in the continuous therapy group (30%). In our study, one patient This investigation vu supported by the Oscar Mayer Foundation and Family Trait. moptysis. Repeat lung scans in the (2%) died of hemorrhage, which is latter two patients did not show evi similar to the mortality (2%) in the dence of emboli (one patient on inter report by Salzman et al.' As in a pre Reference* mittent treatment and one on contin uous treatment). The first patient had a pulmonary embolus proved by pul monary arteriogram while on contin uous heparin therapy for dissem inated intravascular clotting. This patient had an aPTT of 49 seconds on the day before the pulmonary em bolus, and antaPTT taken at the onset of symptoms was shortened to 22 sec onds. No change in the mode of con tinuous heparin therapy had been in stituted in the interim. This suggests vious report,* our bleeders were sig nificantly older than nonbleeders, but we could find no female pre ponderance. The use of antiplatelet drugs prob ably is unwise in patients on full-dose heparin treatment, and aapirin may have been a predisposing factor in the two patients who bled heavily when receiving intermittent treat ment. The number of patients receiv ing aspirin in the continuous therapy group was similar to that in the inter- 1. Barritt DW, Jordan SC; Antiooagulant drugs in treatment of pulmonary embolism; A controlled trial. Lancet 1:1309-1312, i960. 2. Salzman EW, Deykin D, Shapiro RM, et al: Management of heparin therapy: Controlled pro spective trial. N Engl J Med 292:1042-1050,1975. 3. Basu 0, Gallos A, Hirsh J, et al; Prospective study of the value of monitoring heparin treat ment with the activated partial thromboplastin time. N Engl J Med 287:824-387,1972. 4. Stock SL, Warner N: Heparin in acid solu tions. Br Med J 3:307,1971. 6. Mant MJ, O'Brien B, Thong KL, et al: Bleed, to be published. 6. Jick H, Slone D, Bonk IT, et al: Efficacy and toxicity of heparin in relation to age and sex. N Engl J Mtd 279:234-286, 1968. JAMA, Sept 20, 1976-Vol 236, No. 12 Heparin Therapy--Glazier & Crowell 1367 j i AP00024391 j.v naire (n= 108).-These subjects were selected on the basis of their answers to a questionnaire used in the poly-vinyl chloride production plant to de tect early AOL. All available subjects who had given one or more affirma tive answers to the questionnaire (Q+) were examined, a total of 66 subjects. Seventy-one subjects were examined from a random selection of those who had given negative an swers to all questions (Q-). Data on 29 of the 137 subjects examined were excluded because of unreliable obser vations caused by skin injuries or deep pigmentation, leaving 108 em ployees (50 Q+ and 58 Q-) in this group. Control Manual Workers (n = 50).The possibility that repeated minor injuries associated with manual labor might contribute to the abnormal findings seen in VC workers led to the examination of workers from compa nies making neither VC nor polyvinyl chloride and hospital employees work ing as carpenters, truck drivers, and maintenance men, a total of 60 work ers. All were men and ranged in age from 19 to 62 years (mean age, 43 years). METHODS Wide6eld Capillary Microscopy.--The technique of wide-field capillary micros copy as previously reported1* consists of the observation of selected skin sites under immersion oil, using a stereo wide-field mi croscope at magnification X12. Wide-field microscopy permits the detection of microvascular patterns, such as the presence and distribution of dilated or deformed mi crovessels and the absence of capillary loops, without attempting to measure ab solute vessel dimensions. The technique is rapid and suitable for mass screening. Twelve standard sites were examined in each subject: four nailfolds and two each of the following sites: dorsum of the distal phalanx other than the nailfold, dorsum of the middle phalanx, dorsum of the proxi mal interphalangeal joint, and the finger pad. Photography of Microvessels.-The widefield photographic technique as previously described" was used to photograph at least one finger of each subject. AU photographs were coded for blind comparison of capil lary findings in VC workers and control subjects. Clinical and Occupational Data.--All sub jects were interviewed as to length of em ployment, job assignment, general state of health, presence or absence of Raynaud phenomenon, and smoking and drinking Table 1.--Clinical Findings and Complaints of Group 1 Vinyl Chloride Workers' Ho. of Subjects No. of Sub- With Capillary iects Examined Changesf Severe AOL with Raynaud phenomenon 64 Scleroderma-like skin lesions with Raynaud phenomenon 54 Liver abnormalities} 6S Subtotal 17 13 Healed AOL with Raynaud phenomenon 60 Healed AOL without Raynaud phenomenon Raynaud phenomenon alone 10 93 Nonspecific skin rashes without Raynaud phenomenon Miscellaneous complaints Subtolal 7 ' 27 1 0 4 Total 44 17 "AOl Indicates aeroosteolyslt. fOf 17 subjects with abnormal capillaries, ten had scattered changes (8/13 and 2/4 of the two groups above) and seven had isolated changes. JTwo patients with angiosarcoma of the liver and one each with (1) portal fibrosis and en larged spleen, (2) portal fibrosis with early cirrhosis and fatty metamorphosis. (3) thrombosis of the splenic vein, mild liver damage, and reticuloendothelial cell hyperplasia, (4) fatty metamorphosis with focal capsular and subcapsular fibrosis and splenomegaly with vascular congestion. Table 2.--Comparison of Capillary Observations Between VC Workers and Controls* 8cittered capillary abnormalities Abnormal capillaries present but lew in number No capillary abnormalities seen Total Employee* el VC Industry 11 27 70 108 Manual Worker* Not Exposed to VC 0 3 47 so *VC Indicates vinyl chloride. Difference In the prevalence of capillary abnormalities be tween VC workers and controls is statistically significant (x1 = 15.50, PC.001). habits. A limited physical examination was performed. All data were coded for later analysis and were unknown to the capil lary observer and photographer. RESULTS Group 1 (VC Workers Selected by Symptoms).--Examination of group 1 subjects showed scattered capillary abnormalities similar to those found in scleroderma (Fig 1 and 2). Defi nitely dilated capillary loops were seen in the nailfold and in other fin ger sites, contrasting with the sur rounding normal vasculature. Pale avascular areas were frequently ob served. The capillary abnormalities were usually more discrete and less numerous than those seen in most pa tients with scleroderma. They were seen more frequently in subjects with severe AOL, liver abnormalities (as defined in Table 1) and scleroderma like lesions (13/17) than in those with healed AOL, nonspecific skin rashes, or .Raynaud phenomenon alone (4/27; X** 14.21, P<.001). Moreover, 5 of 6 subjects with liver abnormalities ver ified by biopsy specimens had microvaseular abnormalities (Table 1). In a few subjects, lesions resem bling mini versions of clinically vis ible papular skin lesions were ob served microscopically (Fig 3, left). In two subjects with active AOL, capil lary hemorrhages were observed un der the proximal nail plate in addi tion to the common type of "splinter hemorrhages" under the distal nail plate (Fig 3, center). Group 2 (VC Workers Selected by Questionnaire).--Dilated capillaries were observed in 88 of 108 group 2 workers; both the number and distri bution of dilated capillaries differed from those observed in most sclero derma patients. Many group 2 work ers had isolated, extremely dilated capillaries (Fig 3, right and Table 2). No correlation was found between the length of time employed as a reactorcleaner (or the length of time with the company) and the capillary abnor malities. Office and supervisory per sonnel who had never been produc- JAMA, Sept 20, 1976--Vol 236, No. 12 Capillary Abnormalities--Marlcq et al 1368 AP00024392 Fig 1 .--Top left, Wide-field view of nallfold area in normal sub ject. Bottom left, Nallfold capillaries of patient with sclero derma. Bottom right, Nailfold capillary pattern in worker of polyvinyl chloride production plant. Note similarity with pat tern seen in scleroderma patients. Fig 2.--Left, Dorsum of middle phalanx in normal subject. Center, Cluster of dilated capillaries in earns anatomical site in patient with scleroderma. Right, Vinyl chloride worker with pattern similar to that seen in scleroderma. Fig 3.--Left, Miniplaque on dorsum of distal phalanx In VC worker with aeroosteolysis (AOL). Cen ter, Capillary hemorrhages under both distal and proximal nail plate in a vinyl chloride (VC) worker with AOL. Right, Single extremely dilated capillary in nallfold of VC worker with liver abnormalities. 1370 JAMA, Sept 20, 1976--Vol 236, No. 12 Capillary Abnormalities--Maricq et al AP00024393 tion workers had fewer microvascular abnormalities (3/22) than production workers (35/86, x'"4.49, P<.05); however, production workers showed no differences in capillary abnormal ities according to job category. There was no significant difference (x\," 1.32, P>.5) between employees who had been classified as Q + or Q- on the basis of the questionnaire. It was noted, however, that there was little or no correlation between the re sponses regarding Raynaud phenom enon obtained from the questionnaire and those obtained from the inter view at the time of examination. Physical examination disclosed two workers with AOL (one "active" and one "healing" by roentgenographic examination) and papular skin le sions, and ten workers with hepato megaly (4 cm or more below the right costal margin). The subject with ac tive AOL showed scattered capillary changes, the subject with healing AOL had a "normal" capillary exami nation. There was no correlation be tween hepatomegaly and capillary findings. Comparison of VC Workers With Control Subjects.-Capillary abnor malities in group 2 VC workers were significantly more frequent than in the control manual workers examined (xJ = 15.50, P < .001) (Table 2). Analysis of Photographic Data.-- Three hundred seventeen uniden tified photographs of VC workers were mixed with 136 photographs of control subjects and 130 photographs of patients with scleroderma and re lated diseases; all photographs were rated for the presence of capillary abnormalities. The results demon strated that significantly more VC workers (39/152) than control sub jects (3/50) had capillary abnormal ities (x,"7.69, P<.01). Although ob jective, for technical reasons, photographs are slightly less sensi tive than direct observations in de tecting capillary abnormalities. COMMENT The results of this study demon strate that capillary abnormalities seen in the finger skin by in vivo mi croscopy are present in VC workers more frequently than in control sub jects. These capillary abnormalities are not exclusively related to pe ripheral pathological conditions such as AOL, scleroderma-like lesions of hands, and Raynaud phenomenon, but are also found in subjects with liver abnormalities (as defined in Table 1) and in subjects who have papular lesions elsewhere on the body. Microscopic papular or fibrotic lesions can also be observed in VC workers. In 33% of the subjects who show any capillary changes, the pat tern appears similar to the micro vascular changes seen in scleroderma; in the remaining majority, changes consist of a few dilated capillaries only; very few control subjects show even these minimal capillary abnor malities. Since microvascular changes occur more frequently than overt VCassociated pathologic conditions in VC workers, they may represent an early manifestation of the VC effect. More work is needed to determine the nature and evolution of such mi crovascular abnormalities, their rela tionship to VC-associated disease (as opposed to a transient reaction to VC exposure), and their relationship to the subject's susceptibility to VC dis ease. Further studies are also needed to compare the capillary changes re ported here with those seen in small blood vessels and endotheiia of ani mals exposed to VC.11 Capillary mi croscopy might then be used as a re producible, noninvasive technique to monitor VC workers and to identify VC-associated peripheral and visceral disease in susceptible individuals at 1 an early and possibly preventable stage. This investigation was supported in put by the Charlotte snd Sidney Lifschultr Foundation, National Institutes of Health grant AM 18904, the South Carolina State appropriation for re search, and the RGK foundation. Irving J. Selikoff, MD. supplied 24 control industrial workers for capillary examination. Karen Menth, Eleanor Gsttoni, and Villu Marioq provided technical assistance. 1. Harris DK, Adams WGF: Acro-oeteolysis occurring in men engaged in the polymerization of vinyl chloride. Sr Med J 3712-714,1967. 2. Wilson RH, McCormick WE, Tatum CF, et ml: Occupational acroosteolysis: Report of 31 cases. JAMA 201*77-581,1967. 8. Dinman BD, Cook WA, Whitehouse WM, et ah Occupational acroosteolysis: I. An epidemio logical study. Arch Environ Health 22*1-73, 1971. 4. Falk H, Creech JL, Heath CW, et ah He patic disease smong workers at a vinyl chloride polymerization plant. JAMA 230*9-63,1974. 5. Lange CE, Juhe S, Stein G, et al: Die aogenannte Vinyl-chlorid-Krankheit-eine berufsbedingte Systcmsklerose? Int Arch Ar heitmed 32:1-32, 1974. 6. Lilia R, Anderson H, Nicholson WJ, et al: Prevalence at disease among vinyl chloride and polyvinyl chloride workers. Ann NY Acad Set 2462241, 1975. 7. Marsteller HJ, Lelbach WK. Muller R, et al: Unusual iplenomegalic liver disease as eri- References denced by peritoneoscopy And guided liver biopsy Among polyvinyl chloride production workers. Ann NY Aead Set 24695-134, 1975. 8. Miller A, Teiratein AS, Chuang M, et al: Changes in pulmonAry function in workers ex posed to vinyl chloride snd polyvinyl chloride. Ann NY Acad Sci 246:45-52, 1975. 9. Sadu I, Prodsn L, lies E, et si: Clinics! manifestations in vinyl chloride poisoning. Ann NY Aead Sci 246*3-69. 1975. 10. Thomas LB, Popper H, Berk PD, et al: Vinyl-chloride-induced liver disease: From idio pathic portal hypertension (Band's syndrome) to apgioaareomas. N Engl J Med 292:17-22,1975. 11. Viola PL, Bigotti A, Caputo A: Oncogenic response of rat skin, lungs, snd bones to vinyl chloride. Cancer Ret 81:516-522, 1971. 12. Msltonl C, Lefemine G: Carcinogenicity bioaasavs of vinyl chloride: Current results. Ann NY Aead Sci 246:196-218, 1975. 13. Markowitz SS. McDonald CJ, Fethiere W, et al: Occupational acroosteolysis. Arch Derm 106219-228,1972 14. Rodnan GP: Progressive systemic sclerosis (scleroderma) and calcinosis, in Hollander JL, McCarty DJ Jr (eds): Arthritie and Allied Cmditime, cd 8. Philadelphia, Lea dt Febiger, 1972, pp 962-1005. 15. David E, Landau J: Clinical Capillary Miavecopy. Springfield, 111, Charles C Thomas Pub lisher, 1966. 16. Marioq HR, LeRoy EC: Patterns of finger capillary abnormalities in connective tissue dis ease by widefield microscopy. Arthritic Rheum 16:619-629,1978. 17. Marioq HR, Spencer-Green G, LeRoy EC: Relation entre la distribution des caplllaires anormaux et le degrd d'envahiaaement syatdmique dans la addrodermie (progressive gindratisde). J Sci Med LiUt 93283-286,1975. 18. Maricq HR: "Wide-field" photography of nailfold capillary bed and a scale of plexus visu alization scores (PVS). Micrmaec Ree 2:335-340, 1970. JAMA, Sept 20, 1976-Vol 236, No. 12 Capillary Abnormalities--Maricq et al 1371 AP00024394 J Opwviwii wv '{ w.-.w l Drug Addiction Among Physicians The Virginia Experience Robert C. Green, Jr, MD; George J. Carroll, MD; William D. Buxton, MD Drug addiction among physician* appears to ba an occupational haz viduals, 5; patients, 2; other state ard, with chronic pain, depression, and the easy availability of drugs major boards, 2; and Medical Society of Vir factors leading to addiction. In this study of 46 cases of physician addicts ginia, 1. handled by the Virginia Stale Board of Medicine, meperidine hydrochloride Regrettably, legislation passed in (Demerol) was the most frequent addictive agent The Virginia disciplinary the 1974 Virginia Assembly no longer and therapeutic plan for addicted physicians was effective lit successfully makes involuntary commitment of rehabilitating and returning to medical practice 72% of the 46 physician ad drug-addicted physicians a manda dicts reported to the board from 1949 to 1974. tory reportable offense to the state (JAMA 236:1372-1375, 1976) board. Reporting of these cases is now up to the treating psychiatrist's judgment, so there may well be fewer AS DRUG addiction in the general Our study may seem small, but the cases coming to the board's attention. population has increased, awareness board believes these 46 cases are Physicians' ages in initial reports of addiction as an occupational .haz merely the "tip of the iceberg." Too to the board ranged from 27 to 73 ard among physicians has also devel often, the problem is handled locally, years, with a median of 45 years. It oped. In 1957, the US Commissioner by revoking hospital privileges or by appears significant that a stair-step of Narcotics estimated that 1 of 100 inducing the addicted physician to progression developed, peaking in the physicians was addicted, as compared move to another state, without at 45- to 49-year group with 13 of the 46 with 1 of 8,000 in the general popu tempts at treatment and rehabilita cases. The incidence in the older age lation.1 In New York state alone in a tion. group was markedly lower. 25-year period, 0.5% of all licensed physicians were reported as addicts to RESULTS OF VIRGINIA STUDY The number of years in practice re lates directly to the frequency of ad the Bureau of Narcotics Control.* A From 1949 to 1960, less than one diction (Fig 2). Median duration of 1969 estimate* of 300 new physician case per year was reported to the practice was 18 years, followed by a addicts per year is probably now too board. Since 1960, the yearly inci gradual decline in incidence of drug conservative. dence increased to a peak of five cases dependency. This communication analyzes 46 in 1968, but has gradually declined Information on duration of addic cases of drug-addicted physicians since. One case was recorded in 1974, tion prior to its being reported was ** handled by the Virginia State Board although two additional cases were available in only 32 cases, but 16 were of Medicine from 1949 to 1974, with reported but not included in this reported within a year of onset It is emphasis on background, personality study (Fig 1). noteworthy that some addicted physi disorder, and probable causes of ad The majority of initial reports on cians could go on for IQ, 16, and, in diction. We also discuss the board's physician addicts in Virginia came one instance, 18 years, without being disciplinary, therapeutic, and rehabil from state and- federal narcotics in apprehended. itative scheme. spectors, with state mental institu Median age of onset of addiction tions and hospitals the next largest was 43 years, with seven physicians From th Virginia State Board of Madicina, Portsmouth. Reprint requests to 230 W Boscawan St. Win chester, VA 22601 (Or Green], source. Their distribution is as fol lows: state and federal narcotics in vestigators, 26; institutions, 10; indi in the 45- to 49-year group (Fig 3). Eight physicians' ages ranged from 22 to 34 years, and two were in the 1372 JAMA, Sept 20, 1976--Vol 236, No. 12 Drug Addiction--Green et al I AP00024395 i 602 THE LANCET, SEPTEMBER 18, 1976 symptoms of influenza. Both women had primary in fluenzal pneumonia which was at its peak about the 7lh day from the first signs of infection. One woman died on the 12th day, and influenza virus was recovered from lung, liver, spleen, tracheal swabs, plasma, and buffy coat. It now seems clear that virsemia can be present in certain cases of influenza from the day before the first respiratory symptoms'0 throughout the first week of ill ness.3* The virus can be present and multiply in lung, liver, and skeletal muscle of ferret and mouse in the absence of light microscopical evidence of viral infec tion." 31 41 A myopathy can complicate influenza infection on about the 7th day.11-1' Enzymatic evidence of a myo pathic lesion has been identified in some patients with Reye's syndrome.*4233 34 * 36 37 38 39 40 41 We believe that certain cases of primary influenzal pneumonia in previously healthy persons, the myopathy associated with influenza, and Reye's syndrome, all of which become evident about the 7th day after the initial respiratory signs of influenza infection, may represent "7th day" dissemination of influenza virus to the affected tissues. This hypothesis has the merit that it can be tested with simple tools already at hand. Requests for reprints should be addressed to). C. P. REFERENCES 1. Reve, R. D. K.* Morgan, G., Baral, J. Lancet, 1963, ii, 749. 2. Panin, j, C., Schubert, V. K., Panin, J. S. Nets Engl. J. Med. 1971, 285, 1339. 3. Pmin, J. C-, Partin, J. S., Schubert, W. K., McLaurin, R. L.J. Neuropath. exp Neurol. 1975,34,425. 4. Partin, J. C. Panin, J, S., Schubert, VC. K., McAdami, A. J., Meljurin, K. I.. Pediat. Res, 1976,10(4), 431. 5. Corey, L., Reuben, R.in Reye'i Syndrome; p 179. New York, 197). 6. Noble, G. R., Corey, L., Rubin, R. J. ibid, p. 189. 7. Reynolds, D. NX'., Riley, H. D,, Jr, Lat-'tml, D. S., Yorae, H., Stout, L> C. Carpenter, R. L. J. Pedia1.1972,19,429. I. Lirmemann, C. C. Jr, Shea, 1.., Kauffman, C. A., Schiff, G. M., Panin, J. C,, Schubert, W. K. Lancet, 1974, i, 179. 9. Powell, H.C., Rosenberg, R. N,, McKellar, B Ardts Neurol. 1973, 29, 135. 10. Shope, R. E.JT. exp. Med. 1934,40,49. 11. Dowdle, NX'. K,, Schild. G. C. in The Influenza Viruses and Influenza (edited by E. n. Rilboume); p. 243. New York, 1975, 12. Lundberg, A. Acta peedtat. stand. 1957,46, 18. 11. Middleton, P. J., Alexander, R. M., Stymanski, M. T. Lancet, 1970, ii, 533. 14. Mejltienkier, j. D., Safroit, A. P., Hetty, J. J. Artks Neurol, 1973,29, 441. 1). Dietzmift, D. E., Schaller, J. G., Ray, G., Reed, M. E. Pediatrics, Spring- field, 1976,57,255, 16. Schubert, U'. K., Panin, J. C,, Panin, J. S. in Progress in liver Disease (edited by H. Topper, and K Schaffncr); p. 489. New York, 1972. 17. Loveioy, F. H., Smith, A. 1... Brein.n, M. J., Wood, J. R, Victor, D. I.. Adams. P. C.Am J. Bit. Child. 1974,128, 3. 18. Plum, F., Posner, J. B. Diagnosis of Stupor and Coma; p. 56. Philadelphia, 1972. 19. Hong, R., schuben, W. K.Am.J. Du. Child. I960,100, 42. 20. Schubert, NX'. K., Bobo, R. C., Partin, J. C., Panin, J. S. Diseise-A-Month, 1975, (Dec.). 21. Burnet. F. M. Br.J. exp. Path. 1940,21, 147. 22. Almeida, J. I)., VPatenon, A. P. Adv. Virus Ret. 1969, 15, 307. 23. Schmidt, N. J., Ixnnette, E. H in Viral and Rickettsial Infections of Man (edited by F. [_ Horsfall. and 1. Tamm);p. 1189. Philadelphia, 1965. 24. Choppin; P. W., Compani, R. V'. in The Influenza Viruses and Influenza (edited bv E. D. Kilbeurne);p, 15. New York, 197525. Turnel, A. I'., Ixvinsohn, M, W,, l)erakhshin, 1., Gutierrez, Y. Areht Neurol. 19/S, 12,624. 26. Unneminn.CC, Jr, Shea, I... Pamn, J. C, Schuben, W. K., Schiff, G. M. Am.J.Epidem. 1975,101,517. 27. Brown, T., Hut, G., Lansky, l.., Bovc, K., Brown, H., Schuben, W. K., Schevc, A., Panin, J. C, Lloyd-Siill, J , Ryan, M. New Engl. J. Med. 1976,294,161. 28. Norman, M. G., Ixwden, J. A., Hill. D. E., Hannatvne, R. M. Can. mcd. Ats.J. 1968,99, 549. 29. Smith, '4'., Andrcwet, C. H., Laidiaw, P. P. Lancet. 1933, ii, 66. 30. Maxwell, E. S-, Ward, T. G* Van Metre, T. E., Jr, J. din. invest. 1949,28, 307. 31. Toms, G. L., Bird, R. A., Kingtman. S. M., Sweet, C, Smith, H. Br.J. txp. Path. 1976, 57, 37. References continued at foot of next column PORTAL HYPERTENSION IN VINYL-CHLORIDE PRODUCTION WORKERS P. M. Smith I. R. Crossley Department ofMedicine, Llandnugh Hospital, PenanH, South Glamorgan, Wales D. M. J. Williams British Petroleum Chemicals International Limited, Sully, South Glamorgan, Wales Summary Portal hypertension was seen in seven patients who had been involved in the production of vinyl-chloride monomer for 4--15 years. Four presented with bleeding asophagcal varices. In liver biopsy specimens non-cirrhotic portal fibrosis was inconspicuous and was seen more clearly on wedge rather than needle biopsy specimens. The intrahepatic venous pattern was hardly disturbed. Three patients have done well following shunt surgery. In one patient an angiosarcoma developed, but fibrosis was a more common lesion and was probably not pre-malignant. Introduction Forty-seven cases of hepatic angiosarcoma have so far been identified in workers exposed to vinyl chloride,1 but there have also been reports of aero-osteolysis, scleroderma-like changes, Raynaud's phenomenon,2'4 pulmonary ventilation disturbances, fibrosis of the por tal tracts,5 portal hypertension, splenomegaly, and thrombocytopenia.6 In the past 9 years we have seen seven vinyl-chloride-monomer (v.c.M.) production workers with portal hypertension in a large chemical plant producing over 100 000 tonnes of poly-vinylchloride annually, and we describe our findings. Case-reports The seven patients were aged 36-57 years at the time of presentation, having been exposed to v.c.M. gas for 4--15 years, for at least part of the time as a polymer reaction cleaner (table 0- Four had a hsemaiemesis from oesophageal varices and two of these were known to have had splenomegaly and thrombocytopenia beforehand because of prior investiga tion for abdominal pain and a skin rsh respectively. One pa tient, who had had a 15-year duodenal ulcer history, had an enlarged fibrotic liver at laparotomy and targe collateral veins, and later died from an hepatic angiosarcoma. Two subjects were found to have thrombocytopenia during a survey, and this was later shown to be due to' hypcrsplenism associated with portal hypertension. Six of the patients had hepatomegaly and four a palpable spleen. liver function tests showed non-specific and relatively minor abnormalities (table u). One patient was minimally iaundiced, another had a raised serum-alkaline-phosphatasc, PROFESSOR PARTIN AND OTHERS: REFERENCES--continued Si Andcnon, R. Am.}, /Mi*. IVSt. IS, >/'/. 33. LsiUliw, F.I>. Untet, 1935,i, till. 34. Davenport. P. M. Beet. Rev, 1961,25, 294. 35- Hrighiman, f. J., Trask, J. D. Am. J. Dii. Child. 1936, 52, 67. 36. Smocodmiiev, A. A.,Otfrovtkavi, S.M.J. PatJk. Bad. 1937,44, 559. 37. Vagner, R. R. Virology, 1955, 1,497. 38. Rasarab, 0., Smith, II. Br.J. txp. Path. 1970,51, 1. 39. Lehmann, N. I., (rust, I. D. Mtd.J, Aust. 1971, ii, 1166. 40. Stanley, li. I)., Jackson, G. G. Trans. Am. Ass. Phyns, 1966,79, 376. 41. Smith, VC., Andrewev, C. H,, laidiaw, P. P; Bt.J. exp. Path. 1935,16, 291. 42. Roe, C R., Schonberger, 1.. IT, Gclbach, S. H., Vies, L A., Sidbury, J. B., ft.Pedtatnes,Springfield, I975,55i 119. AP00024396 THE LANCET, SEPTEMBER 18, 1976 603 TABLE I--DETAILS OF 7 MALE V.C.M. WORKERS WITH PORTAL HYPERTENSION Case A(sc liipoture no (yr) (yri Presentation 1 4) 2 48 3 34 4 36 5 57 6 32 7 43 15 Abdominal pain. Hsmaiemests. 6 Skin rath, ilgmatemetis. 6 1 licifiatcmcm. 4 Duodenal ulcer. 13 Hatma kinesis. 8 thrombocytopenia on survey.' ID Hwombocytopema on survey. Uvet (cm) 4 4 4 4 6 0 4 Spleen (cm) 4 2 4 0 0 0 4 and two had raised transaminase levels. Four out of six tested had an abnormal bromsulphalein retention. Plasma-protein values and serum electrophoresis were normal except for one low albumin level. Low platelet-counts of 39 000-80 000/mm1 were present in five out of six subjects. Tests for antinuclear factor, hepatitisassociated antigen, and mitochondrial and smooth-muscle antibodies were negative or very weakly positive, except for case 2, who had smooth-muscle antibodies detected in his serum in a titre of 1/64. No patient gave a history of hepatitis, contact with hepatotoxins such as arsenic, or ingestion of known hepatotoxic drugs, but one (case 5) drank to excess, consuming a bottle of wine a day. lntrasplcnic pressure in cases 2, 5, 6, and 7 was raised it 30, 25, 30, and 24 mm I)g. The wedged hepatic vein pressure in cases 6 and 7 was slightly increased at 12 and 14 mm Hg re spectively. Splenic venography was performed in every case, demonstrating the presence of a patent portal vein and tcsophageal varices. Despite this, the intraspienic venous pat tern appeared normal or only very slightly abnormal in four patients. At laparotomy the liver appeared finely nodular and cir rhotic, but biopsy revealed a non-cirrhotic fibrosis in four pa tients (cases 1, 3, 5, and 7) and slight fibrosis in cases 2 and 6. There was loss of portal-vein branches in cases 2 and 7. The degree of fibrosis in most patients was relatively minor, mainly involving the portal tracts only. In case 2 a needle biopsy speci men was normal, and the fibrosis was delected only when a wedge biopsy specimen was taken during a portacaval shunt. Case 4, who later developed an angiosarcoma, showed a remarkable focal severe dilatation of the sinusoids, and has been separately reported.' Of the four patients who bled from oesophageal varices, one died two months after an oesophageal transection from hepatic failure and Etcherickia coli septicxmia. The other three (cases 2, 3, and 5), have done extremely well after end to side porta caval shunts, surviving so far for 3, 6, and 8 years respectively. All are on an unrestricted protein intake and show no signs of .encephalopathy apart from a trace of hepatic fetor. Two have returned to full-time work. Case 6 has recently had a small varicea! hsmateinesis and is being considered for surgery. Discussion Much publicity followed the discovery of angiosar coma in workers exposed to vinyl chloride.* but it it our experience that non-cirrhotic portal fibrosis with as sociated portal hypertension is a commoner lesion. The fibrosis has not yet proved malignant in that six of our cases have been followed for 3-8 years with no deterio ration in liver function; all have been removed from con tact with v.c.m. The three workers who have undergone a portacaval shunt have fared particularly well, suggest ing that hepatic parenchymal function is preserved. The apparently'norma! imrahepatic vascular pattern seen in some of our patients on splenic venography, and the high intraspienic pressure with a slightly raised wedged hepatic vein pressure, indicating a predominantly presinusoida! obstruction to portal flow, are similar to the findings in non-cirrhotic portal fibrosis seen in India10 and in chronic arsenic ingestion.11 Hepatic fibrosis was also observed in v.c.m. workers by Lange et al.,1 who described capsular and portal fibrosis, oesophageal varices, splenomegaly, and thrombo cytopenia in addition to thickening of the collagen bun dles of skin. Thomas et al.1J found in 14 workers exposed to v.c.m. an inconspicuous intralobular and a conspicuous capsular fibrosis in tumour-free portions of liver, with an increase in perisinusoidal reticulin. It has been postulated that a metabolic product of vinyl chloride binds to plasma-protein producing a conforma tional change within the protein molecule.1' This acts as an antigen that stimulates antibody production. Anti body and antigen then form a soluble complex, which is cryoprecipitable and can activate complement, and can result in platelet aggregation, thrombocytopenia, and vas cular occlusion. The vascular occlusion would stimulate new collagen biosynthesis.14 While this mechanism may be responsible for hepatic fibrosis and portal hyperten sion, we believe that the thrombocytopenia is a manifes tation of the hypersplenism, as reduced haemoglobin and white cell levels have been seen in some instances, and there is no improvement on removal from contact with V.C.M. It has been suggested that one of the results of pro longed exposure to v.c.m. may be splenomegaly due to stimulation of splenic cells with a consequent increase in splenic and hepatic blood-flow.12 Portal hypertension may result from an inability to accommodate this in creased hepatic blood-flow because distension of portal vein branches is prevented by portal fibrosis, and thal of the sinusoids by capsular and subcapsular fibrosis. A similar mechanism may also operate in Banti's syn drome. Further investigations to determine the contribu- IAUi.1- l| - MlOCIIt.UlLAl. AND IIAtMATULOUICAL DATA IN V.C.M. WOXKKRS WITH l*OKTAl HVI'fcKfltXMOS Case no. 1 2 3 4 5 6 7 A>Wu/ Bilirubin (umol/l) 44 17 17 17 17 18 11 17 Alkaline phosphatase U-l/l) 3(1 44 15 35 US 31 32 15-45 White blood cell count Aspartate transaminase 1 10 38 64 64 20 15-32 Albumin (8/1) 38 36 39 31 24 37 34 Globulin (|A) 24 17 20 30 31 32 34 35-50 15-45 8.j.r.=bromoiulphaiein. B.h.F. cu 15 21 17 5 7 19 5 (45 min) lib (8/41) 12 2 10 0 173 14 0 120 IS 4 13-7 u.ax. (/mm1) 6900 12000 4700 4000 1300 6100 4700 Platelets (/mm*) 56 000 80 000 170 000 $4 000 61 000 39 000 AP00024397 604 THE LANCET, SEPTEMBER 18, 1976 tion of splenic blood-flow to the portal hypertension are planned. With improvements in technology, workers should no longer be exposed to levels of v.c.m. gas above 5 p.p.m. and it is hoped that no new cases of portal hypertension and hepatic fibrosis will be seen. However, we are left with the problem of how to screen workers exposed to high concentrations of gas in the past. As hepatic paren chymal Function is preserved, tests that rely upon con ventional liver function tests have proved disappointing but we have identified two workers with portal hyper tension through thrombocytopenia secondary to hypersplenism. However, we believe that conventional tests are not sufficiently sensitive and preliminary work suggests that grey-scale ultrasonography may be more successful in detecting abnormalities.15 Wc thank Prof. P. J. Schcuer and Dr D. M. D. Evans for their in terpretation of the liver histology and Dr G. de B. Hinde for perform ing the splenic venograms. Requests for reprints should be addressed to PM.S. dins has a central role in the pathogenesis of Banter's syndrome. Introduction Banter's syndrome is an uncommon disorder characterised by hypokaUemia, hyperreninzmia, hyperaldosteronism, juxtaglomerular hyperplasia, normotension, and resistance to the pressor effects of angio tensin it. The pathogenesis is unknown and treatment has been largely unsatisfactory. It has been suggested1-3 that overproduction of renal prostaglandins is of patho physiological importance. This! study further examines the role of prostaglandins in the pathophysiology of Banter's syndrome and describes the successful use of aspirin in the treatment of a patient with this disorder. Case-report A 22-month-old, 7 kg girl was referred to Walter Reed Army Medical Center following a 13-month history of muscle wcak- REFERENCES 1. Yeomtn, E. Penonil commumctiion. 2. Walker, A. E. Pree. JI. So*. Mcd. 1976,69, 21. 3. Cerdier, J. M., Fievei, C., Lefevre, M. J., Sevrin, A. Cak. mid. Tratml* 1966,4, 3. 4. Sucui, 1.) Drcjnun, 1., Valajkai, M.Mtdn* interna, 1963,15( 967. 5. Lange, C. E., Juke, $., Stein, G., Veltmao, G. Ini. Arch. Arbtiumtd. 1974, 32,1 6. Juhe, S., LaAge, C. ., Stein, G., Vcltman, G. Dt. mcd. XPschr. 1973, 91, 2034. 7. Smith, P. M., Williams, D. M. j., Evans, D. M. D. N.V. Acad. Mcd. 1976,52,447. t. Creech, J. L, Jr., Johnson, M. M.Joccnp. Mcd. 1974,14, 150. 9. Lancet, 1974, i, 1323. 10. Sams, S. K, Bhargava, $., Gopi Nath, N., Talwir, J. R., Nayak, N. C. Tendon, B. N., Wig, K. l.Am.J.Med. 1971,51, 160. 11. Morris, J. S-, Schmid, M., Newman, S., Schcuer, P. Sherlock, S. Gat- troentcrology, 1974, 64, S6. 12. Thomas, L. B., Popper, H., Berk, P. D,, SelikolT, I., Falk, H. Ncu. Engl. J.Mtd. 1975.292. 17. 13. Ward, A. M., Udnoon, S., Watkins, J., Walker, A. En Darke, G S. Br. mcd. J. 1976, t, 936. 14. Jayson, M. 1. V., Bailey, A. J., Lloyd-Jones, K. Prec. ft. Sac. Mcd. 1976, 69, 295. 15. Taylor, K. J. W., Barrett, J. J., Williams, D. M. J., Smith, P. M., Duck, B. W. ibid. 1976,69, 292. 120 90 aol PROSTAGLANDINS AND ASPIRIN THERAPY IN BARTTER'S SYNDROME L. Norby W. Flamenbaum R. Lentz P. Ramwell Department of Nephrology and Pediatrics, Walter Retd Army Institute of Research and Medical Center, and Department of Physiology, Georgetoun University Medical Center, Washington, D.C., USA. Summary A young patient with Banter's syn drome was treated for three months with 100 mg/kg/day of aspirin to inhibit prostaglandin syn thesis. Clinical symptoms resolved and serum-potassium increased from 2-9 to 3-5 mmol/1. Urinary excretion and plasma concentration of prostaglandins E and F were significantly reduced during aspirin therapy. Plasmarenin activity declined from 85 to 20 ng/ml/h (normal 1-5-4 ng/ml/h) and hyperaldosteronism was corrected. These results suggest that overproduction of prostaglan- - 30 <C 0S 0AY Fig, 1--Effect of uplrifi oa pillmi-reala activity, aldoatcroac excretios, ud potauium balance. ness, growth retardation, polyuria, and polydipsia. Develop ment was normal. Hypokalemia (serum-potassium 2-4 mmol/l) was noted three months before admission and per sisted despite 40 mmol/day of potassium-chloride supplements. Physical examination was unremarkable except that height and weight were both below the first percentile. Blood-pressurc was 90/60. A diagnosis of Banter's syndrome was based on persistent hypokalsemia with inappropriate urinary potassium losses, raised plasma-renin activity, increased urinary aldos terone excretion, and exclusion of other known causes of hypokalemia. In response to S mmol/day 3odium restriction the patient excreted less than 1 mmol/day in the urine. After 5 days of oral salt loading, plasma-renin activity was sup pressed from 74 to 47 ng/ml/h. A percutaneous renal biopsy specimen disclosed vascular thickening with juxtaglomerular transformation of ancriole cells, increased number of imma ture glomeruli, and atrophy of macula-densa cells. The renin AP00024398 THE LANCET, SEPTEMBER 18, 1976 605 600r URINE pce Ng/QAYI 600 |-------- ASPIRIN-------1 lOO/mg/kg/OAY t 400 PLASMA POE* Pg/Ml 0 URINE PGf Ng/OAY 0 Fig. 2--'Acute effect of aspirin on urine end plaamn preecaflnndiae. activity of a juxtaglomerular apparatus isolated from the biopsy specimen4 was 181 ng/h. This value is three limes greater than any other human specimen we have analysed (un published observations). Materials and'Methods During the balance study (figs. 1 and 2) the patient received a metabolic diet containing 20 mmol of sodium and potassium per day. Intake and urinary excretion of electrolytes were monitored daily; fecal losses were not measured. Blood-sam ples were obtained at a A M. with the patient supine. Plasmarenin activity and urinary aldosterone were measured by radioimmunoassay. Immunoreactive prostaglandins of the E-type (r.c.E) and F-type (p.g.F) were determined in plasma and urine as previously described.' All prostaglandin analyses were done in duplicate and the variation between paired sam ples did not exceed 10%. Results The results shown in figs. 1 and 2 demonstrate the acute effects of aspirin. During control observations (days 1-S) the patient's weight remained constant and she was hypokafemic and in negative potassium balance. Plasma-renin activity was markedly raised at 119 and 72 ng/ml/h (normal 1-5--4 ng/ml/h). Urinary aldosterone excretion, uncorrected for body surface area, averaged 14 pg/day (normal adult <20 pg/day). Daily urinary excretion of f.g.E and p.g.F averaged 225 and 252 ng respectively. Plasma-p.G.E ranged from 224 to 363 pg/ml and plasma-p.G.F was 218 pg/ml. The pa tient was given 100 mg/kg/day of aspirin in divided doses beginning on day 6. The same measurements were taken for five consecutive days beginning on day 8. Dur ing this interval the serum-salicylate was 15-20 mg/dl. After beginning aspirin, as shown on the right hand por tion of fig. 1, the patient gained weight, potassium balance became positive, and serum-potassium in creased. Corresponding to the 0-6 kg weight-gain was a combined cumulative positive balance for sodium and potassium of 75 mmol. Plasma-renin activity decreased to 22 and 10 ng/ml/h and urinary aldosterone excretion was reduced by approximately 50%. The* effects of aspirin on plasma and urine prostaglandins are shown in fig. 2. Plasma and urine p.g.E were reduced by approxi mately 90% whereas plasma and urine p.g.F declined to 50% of control values. After completing this phase of the study, the patient was discharged from the hospital and maintained on the same dose of aspirin. The same parameters were fol lowed at weekly intervals for 10 wk and these results are presented in the accompanying table. During this time the patient continued to gain weight. Muscle weakness and polyuria resolved. Appetite and activity level in creased. Serum-potassium averaged 3-6 mmol/1 on aspirin, as compared to 2-9 mol/1 during control obser vations. Plasma-renin activity continued to be sup pressed, although not completely into the normal range. Urinary aldosterone excretion remained significantly below pretreatment values throughout the period of observation. In response to aspirin, mean urinary p.g.E excretion declined from 225 to 25 ng/day and p.g.F dec reased from 252 to 99 ng/day. Plasma-p.G.E declined from 293 to 23 pg/ml and plasma-p.G.F was reduced from 218 to 110 pg/ml. After ten weeks of treatment, aspirin was temporarily discontinued for four days. This resulted in a 0-5 kg weight-loss, negative potassium balance, and a fall in serum-potassium to 2-6 mol/1. Plasma-renin activity in creased to 85 ng/ml/h and urinary aldosterone increased two fold. Urinary excretion of p.g.E and p.g.F returned to pretreatment values as did plasma-p.G.F. In contrast, plasma-p.G.E increased only slighily. At the time of writ ing the patient has been on aspirin for 3 months and scrum-potassium is 3-5 tnmol/1 without potassium sup plements. Discussion Fichman et al.J reported the first successful use of indomethacin in a patient with Banter's syndrome. In their patient indomethadn caused a fall in plasma-aldasterone, plasma-renin activity, and prostaglandin A2. Vascular insensitivity to angiotensin n was reversed and potassium balance improved. Banter et al.1 reponed in creased urinary excretion of p.g.E, and p.g.F, in 5 pa tients with this disorder. When these subjects were DATA THOM TATIANT DURINO CONTROL PERIOD AND CHAONIC ASPIRIN THERAPY Vciftu Scrum*poiaium PlasnU'ftnio aaivity Urinc*ildmteronr SmiRMtiicylaie Urine-FU-W Plumi-F-O F Plaimi-r.c.F - (ramol/l) (ng/ml/m) (o^dty) (mg/dl) ing/ilay > injAiayi (pg/tnl) (Pg/tnlJ Comrol period Mem iii-E-M.)- Aspirin*therapy period Mem +(J4J*.) T 7*0i0*2 10+0-1 <001 2*tO*l 3<i0-* <0*01 J11 29tS <001 1442 642 <o-oV 0 15 1*1-5 22$ + $6 25 r* <0 01 252*59 99tii >005 293i69 23 + 5 <001 21*i21 110.12 <001 After five control otuemtiona thr pallet* wu pen 100 tni/t|/dey of aapitin. Obttrmlisiu were then made at weekly interval, for 10 wk. 606 THE LANCET, SEPTEMBER 18, 1976 treated with inhibitors of prostaglandin synthetase, serum-potassium rose and both plasma-renin activity and urinary aldosterone excretion decreased. Verbeckmoes et al.2 also noted a favourable therapeutic response to indomethacin but' prostaglandin measurements were not reported in their subject. The present study provides additional evidence supporting a central role for pro staglandins in the pathogenesis of Bartter's syndrome. In this patient aspirin therapy was associated with a sig nificant fall in urine and plasma prostaglandins, reduc tion of renin and aldosterone production, and correction of hypokalaemia. It seems unlikely that these results re flect some action of aspirin other than inhibition of pro staglandin synthetase. The changes in urine and plasma prostaglandins were clear cut and aspirin does not usually cause significant alterations in sodium or potas sium metabolism. The stimulus for increased prostaglandin production and the mechanism relating prostaglandins to other aspects of this disorder are not understood. Factors which are known to affect renal prostaglandin produc tion include renal nerve stimulation, catecholamines, bradykinin, angiotensin, ischsemia, and the state of sodium balance. The normal pulse-rate, blood-pressure, and vascular resistance6 in these patients, does not sup port overactivity of the adrenergic nervous system as the stimulus for increased prostaglandin production. Vinci et al.T reported a decrease in plasma-bradykinln and urine-kallikrein in several patients with Banter's syndrome after inhibition of prostaglandin synthetase. This suggests that kinins do not stimulate the overpro duction of prostaglandins in Banter's syndrome, but rather are secondary to increased prostaglandin syn thesis. Similarly, in both our patient and those reported by Banter et al.1 and Fichman et al.,3 inhibition of pro staglandin synthetase resulted in a fall in plasma-renin activity suggesting that this disturbance is also second ary to overproduction of prostaglandins. Renal sodium handling in Bartter's syndrome is a controversial issue. Despite the fact that many of these patients conserve sodium normally, several workers have proposed primary defects in sodium absorption in the proximal ascending limb or distal portions of the nephron.*-11 Many of these proposed defects in sodium trans port may have been secondary to prolonged potassium depletion.13 In two reports of patients with this dis order7 14 renal sodium absorption was normal following correction of potassium deficiency. Our patients showed normal renal sodium conservation and displayed no evi dence of extracellular volume contraction. This argues against negative sodium balance as a stimulus for in creased prostaglandin production. Irrespective of the stimulus for increased synthesis, several observations suggest that prostaglandins have an important role in the vascular resistance to angiotensin it. This vascular defect is an order of magnitude greater in these patients than in any other disorder and is usually not corrected by expansion of the extracellular volume or replacement of potassium deficiency.12 14 Several investigators have presented evidence in animals1516 and humans17 showing that prostaglandins are important modulators of the vasoconstrictor response to angiotensin it. Verberckmoes et al.2 and Fichman et al.3 have reported reversal of angiotensin 11 insensitivity in Bartter's syndrome after treatment with indomethacin. Before the use of prostaglandin inhibitors, the thera peutic focus in Bartter's syndrome was correction of hypokalscmia. Therapy with renin inhibitors, aldos terone antagonists, and potassium supplements alone or in combination have been unsuccessful in maintaining normokalsemia for a prolonged period. In our patient treatment with aspirin alone resulted in resolution of clinical symptoms and correction of hypokalaemia. There were no adverse side-effects. These observations are particularly relevant to the treatment of Bartter's syndrome in paediatric patients where the safe use of in domethacin and other inhibitors of prostaglandin syn thetase has not been established; We are grateful to Dr Sheldon Sommers for evaluating the renal biopsy and to Ms C. Armstrong for performing the prostaglandin assays. Requests for reprints should be addressed to L. N.t Department of Nephrology, Walter Reed Army Institute of Research, Washington, D.C. 20012, U.S.A. REFERENCES 1. Barttcr. (>.. Gill, J., F'roltch, J.. Bowden, R llollifuld, J., Reiser, H, Oates, J .Sevtwrih, H., Taylor. A. Clin. Res. 1976,24, 490A. 2. Verbeckmocs, R., Van Damme, B.. Clement, J., Amery, A., Michielsen, P. Kidney Int. 1976,9, 302. 3. Fichman, M., I dfer, N., /.ia, P., Speckart, P.. Golub, M., Rude, R. Am.J. Med. 1976.60,715. 4. F'lamcnbaum, W., Hamburger, R. f. din. Invest. 1974,54, 1373. 5. Ramwell, l\ W., Heicher, J. R.. Ftamenbium, W. Proceedings of the V[tb International Congress on Phirmoeology Helsinki, July, 1973. 6. Norby, l_, Mark, A., Ka!oyanidet,G. CI/n. Nephr. (in the press). 7. Vinci, J., Tetles, D., Bowden, R.. lo, J., Reiser, H., Ridfon, N., Taylor, A., Gill, J., Bartier, V. Clin. Fes. 1976,24, 414A. S. Cannon, P,, Learning, J., Sommers, S , Vinters, R., Uragh, J. Medicine, Baltimore, 1968,47, 107. 9. Goodman, A.. Vagnuect. A., Hartroft, P. Net* Engl. J. Med. 1969, 261, 1435. 10. White, M. Archs tntern. Med. 1972,129, 41. It. Chaumoum. C. 1-evi. J., Better, O., (Islander, l~, Renduli, A. Ftdiat. Fes. 1975,7. 99. 12. Ilartier, F,, Dcba, C., Kawasoki. T., Gill, J. Kidney Jut, 1974, 4, 272. 13. Rubidtu, M.J, c/m. Invcri. 196 f, 40, 2215. 14. Soloman. R., Brown, R. Am.J. Med. 1975, 59, S75. 15. Aiken, J., Vane, J.J. Pharm. exp. The*. 1973, 184, 678. 16. Hcrbaczyneska-Cedro, K., Vane, J. Circulation Res. 1973,33, 428. 17. Golub, M., Speckart, P., Horton, R, Clin. Res. 1975,23, 128A. TOTAL HIP REPLACEMENT IN RENAL TRANSPLANT RECIPIENTS WITH ASEPTIC NECROSIS OF THE FEMORAL HEAD L. Are Gustafsson Marvin H. Meyers Thomas V. Berne Department of Surgery, University ofSouthern California School of Medicine and the Los Angeles CountyUniversity ofSouthern California Medical Center, Los Angeles. California 90033, US.A. Summary Aseptic necrosis of the femoral head is a complication of renal transplantation which seriously delays the rehabilitation of the patient despite otherwise successful transplantation. Of 187 renal allograft recipients, 8 underwent 14 total hip re placements. The most severe postoperative problem was an easily reducible dislocation. All patients were relieved of their severe preoperative pains and all were greatly improved in mobility and strength. Renal function was not altered in any patient..It is therefore suggested that total hip replacement be recommended in such cases at AP00024400 . 'hlV-L~Z/UAA/ Original Contributions Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant Henry Falk, MD; John L. Creech, Jr., MD; Clark W. Heath, Jr., MD; Maurice N. Johnson, tyD; Marcus M. Key, MD Eleven cases of hepatic disease, including seven cases of hepatic ang iosarcoma, have been Identified to date among men employed at one vinyl chloride polymerization plant. The earliest diagnosis was made in April 1964. The two most recent cases, both angiosarcoma, were diagnosed in February 1974 as a result of systematic medical screening for liver abnor malities among workers at the plant. Ages at diagnosis have ranged from 36 to 58 years for the seven patients with angiosarcoma and tram 28 to 56 years for the four patients with nonmaiignant disease; durations of employment before diagnosis have ranged from 12 to 28 years and from 5 to 29 years. All 11 persons had worked In close and continuous contact with various phases of the vinyl chloride polymerization process. Review of pathologic material suggests the presence in both tumor and nontumor cases of portal fibrosis and atypical sinusoidal lining cells. A direct causal relationship between ex posure to vinyl chloride monomer and pathologic findings Is postulated. (JAMA 230:59-63, 1974) CREECH and Johnson' recently re ported the occurrence of three cases of angiosarcoma of the liver among workers at a polyvinyl chloride (PVC) production plant in Louisville. Be cause this tumor is extraordinarily rare (only about 25 cases are esti mated to occur each year in the entire United States), the existence of such cases in this particular setting See also p 64. strongly suggests a causal relation ship to some phase of the PVC pro duction process. Recent animal stud ies being conducted in Italy support the concept that exposure to vinyl chloride monomer (VCM) may be the mechanism involved (C. Maltoni et al, unpublished data). Beginning in late January 1974, in- From the Cancer and Birth Defects Division. Bureau of epidemiology, Center for Disease Control. Atlanta. Ga (Drs. Falk and Heath), the B. F. Goodrich Company, Louisville, Ky (Dr. Creech) end Akron, Ohio (Dr. Johnson), and the National Institute for Occupational Safety and Health, Center for Disease Control, Rockville, Md (Dr. Key). Reprint requests to Cancer and Birth Detects Division, Bureau of Epidemiology, Center for Disease Control. 1600 Clifton Rd, Atlanta, GA 30333 (Dr. Falk). tensive efforts have been devoted to clinical and epidemiologic studies of past and present workers at the Louisville plant, as well as at other PVC production plants elsewhere, in order to define more precisely the ex tent of health risks among vinyl chlo ride workers. This report summarizes findings to date with respect to both malignant and nonmaiignant hepatic disease among workers at the Louis ville plant, particular emphasis being given to epidemiologic features. BACKGROUND The production of PVC by polymer ization of VCM began in Germany about 40 years ago, with production in the United States starting about five years later. The industry grew rapidly after World War II, and growth has continued in recent years at a rate of about 14% per year. Cur rently in the United States, 14 plants employing about 1,500 workers pro duce VCM, while 37 plants employing about 5,000 workers polymerize PVC from VCM. Current annual produc tion of PVC in the United States is estimated at approximately 4.4 billion pounds, or 25% of world production. The B. F. Goodrich PVC polymer ization plant in Louisville first began operations in 1942. The number of persons employed at the plant di rectly in PVC polymerization has steadily increased, reaching, a fairly stable level of 250 to 300 workers by the late 1950s. At present, in addition to 271 persons engaged directly in PVC polymerization, about 850 per sons are employed at the plant in other activities such as synthetic rub ber production, compounding and milling operations, managerial and clerical positions, and maintenance work outside PVC polymerization areas. Until 1966, VCM as well as PVC was produced at the Louisville plant. Since that time, however, all VCM used at the plant has been shipped by tank car from other facilities. The VCM is unloaded, stored, and then piped into large polymerization re actor vats through an essentially closed system. Each reactor receives a measured amount of VCM as well as appropriate catalysts, stabilizers, emulsifiers, and additives (and other monomeric compounds if copolymers or terpolymers are being produced), and the reaction is carried to the de sired end point Polymerized material is dropped into secondary tanks from which unreacted VCM is recovered and recycled through a closed system; it then enters tertiary tanks from which it is concentrated, dried, and packaged. The end product consists of three different materials: (1) PVC resin (a powder with the texture of refined sugar), (2) PVC paste (a very fine powder with the texture of pro cessed flour), and (3) PVC latex (a stable suspension of PVC in liquid). For workers in PVC polymeriza tion, the point of greatest probable exposure to VCM occurs after poly- JAMA, Oct 7, 1974 e Voi 230, No 1 Hepatlc Disease--Falk et al. 59 AP00024401 f and Pathologic Findings of Cases of Liver Disease Physical Findings 1/64-Rlght upper quadrant tenderness 8/67-Epigastric mass and splenomegaly 1/70-No abnormalities 5/70-Hepetosplenomegaly t2/63-Na abnormalities 5/65-Splenomegaly 7/73-Hepatosplenomegaly 2/74-No abnormalities 2/74-No abnormalities Hepatic Work-up* 1/64-Moderate elevation in TB. SGOT 8/67-Mlld elevation in AP, SGOT. LDH Platelet count. 33.000/cu mm Liver scan; large defect, splenomegaly 5/70-Mlld elevation In TB. AP, SGOT. LDH Liver ecan; large defect Esophooram: varicaa S/65-Elevated SGOT Eaophagoscopy: varices 7/73-Marked elevation in AP Mild elavation In TB. SGOT Liver scan: diffuse disease, hepatosplenomegaly 2/74-Mild elevation in LDH Liver scan: possible defect 11 /73-Mild elevation In TB. AP Liver scan and angiogram; 4-cm defect Pathologic Pindinget 1/64-OLB: slight focal hepatitis 3/64-NLB: unchanged 4/64-PM: hepatic angiosarcoma with metastases 8/67-OLB: angiosarcoma 1 /68-PM: hepatic angiosarcoma with spread to diaphragm and abdominal wall 5/70-OLB: angiosarcoma No PM 5/70-OLB: toxic hepatitis 10/70-NLB: hepatitis, cirrhosis 10/70-OLB: slight hepatitis 3/73-PM; hepatic angiosarcoma 7/73-NLB: fibrosis OLB: periportal Inflammation and fibrosis 12/73-PM: hepatic angiosarcoma spread to duodenum 2/74-OLB: angiosarcoma 2/74*OLB: angiosarcoma and extensive portal fibrosis, subcapsular fibrosis 11/68-Splenomegaly . ' 9/71-Hepatosplenomegaly 8/73-Splenomegaly 9/73-No abnormalities 10/68-Marked elevation In AP, BSP Mild elevation In SGOT Esophagram: varices 9/71-Mild elevation In TB, SGOT 10/71-Liver scan: splenomegaly 3/73-Moderate elevation In TB, AP Liver scan: splenomegaly, small liver 9/73-Moderate elevation in SGOT Mild elevation In TB. LDH 11/68-OLB: portal fibrosis, subcapsular fibrosis 12/71 -OLB: portal fibrosis, subcapsular fibrosis S/73-OLB: slight portal fibrosis 9/73-OLB: chronic hepatitis with focal fibrosis *TB Indicates total bilirubin; SGOT, scrum glutamic oxaloacetic transaminase; AP, alkaline phosphatase; LDH, lactic dehydrogenase; BSP, sulfobromophthalein. tOLB indicates open-liver biopsy; NLB, needle liver biopsy; PM, postmortem examination. (case 6), or angiosarcoma with exten tients (cases 8 through 11) range from liver biopsy to have some degree of sive portal fibrosis (case 7). In cases 1 28 to 56 years (average age, 46.5). Ini hepatic fibrosis. Pathologic review of and 2, large hepatic masses were tial clinical manifestations varied specimens available from these sug present at initial evaluation. The con widely: one patient (case 8) had gas gests a close histologic similarity to ditions of three patients (cases 1, 4, trointestinal bleeding; one (case 9) the manifestations of portal fibrosis and 5) were not diagnosed until au had chest pain and weight loss; and and sinusoidal changes described in topsy, despite multiple liver biopsies. two patients (cases 10 and 11) had the cases of angiosarcoma. Three pa Preliminary pathologic review sug unrelated problems. On physical ex tients (cases 8, 9, and 11), as well gests that in all 5 patients for whom amination, hepatosplenomegaly was as one patient in the angiosarcoma nonmalignant hepatic tissue is avail present in one patient (case 9), sple group (case 7), were found at surgery able (cases 2, 3, 4, 6, and 7), similar nomegaly alone in two (cases 8 and to have a peculiar white, speckled ap nonmalignant hepatic lesions exist, 10), with normal findings in one pa pearance to the surface of the liver, consisting of portal fibrosis, sinusoi tient (case 11). Three patients under which on pathologic section was seen dal dilation, and atypical sinusoidal lining cells. Conceivably, such lesions may represent a precursor stage in went splenectomy either for marked splenomegaly (case 9) or as part of splenorenal shunt procedures (cases 8 to reflect diffuse subcapsular fibrosis. EPIDEMIOLOGIC FINDINGS the development of hepatic angiosar and 10). Results of liver function tests The seven men with angiosarcoma coma. varied widely and showed no consis had been employed at the plant for 12 Nonmalignant Hepatic Disease tent patterns in relation to clinical to 28 years (average, 18.0), and the manifestations. four with nonmalignant disease, be I. Ages at diagnosis for these four pa All four patients were found on tween 5 and 29 years (average, 20.6) JAMA, Oct 7, 1974 Vol 230, No 1 >t Hepatic Disease-Falk et al, 61 i I AP00024402 helpers whose principal job is to clean reactors. As can be seen in Table 3, ten of the 11 patients worked at some time as helpers. While virtually every employee at the plant has worked as a helper before being promoted to more /Advanced workx the average work du-' \ ration (le, time from starting work at the plant to date of diagnosis) was 23.5 years for the five patients who spent nine months or less as helpers , (and 15.1 years for the six patients who spent 20 months or more. This suggests indirectly a possible rela tionship between intensity of expo sure and latent period for liver dis ease. All 11 patients, or members of their immediate families, were indi vidually interviewed regarding past hepatic disease and possible exposure to hepatotoxic agents. None of the patients had a history of hepatitis or of exposure to hepatitis, and none had taken hepatotoxic drugs. Three pa tients (cases 1, 7, and 9) may have had significant alcohol intake. None, except for the patient in case 7, re called exposure to possible hepato toxic chemicals outside the work en vironment, in particular to either arsenic or thorium dioxide, two chem icals previously implicated as causes of hepatic disease and hepatic angio sarcoma in humans.*'' Patient 7 gave a history of exposure to arsenical in secticides on the family farm be tween the ages of 6 and 15 years; he both mixed and sprayed the insecti cides two to three times a year for about three hours on each occasion. In no case was there any history of acroosteolysis. COMMENT Before the report by Creech and Johnson,1 the only evidence that VCM might be oncogenic came from ani mal experiments. In 1971, Viola et al* published data suggesting oncogenic ity of VCM when inhaled by rats at very high doses; tumors of many tis sue sites, including lung, bone, and skin were recorded. Preliminary re sults of a more recent animal study in Italy by Maltoni et al (unpublished data) appear to indicate that angio sarcoma of liver as well as of other tissues can be induced in rats by at mospheric levels of VCM that are not uncommon in the human workplace environment In light of these obser vations, it appears likely that expo sure to VCM is responsible for the Louisville cases. Further support for this hypothesis is needed, of course, from additional epidemiologic data concerning workers at other PVC and VCM plants. Further toxicologic analyses are also needed to address the possibility that the active onco genic material may be some metabo lite of VCM instead of VCM itself. Whatever the precise mechanism for oncogenicity and hepatotoxicity of VCM, the Louisville data suggest that relatively high levels of VCM ex posure and relatively long intervals since first exposure (20 years or so) are involved. This does not rule out the possibility of less marked health effects at lower doses or at shorter in tervals of exposure, but it does for.the present focus attention on the imme diate problem of assessing the health status of persons exposed in the re mote past to high doses. None of the Louisville cases involved men work ing at the plant less than six years before diagnosis, and it can be safely assumed that levels of VCM exposure during earlier years of PVC produc tion were considerably higher than at present because of less stringent work practice procedures and less at tention to minimizing possibilities of VCM exposure in places of work. In humans, both thorium dioxide and arsenic have previously been re ported as causes both of hepatic dis ease and of angiosarcoma of the liver. In only one case at the Louisville plant (case 7) was there any history of exposure to either of these two ma terials (arsenic in insecticide spray). Likewise, there is little or no evidence among the Louisville cases that ex cessive alcohol intake plays any accel erating or potentially cocareinogenie role or that any direct relationship exists between acro-osteolysis and liver disease. Various data now suggest that VCM (or some derived metabolite) may produce in addition to angiosar coma of the liver a nonmalignant he patic disorder characterized by portal fibrosis and portal hypertension. This is suggested by the fact that such fi brosis was present in at least five of the Louisville tumor cases and was in addition observed in four other Louis ville vinyl chloride workers without tumor. Recent observations in Ger many,' together with earlier reports from Eastern Europe,1' suggest that hepatic fibrosis and portal hyperten sion represent an occupational dis ease not uncommon among vinyl chlo ride workers. Conceivably, such fibrotic liver disease represents a pre malignant'state. If this proves to be so, the early detection of such liver disease may be of greater industrial and public health importance than de tection of tumor itself, both because hepatic fibrosis may well emerge as a more frequent condition than tumor in vinyl chloride workers, and because it remains a possibility that very early liver abnormalities may be re versible or nonprogressive after workers have been removed from high-risk areas. Lasslo Mskk, MD, Lynn Ogden, MD, Edward Fadell, MD, Anne Rlcnman, MD, Curtis Song ster, MD, George Schrodt, MD, Edward Callahan, MD, Jerry Clanton, MD, James Eurfees, MD, Stanley Scipel, MD, and Will Ward, MD, sup plied records and pathologic materials. Louis Thomas, MD, and Hans Popper, MD, re viewed pathologic material. References 1. Creech JL Jr, Johnson MN: Angiosarcoma of liver in the manufacture of polyvinyl chloride. J Occup Med 16:130-151,1JII 2 Wilson RH, McCormick WE, Tatum CF, et al: Occupational acro-osteolysis. JAMA 201:677578,1967. 3. Dodson VN, Dinman BD, Whitehouse WM, et ah Occupational acro-osteolysis: IIL A dinical study. Arch Environ Health 22:83-91,1971. 4. Da Silva Horta, Cayolla da Motta L, Abbatt JD, et al: Malignancy and other late effects fol lowing administration of Thorotreat. Lancet 2201-205, 1965. 5. Da Silva Horta, Cayolla da Motta L: Fol lowup study of thorium dioxide patients in Portogal. Ann MY Acad Set 145:830-842 1967. 6. Regelson W, Kim U, Oapina J, et al: Hemangioendothelial sarcoma of liver from chronic arsenic intoxication by Fowler's solution. Cancer 21*14-522,1968. 7. Morris JS. Schmid M, Newman S, et al: Ar senic and nondrrhotic portal hypertension. Ga.trventerology 64*6-94,1974. 8. Viola PL, Bigotti A, Caputo A: Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Jtu 81*16-522 1971. . Msrsteller HJ, Leiback WK, Muler R, et al: Chronisch-toxischc Lebenchsden bei Arbeitem in der PVC-Production. truck Med Wochentchr 98*311-2314, 1978. 10. Soda F, Drejmsn I, Vslssksi M: Etude des maladies dues an chlorure de vinyle. Med Lav 58*61-271,1967. JAMA, Oct 7, 1974 Vol 230, No 1 Hepatic Disease--Falk et al. 63 AP00024403 Fig 1.--Liver disease detection protocol. mality was found, the 12-factor anal ysis was repeated in three weeks. If the single abnormality presisted, or if two or more liver-related abnormal ities were found on the initial labora tory tests, the patient underwent a comprehensive examination consist ing of tests for the following values: a 12-factor automated chemical analy sis, serum electrophoresis, LDH isoenzymes, fractionated alkaline phosphatase, direct and indirect bili rubin (if total level was elevated), se rum glutamic pyruvic transaminase (SGPT), y-glutamic transpeptidase (GGTP), isocitrate dehydrogenase (ICD), a-fetoprotein (fetoglobulin), and carcinoembryonie antigen (CEA). Also, a complete blood cell, count (CBC), platelet count, chest roentgen ogram, and liver and spleen scan were done. Abnormalities found in the initial 12-factor chemical analyses indicat ing the need for comprehensive pro Table 1.--Liver Enzyme and Bilirubin Results Test Alkaline phosphatase. mU (milliunlts)/ml y-Glutamlc trans peptidase. mU/ml Serum glutamic oxaloacetic transaminase, mU/ml Bilirubin, ms/100 ml Serum glutamic oxaloacetic tranaaminaae, mU/ml Isocltrate dehydrogenase, mU/ml Lactic dehydrogenase, mU/ml No. of Patients Studied 72 70 68 72 73 59 72 No. (*A| With Abnormal Values 35 149.4) 31 (443) 19 (29.8) 19 (26.4) 13 (18.0) 9 (18.0) B (11.1) Normal Range 3-85 6-23 4-25 0.15-1.0 12-40 0-7 90-225 Abnormal Range 87-135 29-575 27-559 1.1-2.6 48-150 338 237-475 files were grouped according to the following work areas: (1) PVC pro duction, (2) synthetic rubber produc tion, and (S) all others (including maintenance, shipping, laboratory, and ot{ter salaried employees). Liver Abnormalities Dictated Hospital Studies If abnormal scan or other findings suggested serious liver disease or the possibility of angiosarcoma, the pa- JAMA, Oct 7, 1974 Vol 230, No 1 Liver Damajje-Makk et al -65 AP00024404 1 Tibi# 3.--Tissue, Roentgenologic, and Laboratory Examination Correlation . Liver Seen 1 Riling defect end cirrhosis Riling defect, both lobee i Suggestive of cirrhosis Suggestive of cirrhosis Suggestive of cirrhosis Normal ) Normal Alkaline Phosphate t t t t t t t y-Glirtamlc Transpetidase tt t t t t N N Level of Serum Glutamic Pyruvic Transaminase t Bilirubin t NN tt tN NN NN Nn Serum Glutamic Oxaloacetic Transaminase t Lactic Dehydrogenase Nt tt t N 11 NN NN NN NN Platelet Count 4 t N N N N N factor chemical analysis. Of these, 75 isoenzyme 1. Splenomegaly with abnormal liver (6.3%) had either two liver-related ab Fetoglobulin determinations gave scan was present in three patients. normalities on the initial screening or normal results in all cases. Results of Hepatic panangiography was per one such abnormality that persisted. carcinoembryonic antigen radio formed in seven patients. By pan These patients had comprehensive immunoassays were normal in all pa angiography, two of the liver scan evaluations. One patient had initial tients, with one marginal result in a filling defects were found to be tu screening in the plant, further eval patient who smoked. Complete blood mors (Fig 3 and 4). The anomalous po uation and biopsy elsewhere, and cell counts disclosed normal values for sition of the gallbladder in one case, then was transferred to our institu hemoglobin, hematocrit, red blood cell and dilatated short gastric and collat tion for further studies. Results of al (RBC) count, and corpuscular indexes eral portal veins in another correlated kaline phosphatase, GGTP, SGPT, bil irubin, SGOT, ICD, and LDH examinations are listed in Table 1. in each patient. Four patients had slight to moderate granulocytosis. One patient showed relative lym with the defects on the scans. The other angiograms were normal. Of the six splenic arteriograms, Significant Biochemical Findings phocytosis. Twenty-three patients two showed splenomegaly; one of showed slight, and seven moderate, these with multiple splenic aneu Other tests of the comprehensive liver profile yielded the following sig nificant results: moderately elevated monocytosis. Three patients had mod erately increased eosinophil counts. One patient had thrombocytopenia rysms and the other, intrasplenic ar terial strictures. The other four were normal. total protein values were found in with a platelet count of 98,000/cu three patients with a similar eleva mm, and another had thrombocytosis Surgical Findings tion in 7-globulin content. Serum with a count of 750,000/cu mm (nor Exploratory laparotomy was per electrophoresis showed slight to mod mal range, 140,000 to 440,000). The formed in seven patients; in two, an erate increase in y-globulin in 11 pa number of 12-factor abnormalities giosarcoma was found. Resection tients. The /3-globulin value was and comprehensive profiles according could not be carried out in one patient slightly lowered in six patients. to work areas is listed in Table 2. because of the extent of neoplasm. In In 32 patients with elevated alka line phosphatase values, 15 had in Radiological Studies the other patient, angiosarcoma was limited to one lobe, but because of creased liver fractions. In 37 patients Seventy-four patients had liver portal fibrosis in the other lobe, resec with normal levels of alkaline scans: 11 of these scans (14.8%) were tion was deferred. phosphatase, fractionation was also abnormal, and 2 were borderline. Six In three patients, varying degrees performed. Thirteen showed relative patients had filling defects (Fig 2). of portal fibrosis was found on lap elevation of liver fractions. Eight of Three of these and another five pa arotomy. Of the remaining two pa 72 patients (111%) showed elevated tients had irregularities suggestive of tients, one had a cholecystectomy, the values for total LDH. Four of these cirrhosis. Spleen scans were per other an incisional hernia repair. had isoenzyme 4; two, isoenzyme 5; formed concurrently with liver scans Liver biopsy was performed con and one, isoenzyme 3 elevation; one in 71 patients. comitantly on each. Both of these liv had normal isoenzyme proportions. Three patients had had splenec ers were essentially normal on biopsy Of 64 cases with normal LDH val tomies in the past. Nine patients and on laboratory testing. Correla ues, 44 showed relative elevation in (12.7%) had enlargement of the spleen tion of biopsy, roentgenographic, l level of isoenzyme 4 (68.7%); two, rela to 22 cm (normal spleen size is consid and laboratory findings is given in tive isoenzyme 5; and one, relative ered less than 14 cm in length5). Table 3. JAMA, Oct 7,1974 Vol 230, No 1 Liver Damage--Makk at al - 67 AP00024405 i, i > ^ /1? rvr f- Vo!. 292 No. 1 ' t- VINYL-CHLORIDE-INDUCED LIVER DISEASE -- THOMAS ET AL. 17 VINYL-CHLORIDE-INDUCED LIVER DISEASE From Idiopathic Portal Hypertension (Band's Syndrome) to Angiosarcomas Louis B. Thomas, M.D., Hans Popper, M.D., Ph.D., Paul D. Berk, M.D., Irving Selikoff, M.D., and Henry Falk, M.D. Abstract Histologic examination of liver tissue (eight autopsy and 18 biopsy specimens) and five spleens from 20 workers with vinyl chloride polymerization showed hepatic angiosarcomas in 15. In addition, a peculiar pattern of progressive portal-tract, inconspic uous intralobular and conspicuous capsular fibrosis was observed in the five workers without angiosarco ma, in all the seven patients with angiosarcoma from whom tumor-free portions of the liver were available, and in two tumor-free biopsies from patients subse quently found to have angiosarcoma. The fibrosis was accompanied by splenomegaly. Hypertrophy and hy perplasia of both hepatocytes and hepatic and splenic mesenchymal cells were also seen. The histologic simi larity to chronic inorganic arsenical poisoning, in which angiosarcomas also oqcur, and to idiopathic portal hypertension (Banti's syndrome) suggests that the latter syndrome at times results from unknown tox ic, possibly environmental, chemicals. (N Engl J Med 292:17-22,1975) 1 I ' HE development of hepatic angiosarcomas in work-*- ers exposed to vinyl chloride gas in the manufacture of polyvinyl chloride has been well documented.'* Histo logic study of hepatic and splenic specimens taken from such workers suggests to us, moreover, that other diseases that have not in the past been related to industrial ex posure may be involved. Specifically, a case will be made for the concept that splenomegaly with portal hyperten sion associated with slight hepatic fibrosis, previously des ignated as Band's syndrome,4 may be the result of ex posure to known or unknown chemical agents. History of the Vinyl Chloride Liver Injury Polyvinyl chloride, one of the most widely used syn thetic plastics, has been manufactured for more than 40 years by polymerization of gaseous vinyl chloride in the United States and in many other countries. Concern about untoward side effects in workers had centered primarily on a disease, acro-osteolysis,* which is charac terized by Raynaud's syndrome, dermal induration and bone lesions. Two reports* 7 also dealt with nonspecific alterations of hepatic structure and function, and a lesion designated "chronic epithelial hepatitis" was found in about 25 per cent of the examined workers in Russia.* Hepatic abnormalities, which did not attract major atten tion, became far more important because of the discovery of three cases of angiosarcomas of the liver, an otherwise very rare tumor, in workers in a polyvinyl chloride pro duction plant in this country.1 The introduction of a sur veillance system in this plant detected a total of seven cases of hepatic angiosarcoma.*'* Animal experiments confirm the relation between hepatic angiosarcoma and exposure to gaseous vinyl chloride. In 1971, Italian investigators re ported that prolonged inhalation of vinyl chloride pro duced carcinomas of the zymbal gland in rats,* and From the Laboratory ot Pathology. National Cancer Institute. National Institutes of Health, Bethesda, MD, the Mount Sinai School of Medicine of the City Universityof New York, NY, the Section on Diseases of the Liver, National Institute of Arthritis, Metabolism and Digestive Diseases. Na tional Institutes of Health. Bethesda. MD. and the Cancer and Birth Detects Division, Bureau of Epidemiology, Center for Disease Control, Atlanta, GA (address reprint requests to Dr. Thomas at the National Cancer Institute, Bldg 10. Room2A29. Bethesda. MD200M). Dr. Popper is Fogarty Scholar in Residence. Fogarty I ntemational Cen ter, National Institutesof Health. Maltoni1* subsequently described angiosarcomas of the liver and other organs as well as nephroblastomas in rats exposed to vinyl chloride gas. Recently, angiosarcomas of the liver have been seen in mice after exposure to as little as 50 ppm of vinyl chloride.1 ` When specimens of human angiosarcoma from vinyl chloride polymerization workers were reviewed in the Laboratory of Pathology of the National Cancer Institute, the lesions in areas of the liver not involved by an giosarcoma appeared similar to alterations recently re ported in vinyl chloride workers in Germany.1* In these patients inconspicuous portal and perisinusoidal fibrosis was associated with impressive heptic capsular fibrosis as seen by peritoneoscopy. Hepatic-function tests revealed variable abnormalities. Clinical manifestations included portal hypertension with splenomegaly, thrombocyto penia and bleeding esophageal varices. This German report focused our interest on the appearance of the liver not only in patients with hepatic angiosarcomas but also in other workers with hepatic fibrosis exposed to vinyl chlo ride who had been diagnosed as having cirrhosis because of portal hypertension, variceal hemorrhage and spleno megaly. By coincidence, there have been several recent reports of portal hypertension without obvious cause in patients with psoriasis who had teceived an inorganic arsenic prepration, Fowler's solution, for prolonged peri ods.13'1* These observations were interesting in view of earlier reports from Germany and France of heptic an giosarcomas developing in vintners exposed to insecti cides containing inorganic arsenic.1* Roth's report in cluded 47 workers with chronic arsenic intoxication. Of these, four had heptic carcinomas, five had sarcomas (an giosarcomas), and 13 of 27 autopsied by him had unusual tyjjes of cirrhosis.16 Material Studied The observations recorded here are based on the study of he patic tissues obtained from 20 workers who had industrial ex posure to vinylchloride far prolonged periods, usually exceeding two years and extending up to 18 years. The tissues were derived from 15 patients with angiosarcoma and five with hepatic fibrosis but without angiosarcoma. The majority of these specimens were --initially obtained, studied, and in some cases reported by oth ers,1-* but all were submitted to the Laboratory of Pathology, Na- AP00024406 X v f Vol. 292 No. 1 V1NYL-CHLOR1DF.-INDUCED LIVER DISEASE - THOMAS ET AL. 19 pressed the surrounding parenchyma, and in one tumor, anaplastic sarcoma cells invaded portal-vein branches. Focal dilatation of sinusoids with proliferation and en largement of the sinusoidal cells associated with perisinusoidal fibrosis may represent an early stage in the evolu tion of angiosarcoma. This lesion was noted in six of the seven available tumor-free liver specimens from patients with angiosarcoma, and in the two biopsies taken before angiosarcoma was detected. It was also observed in two pa tients in whom angiosarcoma has not developed, and in whom, accordingly, careful follow-up observation is indi cated. In the patients with angiosarcoma, some of the si nusoidal lining cells had enlarged, atypical nuclei and bul ky cytoplasm that was PAS negative in contrast to many of the lining cells in the uninvolved parenchyma (Fig. 2). There were gradual transitions between the areas of focal dilatation of sinusoids to multiple, microscopic-sized an giosarcomas with a sinusoidal pattern. In three patients other organs besides the liver were in volved by angiosarcoma: the duodenum in one, the lung in a second, and the lung, heart, kidney and lymph nodes in a third patient. Hepatic fibrosis All the livers without angiosarcomas and the unin volved portions of the livers with angiosarcomas showed Figure 2. Focal Dilatation o( Sinusoids, with Hyperplastic Hepatocytes (Arrow) and Excess Sinusoidal Lining Cells, Some with Hyperchromatic Nuclei (This Appearance Con trasts with That of Normal Sinusoids in the Right Lower Corner)(Hematoxylinand Eosin Stain x 160). the same basic changes, the extent of which varied widely within each specimen. Excess fibrous connective tissue caused variable enlargement of most portal tracts (Fig. 3A). A distinctive feature was the tendency for single hepatocytes and groups of hepatocytes at the margins of the portal tracts to be separated from adjacent hepatic cord cells and surrounded by this progressive fibrosis. Proliferation of bile ducts was noted in these fibrotic portal tracts together with a variable infiltration of lymphocytes. Occasionally, the walls of portal-vein branches showed focal fibrosis. Adjacent portal tracts were often connected by connective-tissue septa in thi sense of a perilobular Fibrosis and some thin septa traversed the parenchyma and extended irregularly toward central veins. In two cases, both with angiosarcomas elsewhere in the liver, these septa separated parts of the parenchyma to produce nodules. The hepatic capsule showed focal thickening and nodular fibrotic areas that often extended into subcapsular hepatic tissue to connect with adjacent portal tracts. The sinusoidal lining cells were focally increased in number, especially in areas where sinusoids were dilated. Intralobular, perisinusoidal fibrosis, though inconspicu ous in hematoxylin-eosin-stained sections, could be dem onstrated in these areas by special stains for reticulin and collagen fibers. In other areas, groupsof hepatocytes, with no particular intralobular localization, varied conspicu ously in cellular and nuclear size, many of them being dis tinctly enlarged (Fig. 3B). Binudear and multinucleate hepatocytes, some with much cytoplasm, were intermixed with a few hepatocytes that were smaller than normal. De generation and necrosis of hepatocytes, although present, were not more conspicuous than in any routine surgical specimen. In the second biopsy specimen, obtained in one patient two years after cessation of exposure to vinyl chlo ride, these abnormal changes in both hepatocytes and si nusoidal lining cells had disappeared, but the capsular, portal-tract and intralobular fibrosis persisted. Splenic Changes The available spleens were large, weighing 560 to 1050 g, and showed on cut section enlarged Malpighian follicles and a firm, beefy red pulp. Microscopically, the follicles were hyperplastic and had large germinal centers and wide perifollicular zones (Fig. 4). Occasionally, fresh hem orrhage was found around penicillary arteries and folli cles. The red-pulp sinuses were widened and often lined by a continuous layer of cuboidal reticuloendothelial cells. The slightly thickened pulp cords contained many lym phoid and histiocytic cells as well as erythrocytes. Fibrosis of the red pulp was absent or inconspicuous, and hemosiderinophages were only occasionally seen. Discussion The relatively frequent development of hepatic angio sarcomas in workers engaged in the polymerization of vi nyl chloride has now been established. In this report the emphasis is on a peculiar hepatic fibrosis in such workers and its relation to the angiosarcomas. This fibrosis, which appears to represent a second hepatic lesion attributable to vinyl chloride exposure, was found in the tumor-free AP00024407 Vol. 292 No. I VINYL-CHLORIDF.-INDUCED LIVER DISEASE -- THOMAS ET AL. 21 of the spleen suggests a stimulating effect by vinylchlonde or its metabolites on several types of hepatic and splenic cells. The prominence of the enveloping growth pattern of the tumor cells, rather than a nodular growth pattern, is characteristic of hepatic angiosarcomas that develop after vinyl chloride exposure. Many cases of hepatic angiosar coma of unknown cause fail to show this prominence.1*18 On the basis of experience to date absence of this promi nence in a given case militates against vinyl chloride as the origin. These observations may be of assistance in epide miologic surveys. Enveloping features are prominent in angiosarcomas induced by thorium dioxide suspension (Thorotrast) in which large and irregular areas of fibrosis are observed.18-*1 Similar features are emphasized in an giosarcomas induced by inorganic arsenicals,1811 which, like vinyl chloride, may also produce inconspicuous he patic fibrosis. Through the courtesy of Drs. P. J. Schcuer and M. Schmid, we were able to study histologically three cases of mild hepatic fibrosis with portal hypertension after long-term treatment of psoriasis with Fowler's solu tion, two of them reported.11 We observed hepatic changes similar to those in the patients exposed to vinyl chloride -- namely progressive portal fibrosis, mild intra lobular fibrosis and focal hypertrophy and hyperplasia of hepatocytes. Figure 4. Surgically Removed Spleen of a Worker with Mod erate Hepatic Fibrosis. Note hyperplastic Malpighian follicles, which merge and ex hibit large germinal centers. Sinuses are widened, and redpulp cords are slightly thickened (hematoxylin and eosin stain x 40). Inconspicuous hepatic fibrosis accompanied by spleno megaly and portal hypertension is the syndrome de scribed by Banti* An identical clinical picture is observed in some workers exposed to vinyl chloride. In contrast to splcrtomegalic cirrhosis, in which the splenic enlargement is caused by the hemodynamic alterations in the cirrhotic liver, in Band's syndrome the splenomegaly precedes the hepatic fibrosis or is associated with inconspicuous fibro sis. It may, however, be followed by cirrhosis eventually.13 Most cases of Banti's syndrome are associated with portal hypertension and are now usually designated as idiopath ic portal hypertension.14 Some authors associate the por tal hypertension with a primary** or secondary1* altera tion of the wall of the portal vein or its branches (hepatoportal sclerosis), but this lesion was not conspicuous in our cases. Conditions resembling Banti's syndrome have been produced in animal experiments by stimulation of splenic lymphoid and reticuloendothelial cells.*7 It is possible, therefore, that one of the results of prolonged exposure to vinyl chloride maybe splenomegaly due to stimulation of splenic cells, with a consequent increase in splenic and he patic blood flow. Portal hypertension might be the result of an inability to accommodate this increased hepatic blood flow because distention of portal-vein branches is prevented by the portal fibrosis, and that of the sinusoids by the capsular and subcapsular fibrosis. Whatever the origin of the idiopathic portal hyper tension or Banti's syndrome, its apparent induction by prolonged exposure to vinyl chloride or inorganic arseni cals suggests that unidentified chemicals may cause other similar conditions in which the cause is unknown. Such cases are observed sporadically in the Western world1*-1* but are rather frequent in India,**-*8 Uganda,38 North Af rica31 and Japan, where they are studied extensively.3* This geographic distribution is an additional reason for suspecting environmental factors. In Banti's syndrome of unknown origin development of angiosarcoma has not been reported, but this fact does not negate the possibility that chronic exposure to vinyl chloride or inorganic arsenicals will produce either Banti's syndrome or hepatic an giosarcoma or both. References 1. Creech JL Jr. Johnson MN: Angiosarcoma of liver in the manufacture of polyvinyl chloride. JOccup Med 16:150-151,1974 2. Block IB: Angiosarcoma of the liver following vinyl chloride expo sure. JAMA 229:53-54,1974 3. Lee FI. Harry DS: Angiosarcoma of the liver in a vinyl-chloride work er. Lancet 1:1316-1318,1974 4. Banti G: Splenomegalie mit Lebercirrhose. Beitr Pathol Anat 24:21-33, 1898 5. Lange CE. JDhe S. Stein G, et at: Die sogennante VinylchloridKrankheiteine berufsbedingte Systemsklerose. Int Arch Arbeitsmed 32:1-32,1974 6. Puschin GA: Affection of the liver and bile ducts in workers engaged in the production of some types of plastics. Sov Med 2*(2):132-I35, 1965 7. Suciu I, Drejman I, Valaskai M: Etude des maladies dues au chlorine devinyie.MedLavoro58:261-27l, 1967 8. Falk H, Creech JL Jr, Heath CW. et al: Hepatic disease among workers at a vinyl chloride polymerization plant. JAMA (in press) 9. Viola PL. Bigotti A, Chputo A: Oncogenic response of nt skin, lungs, and hones to vinyl chloride. Cancer Res 31:516-522.1971 10. Maltoni C, Lefemine G: Le potenzialiu dei saggi sperimenlali nella predizione dei rischi oncogeni nmbientali: un esempio: 11 cloruro di vinile. Accad Nazionale dei Lincei 56:1-11,1974 AP00024408 f 936 > fvrSd.in.ftt appeared tn abstract form in the Scottish Medical Journal Janutry 1974. Requests for reprints should be sent to Dr R M Pearson. References ' Lcishman, A W D, and Sandler, G, Angiolagy, 1967, 18, 705. * Dorpli, S, and Binder, C, Acta Mediea Scandinavica, 1971, 185, 443. * Moioksc, M, Mulcsan, G, and Columbi, A, European Journal of Pharma* colony, 1975, 8, 21. * George, C F, ti al, Archives of Internal Medicine, 1972, 130, 361. 1 Bulpiti. C J, Dollcrv, C T, and Came, S,Journal of Chronic Diseases, 1974, 27, 309. * Jack, D B, and Reiss, W, Journal of Chromatography, 1974,88, 173. 1 Armitage, P, Statistical Methods in Medical Research, p 226. Oxford, Blackwell Scientific, 1971. BRITISH MEDICAL JOURNAL 17 APRIL 1976 ** RruckcnritJfic, A, M,and Dollery, Cl, Lancet, 1966, 1, 1074. * Zaccst, R, Gilmore, Li, and Koch-\XV*er, J AVr<* Kttgfand Jottmai of Medicine, 1972, 286, 617. ,M Sweet, C S, and Mandradjieff. M, Clinical Science, 1979, 48, 147. Buhlor, 1', cl al, American Journal of Cardiology, 1973, 32, 511. 11 Salvetti, A, et el, Journal of Nuclear Biology and Medicine, 1973, 17, 142. u George, C 1*\ in Biological Effects of Drugs in Relation to their Plasma Concentrations, cd D S Davies and H N C Prichard, p 128. London, Macmillan, 1973. u Cuthbert, M I;, and Collins, R F, British Journal of Clinical Pharmacology, 1975, 2, 49. li Reiss, W, Hucraelcr, H, and Raschdorf, F, Xenohiotico, 1974, 4, 365. ,fi Rrunner, O, Imhof, P, and Jack, D, European Journal of Clinical Pharma coiogy, 1975, 8, 3. 17 Leonetti, O, et al, Clinical Science, 1975, 48, 491. u Veterans Administration, Journal of the American Medical Association, 1967,202, 1028. Immunological mechanisms in the pathogenesis of vinyl chloride disease A MILFORD WARD, SOPSAMORN UDNOON, J WATKINS, ANNE E WALKER, C S DARKE British Medical Journal, 1976, 1, 936-938 Summary Vinyl chloride (VC) disease is a multisystem disorder incorporating Raynaud's phenomenon, aero-osteolysis, thrombocytopenia, portal fibrosis, and hepatic and pulmonary dysfunction. Immunological and immuno chemical investigations showed the presence of circulat ing immune complexes in 19 out of 28 patients with the disease and In a further two out of 30 workers exposed to VC. The immunological data were reviewed in relation to the clinical picture of the disease and to the available evidence on the metabolism of VC. The results suggest that VC disease is an immune complex disorder and that the immune response is initiated by the adsorption of VC or a metabolite on to tissue or plasma protein. Introduction Vinyl chloride (VC) is known to be oncogenic,'1 but hepatic angiosarcoma may not be the most serious of its medical hazards. Aero-osteolysis was first described in 1966 in workers employed in the polymerisation of VC," and since then other cases have been described.4 * In all these reports the affected workers helped in cleaning the autoclaves after the polymerisation process. Stewart et at,* however, reported a case in a worker who had never done this job. Their report widened the possible exposure to toxic levels of VC to other workers in the industry. The descriptions of aero-osteolysis do not describe adequately Protein Reference Unit, Department of Immunology, Hallmmshire Hospital Medical School, Sheffield S10 2RX A MILFORD WARD, mb, mkcfath. director SOPSAMORN UDNOON, bsc, research student J WATKINS, bsc, pud, principal scientific officer Chesterfield Royal Hospital, Chesterfield ANNE E WALKER, md, mrcp, consultant dermatologist Respiratory Function Unit, Royal Infirmary, Sheffield C S DARKh, md, men, consultant n ftpA.;\ iol fh - `n* .. the full extent of the disorder as it may affect VC workers. The syndrome for which Lange et aV suggested the name vinyl chloride disease includes sclerotic changes in the skin, osteolysis, circulatory disturbances, thrombocytopenia, portal fibrosis, and impaired hepatic and pulmonary function. Despite considerable effort in many countries little is under stood about the aetiology and pathogenesis of VC disease. We therefore outline the immunological investigations performed on 58 workers from a VC polymerisation plant. The results explain the pathogenetic mechanisms of the disease and allow some conclusions to be drawn about its aetiology. Patients The 58 patients were referred to a hospital clinic from a single VC polymerisation plant. They represented 18",. of the past and present work force (320). Referrals of those still working at the plant were from either the factory medical officer or local general practitioners; ex-employees were referred after investigations made by the Employ ment Medical Advisory Service. For the purposes of the study the patients were divided into four clinical groups. Group l patients)--These patients were disabled by pain in limbs and hands and by dyspnoea. AH had symptomatic Raynaud's pheno menon, which in five was clinically demonstrable; the others had severely cold hands. Four had scleroedcma of the hands or face, four a mild clawing deformity of the hands, and two mild radiological evidence of aero-osteolysis. Group 2 (19 patients)--This group was moderately disabled and muliisymptomatic, excessive fatigue, limb pain, and paraesthesiae being the most common complaints. Raynaud's phenomenon was symptomatic but never observed, although many patients were found to have cold hand> or feet. Sclcroedcma-iike changes of the face were evident in two patients. No radiological abnormalities were seen. Group 3 (25 patUnts)--These patients were not disabled and were continuing in active employment. They presented with miscellaneous symptoms that were not confirmed by visible abnormalities or overt clinical signs. Group 4 (5 patients)--'This group was asymptomatic, the patients having been referred because of expressed concern or because they wished to seek other employment. The patients1 ages ranged from 23 to 59 years with a mean of 39*7 years. There was no significant difference between the groups. The duration of exposure to VC varied from six to 75 months with a mean of 39 months. As the degree of exposure varied with the job at the plant the patients were divided into high- and low-exposure groups, the former being workers in the reactor building and dry-bagging AP00024409 IlHI 1 IMI MUIU.Al. JUUKNAL ii Al'KlL |V|0 / giant, and the latter maintenance fitters and warehousemen. Fifty of the patients (86".,) had at sometime worked in the reactor building or dry-ax'.kging plant. Altogether 46 patients (79",,) admitted to having suffered from VC narcosis on at least one occasion; the incidence of admitted narcosis tended to decrease with diminishing severity of the disease--group 1, 100",,; group 2, B0",, ; group 3,76",,; group 4,60",,. The differences do not approach statistical significance. Methods hmmmoglobitlim were estimated by an automated immunoprccipitin technique. Complement was determined in fresh F.DTA plasm*, C3 and C4 being estimated by single radial immunodiffusion and conversion assessed by two-dimensional immunoelectrophorcsis. Cryoproiciu studies were performed on warm-separated eitrated plasma, the cold aliquot being kept at 4 C for seven days before separation of the cryoprecipitaie. Rheumatoid factor was determined by passive haemagglutination of sensitised cells, and the presence of antilissue antibodies by indirect immunofluorescence. Lymphocyte transformation was assessed by a modification of the technique of Schcllckcns and Eijsvoogel.' Lymphocytes were separated from heparinised whole blood by a Ficoll-bascd centrifugation method.' Duplicate culture tubes were set up containing culture medium, autologous plasma, and cither 0 2 ;,g purified phytohacmagglutinin (PHA) (Burroughs Welcome) or 2 pg purified protein derivative of tuberculin (PPD). Control cultures were set up using lymphocytes from healthy donors. All cultures were incubated at 37 C for 72 hours (PHA) and 120 hours (PPD). Transformation was assessed by the incorporation of tritiated thymidine into active DNA synthesis. Similar cultures were set up with test lymphocytes supple mented with normal plasma and normal lymphocytes supplemented with test plasma to assess possible plasma inhibitory effects. Lympho cyte sttbpopnlations were defined as spontaneous E-rosette-forming cells or T cells'* and as mcmbrane-Huorcsccnr cells or B cells.11 Absolute numbers of T and B cells were determined as a percentage of the total lymphocyte count. Biopsy specimens were snap-frozen and cryostat sections stained for IgG, IgA, and IgM, C3 and C4, and fibrinogen/fibrin by direct immunofluorescence. Results The major abnormalities observed were a polyclonal increase in one of the immunoglobulin classes, usually IgG; the presence of mixed cryoglobulins, including IgG, C3, and fibrinogen; and in-vivo conversion of both C4 and C3. Table I shows the mean values and ranges of immunoglobulins, C3, C4, and lymphocytes for each clinical group together with the numbers of patients with cryoprotcins and showing C3 conversion. The abnormalities were present in almost ail cases in group 1, and showed a progressively decreasing incidence in ; 937 the other groups. The presence of circulating immune complexes was inferred when there was evidence of mixed cryoglobulins, in-vivo conversion of complement, and depressed values for C3 or C4; the presence of immune complexes showed a close relationship with Clinical grouping, with 88''.., 58",,, 8",,, and 0",, respectively for the four groups. Eight of the 58 patients were classified as of low exposure, six of them being in group 2. No abnormalities were detected in these Six patients. When the low-exposure workers are removed the inci dence of circulating immune complexes in group 2 (85";,) closely approximates that in group 1. Autoantibody screening (table II) showed no abnormalities except for a high incidence of antinuclear antibodies in group 1. These were ail of low litre (IgG 1/20-1/50) and probably resulted from tissue damage rather than an autoaggressivc disease process. The only other observation of note was the presence of thyroid autoantibodics in three patients in group 2. There was evidence of a reduction in the T eel! population with a slight increase in B"cells; this was most pronounced in group 1 but was also evident in group 2 and to a less extent in group 3. In-vitro stimulation showed few abnormalities except for an apparently increased frequency of reduced or absent responses to PPD. PHA transformation studies showed minor degrees of suppression, and inhibition by plasma was not seen. Direct immunofluorescence studies of biopsy specimens of skin (10 eases), muscle (one case), and lung (one ease) showed aggregates of IgG, C4, C3, and fibrinogen/fibrin in the lumen of vessels and adherent to the vascular endothelium. IgG, C4, C3, and fibrin were also detected in the media and subintimal regions of small and medium-sized arterioles. Discussion The major immunological features of VC disease in this series are hypcrimmunoglobulinacmia, cryoglobulinaemia and cryofibrinogenaemia, and in-vivo conversion of complement involving the classic activation pathway via C4 and C3. There was additional evidence of a cellular disturbance in the form of a reduced T cell population and a modest increase in B cells. The presence of non-organ-specific antiiissuc antibodies in those most severely affected clinically may have related to tissue damage rather than to a pathogenetic mechanism. Immunofluorescence studies of the biopsy material confirmed the immunochemical evidence of circulating immune com plexes svith the identification of immunoglobulin, complement, and fibrin/fibrinogen in the lumen of vessels, adherent to endo thelium, and in the vessel wall. An immune complex or cryoglobulinaemic vasculitis would be consistent with the findings of a perivascular inflammatory infiltrate as described by Markowitz table t--Mean vatues and ranges for immunoglobulins, complement, and lymphocytes in ,ach group and numbers of patients in each group with cryoprotcins and shotting C3 conversions ]i j Immunoglobulin* (f/1) . patients 1 !| j IgA l i 9 ; 14-3 (10*0-19 0)' 2*8(1 *8-4*7) 2 I 19 119(83-18 5). > 2*4 (0 6-5-0) 3 1 25 1 9-5 (6-8-11*4) t 2-0 (0 9-41) 4 | 5 12*1 (9-5-160) : 3*6 (1*1-5 2) Normal value* 1 9-5 (6-0-14-0) j 1-8 (0-5-3 0) i1 IgM 1-3 (0-4-2-7) 0*9 (0*3-21) 0-9 (0*4-1 *9) 1-0 (0-5-1-4) 0 9 (0 5-1*7) S'Mcj 4 k-g w C b 85 13 13 21 Complement (g.'l) C3 ; C4 =Ii nt- o, Ya |S 8Q | Lymphocyte* x 1C Vi Total T cells B cells 1*0 (0*6-l*5) 0 45 (0-19-110) 0*8 (0-6-1-3) 0-38 (0-22-0 7) 0-8 (0 5-1*2) 0 45 (0 32-0 69) 0*6 (0 6-1-0) ! 0 38 (0*27-0*46) 1*2 (0*8-1*7) j 0 45 (0*30-0 75) 6 ; 1*8 (1*0-3 I) 9 ! 2 4 (1-3-5-4) 10 : 2 0 (0-7-3 0) 3 i 2*5 (2 0-3 0) 2 0(1 5-2 5) 1 04 (01-1*5) 0*5 (01-1 *8) 0 4 (O 1-1*0) 0*4 (0*3-0 4) 10 (0 6-1-3) 0 6 (0 1-1-5) 0*7 (0-3-1*5) 0*5 (0 1-0 8) 0 5 (0 3-0 6) 0*7 (0*4-10) TABLE ii--Results of autoantibody screening in the four groups of patients Group ^ i 2 3 A Total i j 1 j No of patients 9 19 25 ' 58 No with factor 11 2 Nuclei (ANA) 8 2 10 No with intitissue antibodies to: Mitochondria (AMA) Smooth muscle Gastric parietal cells 11 2 1 1 1 i 4 1 1 ] 1| Thyroid 3 3 AP00024410 938 * BRITISH MliblCAL JOURNAL 17 APRIL 1976 ct al,` although the severe narrowing of dermal vessels with subintimal fibrosis described by Harris and Adams' could haptenie effect in the incitement of antibody synthesis. The theoretical interaction between VC and plasma proteins was be construed as an end-stage phenomenon of a similar process. Both histological features were present in this series, although partially confirmed by Oster1" in his observations of the binding of VC or its metabolite to serum albumin. the subintimal proliferation was seen only in the cases of The association of the observed results with the experimental longest duration. The tendency towards thrombocytopenia noted in many patients could be construed as confirmatory metabolic data allows the construction of a theoretical model for the pathogenesis of VC disease and explains many of the of an immune complex disorder with complement activation.1* features of this multisystem disorder. A metabolic product of The persistence of disease activity as evidenced by the presence VC, presumably the dioxide, binds to plasma protein, producing of cryoprccipitafe and conversion of complement in workers either a haptenie group or a conformational change within the who had been removed from exposure to VC for more than six protein molecule. This acts as an antigen that escapes tolerance months is in accord with the observations of Veltman ct a/.'* Despite these pronounced abnormalities a search of published and stimulates B cell proliferation and immunoglobulin produc tion. The antibody and antigen interact to produce a soluble work on VC disease failed to disclose any similar investigation, complex that is both cryoprecipitable and capable of initiating most reports having concentrated on the hcpatotoxic effects of VC and its oncogenic properties. Danishcvsky and Egorev" the complement sequence by the classic activation pathway. The cryoprecipitate and reactions secondary to complement showed that tissue damage from VC could produce allergenic activation will produce platelet aggregation and apparent throm substances. Hcrvicux and Tessicr'' had shown that a chemical bocytopenia, fibrinogen/fibrin conversion, and vascular occlu dermatitis could be induced among workers in the industry. sion. The vascular occlusion, either temporary or permanent, is V One patient in group 1 complained of skin irritation a year enough to explain the observed clinical, radiological, and histo before, with evidence of Raynaud's phenomenon and dyspnoea, logical findings in the skin, skeletal and soft tissues, and lungs. and gave a positive reaction to a patch test with polyvinyl chloride (PVC) powder. One other patient (group 3) had also By producing ischaemia the vascular occlusion would also stimulate new collagen biosynthesis, as observed by Jayson et at.-' complained of severe generalised pruritus. This final episode in the pathogenesis of the disease process In a detailed study of 13 patients employed in PVC production would then be further augmented by the interaction of collagen with circulatory disturbances, thrombocytopenia, splenomegaly, and complement,** ** resulting in further activation of the com and hepatic malfunction. Lange et a!' concluded that there was plement pathway and thus reinforcing the terminal recycling no evidence for an autoaggressive disorder. A more recent phenomenon. report'1 detailed the investigation of 70 patients from a work The frequency with which abnormalities were detected in force of 128 at a single plant, the findings of which did not exposed workers in this series, especially in those groups (2 and change the earlier conclusions. These workers did make the 3) in which there were few or no overt clinical signs, suggests point, however, that although skin and bone changes may that the disease process may be more common than is generally disappear when the patient is removed from contact with VC, the supposed. Further studies are in progress to investigate the total thrombocytopenia may persist for 12-18 months after exposure. exposed population at the factory and to study populations from In a study of 36 workers exposed to VC who were shown to other related industrial plants. have Ieucopcnia, thrombocytopenia, and splenomegaly, Suciu et al" showed an increase in the -.-globulin in 11. Although they found only a 6",, and 2-9"., incidence of clinical Raynaud's References phenomenon in two series of VC workers, they noted that the phenomenon cleared spontaneously.on removal from exposure and that the different incidence figures were associated with a 22-fold decrease in VC levels. They also quoted a much higher (66"., and 55",, respectively) incidence uf vasospastic changes in discussing the significance uf the splcnogmealy, they suggested that VC acted as an irritant in the reticuloendothelial system to produce a reactive splenic enlargement. Marsteiler ct at,'* in an extensive investigation of 50 VC workers, introduced some immunological tests into the initial protocol but abandoned them when they failed to give positive results. No comment was made on the presence or absence of hypergammaglobulinacmia. These workers also suggested the possibility of reticulo endothelial stimulation as a cause of the splenomegaly and the hepatic littoral cell hyperplasia, although they regarded the latter as a possible pathogenetic factor in the development of hepatic angiosarcoma. The metabolism of .VC in man is not fully understood. Hefner et at" showed that in rats metabolism is by the alcohol dehydrogenase pathway at low concentration, and via an inter mediate chlorocthylcnc oxide at higher concentrations. William son'* proposed that the intermediate product is more likely to be a cyclic dioxide. Both the oxide and dioxide would be highly reactive molecules and would be able to bind to free sulphydryl and amino radicals. The incorporation of these into protein synthesis would give a conformationally altered molecule that would be antigenic. The inclusion of this intermediate product with its chloride residue in a protein molecule would have a 1 Lamer, 1571, 1, 1321. 1 Hriliib Medical %um.il. 1971, 1, 590. I Cordier, J ,\1, ct al, Colliers dc Mcdeeine, 1906, 4, 3. ' Harm, 13 K, and Adams, \Y G 1-, British Medical Journal, 1967, 3, 712. I Markowitz, S S, e: at. Archives of Dermatology, 1972, 106, 219. * Stewart. J I), Williams, I) M ], and McLaehlan, M S F, Journal of Stieiol and t leeuparionjt Medicine. 1975, 23, 103. T Lange, C li, et al. Infernalionates Archie Jitr Ai behzntedizin, 1974, 32, 1. * Schellekcm, P T A, and liijsvoogc], V P, Clinical and Experimental Immunology, 1968. 3, 571. * Bovum, A, Scandinavian Journal of Clinical and Laboratory Investigation, |'96H. 21, supp! No 97. p 9. '" Steel, C M, Leans, J, and Smith, M A. British Journal of Hacntuivlogy, 1974, 28, 245. " Papamichail, M, Brown, J C. and Holborow, L J, Lauccl, 1971, 2, H50. '* Pfueller, S 1., und Lvischer, U l-\ Intmmioehenustry, 1972, 9, 1151. II Veltman, G, et at, Annals of the ScscYork Academy of Sciences, 1975, 246, 6. " 13anishevsky, S 1- and liporev, N M, Ciigiena Trudo i Professional1 nyc /obolevaniya, 1961, No 9, p 26. 11 Hereieux and Tessier, Archives des Maladies Profassionnclhs, dc Mcdccinc dn Travail s'l tie Sceurite Saeiale, 1959. 20, 61. ,h Sueiu, 1, el al. Annals of the XeieYork Academy of Sciences, 1975, 246, 53. " Miltsleller, II J, ,7 al. Annals of the XeteYorh Academy of Sciences, 1975, 246, 95. "* Uelder, It li. Watanabe. P (i, and Gehrinp, P J, Annals of lire XeeeYark Academy of Sc,.tics, 1975, 246, 135. '* Williamson. K, ProcccJine, of the Royal Sm'iety of Medicine, 1976. 69, 281. *" Oster, (1, Annals of the XecvYork Academy of Sciences, 1975 , 248, 149. 11 Jayson, ,\1 I V. Hailey, A J, and l.lnyd-Jones, K, Proceedings of the Royal Society of Medicine, 1976, 69, 295. ** Jobin, F, and Tremblay, F, Thrombosis et Diathesis itaemorrhagica, 1969, 22, 450. II Takahashi, M, Kawachi-Takahasbi, S, and Matsuura, M, tnlernatianal Archivet of Allergy and Applied Immunology, 1975, 48, 642. h AP00024411 AP00024412 }rent Intelligence NOTICE: THIS MATERIAL MAY BE PROTECTED BY COPYRIGHT LAW (TITLE 17 IIS. CODE) r jigiosarcoma of the Liver in jnyl Chloride/Polyvinyl Chloride Workers 77 Update of the NIOSH Register Spirtas, Dr. P.H., and Rose Kaminski, M.S. Hyg. 'the December 1974 issue of the lournal of Occupational "Seine (16:809), NIOSH presented data on 26 cases of liver iarcoma among vinyl chloride workers, 21 of whom i in the polymerization process. The present update pro- details on 64 cases of angiosarcoma among vinyl chloride rriration workers reported as of October, 1977. Con1 surveillance of these workers is encouraged for the pur^jof furthering our knowledge of this disease process in on to vinyl chloride exposure. ice the original publication of NlOSH's registet of 26 cases of ang'otarcoma associated with occupational exposure to vinyl L^de or polyvinyl chloride (VC/PVC).' evidence has continued cumulate which further confirms this association. In the May issue of lOM.1 an additional 12 cases were presented. Since tgxiate. 33 more cases have been reported to NIOSH and one ...has been discounted (U.S. Case No. 14) as of October. 1977. yields a total of 70 cases known to NiOSH, 64 of whom were rization workers. Details of the 64 cases among polyation workers are provided in Table 1. Data on non-poly ration workers is not presented in this update since there has essentially no change. (One West Cerman case was added ** previously published table of six cases among non-polyution workers,1 while one U.S. case (No. 14] was removed use the diagnosis could not be confirmed.) Jtble 1 consists of 23 U.S. cases and 41 foreign cases from 11 *>ent countries. The 23 cases identified among U.S. polytion workers represents approximately 10% of all liver -sarcomas identified in the U.S. since 1964. This percentage de is based on the number of cases identified by the Chronic **s Division, Bureau of Epidemiology, CDC, through their ^w*dg case finding effort for liver angiosarcoma for the years , Wwm tHecis Section. SB/OSHEFSINIOSH Robert A Till laborjlonej. '-"`'"bia Rarkway. Cincinnati OH 4522S. d Occupational MeUicins/Vol. 20, No. 6/June 1978 1964-1974.* In the course of their study. CDC identified approxi mately 240 cases (confirmed by a panel of pathologists), of which 75% occurred during the 1964-1974 study period. Some of the information presented in Table 1 is summarized below. The numbers in parentheses represent the medians which were calculated front the data in the previous updated (1) The ages at diagnosis ranged (rom 37 to 71 years with a median of 49 years (44 years). (2) The latency periods. I.e., years from first expo sure to diagnosis, ranged from nine to 38 years with a median of 21 years (17 years). O) The total years exposed ranged from four to 31 years with a median of 18 years (16 years). A distribution of the cases by date of diagnosis is presented in Fig 1. Although it is difficult to interpret the aggregation of dif fering sources of data, and while the data for 1977 is incomplete, this figure suggests a gradual increase over time in the reported cases of VC-induced angiosarcoma. During the past 17 years (1961-1977), a total of 23 cases have been identified in U.S. poly merization workers. Fifty-two percent of these cases had been diagnosed over the past five years. The rise in the number of cases diagnosed in these workers during the 1970's is probably the reflection of two events: 11) the increased surveillance of VC/PVC workers during this time and (2) the growth of the VC/PVC in dustry after World War II. The 20 to 30 year lag between the growth of the industry after World War II and the increase in the number of diagnosed angiosarcomas among VC/PVC workers ob served during the 1970's roughly coincides with the median latency period (21 years). In regard to some of the observations made from the data in Table 1, It will be interesting to note whether certain trends con tinue. For instance, the age at diagnosis and latency period appear to be increasing. Some possible explanations for these phenomena are (II the initial cases may have had heavier exposures, (2! the ini- The results of CDCs case finding effort were obtained through personal communi cation with Dr. Henry Fak. Chrome Disease Division. Bureau of Epidemiology. CDC. Atlanta. Ceorgia. v *27 ^-4 n `rsfc -T. 3: !i AP000244\3 mm MRQ$pct mcdicai ro w s N L IM U tH OF A H O lO S A .K C .C M fcS rr 4i 1 Mr f Tbl 1. -- Reperted Cam of Angiosarcoma af tha Liver Among Vinyl Chloride Polymerization Worker*. Country Belgium Canada Canada Canada Canada Canada Canada Canada Canada Canada Canada Caechodovakta & echosk*Jua Fed. Reo. Germany Fed Reo Germany Fed. Reo Germany Fed. Reo. Germany Fed. Reo Germany Fed. Reo Germany Fed. Reo Germany Fed Reo Germany Fed Reo Germany Fiane* France France France France France France France Great Britain Great Britain Italy Itaty Japan Norway Sweden Sweden Sweden USA USA USA USA USA USA. USA USA U.SA USA USA USA USA USA USA USA USA USA USA USA USA USA USA Yugostana Yugoslavia Total Reported Cases 64 Case Birth Ho. Data tit VC r FVC Exposure 01 000040 00-00-00 or 1215-13 00-0044 02* 03-0614 004043 03' 022619 00-0041 04* 044)519 004045 05* 05-07-11 004444 06* 12-15-19 004047 07* 11-09-19 004046 OB 0513-20 0040-61 09 07-19-21 ( 004046 10 05-16-15 or 00-00-2* 0440-53 0040-57 02' 00-00-26 0040-51 or 06-04-30 02* 07-26-31 1041-56 10-14-57 04 09-04-30 0416-57 05* 01-01-32 07* 09-29-26 1216-62 04-15-54 08* 10-19-17 04 19-54 09* 12-13-34 1242-59 10* 07-2529 10 LO SS 11* 1229 36 010241 or 04-15-24 02 0603)) 03* 0040-19 010046 0746-59 004046 04* 01-77-27 10-1949 05* 01-29-38 0040-65 06* 04-14-34 0000-58 07 0040-27 08* 0441-34 0741-50 0523-57 or 04-2041 00-00-44 03 0642-37 02* 11-13-20 03* 03-14-20 0240-66 0040-S7 0040-53 01 0841-22 0440-53 or 1223-15 or 0623-27 03* 061010 04* 11-16-14 0340-50 08-14-51 054047 004046 or 10-17-23 120948 02* 08-19-33 11-15-55 03* 0525-15 112845 04* 01-1524 0746-52 05* 0125-12 06-1944 06* 1123-28 01-17-62 07- 054322 08-2744 OS' 054620 104746 09* 1148-31 10* 08-16-13 05-2845 0612-51 It* 05-2749 12* 11-17-18 13' 1241-21 10-1446 09-1349 12-1142 16* 1144-27 17* 05-06-31 054850 06-23-55 18* 042228 19* 0000 IS 0915-54 004043 20' 08-31-17 0040 55 21* 094249 22' 1042-23 124046 07-1147 23* 0040-23 24* 0547-17 25* 0847-10 094-58 0040-39 024047 or 0445-14 02* 11-15-31 0040-53 004050 Btagnetie af Angiesarcoma 004040 0040-55 00-00-57 0040-62 0040-67 0040-61 0040-71 0040-72 004073 0040-74 0040-76 0040-73 0040-66 0919-61 09-25-70 0040-74 0040-75 0040-75 0040-75 06-16-76 06-26-77 00-00-77 0MI47 010875 014075 0144-76 0400-76 0940-76 0740-76 124376 124072 1240-74 12-1372 07-1075 08-2174 122071 080074 031976 05-1277 0303-73 054070 1219-73 08-1947 04-0964 0240-74 004068 084041 034174 054048 034070 054269 054074 004069 10-11-74 0040-75 06-19-75 01-30-76 0040-77 0140-76 0446-73 05-2777 031077 044873 0712-73 A*. at fitfgntM 00 41 43 42 48 57 51 S3 S3 53 61 46 40 38 39 44 43 49 58 42 47 41 43 63 55 49 38 42 49 42 n 37 43 55 . 52 56 43 6S 62 49 37 58 43 52 46 45 41 43 55 61 50 52 41 43 46 60 58 67 52 50 60 67 59 42 Year* from lit Cxpasura to Magnesia 00 11 U 21 22 24 24 26 12 a\ 23 16 15 12 13 17 13 21. 22 17 22 1$ 21 15 29 26 11 18 26 * 19 28 09 15 22 22 22 19 29 31 24 14 28 IS 20 12 24 15 29 17 23 20 32 19 19 21 32 21 30 29 15 38 30 20 23 Total Yean Expcaur 00 11 14 20 22 05 23 25 0$ 26 14 16 15 12 12 17 12 12 21 15 22 10 19 12 29 26 10 17 23 13 22 04 06 21 22 21 18 21 31 21 13 28 15 20 12 17 15 24 17 23 19 26 04 19 U 22 18 21 28 14 26 20 20 18 Otttli 01-24-75 06-2975 0144-76 051376 09-1276 0742-76 01-30 77 120072 12-24-74 120072 0M0 75 10-24 7 5 0144-72 10-2070 03-1976 05-1277 0303-73 09-2871 12-1973 0147-68 0449-64 07-24-75 0323-68 08-2961 0340-75 * 05-1068 03-1&70 054269 0744-74 03-2769 Ative 114275 0446 76 01-3077 014277 124476 04-06-73 0577-77 03-10 77 0441-73 07-12-73 9 jjj rS i 58 s W 1 V * *: j,' ~ `Diagnosis was mcroscooialty arttmed "00" ndKates unknown data 428 Angiosarcoma of the Liver in VC/PVC Workers/Spirtas and Karnirt AP00024414 One of the values in maintaining this register of cases among vinyl qhloride workers is that it provides a base from which hy potheses for future research can be generated. However, the ex tremely rare occurrence of liver angiosarcoma lends great impor tance to the need for as complete reporting of the cases as possi ble if we are to develop a better understanding of liver angiosar coma in relation to vinyl chloride exposure. The occupational health community is asked to report all new or as of yet un reported cases of angiosarcoma among VC/PVC workers to either Dr. Robert Spirtas or Ms. Rose Kaminski. Surveillance Branch/ DSHEFS/NIOSH. 4676 Columbia Parkway, Cincinnati, OH 4S226, (513) 684-3284. In addition to vinyl chloride's association with angiosarcoma of the liver, recent studies have also found vinyl chloride to be asso ciated with excesses in brain cancer and neoplasms of the respira tory and lymphatic systems,1 4 5 More studies are needed to assess these findings. For an overview of the literature concerning the health effects associated with VC/PVC exposure, the reader is referred to Volume 246 of the Annals of the New York Academy of Sciences,1 YEAR OF DIAGNOSIS Fig 1. -- Number of coon of VC/PVC rolatod oogionreomat roportod to NIOSH by - of diipiosls. (Tftit roprotenti only 63 of the (4 cooei known to NIOSH tint! ! nation on diifnoili it minim for one cast.) cases were more biologically susceptible, or (3) random flue' tion. Analysis of the latency period for this disease in vinyl oride workers should be a fruitful area for future study since Itiple etiologies do not seem to be a factor in these cases and exposure periods can be fairly well defined via employment 'lories.' setnt preliminary finding, by NtOSH*, Division of Biomedical and Behavioral ce suggest that the Interaction of disulfuram with the eneymarlc detoxification of pflene dibromide tesults In increased carcinogenicity (including angiosarcoma of the I et laboratory rats. The Authors wish lo thunk Or. |.*Wiiliam Lloyd. Dr. Henry Fak and Mr. Richard Waxweiler ior their constructive entesmv References 1. Lloyd |W: Angiosarcoma of the liver in vinyl chloride/polyvinyl chloride workers. /OM 16:809, 1974. 2. Lloyd |W: Angiosarcoma of the liver in vinyl chloride/polyvinyl chlo ride workers. /OM 17:333-334. 1975. 3. Waxweiter R). Stringer W. Wagoner |K. et a(: Neoplastic risk among workers exposed to vinyl chloride. Ann NY Acad Sc/ 271:40-48. 1976. 4. Tabershaw IR and Caffey WR; Mortality study of workers in the manu* facture of vinyl chloride and its polymers. /OM 16:509-518, 1974. 5. Monson RR, Peters 1M. and Johnson MN: Proportional mortality among vinyl chiaide workers. Lancet 2:397. 1974. 6. Toxicity of vinyl chloride-polyvinyl chloride. Ann NY Acad 5ci 246, 1975. !_ ' 1, Angina as a Diagnostic Predictor It turns out that a history of typical angina is as good a predictor of ischemic heart pain or coronary disease as any other data we can get, including the exercise electrocardiogram, if you get this typical history your chances of finding significant obstructive coronary disease are 80 or 90%, depending, of course, on the "expertise" of your evaluation, if you find atypical features, on the other hand -- something isn't quite right and you're not sure it's really angina -- then the chances are going to fall off to only about 50% that such patients will have.obstructive coronary disease. And if you're pretty certain that this is not ischemic pain, then the incidence of coronary disease falls to about 10 or 20%. So the ability to take a history and identify the typical anginal syndrome is your best way of determining whether the patient is likely to have obstructive coronary disease We're all anxious to use exercise electrocardiography and other studies as supplements, but there's no specific test available that Is any better than a good and careful history. -- From "B6side Candue Diagnosis by t. O'Neal Humphries, in Emergency Medicine, lanuary. 1976. : lul of Occupational Medicine/Vol. 20, No. 6/June 1978 629 AP00024415 Z iort notes and case reports NOTICE This material may ba Vinvlchloride induced heE5^t?8? ^ copyright were normal. Ultrasound showed a large dense tumour oi'thc right angiosarcoma fiw ITltH 17 Li 2 Code) hepatic lobe with areas of necrosis. The "Tc hepatic scan confirmed ' u/ the presence of an ill-defined filling defect in the inferior part of the right lobe with two small defects at the level of the hilum. The liver computerized tomography confirmed the integrity of the Y.A. Louagie, P. Gianello, P.J. Kestens, F. left lobe. A selective angiography of the ccliac artery revealed a hyper_ Bonbled and J.G. Haot vascularized tumour ofthe right hepatic lobe (Figure I). Department of Surgery of the Alimentary Tract, Louvain-en-Wduwe Medical School, and St. Luc Hospital, 1200 Brussels, Belgium At a right tltoracophrcnolaparolomy the tumour was found to be confined to the right lobe. An extended right lobectomy was then performed. The postoperative recovery was uneventful and the patient was sent home with a monthly administration of Vincristine Correspondence to: Dr Y.A. Louagie, 20 Avenue d'Huart (bte 3), 1150 Brussels, Belgium The relationship between vinylchloride exposure and human angiosarcoma ofthe liver (ASL) received attention in 1973 when a case of this rare tumour was diagnosed at autopsy1. Case report A 39-year old man was first seen in July 1979 with pain in the right upper quadrant. The liver was palpated at the right costal margin. Oral cholecystography and barium swallow were normal and liver function tests were in normal limits. From 1965 to 1970 he had cleaned reactors used for the polymerization of the vinylchloride monomer in PVC and was thus exposed lo high amounts. He was admitted 3 months later with persisting right upper quadrant pain, loss of appetite and fatigue. The liver edge was by then hard and 4 cm below the costal margin. The ESR was accele rated (80 mra/h) and alkaline phosphatases and GGTP were elevated. Carcino-embryonic antigen (CEA) and o fcloglobulin (I mg IV) and Adriamyctn (150 mg/m' IV) which was discontinued in June 1981. Repealed controls up lo September 1981 by liver scan and computerized tomography remained normal. The patient is still jn good health 38 months after the resection. Pathology The resected specimen was 2050 g. On macroscopical examination, the main tumour (13-5 x 8 cm) was yellowish and spongy and contained cystic and haemorrhagic zones. A second smaller haemorrhagic mass was found at the inferior aspect of the right lobe surrounded by numerous purple masses. Mucroscopically. the main tumour showed large areas of necrosis and haemorrhagic pscudocvstic spaces [Figure 21 These spaces were surrounded by areas of dense vascular proliferation. The sinusoids were lined with variably sized irregular sarcomatous cells with hypcrchromalic nuclei. Elsewhere, blood-filled spaces were surrounded by sarcomatous cells. The sarcoma cells encompassed adjacent liver cells and bile ductules and infiltrated the paren chyma. The pathological diagnosis of muliiccniric angiosarcoma was made. The rest of the liver was normal except for some moderately enlarged portal tracts. Progressive fibrosis separated hcpatocytcs at the margins of the portal tracts from adjacent hepatic Figure 1 Selective angiography of the coeliac artery. The hyper- vascularized iumtntr is supplied by an anterior branch furrow) ofthe right hepatic artery Figure 2 Photomicrography of the main tumour sharing blood filled spaces surrounded h r sarcomatous cells, tHaemaioxvlin eosin, x I60l AP00024416 cord cells. Anisocaryosis and anisocylosis were frequent. A crosssection biopsy of the left lobe showed normal tissue with slight hcpatocytic anisocaryosis. The lymph nodes taken from the liver hilum were hyperplastic. The main features of this tumour were its multicentricity and the presence of mild fibrosis. Discussion The occurrence of liver angiosarcoma in vinylchloride polymerization workers was reported in 197414. Prolonged exposure and long interval from initial exposure is required before liver disease becomes apparent. The average interval is 12 years (range 6-29)1. Our patient was exposed for 5 years and became symptomatic 14 years later. It is a rapidly progressing fatal disease, especially in adults. The clinical features include rapid liver enlargement with haemorrhagic ascites, fast deterioration and cachexia usually with death within 6 months. The treatment is disappointing and chemotherapy and radiation of palliative value only. If the diagnosis is made early, the disease is localized and there is no associated liver fibrosis or portal hypertension, resective operation might Short notes and case reports prove to be curative. However, there arc few reported cases of successful operative removal of hepatic angiosarcoma and the longest survival has been 16 months4. In our case the tumour was confined to the right lobe and there was no sign of the extensive fibrosis. So far the patient is apparently free of disease after 38 months. This is, to our knowledge, the longest published survival. References 1. Creech JL Jr, Johnson MN. Angiosarcoma of the liver in the manufacture of Polyvinyl Chloride. J Occup hied 1974; 16: 150-1. 2. Block JB. Angiosarcoma of the liver following vinyl chloride exposure. JAMA 1974; 229:53-4. . 3. Heath CW, Flak H. Creech JL Jr. Characteristics of cases of angiosarcoma of the liver among vinyl chloride workers in the United States. Ann NY Acad Sci 1975; 246:231-6. 4. Adam YG. Huvos AG, Hajdu SI. Malignant vascular tumours of the liver. Ann Surg 1972:175:375-83. Paper accepted 27 July 1983 Brachiodistal vein arteriovenous fistula with valve destruction as a secondary access procedure for haemodialysis S.D. Parvin, M.R. James, P.S.Veitch and P.R.F. Bell Department of Surgery. University of Leicester. Clinical Sciences Building. Infirmary Square, Leicester. LEI 5WW, UK Correspondence to: S. Parvin, Department of Surgery, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK With the increasing use of dialysis and the length of time which has passed since programmes began, the number of patients experiencing problems with access are either increasing or will increase in the future. It was hoped that the wide variety of artificial grafts would overcome the access problem by allowing their prolonged use when primary fistulae failed. Unfortunately, the longterm patency of these grafts is in some doubt14, and consequently wherever possible the patient's own vessels should be used1*4. This paper describes a technique which can be useful in achieving this aim where other methods have failed. Method The patient is usually one who has had several previous attempts at fistula creation in the wrist or elsewhere. No obvious vessels are available, and the first step is to assess the veins carefully by a combination of clinical examination after placement of appropriate light tourniquets on the arm, and superficial venography, which will often show impalpable veins. In the unlikely event that there are no vessels available for the operation to be described, an anifical graft may have to be used. Using an axillary block or general anaesthesia, the patient's arm is prepared from the axilla to the wrist. The brachial artery is explored through an S-shapcd incision passing across the brachial skin crease. Before the artery is exposed, a search is made for any veins that may be present in the area under the incision. The best vein is the basilic, but if not available it is usually possible to find a vein though it may be some distance from the brachial artery. Once a vein has been found, the brachial artery should be exposed and the vein mobilized in such a way that approximation to the artery is possible without tension. Once this has been done, the patient is given Heparin (5000 units) and a size 3 Fogarty embolectomy catheter is passed down the vein from elbow level distally in order to define the path of the vein and its distal extremity. When the catheter has passed as far as it can go, preferably to wrist level, the vein containing it is exposed and controlled with slings. The vein at the elbow should next be turned across and anastomosed end-lo-side to the brachial artery using 5/0 or 6/0 prolene. The posterior wall of the anastomosis is completed leaving the anterior wall open (Figure I). Any large branches passing from the main vein deeply into the forearm near to the fistula are ligated and divided. The smallest of the valve strippers (Lcpine 6 Lyon Ltd) is now passed through a small transverse incision made in the chosen vein near to the wrist. Upward progress ofthe instrument can be assessed by palpation until it reaches the anastomosis (Figure 1). The stripper is then gently withdrawn using a circular movement to cut through the valve leaflets. This process is repeated until the biggest stripper consistent with the size of the vein is passed without Figure t The stripper is being withdrawn using a gentle circular movement to destroy the valves. _ 32.3 J AP00024417 Biology of Cancer and the Cancer Cell: Normal and Abnormal Regulation of Cell Reproduction DavidM. Prescott, ph.d. The distinguishing property of the cancer cell is its capacity to multiply and invade in situations where normal cells are restricted. The extraordinary research effort of the last decades to translate this primary difference be tween cancer and normal cells into unique and specific biochemical dif ferences is based on the very reasonable premise that the discovery of a unique biochemical property for all cancer cells is virtually an essential step in the even tual design of completely and absolutely specific anticancer chemotherapeutic agents. Unfortunately, the efforts to achieve such translation have so far largely failed, but in the course of this "failure," an enormous amount has been learned about cell chemistry, physiology and reproduction. We have learned to make a sharp distinction between abnormal cell repro duction and abnormal regulation of cell reproduction. The failure to recognize this difference has sometimes been a source of confusion, has inspired some fruitless research and has led to point less controversy. Neoplasia is defined as the formation of any new and abnormal growth, but there is no evidence that in a neoplasm the cells grow and divide by any other than the same rules and mechanisms by which cells grow and divide in any normal tissue or in any other situation (e.g., in culture). Thus, the abnormality in neoplasia is present not in the processes that con- Dr. Prescott is Professor, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado. Revised and updated from a paper presented atthe Sixth National Cancer Conference, 1968. AP00024418 A Spirit of Adventure: A Personal Tribute to Wendell G. Scott, M.D. (1905-1972) The onrushing flood offuture possi bilities brings with them a spirit ofad venture; a needfor bold, imaginative thinking; a willingness to gamble on provocative new concepts; the courage to break awayfrom entrenched view points, and the aggressiveness to bring them into reality. --Wendell G. Scott, m.d. The Chang ing Concepts in Control and Preven tion of Cancer. Presidential Address to the American Cancer Society, October28, 1964. It was exhilarating just being in his presence. His quick smile and sponta neous chuckle induced an atmosphere of friendship and warmth. His bright and alert look, the way he leaned forward to greet you, said that here was a man of ac tion. He was impatient, although always courteous anddiplomatic, about unnec essary delays, postponements and ob stacles to rapid achievement, and a me ticulous planner of the details and orga nization needed to get the desired re sults. This was Dr. Wendell G. Scott --a man of extreme capability and considerable achievement. We greatly respected his stature in the world of medicine, yet it somehow seemed right to call him "Scottie." He was President of the American Cancer Society; Professor of Clinical Radiol ogy, Washington University School of Medicine; President of the American Roentgen Ray Society; Editor of the journal Cancer; Chancellor of the American College of Radiology; Con sultant to the Surgeon General with the rank of Rear Admiral in the Navy; Member of the National Cancer Advi sory Board; recipient of the American Cancer Society's highest citation, the Annual National Award--and held a host of other titles and committee re sponsibilities which, if listed, would challenge those of lesser energy. It was said that he ha^i 24 jobs a day--one for each hour. When Scottie accepted an assignment for the American Cancer Society, things began to happen, and quickly. He was a man soon in motion. Telephones buzzed, visits were arranged, meetings called, committees appointed, confer ences organized, documents drafted and the goals achieved in seemingly im possible short periods of time. The en thusiasm he generated for professional education, the earlier diagnosis of cancer and the use of modern tech nological resources captured all who were near him. Even as an intern at St. Louis' Barnes Hospital in the 1930's it was said, "Give Scottie the job---he works harder than anybody else. " Most of us change with the years; Dr. Scott's appetite for hard work did not. Although Scottie served a variety of organizations and pursued a wide range of interests, we never had the feeling that he was being shared with others. This trait of total commitment was typi cal of everything he chose to do, and we could always rely on him for a solid and outstanding performance. He lived by the adage, "We work not only to pro duce, but to give value to time. ' ' Dr. Wendell G. Scott lost only one battle in his life--to the disease he was so eager to control. /jrf&MA. M.D. 261 AP00024419 4 stitute growth and division, but in some degree of loss of regulation of cell re production. The degree of loss of such regulation is, from one type of neo plasm to another, highly variable but apparently rarely complete. This is so because neoplastic cells, with few possible exceptions, do not reach the high rates of proliferation that are quite usual for certain normal cell types (e.g., stem line cells in blood-forming tissue, or progenitor cells in certain epithelia). . Various hypotheses have been evolved based on the presumption that growth and division in a neoplastic cell are in themselves intrinsically abnormal, or that neoplasia results from altered patterns of such housekeeping activities as energy metabolism or deoxynucleoside triphosphate synthesis, but such hypotheses are clearly not in keeping with the specific experimental findings. In addition, they are not consonant with the present large body of knowledge about the biochemistry, genetics, physiology and cytology of cell reproduction. Neoplasia is the result of a heritable lesion or defect in the still unidentified mechanism that regulates the initiation of chromosome replication (i.e.. the entry of a cell into the synthesis of deoxyribonucleic acid-- DNA). In cancers, the variable degree of loss of control over cell reproduction, ob; served as different growth rateT. is ac companied by two additional, primary changes: (1) a failure to a greater or lesser degree of the cells involved to develop or maintain a fully differen tiated, maiure state: and (2) a loss of repression of cell migration that is ex pressed to a greater or lesser degree as an ability to invade normal tissues. A variety of observations point up the specific inter-relatedness of these three phenomena (i.e., loss of regulation of"'] "cell proliferation, adeficiency in dif ferentiation, and a loss of repression of/ 'ceil migration), but the elucidation of the molecular mechanisms that underlie the causality of this inter-relatedness is still to be reached. Ail three of these basic changes in cell behavior may be initiated by a single common causative event. It is-well known that the more highly differentiated the cancer cell, the more slowly it grows, and vice versa. This is one of the observations that sup ports the thesis that an integral part of differentiation is the establishment of a rigid control system over cell reproduc tion. Incomplete differentiation of a cell --for example, of an hepatocytc-- means not only pbsence of some normal hepatocyte properties, but includes an incomplete differentiation of regulation of cell reproduction. The greater the deficiency in differentiation, the greater the deficiency in the regulation of cell reproduction. In basic research in cell biology, the study of the loss of regulation of cell reproduction logically proceeds from a prior understanding of the molecular basis for the intact regulatory mechanism in normal tissues. This latter under standing depends, in turn, upon afirm and detailed knowledge of the processes that constitute cell growth and cell division. The discussions in this paper deal, therefore, with the components of cell growth and reproduction that make up the life cycle of every proliferative cell, normal or abnormal. On this basis we proceed to the normal interruption of the cell life cycle by which the regula tion of cell reproduction is accomp lished. And finally, we consider briefly the possible basis for the loss of such regulation. The Cell Life Cycle All of the events of cell reproduction arc encompassed within the period called the cell life cycle. The life cycle extends from the completion of one cell division to the completion of the next, and it contains all of the events nec essary to produce a doubling in the structural elements and functional ca pacities of the cell, and--what is espe- 263 AP00024420 daily important in this discussion--it includes those events that are particu larly and directly preparations for cell division. For present purposes, rel atively little attention need be given to growth or development of the cyto plasm, because the pace of cell progress through the life cycle is governed principally by the nuclear events of chromosome replication and segregation (mitosis). All other growth activities are ultimately geared directly or re motely to the accomplishment of these chromosomal processes, and the regu lation of cell reproduction is precisely a question of the regulation of chromo some replication. The cell life cycle, which is divided into four subsections on the basis of chromosome replication and segrega tion's summarized in the diagram. (Fig. 1.) The first subsection, which occupies the first part of interphase, is called the Gt phase, and it extends from the completion of the previous cell division to the beginning of chromo some replication. The essence of chromosome replication is deoxyri bonucleic acid synthesis, and the be ginning of DNA synthesis defines the end of the Gt phase and the initiation of the S (synthesis) phase. The S phase is usually six to eight hours in mammalian cells, and is followed by an interval of usually two to three hours, called the Gj phase, which separates the end of chrom osome replication (DNA synthesis) from the detectable beginning of prophase in mitosis and cell division. The period of mitosis or division is called the D (division) phase and commonly lasts less than an hour. The terminology of G i, S, Gj and D was originated by Howard and Pelc1, who first clearly de fined the existence of the four subsec tions of the cycle from studies on plant root cells, although it had been shown previously by others that DNA synthesis occurs during interphase. * Although much has been learned about the properties and contents of all four of the cycle subsections, the definitions of the Gi and G2 phases continue to be essen tially negative (i .e., no specific molec ular events have yet been discovered which define or explain the existence of these two phases). They stand as time gaps between the clearly defined D and S phases. The transition of a cell from Gi to S is a critical point in the cycle. Once DNA synthesis begins, a cell ordinarily pro ceeds quickly to the completion of di vision and the two resultant daughter cells enter th^next Gi phase. Cells do not normally cease reproductive ac tivity by becoming arrested in either S, G] orD, although, in certain circum stances, both S orGj, and even D, can be very long. Because control of cell reproduction is essentially always achieved by action in the G, phase, and never in either the S or G, phases, little more will be said about these latter two. There are many important unanswered questions about events within S and G2, but these are not immediately germaine to the problem of regulation of cell reproduction. In mammalian cells, Gj is probably concerned primarily with synthesis of the macromolecules needed for assembly of the mitotic apparatus and with the beginning of condensation of the chromosomes into their mitotic configuration. The individual cell cycle is made up of a succession of many steps. The problem is to determine the nature of these steps and the means by which these steps are held together by cause and effect relationships in a way to produce the ordered continuity known as the cell cycle. The identification of the four major subsections of the cycle (i.e., G,, S, Gi, and D) represents the beginning of the decipherment of this cause and effect sequence. It is likely that only a small fraction of the total genetic ap paratus of a cell is concerned with these matters of continuity of the cell life cycle. 264 AP00024421 Fig. 1. A diagram of the major components of the cell li/e cycle. In i mammalian cell in culture, Gi is typically 4 hours; S, about 7 hours; G,. 2 to 3 hours; anddivision (D), about I hour. Cell reproduction in a tissue is regulated by the arrestof cells inthe G\ phase. The lesion in cancer is the failure of the neoplastic cell to be properly restrained in the G, period. The preceding section presents a gen eral view of Ihe composition of the cell life cycle. The sections to follow deal with certain aspects of the cell cycle with particular attention to possible mecha nisms for regulation of cell reproduction. TheGi Phase As already pointed out, the presence of the G, phase is not defined by any known events but is the time gap be tween the end of cell division and the initiation of DNA synthesis. Consider able information has, nevertheless, been obtained about its properties. The existence of Gi has sometimes been ascribed to preparations for DNA synthesis; in particular, the synthesis of enzymes needed to produce pools of the immediate precursors of DNA--i.e., the four deoxynucleoside triphosphates, and the synthesis of DNA polymerases and possibly other proteins concerned with configurational modifications of DNA in connection with replication. Some of these enzymes are, in certain cells, synthesized just before the in itiation of DNA synthesis, an observa tion that supports the hypothesis already stated that a sequential cycle of gene activations underlies the cycle. But, at the very most, only the terminal part of G, can be explained by these enzyme syntheses, and the explanation for the existence of Gt needs to be sought in other events. 265 AP00024422 Experiments with inhibitors have shown that both RNA and protein syntheses are necessary for a cell to progress through the G, phase, but this type of information is too nonspecific to afford insight into the reasons for G t. Measurements of cell mass in mouse cells in culture have shown that the larger the cell at the beginning of G i, the shorter the subsequent G! period.' The measurements demonstrate that the mass of the cell, or some component of cell mass, has an important bearing on the rate at which the G, events are accomplished, but unfortunately it does not contain any specific clue about the nature of these events. This relationship between cell mass and the length of G may be interpreted to mean that Gi is principally a period of cell growth, with the corollary that the mechanism that activates DNA synthesis is itself sensi tive to total cytoplasmic mass or, more likely, to some specific part of cyto plasmic mass; for example, the amount of a particular protein. This important corollary is borne out by the observa tion in these experiments that in a popu lation of cells there is less variation of mass among those cells initiating DNA synthesis than among those cells begin ning the G, phase. This suggests that initiation of DNA synthesis is related to the attainment of a given cell mass or a given amount of some particular part of cell mass. Any explanation of the Gj phase must take into account the fact that a measurable G, phase can be absent in the life cycle of some cells. Forexample, ascites tumor cells in mice have no G, period under certain conditions permitting rapid growth,4 and rapidly proliferating stem line cells of erythropoieses may have little or no G, phase. Cells in early cleavage stages in at least some animals lack G,, although G, be comes atypical part of the life cycle for almost all cells of the embryo at later stages of development. Finally, a line of hamster cells has been described that grows without a G, period. This is a particularly interesting example be cause this hamster cell line was almost certainly derived by a basic (genetic?) change from a hamster cell in which G , was a regular feature. Perhaps itmight be tentatively concluded that under conditions that favor very rapid cell proliferation, the cell cycle isshortened by reduction of the Gt phase to an im measurably brief interval, or eliminated altogether. Unfortunately, we have no solid clues as to the underlying cause of G, elimination. Whatever the reason that Gj is present under some conditions and not under others, the observations reveal the im portant fact that Gt is expendable as a time period with respect to maintenance of continuity of the cell life cycle. In general, the facts are consistent with the proposition (developed below) that the G| period is brought about by the con trolled interruption of the cell life cycle --an interruption by which regulation of cell reproduction is achieved. The most striking property yet dis covered for the G t phase is its vari ability in length within a population of cells of any given type. This was first clearly noted for clonal populations of cells proliferating in culture with a con stant average cell cycle time. AH cells show cell cycle times that are normally quite variable even though the average generation time for the population re mains constant. The variability is due primarily to variation in the length of the G, phase with relatively little or no fluctuation ordinarily present in the lengths of the S,Gt orDphases. The observation has been extended to situa tions in which the average generation time for cells of a uniform type can be lengthened by a shift to slightly less favorable culture conditions. In such cases the increased generation time is accounted for largely, if not entirely, 266 AP00024423 by an expansion of the G, phase with little or no increase in the durations of S, G2 or D. This is not to say that in creases in S. G* or D do not occur under some conditions, but the major change is always in the length of Gi'. An ex treme extension of Gj occurs in cells in culture that cease proliferation during stationary phase of culture growth; all the cells under stationary phase condi tions are arrested in the Gj state. ' Especially pertinent is the arrest in Gt of mammalian cells in culture as a consequence of contact inhibition. It is a well-established observation that normal (diploid) mammalian cells in culture cease migration and cell division when the cells become so crowded that large areas of contact between cells are estab lished. Regulation of Cell Reproduction During cleavage stages of develop ment, cell reproduction is extremely rapid. During this period of minimum restraint on cell reproduction, the cell life cycles are foreshortened by the elimination of any measureable Gt period. At a point in the development of the embryo not yet determined, the rate of cell reproduction begins to decline in various groups of cells that have entered very early differentiation; there is some evidence that the reduction in reproduc tive rate is achieved by the introduction of a G i phase into the cells cycles. Finally, in the adult organism the degree of restraint operating on cell proliferation becomes stabilized, although it differs enormously from one tissue to another in a way that conforms precisely to the need for new cells to replace those lost by death. The degree of restraint in each tissue is exactly reflected in the time that the average cell requires to complete one life cycle. Average life cycle times, therefore, vary from many thousands of hours--in a vety slowly renewing tissue such as liver--to 12 or less hours in stem line cells serving the very rapid turnover of erythrocytes. The normal demand for new erythro cytes in man is roughly 2.5 million per second, which means the completion and initiation of 2.S million cell life cycles every second. The dynamic state of the regulatory mechanism that maintains the different and appropriate rates of cell reproduc tion in each adult tissue is dramatically apparent in the changes in the rates of cell reproduction brought about by wounding of epithelia, partial hepatectomy, acute loss of blood cells, or similar disturbances to renewable tis sues. The rate or cell proliferation is greatly accelerated in such cases of acute cell loss until the cell population is restored to the normal level. The analysis of the life cycles for cells in a variety of renewing tissues has produced an important insight into the normal regulatory mechanism. Forexample, in the alimentary tract of the mouse, the average cell cycle time for the epithelial stem cells varies from 17 hours in the ileum to 36 hours in the colon, to 85 hours for buccal mucosa, to 181 hours in the esophagus. * It is a strik ing fact that these differences in cell cycle times are due to a change in the duration of only the Gt period of the cycle. The combined duration of the S, G2, and D phases is approximately 10 hours in all cases, while the duration of Gj varies from approximately seven hours in the ileum, 25 hours in the colon, 75 hours for buccal mucosa, to 171 hours in the esophagus. Under some circumstances, such as suboptimal nutrient conditions (for example, because of a poor blood supply),thelengthsofS.Gj andDmay be increased. But it is clear that the regulation of the rate at which tissue cells are allowed to reproduce is exer cised by the retention of cells in the G, state for a shorter or longer period. Cellcycle analyses are now available for the cells in a number of normal renewing tissues as well as in neoplasms, and they 267 i AP00024424 bear out the generalization. Finally, in nonrenewing tissues of the adult (e.g., skeletal muscle cells and neurons) the differentiated cells are permanently arrested in the Gi state. This role of Gi in the regulation of cell reproduction in renewing tissues and neoplasms is in striking accord with the established fact that in free-living cells of all types and mammalian cells grown in culture the natural arrest point for cell reproduction is in the G, phase. On this basis, it is reasonable to postu late that the regulation of cell repro duction in multicellular organisms has evolved out of the mechanisms by which cell reproduction is curtailed in free-living cells under natural (but un favorable) conditions. The discovery of this property of the Gi phase represents a major and most significant advance in our understand ing of cell reproduction and has brought into much sharper focus the whole prob lem of the regulation of cell reproduc tion and, most important, the loss of such regulation in neoplasia. In this regard, it is pertinent to bring up once more the phenomenon of con tact inhibition ,_We have very little idea of why or how contact between cells prevents their entrance into chromosome replication, but it is a most impressive fact that these cells can be released from inhibition by transformation with cer tain RNA or DNA viruses (e.g., polyoma virus, simian virus 40, Rous sarcoma virus).7 Viral transformation of diploid, normal cells is measured as a release from contact inhibition of hath migration and reproduction. One of the first signs that transformation has oc curred is the exodus of cells from Gt with the initiation of DNA synthesis. Shortly following the initial events of viral transformation, the cells undergo a pronouncedjaryotype chang<hfrom diploid to aneuploiin- The changes that occur in transforma tion (i .e., release from inhibition of migration and reproduction and devel- opment of ancuploidy) parallel strikingly the changes that convert a normal cell into a neoplastic cell. Because trans formation can be induced not only by viruses but by radiation or carcinogenic chemicals, and because at least some cells transformed in culture are capable of producing tumors when injected into animals, it can hardly be doubted that cell transformation in culture is essen tially the same process by which cancer develops in vivo. The breakdown in the control of cell proliferation must, as a corollary to the discovery of the Gt arrest, or Gj exten sion of the cell cycle, stem from a loss of sensitivity in one or more Gi events to a tissue-specific regulator substance (co-repressor). (Fig. 2.) Such regulator substances are still undefined in spite of ahard search, butavarietyof observations on normal tissue develop ment, experiments on partial hepatectomy, unilateral nephrectomy and skin regeneration, and other studies ail compel us to believe that they exist. Regulator substances are usually dis cussed in the context of the autoregula tion hypothesis, which is composed of the following elements. It is clear that the differentiation of any cell type for performance of a particular, specialized function (for example, hepatocytes) at the same time includes the differentia tion of a highly characteristic regulation of the reproductive rate for that given cell type. Quite obviously, differentia tion of a fully functional hepatocyte can only be successful if this is accompanied by differentiation of the appropriate rate of cell reproduction (i.e., a rate suf ficient to replace liver cells lost by death). If this differentiation of repro ductive rate is incomplete or otherwise defective, a hepatoma is the result. The differentiation of regulation over the rate of cell reproduction also in cludes a flexibility that allows the cell to accelerate greatly its rate of reproduc tion in response to the acute lossof 268 i AP00024425 Fig. 2. A scheme for the regulation of reproduction of tissue cells. The scheme encompasses and con nects the autoregulation hypothesis and the initiator protein concept. (See text for details). The cell restricts its own reproduction by synthesizing factors (co-repressor and repressor in this figure) that work together to prevent the synthesis of the RNA responsible for production of initiator proteins. The inhibition of the synthesis of initiator proteins pre vents the initiation of DNAreplication. other cells of the same type in any specific but also must be tied in some renewable tissues (e.g., the increase fashion to the functional demands in reproduction of hepatocytes in re placed on the particular cell type in sponse to partial hepatectomy). There order to explain the capacity of renew fore, it has been postulated (Fig. 2) ing tissues to undergo hyperplasia or that, as a part of differentiation, each regression in response to a variable particular cell type establishes and functional demand placed on the tissue. maintains a specific microenvironment The co-repressor must also be liable for itself by continuous addition to the or actively broken down if the hypo microenvironment of a short-lived co- thesis is to explain the dynamic response repressor that specifically interacts, by \ following such cell losses as partial feedback, to inhibit cell reproduction hepatectomy. jg, : > '` .4# in that particular cell type which The essence of the hypothesis must. produced the particular co-repressor. certainly be correct, although a regulator The production of the postulated co- " substance (co-repressor) has never been I repressor not only must be tissue identified. What is important here is that 4 '$$ '%iy - -* t*<- Vv AP00024426 autoregulation, whatever its molecular mechanisms, works by holding cells in the G! phase of the cell cycle, and breakdown in a link of the autoregulatory mechanism results in neoplasia by allowing the entrance of cells into DNA synthesis. 'Without knowing any of the molecular de tails in autoregulation, we can hardly speculate about the specific details of their breakdown. Nevertheless, there are important indirect clues. It is well established that a change in chromosomal constitution is a general accompaniment of the development of neoplasia whether the initiating stimulus is chemical, viral, radiation or unidentified (spontaneous). The karyotype changes are usually extensive and variable from neoplasm to neoplasm, and it has not been possible (with one or two exceptions) to associate any regular pattern of change with any particular neopl asm. It has been argued that the gross karyotype changes are secondary to the transformation of a cell to a neoplas tic state and that the initial primary change is more subtle. It is, in fact, difficult to understand why gross chromosomal changes would be necessary to produce the G t phase breakdown in control of cell reproduction. A human tumor cell line with an apparently normal diploid karyotype has been reported.8 In a few neoplasms the chromosome change that accompanies transformation is indeed small (e.g., the loss of a small piece of chromosome 21, the Philadelphia chromosome, present in chronic myelogenous leukemia). It seems significant that the loss of part of chromosome 21 in chronic myelogenous leukemia is detectable in normoblasts and megakaryocytes but is only effective in producing neoplasia in leukocytes. The observation is in keeping with the hypothesis that the breakdown in the regulation of cell reproduction is an intimate part of the differentiation of the particular cell type (in this case, leukocytes), and, therefore, transformation of different cell types (in this case, normoblasts and megakaryocytes) requires changes in different parts of the genetic apparatus, In those cases of chronic myelogenous leukemia that lack any detectable loss from chromosome 21, it can be argued that the crucial deletion underlying this disease occurs at the very end of the chromosome, and that the deletion is therefore accomplished whether a large or ui^detectably small deletion is involved. The same argument applies to the apparently normal diploid human tumor mentioned in the paragraph above, This argument on the minuteness of the chromosomal change necessary to produce neoplasia gains validity from the important discovery by Gateff and Schneiderman* that the development of an invasive, malignant, transplantable neuroblastoma in Drosophila larvae is due directly to the homozygous presence of a recessive mutation in the left tip of the second chromosome (with no other chromosomal change and certainly no change in karyotype). This clear evidence of a single mutational basis for a neoplasm is remindful of the unrecognized work of Gordon14 many years ago proving beyond doubt the genetic basis of melanoma induction in certain fish. The fact that the neoplastic growth occurs only in neural tissue in Drosophila larvae even though the mutation is present in all tissues, and that the neoplastic growth occurs only in melanocytes in fish even though the same genetic makeup is present in all tissues, strongly indicates that trans formation to the cancerous state is connected to a failure of the particular cell type to differentiate the normal regulatory mechanism that is unique to the differentiation process of that cell type. Obviously, it would be important for our understanding of cancer if the activi- 270 AP00024427 ty specified by the mutant locus in Drosophila could be identified. There appears to be, at present, no feasible way of making such an identification. Thefact that all neoplasms develop by a change in some G t event focuses special attention on this part of the cell cycle. Of particular importance is the transition from the Gi into the S phase, because the initiation of DNA synthesis is the earliest identifiable point at which it is clear that the cell has become committed to another cell division. The ' transformation into a neoplastic state is first detected as the acceleration of en trance of cells into DNA synthesis. The breakdown in regulation of the initiation of DNA synthesis is therefore currently the most specific event by which we are able to explain the breakdown of regulation of cell reproduction. A lesion in the control mechanism for DNA synthesis obviously is the principal issue in the development of neoplasia, but identification of the lesion seems very unlikely before the fully intact control mechanism is elucidated. Most of what is known about the control of DNA synthesis comes from the extensive and ingenious studies of microorganisms. It has been established that initiation of synthesis is brought about by the synthesis of a protein that apparently does not con stitute any of the enzymes known to be necessary for DNA synthesis but instead acts as an initiator of DNA synthesis. What controls the synthesis of this initiator protein is one of several key questions. Mutants have been ob tained in microorganisms in which the ability to initiate DNA synthesis has been lost, with some evidence that the alteration or loss of the initiator protein is the basis for the defect in initiation of DNA replication. Such information and concepts developed for micro organisms are beginning to have a pro found impact on efforts to understand the control of DNA synthesis in higher cells, and it is likely that the develop ment of new approaches to basic re search on cancer will be guided more and more by the discoveries from micro bial studies. Figure 2 contains a speculative diagram of a tissue cell that attempts to encompass and connect, with the least number of postulated elements as possible, both the autoregulation idea and the initiator protein hypothesis. Initiation of DNA synthesis is postu lated to be brought about by an initiator protein, the synthesis of which is controlled at the transcription level (messenger RNA synthesis). The syn thesis of this messenger RNA is re pressed by the complex between a repressor protein and a co-repressor. Both the repressor protein and the co repressor are synthesized continuously, and both are specific for any given cell type; the repressor protein remains intracellular while the labile co-repressor equilibrates with the space of the microenvironment. As long as there is sufficient co-repressor in the system, DNA synthesis is repressed. If the effective concentration of the labile co-repressor in the microenvironment is reduced by acute loss of cells, or is destroyed at an increased rate as a consequence of increased functional de mand on the particular cell type, re pression of DNA synthesis is weakened in some proportion to the decrease in the amount of repressor-co-repressor complex. The active destruction of co repressor at a rate dependent on the functional load is, in this scheme, pro posed as the means by which functional load is communicated to the regulation of DNA synthesis and cell reproduction. Without presenting the details of the argument, it seems most likely that the breakdown in the mechanism that controls DNA synthesis most likely would occur in the regulation of synthesis of repressor protein, or in a 271 AP00024428 mutation of the repressor protein gene itself. The development of the proper amount of regulation may be considered a part of the total pattern of differentia tion of that cell type; the less the dif ferentiation of the cell, the less com plete the development of regulation of repressor synthesis. Inclusion of the property of specificity by cell type for both co-repressor and repressor is sufficient to explain independent regula tion from one tissue cell type to the next and is particularly pertinent to the fact that neoplasia are highly tissue specific. All these hypothetical arrangements may yield a picture that seems just too contrived and fanciful in view of the. dearth of solid information, but most of this scheme has a counterpart in the mechanism of regulation of RNA transcription in microorganisms that is based on firm fact. Elements of the diagram (Fig. 2) have already been published by others. In any case, the elucidation of the molecular mechanisms that control the initiation of DNA synthesis will put within much closer reach a full explana tion of the regulation and the loss of regulation of cell reproduction in mam malian tissues. At this point it is quite clear that the problem of loss of regula tion of cell reproduction in neoplasia is precisely a question of a genetically based loss of control over initiation of DNA replication, and until we solve these pointed problems, the chance of finding a prevention or cure to cancer is extremely remote. References 1. Howard, A., andPete, S. R.: Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation tochromosome breakage. Heredity <Suppl.)6:261-276,1953. 2. Swift. H. H.: The deoxyribose nucleic acid con tentofanimal nuclei. Physiol. Zool. 23: 169-198, 1950 3. Killander, D., and Zetterberg, A.: Quantitative cytochemical investigation of the relationship be tween cell mass and initiation of DNA synthesis in mouse fibroblasts in vitro. Exp. Cell. Res. 40:12-20, 1965. 4. Baserga, R.: A radioautographic study of the uptake of "C leucine by tumor ceQs in deoxyribo nucleic acid synthesis. Biochem. Biophys. Acta 61:445-450,1962. 5. Robbins, E., andScharff, M.D.: The absence of a detectable G, phase in a cultured strain of Chinese hamster lungcell. J. Cell. Biol. 34:684-686,1967. 6. Cameron, I. L., andGieulich, R. C.: Evidence for in essentially constantduration of DNA synthesis in renewing epithelia of the adult mouse. J. Cell. Biol. 18:31-40,1963. 7. Macieira-Coelho, A., and Ponten, J.: Induction of the division cycle in testing stage human fibro blasts after RSV infection. Biochem. Biophys. Rea. Commun. 29:316-321.1967. 8. Moore, G. E., and Sandberg, A. A.: Studies of a human tumor cell line withadiploid karyotype. Cancer 17:170-175,1964. 9. Gateff, E., andSchneidermin, H. A.: Develop mental studies of a new mutant of Drosophila meUmogasttr: lethal malignant brain tumor l(2)gl*. Am. Zool. 7:760,1967. (Abstract 218) 10. Gordon, M.: Variable expressivity of pigment odl gene from zeroefiect to melanotic tumor induc tion. CancerRes. 2:676-686, 1951. i 5. I' 't 272 V f < AP00024429 A Spirit of Adventure: A Personal Tribute to Wendell G. Scott, M.D. (1905-1972) The onrushing flood offuture possi bilities brings with them a spirit of ad venture: a needfor bold, imaginative thinking; a willingness to gamble on provocative new concepts; the courage to breakawayfrom entrenched view points, and the aggressiveness to bring them into reality. --WendellG. Scott, m.d. The Chang ing Concepts in Control and Preven-. tion of Cancer. Presidential Address to the American Cancer Society, Oc tober 28, 1964. It was exhilarating just being in his presence. His quick smile and sponta neous chuckle induced an atmosphere of friendship and warmth. His bright and alert look, the way he leaned forward to greet you, said that here was a man of ac tion. He was impatient, although always courteous and diplomatic, about unnec essary delays, postponements and ob stacles to rapid achievement, and a me ticulous planner of the details and orga nization needed to get the desired re sults. This was Dr. Wendell G. Scott --a man of extreme capability and considerable achievement. We greatly respected his stature in the world of medicine, yet it somehow seemed right to call him "Scottie." He was President of the American Cancer Society; Professor of Clinical Radiol ogy, Washington University School of Medicine; President of the American Roentgen Ray Society; Editor of the journal Cancer; Chancellor of the American College of Radiology; Con sultant to the Surgeon General with the rank of Rear Admiral in the Navy; Member of the National Cancer Advi sory Board; recipient of the American Cancer Society's highest citation, the Annual National Award--and held a host of other titles and committee re sponsibilities which, if listed, would challenge those of lesser energy. It was said that he ha^l 24 jobs a day--one for each hour. When Scottie accepted an assignment for the American Cancer Society, things began to happen, and quickly. He was a man soon in motion. Telephones buzzed, visits were arranged, meetings called, committees appointed, confer ences organized, documents drafted and the goals achieved in seemingly im possible short periods of time. The en thusiasm he generated for professional education, the earlier diagnosis of cancer and the use of modern tech nological resources captured all who were near him. Even as an intern at St. Louis' Barnes Hospital in the 1930's it was said, "Give Scottie the job--he works harder than anybody else. " Most of us change with the years; Dr. Scott's appetite for hard work did not. Although Scottie served a variety of organizations and pursued a wide range of interests, we never had the feeling that he was being shared with others. This trait of total commitment was typi cal of everything he chose to do, and we could always rely on him for a solid and outstanding performance. He lived by the adage, "We work not only to pro duce, but to give value to time.' ' Dr. Wendell G. Scott lost only one battle in his life--to the disease he was so eager to control. f^dttuAA. M.D. 261 AP00024419 Vol. 295 No. 4 CELL-SURFACE ORGANIZATION IN CANCER -- NICOLSON AND POSTE 197 In hospitals, as in all other health facilities, what real ly mattered, above any kind of technical considera tion, was political relevance. From this brief review of the effects of social revolu tion on the public health of Chile, a basic message emerges. The health status of a developing nation de pends upon an equilibrium between economic, social and political factors but with due regard for scientific and technical advances. A solely political or ideologic approach to health and medical problems cannot solve the low standard of health of these populations. Only if the technical and scientific levels of medicine are increased in concert with a balanced and progres sive social change, is it possible to close the large gap between the health levels of Western democracies and those of the developing countries. References 1. Romero H; Desarrollo de la median* y la salubridad en Chile. Rev Med Chil 100:853-876, 1972 2. Fraser Brockington C: Informe sobre U salud public* de Chile. Rev Med Chil 90:374-380, 1962 3. Medina E. Kacmpffer AM. Molina R, et al; Tendencias recienlec de la mortaiidad en Chile. Rev Med Chil 99:468-472, 1971 4. Allendc S: Primer Mensaje de! Presideme Allcndc ante ei Congrcso Pleno. Presidencia de la Republics, Santiago, 1971 5. Moss R: Chile's Marxist Experiment. London, Davies and Charles, 1973 6. Cruz-Coke R, Ooid A: Cambios socio-economicos bruscos e indices de salud publics en Chile (1970-1973). Rev Med Chil 101:996-1001. 1973 7. Medina E: La siluacidn de salud chiicn* en el periodo 1970-1972. Rev Med Chil 102:150-156. 1974 8. Baltra AC: La gestidn economic* en ei gobierno de la Unidad Popular. Orbe Santiago, 1973 9. Waitzkin H, Model! H: Medicine, socialism, and totalitarianism: les sons from Chile. N Engl J Med 291:171-178, 1974 10. Garces JE: El estado y los problemas lacticosenel gobierno de Allende. Sigh XXI Mtxico, 1974 ) 11. Cimara de Diputados. Chile. Comisidn de Salud Publics y Asistencia Social. Informe de la situaci6n del Servicio Nadonal de Salud. Novem ber, 1972. Santiago, Biblioteca de! Congreso, 1972 12. ThebergeJD. The Soviet Presence in Latin America, New York. Crane Russack. 1974 13. Kuusinen OV, Arbatow Y, Beliakov A. ei al: Manual de Marxismo- Leniniimo. Third edition. Fundamentos. Buenos Aires, 1964 14. Editorial: Health Service prospects: an international survey. Lancet 2:770-771. 1973 15. Medina E: La salud: necesidad social del mundo actual. Rev Med Chi! 103:451-463, 1975 16. Servicio Nacional de Salud, Subdepanamento de Estadistica. Anuarios de Mortaiidad, Natalidad, Hospitalizaciones, Recursos y Atenciones 1965-1974. Santiago, Chile 17. Cruz-Coke R: Medicine in Chile, N Engl J Med 291:981, 1974 18. Oficina de Planiflcacidn Nacional (ODEPLAN). La Balanza de Pagos de Chile en 1970-1973. Santiago, 1974 MEDICAL PROGRESS THE CANCER CELL: DYNAMIC ASPECTS AND MODIFICATIONS IN CELL-SURFACE ORGANIZATION (Ein *i Tiuw Pwp(r) Garth L. Nicolson, Ph.D., and George Poste, M.D., Ph.D. ' | 'HE essential features of tumor cells that distinguish them from their normal counterparts are their ability to proliferate in an uncontrolled fashion, invade normal tissues and metastasize to distant sites, though these behavioral alterations are not always coupled. It is now recognized that changes in the sur face properties of tumor cells may be important in de termining aspects of each of these major behavioral characteristics. Within the primary tumor, alterations in the sur face properties of tumor cells contribute to their es cape from many of the controls and social restraints to which normal cells are subject. The proliferation of tumor cells is no longer effectively regulated by cell-tocell contact interactions, and they are increasingly un responsive to growth regulation by serum factors, hor- From the Department of Developmental and Cell Biology, University of California, Irvine, the Department of Cancer Biology, Salk Institute for Biological Studies, San Diego, CA, and the Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, NY (addreas reprint requests to Professor Nicolson at 367 Steinhaus Hall, University of Califor nia, Irvine, CA 92717). Supported by a contract (CB-33879) with the Tumor Immunology Program, Nationtl Cancer Institute, a grant (BMS72-02107) from the U.S. National Science Foundation, a grant (BC-211) from the American Cancer Society and grants (CA-IS122-IA and CA-13393) from the U.S. Public Health Service. mones and other agents that exert their effects after binding to the cell surface. Tumor cells can therefore achieve varying degrees of autonomy from normal growth restraints, and this ability is reflected in un controlled proliferation and progressive enlargement of the primary tumor. Altered surface properties of tumor cells may also result in aberrant cell-to-cell recognition, allowing tumor cells to escape from the control mechanisms responsible for maintaining prop er cell position. In malignant lesions these processes are augmented by metastasis, in which the surface properties of tumor cells are important not only in the invasion of surrounding normal tissue (or tissues) and initial separation of cells from the primary tumor, but also in determining the subsequent pattern (or pat terns) of cell distribution and establishment of meta static foci. Finally, the outcome of the interaction of tumor cells with elements of the host immune appa ratus in both primary and secondary tumors is influ enced in large part by the surface properties of the tu mor-cell population. To understand these complex phenomena, the properties of cell membranes and the surface organi zation of normal and neoplastic cells must be com prehended. Over the last decade knowledge of the AP00024430 108 THE NEW ENGLAND JOURNAL OF MEDICINE July 22, 1970 structure and dynamic organization of cellular mem branes has increased substantially, and a large num ber of surface differences between tumor cells and their normal counterparts have been identified. A full discussion of this vast subject is impossible within the limits of the present article. We will instead restrict our comments to a brief review of recent data ob tained in several laboratories suggesting that the top ographic rearrangement of various cell-surface com ponents may be important in determining the expres sion of certain of the altered surface properties and ab errant behavioral characteristics displayed by tumor cells. Particular emphasis will be given to the control of cell-surface receptors by cytoskeletal elements situ ated in the cytoplasm and the related question of how changes in these trans-membrane control systems' might account for some of the surface modifications associated with neoplasia.* Cell-Surfa.ce Organization Cell membranes are composed of lipids, proteins, oligosaccharides and polysaccharides.3 The most like ly arrangement for these components is one that max imizes the lowest free energy environments for each constituent. For lipids (such as phospholipids), which are structurally asymmetric in the hydrophilic and hydrophobic portions of their structures, hydrocarbon tails in solution tend to associate and exclude water whereas ionic heads tend to seek interactions in the aqueous phase. These interactions favor the formation of a continuous bilayer configuration for membrane lipids that sequesters lipid hydrocarbon tails from the aqueous phase.3'4 The lipid molecules on each side of the bilayer are also arranged asymmetrically in at least certain cell membranes,3 and they are capable of rapid lateral motion in the plane of the bilayer giving most biologic membranes the viscosity of light oil.1 -J-4 Lipids can also segregate in membranes to form spe cific domains with different average compositions, and they can associate with membrane proteins ("bound ary lipid") to form lipoprotein complexes.1 In addi tion, some membrane lipid may exist in a "frozen" gel state segregated from the bulk fluid lipid.4 These various lipid states may be important in organizing cell membranes into specific domains, although avail able evidence1-3'4'4 suggests that most lipid molecules are in a fluid state and arc freely diffusable in the membrane plane. Membrane proteins are basically of two types: those stabilized in the membrane by hydrophobic in teractions (for example, components bound by wa ter exclusion and interaction with lipid hydrocarbon tails); and those stabilized onto the membrane surface by hydrophilic interactions (for example, components surrounded by aqueous environment and charged groups and bound to the membrane by other than hydrophobic forces). Proteins in the former group have been termed integral membrane proteins,1 and this class of proteins appears to be involved in phe nomena such as metabolite and ion transport.''3 Inte gral membrane proteins (and glycoproteins) are glob ular, bimodal proteins that interact in both the hy drophobic (hydrocarbon) and the hydrophilic parts of the membrane.1'3 Thus, they are intercalated into the fluid lipid bilaycr to various depths, depending on their amino acid sequences and three-dimensional folding (Fig. 1). It is important that some integral pro teins and glycoproteins can completely span the membrane1'3'1 and have hydrophilic peptide sequences on Figure 1. Hypothetical Trans-membrane Control over the Distribution and Mobility of Cell-Surface Receptors by Peripheral and Membrane-Associated Cytoskeletal Components (Reproduced from Nicolson' with the Permission of the Publisher). In this schematic model of membrane organization the mobility of integral glycoprotein complexes GP, and GP4 is controlled by outer-surlace peripheral components and also trans-membrane linkages to membrane-associated cytoskeletal elements. In addition, complexes GPj and GP4 could be sequestered into a specific lipid domain indicated by the shaded area, whereas complex GPj exists in a free or "unanchored" state and is capable of lateral motion in the membrane plane formed by the fiu- i Id lipid matrix. MT denotes microtubules, and MF microfilaments (not to scale). AP00024431 Vol. 295 No. 4 CELL-SURFACE ORGANIZATION IN CANCER - NICOLSON AND POSTE 199 both the external and the cytoplasmic membrane faces (Fig. 1). The second group of proteins, termed peripheral membrane proteins,3 seem to be involved in a variety of roles not essential to the basic structure of biologic membranes. Peripheral membrane proteins usually associate with integral membrane proteins and glycolipids by ionic interactions that can be. disrupted by high salt concentrations or chelating agents.5 Remov al of peripheral membrane components does not re sult in destruction or solubilization of the mem branes, whereas removal of integral proteins is usual ly accompanied by dissolution of membrane struc ture.3 The cell membrane in its most simple form can be considered a two-dimensional solution of a mosaic of integral membrane proteins embedded in a fluid lipid bilayer.3 This concept of membrane architecture has two important implications for membrane function. In the first place, the arrangement permits membrane components to be organized asymmetrically, thus al lowing certain membrane components to be localized predominantly in either the outer or the inner half of the membrane.3'5 Membrane glycoproteins and glycolipids, for example, are arranged so that their car bohydrate residues are exposed exclusively at the out er face of the cell membrane (Fig. 1), where they func tion as specific receptors for antibodies, hormones, lectins, viruses and other agents.1'3 Maintenance of membrane asymmetry dictates that components do not continually rotate through the membrane from one side to the other. Restriction of such trans-mem brane rotation (or so called flip-flop) has recently been demonstrated experimentally.* The second important feature of the type of mem brane organization is that components can diffuse lat erally within the plane of the membrane.' '3'*'` This dif fusion offers a potential mechanism for rapid and re versible changes in the topography of specific mem brane components (Fig. 1). Various integral proteins and lipids have been shown to move laterally within the membrane, though at quite different rates.4 The lateral diffusion of lipids is rapid, each molecule ex changing with its neighbor about 107 times a second.* A number of integral membrane proteins and glyco proteins are also able to diffuse laterally within the membrane but at much slower rates than the lip ids,1 M'M whereas certain proteins appear to be rela tively immobile or "anchored" within the mem brane. I *', The distinct mobilities of unique classes of integral proteins and glycoproteins within the plasma membrane suggest that the cell may possess a con trol mechanism that restricts the mobility of some components to maintain ordered topographic dis plays or "patterns" of molecules on the cell surface.10 Such patterns might well be highly specific, differing on individual cell types and specific regions of the same cells, and they might also change during vari ous stages of cellular activity. Topographic molecular patterns could be funda mental to the inherent specificity of cell-surface orga nization. The existence of cell-specific surface pat terns could constitute an important mechanism for determining cell contact and recognition phenomena and for transmission of cell positional information in tissues. In addition, topographic redistribution of surface components might occur in response to various environmental stimuli, offering a mechanism for rapid and reversible modulation of cell-surface properties after interaction of such agents as mito gens, serum factors, hormones, lectins and antibodies with the cell surface. There is an increasing amount of evidence that the translational mobility of certain classes of integral membrane proteins is controlled by cytoplasmic skel etal elements composed of microtubules and microfil aments that appear to be "linked" in some way to in tegral membrane proteins.1-1'7'* This concept has been termed trans-membrane cytoskeletal control1 be cause any regulatory influence exerted by cytoskele tal elements on the mobility of integral membrane components will simultaneously affect components on the outer face of the membrane that are trans membrane linked to this cytoskeletal system (Fig. 1). Microtubules are large, rigid tubular structures with an outside diameter of 25 nm and an inner core 15 nm in diameter. These structures, which are con structed predominantly from a protein subunit called tubulin, have been found in both the nucleus and the cytoplasm (in the latter often associated with the cell membrane) in a wide variety of cells." Cytoplasmic microtubules appear to undergo rapid and revers ible assembly and disassembly, and although con trol of these processes is unclear, calcium ions and cyclic nucleotides appear to be involved." Microtu bules can be induced to dissociate by pressure and low temperatures, and their assembly is inhibited by plant alkaloids such as colchicine and vinblas tine." Microtubules are often found in association with another class of cytoskeletal elements, the microfila ments. The latter are thin (approximately 6 to 8 nm in diameter), double helical filamentous structures that are frequently associated with the plasma membrane in bundles or sheets immediately apposed to the inner face of the membrane.12 The identification of actin, myosin and tropomyosin in association with microfil ament bundles12'13 has contributed to the belief that they are contractile and perform important muscle like functions in cell locomotion, cell-shape changes, cytoplasmic streaming and the movement of various plasma membrane components (see below). Although microtubules and microfilaments are not true cell-membrane components, their control of the mobility and distribution of certain cell-surface recep tors indicates that they are in some way trans-mem brane "linked" to surface components, and this char acteristic justifies their classification as membrane-as sociated components.1 AP00024432 200 THE NEW ENGLAND JOURNAL OF MEDICINE July 22, 1976 Trans-membrane Cytoskeletal Control of Surface Receptors The binding of multivalent ligands to receptors on the cell surface can either cause the receptors to re main relatively dispersed or produce movement of re ceptor-ligand complexes into groups or "clusters" (Fig. 2). In some cases the clusters migrate into much larger "patches," and on some cells the patches even tually form "caps" (Fig. 2). Alternatively, the clusters coalesce into specific regions, where the receptor-li gand complexes are in time internalized by endocytosis.LLH.is Examples of multivalent ligands showing these properties are antibodies1'14''5 and lectinsl'7',` (proteins and glycoproteins that bind to specific classes of oligosaccharide sequences).'* The binding of antibodies directed against cell-sur face immunoglobulin (Eg) receptors on lymphoid cells results in anti-Ig-induced capping followed by endocytosis or "shedding" of the Ig-receptor-antibody complexes.''M''5 Capping of surface-Ig receptors can be inhibited or even reversed by drugs that act on the membrane-associated cytoskeletal system such as cytochalasin B, a fungal metabolite that disrupts micro filaments. Similarly, inhibition of cellular metabolic machinery also blocks cap formation.1'14 These re sults suggest that microfilaments play an active part in ligand-induced capping and are responsible for translocation of the receptor-ligand clusters and patches into caps. The binding of lectins such as concanavalin A (Con A), which recognizes a-D-mannosyl-like residues in oligosaccharides, to the surface of lymphocytes inhibits subsequent capping of surfaceIg by anti-Ig.7'* The inhibition of anti-Ig-induced cap ping by Con A, however, can be overcome by drugs such as colchicine or vinblastine that impair micro tubule function. Nevertheless, if Con A is bound to lymphocytes at low temperature (0 to 5C), it can in duce capping of its own receptors.7'* The interpreta tion of these apparently contradictory results is that both Con A receptors and surface-Ig receptors are linked through a microtubule system that "anchors" these receptors. In the presence of microtubule-dis rupting drugs or under conditions promoting micro tubule depolymerization (such as low, temperature), the anchorage is broken, and anti-Ig, as well as Con A, induces cap formation.7'1 Not all lymphoid cells can be easily capped with Con A, but in some of these cases, addition of colchicine dramatically facilitates capping.7 REMAINS 'DISPERSED' COALESCENCE OF 'CLUSTERS' Figure 2. Alternate Pathways of Ligand-Induced Receptor Redistribution on Fibroblastic Cells (Reproduced from Nicolson1 with the Permission of the Publisher). After ligand binding, initially dispersed receptors can remain dispersed or undergo ligand-induced clustering. The clustered receptor-ligand complexes can coalesce before endocytosis, or they can form "patches" and eventually "caps". AP00024433 Vnl. 295 No. 4 CELL-SURFACE ORGANIZATION IN CANCER -- NICOLSON AND POSTE 201 Non-random movements of cell-surface receptors into specific membrane regions under the influence of the cytoskeletal system has also been documented during phagocytosis in polymorphonuclear leuko cytes.2 Phagocytosis of large particles such as polyvinyltoluene spheres by these cells results in concomit ant loss of lectin receptors from the cell surface. Since the receptors are present initially in a random distri bution, the loss of surface lectin receptors should par allel the loss in cell-surface area after phagocytosis. Normally, this action does not occur, and a larger number of lectin receptors are removed than expect ed from the relative differences in surface areas. How ever, if colchicine is present during phagocytosis, se lective loss of lectin receptors from the cell surface is prevented.2 Further insight into the role of membrane-associat ed cytoskeletal elements in controlling the distribu tion and mobility of a number of integral plasma membrane proteins has come from recent studies us ing tertiary amine local anesthetics. Until recently, these drugs were believed to interact exclusively with membrane lipids, but evidence obtained within the past two years in several laboratories has shown that they also affect intracellular microtubules and micro filaments.,7'" Tertiary amine anesthetics of the cocaine series (dibucaine, tetracaine, procaine, etc.) dramatically en hance lectin agglutinability and lectin-induced recep tor redistribution on fibroblastic cells, but inhibit lectin and antibody-induced capping on lymphoid cells.Local anesthetics are known to partition in to the fluid lipid bilayer, where they associate with acidic phospholipids. At high concentrations local an esthetics are known to enhance the fluidity of mem brane lipids." However, at the concentrations active in producing a variety of effects on the dynamics of cell-surface receptors,17-" local anesthetics have little effect on cell-membrane fluidity.17 The site of action of local anesthetics in modifying the dynamics and trans-membrane control of surface receptors is now thought to be on the membrane-associated cytoskele tal system for the following reasons: agglutination of untransformed mouse fibroblastic cells by lectins does not occur at low lectin concentrations where a rela tively low rate of lectin-induced redistribution oc curs,1* unless the cells are first treated with local an esthetics, which enhance lectin-induced agglutinabil ity and receptor redistribution17-"; local anesthet ics reverse the inhibition of Con-A-induced aggluti nation of transformed mouse fibroblastic cells by col chicine and vinblastine17; lymphoid cell capping is prevented by local anesthetics17-"-20; and local anes thetics reverse the assemblage of performed caps on lymphocytes." The fact that the above properties of local anesthetic-treated cells can be duplicated by treatment with colchicine or vinblastine together with cytochalasin B (colchicine or cytochalasin B alone will not suffice) strongly suggests that colchicine-sensitive microtubules and cytochalasin-B- sensitive microfilaments are both involved in mainte nance of surface-receptor distribution and dynam- ics.1-17-" More direct evidence that local anesthetics modify cytoskeletal elements or their plasma membrane at tachment points has been obtained with electron mi- 1 untrrt*d 2 eolckicino 3 cytochalasin K dibucaino or A colchicino plus cytochalasin S Figure 3. Schematic Representation of the Effect of Colchi cine, Cytochalasin B and Dlbucaine Hydrochloride on the Distribution of Concanavalin A Receptors on Untransformed Mouse 3T3 Fibroblastic Cells (Reproduced from Posts et Al.17 with the Permission of the Publisher). (a) denotes a segment of plasma membrane showing intrinsic membrane proteins carrying concanavalin A receptors em bedded within a lipid bilayer and linked on the inner mem brane face to microtubules (=) and microfilaments ( ---). (b) is a representation of the planar view of the distribution of concanavalin A receptors over the upper surface of the en tire cell. In 1, untreated 3T3 ceils, (a) represents the Integral proteins linked to both microtubules and microfilaments, and (b) concanavalin A receptors remaining randomly dis persed after binding of concanavalin A to the cell surface. In (2) 3T3 cells treated with colchicine, (a) shows that disrup tion of microtubules by colchicine allows epncanavalin-A-induced aggregation of integral proteins that'are still linked to an intact microfilament system, resulting in redistribution of concanavalin A receptors into a single large "cap" as shown in (b). In (3), 3T3 cells treated with cytochalasin B. (a) demon strates that'despite disruption of microfilaments by cyto chalasin B, integral membrane proteins remain "anchored" by their connection to microtubules, ahd binding of con canavalin A to the cell surface does not Induce redistribu tion of concanavalin A receptors, which remain random ly dispersed as shown in (b). In (4), 3T3 cells after treatment with dibucaine or combined treatment with colchicine and cytochalasin B, (a) indicates that disruption of the link age of integral membrane proteins to both microtubules and microfilaments allows concanavalin-A-mediated redis tribution of concanavalin A receptors to form multiple `clusters' and 'patches' distributed over the entire cell as shown in (b). - AP00024434 202 THF, NEW ENGLAND JCK'ltNAI. OF MEDICINE July 22, 1976 --. ^ V MYOSIN MOLECULES c_, .. Z MICROTUBULES Figure 4. Hypothetical Interactions between Membrane-Associated Microtubule and Microfilament Systems Involved In Trans membrane Control over Cell-Surface-Receptor Mobility and Distribution (Reproduced trom Nicolson' with the Permission of the Publisher). This schematic model envisages opposite roles for microfilaments (contractile) and microtubules (skeletal) and suggests that they are linked to one another or to the same plasma membrane inner surface components at specific "nucleation" points. In addition, peripheral membrane components at the inner and outer membrane surface may extend this control over specific membrane domains (not to scale). croscopy. Concentrations of dibucaine, tetracaine or procaine that modify surface-receptor dynamics re sult in cell rounding and disappearance of cell-membrane-associated microfilaments and microtubules.2' The mechanism of local anesthetic action in disrupt ing cytoskeletal systems is unclear, but they could in crease intracellular Ca2+ concentrations to levels suf ficient to depolymerize microtubules by displacing membrane Ca2+. The effects of local anesthetics on microfilament integrity are even less certain, but these drugs might affect Ca2+-sensitive components such as actomyosin complexes, adenylcyclase or guanylcyclasc, Ca2+-requiring ATP-ases, or many other Ca2+dependent processes.'`A' The various effects of the different classes or com binations of drugs discussed above point to a system of trans-membrane control of receptor mobility in which receptors are linked to both microfilaments and mi crotubules and suggest that these two classes of cyto skeletal elements have opposing roles in controlling receptor mobility.'* Specifically, the microtubule sys tem is envisaged as acting as a system of anchoring elements that restrain or limit the mobility of certain receptors. In contrast, the microfilaments are be lieved to act as a contractile system to redistribute clusters of receptors unless they are restrained by the anchoring microtubules. If, as suggested, certain surface receptors are trans membrane linked to both cytoskeletal systems (Fig. 3. f), that action would offer a reasonable explanation for the following observations: disruption of microtu bules by colchicine or vinblastine allows microfila- ment contraction and functional dislocation of sur face receptors from anchoring components formerly AP00024435 Vol. 2< No. 4 CELUSURTACE ORGANIZATION IN CANCER - NICOLSON AND l'OSTE 203 stabilized by microtubules -- in this case membraneassociated microfilaments would redistribute the "un anchored" receptor-ligand clusters into caps (Fig. 3.2); and disruption of microfilaments by cytochalasin B allows ligand-induced clustering unless the re ceptors remain trans-membrane linked to a microtu bule anchoring system -- in this case the anchoring structure would be expected to prevent any redistri bution of receptors and would remain randomly dis persed (Fig. 3.3). Destruction of both the microfila ment and microtubule systems with either local anes thetics or a combination of colchicine and cytochalasin B will result in the formation of random multiple clusters (Fig. 3.4). The synthesis of various results and theories on trans-membrane control mechanisms'27,',, has been incorporated into a unifying model that is consistent with the results discussed here and elsewhere in de tail.Specifically, microtubule and microfilament assemblages are proposed to be interconnected and coupled to similar trans-membrane anchorage points. These anchorage points could serve as regulatory cen ters for the organization of specific membrane do mains. The contractile movements of the microfila ment system would be opposed by an anchorage sys tem made up of microtubules (Fig. 4),' and thus offer a system whereby the cell surface and its receptors would be under fine control by opposing membraneassociated cytoskeletal elements. Cell-surface modifications occurring after neoplas tic transformation are discussed in Part 2 of this se ries. ' References 1. Nicolson CiL; Trans-membrunc control of the receptors on normal and tumor ceils. I. Cytoplasmic influence over cell surface components. Biochim Biophys Acta 457:57-1 OK, 1976 2. Berlin RD. Oliver JM, Ukenu TE. et ai: The cell surface. N Hn^l J Med 292:515-520. 1975 J. Singer SJ. Nicolson GL: The fluid mosaic model of the structure of cell membranes. Science 175:720-731. 1972 4. Edidin M: Rotational and translational diffusion in membranes. Annu Rev Biophys Bioeng 3:179*201, 1974 5. ZVaal RFA, Roelofsen B, Colley CM: Localization of red cell membrane constituents. Biochim Biophys Acta 300:159-182 1973 6. McConnell HM*. Coupling between lateral and perpendicular motion in biological membranes. Functional Linkage in Biomolecular Systems. Edited by FO Schmitt. DM Schneider. DM Croihcrs. New York, Ra ven Press. 1975, pp 123*131 7. Edclman CM, Yahara 1. Wang JL: Receptor mobility and receptor* cytoplasmic interactions in lymphocytes. Proc Natl Acad $ci USA 70:1442-1446, 1973 8. Edclmun GM: Origins and mechanisms of specificity in clonal se lection. Cellular Selection and Regulation in the Immune Response. Edited by GM Edclman. New York. Raven Press. 1975. pp 1-38 9. Edidin M: Two-dimensional diffusion in membranes. Transport at the Cellular Level. Edited by MA Sleigh. Dll Jennings. Cambridge. Cam bridge University Press. 1974, pp 1*14 10. Nicolson GL. Painter RG: Anionic sites of human erythrocyte mem branes. II. Antispcclrm-induccd transmembrane aggregation of the binding sites for positively charged colloidal particles. J Cell Biol 59:.195*406. 1973 11. Wilson L. Bryan J: Biochemical and pharmacological properties of mi crotubules. Adv Cell Moicc Biol 3:21-72, 1974 12. La/aridcs E: Immunofluorescence studies on the structure of actin filaments in tissue culture cells. J Histochem Cytochem 23:507*328. 1975 13. Idem: Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells. J Cell Biol 63:549*561, 1975 14. Ununue HR. Kurnovsky MJ: Redistribution and fate of Ig complexes on surface B lymphocytes: functional implications and mechanisms. Transplant Rev 14:184*210, 1973 1$. de Petris S: Concanavaiin A receptors, immunoglobulins, and 8 antigen of the lymphocyte surface. J Cel! Biol 65:123-146, 1973 16. Nicolson GL: The interactions of lectins with animal cell surfaces. Ini Rev Cytol 39:89*190, 1974 17. Poste C, Papahadjopoulos D. Jacobson K, el al: Effects of local anes thetics on membrane properties. 11. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins. Biochim Biophys Acta 394:520-539. 1975 IB. Poste G, Pupuhadjopoulos D. Nicolson GL: Local anesthetics afreet transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc Natl Acad Sci USA 72:4430*4434, 1975 19. Pupuhadjopoulos D. Jacobson K, Poste Q, et ai: Effects of lo cal anesthetics on membrane properties. I. Changes in the fluidity of phospholipid bilayers. Biochim Biophys Acta 394:504-519. 1975 20. Rj.m <B. Ununue 1'R. kurnovsky MJ: Inhibition of surface capping of m.icromolcculcs by local anesthetics and tranquillisers. Nature 25(1*56-57. 1974 21. Nicolson GL. Smith JR. Poste G: I'tTects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells. J Cell Bio! 68:395-402, 1976 MASSACHUSETTS MKDICAL SOCIETY -- RKGISTRY ON CON TINUING MKD1CAI. KDUCATION To obtain information on continuing medical education courses in the New T.ngland area, write or call, indicating field(s) or specialty in which information is desired, to the Committee on Medical Kducation, 22 'The Tenway, lioston, Massachusetts 02215; telephone (617) 536-8812. AP00024436 Vol. 295 No. 5 CELL-SURFACE ORGANIZATION IN CANCER -- NICOLSON AND POSTE MEDICAL PROGRESS 253 THE CANCER CELL: DYNAMIC ASPECTS AND MODIFICATIONS IN CELL-SURFACE ORGANIZATION (Second of Two Parts) Garth L. Nicolson, Ph.D., and George Poste, M.D., Ph.D. Cell-Surface Modifications on Cancer Cells The central importance of the cell surface in deter mining many features of tumor-cell behavior has served to stimulate a massive research effort to iden tify differences in the surface properties of normal cells and their neoplastic counterparts. This effort has been successful to the extent that an enormous catalogue of differences between normal and tumor-cell popula tions has been accumulated (Fig. 5).22,22 We now have little insight, however, into the mechanisms by which these manifold changes in surface properties arise in tumor cells and how these changes are maintained. Of more immediate importance is the fact that we are still largely ignorant of how individual cell-surface alterations contribute to complex-multi-componcnt phenotypic alterations such as tumorigenicity, inva siveness and metastasis. 22'24 In experimental studies on cancer most investiga tors have chosen to use cloned, untransformed tissueculture cell lines, which can be transformed by onco genic viruses, chemical carcinogens, or radiation to form stable cultured cell lines that can be assayed in vivo for their tumorigenicity. An alternative method has been to remove tumors and surrounding normal tissues surgically for simultaneous study or to collect leukemic cells for comparison to normal lymphoid cell populations. These "in vivo" approaches are compli cated by the fact that most tumor cells arise from un known precursors, making comparisons with other cells difficult.22-25 Because of these problems and the limited availability of uniform cell populations, the main tools of the cancer-cell biologist have thus been model systems employing untransformed/trans formed tissue-culture cell lines, frequently of rodent or avian origin. It should be stressed, however, that many features of cellular organization are greatly affected by cultiva tion of cells in vitro. Awareness of the phenotypic changes that can be imposed on cell populations by cultivation in vitro is therefore of paramount impor tance if such technics are to provide a meaningful ex- From the Department of Developmental and Cell Biology, University of California. Irvine, the Department of Cancer Biology, Salk Inititute for Biological Studies, San Diego, CA, and the Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, NY (address reprint requests to Prof. Nicolson at 367 Steinhaus Hall, University of California, Irvine, CA 92717). Supported by a contract (CB-33879) with the Tumor Immunology Pro gram (National Cancer Institute), a grant (PCM76-18528) from the U.S. Na tional Science Foundation, a grant (BC-211) from the American Cancer So ciety, and grants (CA-15122-1A and CA-13393) from the U.S. Public Health Service. perimental approach to the stqdy of transformation and neoplasia.25 Alterations in Surface. Composition There arc a variety of molecules present at the sur face of cells, such as glycoproteins, glycolipids, glycosaminoglycaris and acid mucopolysaccharides. The latter compounds form the "glycocalix" or extracel lular saccharide-containing zone. Few changes in gly cocalix components seem to be of general importance in neoplasia,25 but research in this area has not been as active as the study of changes in glycolipids and their association with neoplasia. Modifications of glycolipid structure and content after transformation have been fully reviewed by Hakomori25 and Brady and Fishman.27 Two of the rela tively general changes in glycolipid structure have been decreases in the more complex glycolipids and deletion of glycolipid terminal saccharide residues. A loss in ability of glycolipid synthesis to respond to cell contact, as well as an increase in accessibility of gly colipids to antibodies, lectins and enzymes, is also as sociated with neoplasia.25,27 One recent observation of interest is that some spontaneous mouse and hamster transformed cell lines do not show the generalized gly colipid changes such as structural simplification that are characteristic of the transformed state. Several studies have concentrated on changes in surface proteins detectable by lactoperoxidase-catalyzed iodination. With this technic cell-surface pro teins are labeled in their tyrosine residues with 125I (reviewed elsewhere22,23,2`). After labeling, the 125Iproteins are solubilized and separated by electropho resis on polyacrylamide gels in the presence of de tergent. The most prominent change in surface-pro tein exposure is the loss of a high-molecular-weight (210,000 to 250,000 daltons) glycoprotein after trans formation. This glycoprotein has been called a vari ety of names (LETS, galactoprotein a, CSP, compo nent Z, SF210).25,21 Interest in this component sub sided somewhat with the finding by Yamada and Weston2* that it will not restore density-dependent in hibition of growth, a characteristic of normal cells in culture, when added back to transformed cells. How ever, the possibility exists that under these condi tions, the glycoprotein would not associate with the plasma membrane in sufficient amounts or would not be oriented correctly on the cell surface. Alternative ly, it could associate with the cell surface, but fail to establish "linkage" with cytoplasmic cytoskeletal ele ments. AP00024437 254 THE NEW ENGLAND JOURNAL OF MEDICINE July 29, 1976 INCREASED LECTIN AGGLUTIN ABILITY ALTERED SURFACE ENZYMES ALTERED MOBILITY OF COMPONENTS SURFACE CHARGE DENSITY 0 SURFACE ION DENSITY NEW ANTIGENS SECRETION OR 1 SHEDDING LOST ANTIGENS LOST OR MODIFIED GLYCOLIPIDS IMPAIRED INTERCELLULAR COMMUNICATION AND DENSITY INHIBITION OF GROWTH ALTERED PERMEABILITY ALTERED TRANSPORT LOST OR MODIFIED GLYCOPROTEINS ALTERED PHAGOCYTOSIS OR ENDOCYTOSIS MODIFIED ADHESION AND CONTACT INHIBITION OF MOVEMENT Figure 5. Some Cell-Surface Alterations Found after Neoplastic Transformation (Reproduced from Nicolson25 with the Per mission of the Publisher). Fragments of proteins can be removed from the cell surface by proteolytic enzyme treatment and subse quently characterized in detail. Glycopeptides (aver age molecular weight of approximately 4000 daltons) removed from the surfaces of tumor and cultured transformed cells show transformation-related differ ences in elution profiles when the peptides are chro matographed on gel-filtration columns.3* Specifically, the peptides from transformed and tumor cells are of higher apparent molecular weight, but their elution can be rendered similar to the normal glycopeptide elution profile by removal of sialic acid by the enzyme neuraminidase. The glycopeptides appear to be quite heterogeneous, and their characterization awaits fur ther study. One problem with this approach will be the identification of the released glycopeptides for their proper molecular origin on the cell surface. Alterations in Surface Enzymes A variety of observations suggest that transforma tion results in increased levels of cell degradative en zymes such as proteases and glycosidases.22'24'31 Un fortunately, many of these studies were performed without distinguishing between surface-localized, se creted or. total enzyme levels. When pathological tis sue samples are examined, little attempt is usually made to determine leukocyte count or other cell con tamination. Hence, these observations are often not reliable.23 In tissue-culture systems transformation generally increases the levels of specific glycosidases and proteases.22-23 Proteases have been implicated in loss of growth regulation after transformation,>.*.>. and they are thought to be a prominent determinant of several of the surface properties characteristic of the trans formed state such as alterations in lectin agglutinability,14 mobility of surface components, "'23 changes in sugar and phosphate transport, reactivity with an tibodies and others.22-24 When untransformed cells are treated with proteases, they transiently acquire many of the properties routinely possessed by transformed cells.23'24 Holley32 has recently proposed that protease treatment may mimic hormonal stimulation of growth by acting on cell-surface hormone receptors. Addi tional circumstantial support for the view that pro teolytic modification of the cell surface might account for certain altered surface properties found in tumor cells has come from a large body of experimental work showing that the abnormal growth properties dem onstrated by transformed cells in vitro and some of their altered surface properties can be modified by protease- inhibitors.23'24'31 i AP00024438 Vol. 295 No. 5 CELL-SURFACE ORGANIZATION IN CANCER -- NICOLSON AND POSTE 255 Alterations In Transport and Cyclic Nucleotides Transformed cells generally have higher plasmamembrane transport activities than their untrans formed counterparts.21'" Transport of several, but not all, sugars is enhanced by neoplastic transforma tion,53 and transformed cells show greater rates of up take of certain amino acids and amino acid ana logues.22,23 Interestingly enough, many of these trans port modifications can be mimicked in untrans formed cells by treatment with proteases, glycosidascs, serum or hormones.23 Serum addition can re lease quiescent untransformed cells from density-de pendent inhibition and also stimulate sugar, nucleo tide, and phosphate transport.32 Concurrent with some of these transport changes are rapid increases in a cellular cyclic nucleotide, cyclic GMP.'4 The levels of cellular cyclic nucleo tides have been proposed to be the determining fac tor (or factors) in releasing quiescent normal cells from growth regulation and permanently main taining transformed cells in a state of unrestrained growth.34,35 Cyclic nucleotides may serve as intracel lular messages that alter cytoplasmic enzymes, trans port systems and perhaps more directly regulate DNA synthesis.34,35 Alterations In Receptor Mobility There arc several observations indicating that trans-membrane control over cell-surface receptors is altered by transformation. Modifications in lectin agglutinability of tumor cells usually correlate with en hanced mobility of surface lectin receptors. These changes have been monitored with fluorescence or electron microscopy.16,22,33 The literature abounds with proposals that this greater receptor mobility in tumor cells might involve an increase in the "fluidity" of the membrane lipids. Experimental evidence to support this view has yet to be obtained.22'24 Perhaps more important are the recent observations made in several laboratories that the content or organization of membrane-associated cytoskeletal components such as microfilaments and microtubules arc usually re duced in transformed cells compared to their untrans formed counterparts (reviewed elsewhere23). This finding might explain the loss of receptor capping on cells from chronic lymphatic leukemia patients and in other neoplastic disorders.23 Similarly, antigens on transformed cells are in many cases more mobile than similar antigens on un transformed cells.23,36 These results may be due to constant sublethal proteolytic action on tumor cells, because the addition of proteolytic enzymes to un transformed cells also enhances agglutination and re ceptor mobility. ",23,24 Changes in cell-surface recep tor mobility due to alterations in trans-membrane linkage to cytoskeletal components is also likely, be cause local anesthetics or colchicine, in combination with cytochalasin B, can convert untransformed cellsurface receptors to a relatively more mobile state re sembling the situation in transformed cells. ,T,, Other Alterations at the Cell Surface A variety of other-cell surface changes seem to be associated with transformation (Fig. 4).21,23,26'37'3* Cancer cells are characterized by post-transformation appearance of tumor-associated antigens.37,36 These antigens can be fetal or embryonic, viral, or antigens entirely unique to the transformed state, although many of them are present in normal tissues in low concentrations. Tumors also release or shed antigens and other glycoproteins into the surrounding media (see below). These released components can serve as blocking factors that interfere with host immunologic responses to neoplasia, or they can elicit weak hu moral antibody responses that may in fact stimulate tumor growth (tumor "enhancement").40 Tumor cells have lowered requirements for many serum factors essential for cell growth.32 Serum is a complex mixture of vitamins, proteases, hormones and other proteins, minerals and nutrients. A lowered cellular serum requirement for growth and mainte nance can allow cell growth under conditions when normal cells would be quiescent. Cell-Surface Changes and the Biologic Properties of Cancer Cells We now have little insight into which of the many surface alterations of neoplastic cells described in the preceding section are functionally correlated with the in vivo capacity of these cells to grow unchecked, to in vade and to metastasize and which alterations are on ly secondarily related to these complex biologic pro cesses. Some of the more important biologic proper ties of cancer cells may well depend on the dynamics of surface receptors and their cytoplasmic control,2,23 and the remainder of this article is devoted to a brief discussion of this possibility. Escape from Immune Destruction The fact that cancer cells express novel tumorassociated antigens37,36,40 should result in their detec tion and destruction by the host immune apparatus. Theoretically, there arc several mechanisms by which tumor cells might overcome the immune surveillance mechanisms of the host.40,41 Of obvious relevance to the present discussion are examples in which anti genic determinants are shed from the cell surface or are "masked" or redistributed in such a way as to re duce the ability of the hosts ' immune systems either to detect or to destroy (or both) the cell. Antigenic modulation is probably the best known example in which neoplastic cells develop (or, more probably, pre-existing cells possessing those charac teristics are selected for survival in the face of an im mune challenge41) "immunoevasive" surface proper ties. Antigenic modulation occurs when certain tu mor cells are exposed to antibodies against cell-sur face antigens; they then rapidly become insensitive to the cytotoxic effects of complement,42 which is known to require the correct display of cell-surface-bound an tibody molecules.43 Stackpole et al.43 have examined AP00024439 256 THE NEW ENGLAND JOURNAL OF MEDICINE July 29. 1976 the dynamics of surface antigens such as thymus-leu kemia (TL) antigens on mouse lymphoma cells and have found that such antigens that undergo modula tion possess relatively rapid lateral mobility in the membrane and are easily redistributed after antibody binding to form patches and caps. Some endocytosis and shedding of TL-anti-TL complexes also occurs during modulation, but the majority of TL antigens remain at the cell surface under conditions of modu lation. The ease with which TL antigens are re distributed might well account for tumor-cell es cape from complement-mediated immunologic kill ing, because a correct distribution and disposition of adjacent antibody molecules bound at the cell surface is necessary to initiate fixation of complement com ponents. Capping or patching on the one hand prob ably prevents complement binding by aggregating an tigens and antibodies into large, immobilized com plexes so that steric hindrance or antibody molecular distortions are deterred. Alternatively, lack of anti gen-antibody lateral mobility would inhibit close ap proach of surface-bound antibody molecules, again prohibiting complement attachment. Several means of tumor-cell escape from complement-mediated im mune lysis that could result from dynamic changes in ligand-receptor redistribution are shown in Figure 6. Cancer cells probably use other mechanisms (re viewed elsewhere40'4 M`) as well to escape destruction. Cellular trans-membrane control over the dynamics of surface antigens, perhaps mediated via the cytoskeletal systems described above, may be critically im portant to the neoplastic-cell survival, and the altered display of surface receptors on tumor cells may aid in their recognition by the immune system or abet their escape from immune surveillance. Another important means of tumor-cell escape from immune destruction is by shedding of antigens or an tigen-antibody complexes from the cell surface (Fig. 6). These shed components or complexes make up a family of so-called "blocking factors" that interfere with host immunity against neoplasia, and their pres ence in the host above critical levels can result in ef fective neutralization of the cell-mediated arm of im munity against tumor cells.40`44 Alterations in Cell Adhesion; Recognition and Growth Control Several other biologic properties of tumor cells may be influenced by the dynamics of surface receptors and their cytoplasmic control. The property of contact inhibition of cell movement that is lost after neoplastic transformation could be an example of a failure in trans-membrane communication. The locomotion of cells is controlled by their cytoskeletal assemblages, and alterations in cytoskeletal organization mediated through cell-surface trans-membrane receptor link ages to these assemblages could provide an important mechanism for the control of normal cell movement and maintenance of cell position. In this case neo plastic transformation might result in alteration of the surface receptors or patterns of receptors respon sible for contact inhibition, perturbation or cytoskele tal organization or its trans-membrane communi cation to surface receptors, or combinations of the above. The loss of substrate dependency (anchorage de pendence) or necessary attachment of cells to a suit able surface to trigger initiation of normal cell prolif eration seems to be a reliable in vitro criterion for neo plastic transformation and tumorigenicity.45 The at tachment of normal cells via their surface receptors to a suitable substrate may require redistribution of cellsurface receptors into nonrhndom arrays or distribu tions. Communication of this surface rearrangement could occur via trans-membrane linkages to cytoskel etal components, and alterations in this communica tion after transformation might be involved in the re lease of tumor cells from substrate-dependent growth properties. The stimulation of cell growth by serum factors, the most important being serum hormones," provides yet another possible example whereby modification in the dynamics of cell-surface receptors may be implicated in the initiation of trans-membrane signals controlling essential biologic processes. As with the proposal of Cuatrecasas,4* hormone binding to its surface recep tors may not result in direct activation of intracellular enzymes such as nucleotide cyclases; further interac tions between other surface receptors and inner-mem brane surface components may be necessary for a cel lular hormonal response. In this scheme the dynam ics of surface receptors on tumor cells and the ease with which they can be redistributed into larger ligand-receptor complexes may allow neoplastic cells to be triggered to proliferate by levels of hormones and other mitogens that would fail to stimulate growth of normal cells. Finally, the existence of specific topographic "pat terns" of surface macromolecules on different cell types or on cells at different growth or developmental stages could serve as a potential mechanism for cellto-cell recognition and adhesion.'24-4' Disruption or alteration of the normal topographic displays of sur face components on tumor cells might render them increasingly unresponsive or refractory to the normal cellular "signals" responsible for maintaining cell po sitioning within tissue. The progressive loss or com plete absence of such positional controls in tumor cells would enable them to invade surrounding tissues and facilitate the establishment of metastatic foci. Altera tions in cell recognition and adhesion appear to have important roles in the arrest of circulating cancer cells at specific secondary sites.4*'50 How Are Cell-Surface Topography and Trans-membrane Control Modified in Tumor Cells? In pursuing the theme that changes in the mobility and distribution of plasma membrane components make a major contribution to the malignant pheno type,2'" we have still to identify the basic mechanism (or mechanisms) underlying the aberrant surface dy namics expressed by tumor cells. One possibility that AP00024440 Vol. 295 No. 5 CELL-SURFACE ORGANIZATION IN CANCER -- NICOLSON AND POSTE 257 has attracted considerable attention recently con cerns the question of modification of the tumor cell surface by proteolytic enzymes. In the type of mem brane organization described in the first part of this article, protease-induced alteration or loss of compo nents on the outer face of the plasma membrane could well produce conformational changes in the integral membrane proteins or glycoproteins carrying the pro tease-susceptible moieties, and such changes, in turn, could alter their trans-membrane "linkage" with cy- toskeletal elements on the inner side of the mem brane. Dislocation of integral membrane proteins and glycoproteins from cytoskeletal trans-membrane con trol elements would be expected to result in altered mobility and redistribution of these components. The fact that increased receptor mobility and several of the altered surface properties displayed by tumor cells can be detected in normal cells after exposure to pro teases for just a few minutes,u-u.tt.si strongly suggests that expression of these particular surface modifica tions in tumor cells need not involve acquisition of novel cell-surface determinants and may merely re sult from topographic rearrangement of already ex isting normal cell-surface components2 or loss of trans-membrane control over induced rearrange ments. The disruption of pre-existing linkages or top ographic relations of components on the cell surface by proteases could occur as a reversible phenomenon, whose duration would be restricted by the period of active proteolysis on the cell surface. Such a mecha- ESCAPES ESCAPES ESCAPES KILLED!?) ESCAPES(?) ESCAPES KILLEO ESCAPES ESCAPES REMAINS 'DISPERSED' Figure 6. Some Possible Mechanisms for Tumor-Cell Escape from Immune Surveillance That Depend on Ceil-Surfaee-Antigen Dynamics (Reproduced from Nlcolson2* with the Permission of the Publisher). Block indicated by 0 represents low temperature or chemical fixation; local anesthetics or colchicine combined with cytochalasin B; and 0 metabolic inhibitors. The normal pathway of antibody-complement cytotoxicity Is indicated by bold underlining. AP00024441 258 THE NEW ENGLAND JOURNAL OF MEDICINE July 29, 1976 nism might explain the expression of "transformed" cell properties on normal cells during mitosis, and the continued expression of some "mitotic" surface prop erties in tumor cells during interphase" could be in terpreted as indicating that proteolysis is occurring continuously. In either situation, proteolysis could modify cell-surface control mechanisms, leading to a pleiotropic stimulus that initiates a wide range of cellsurface and metabolic alterations. Additional information is clearly required to an swer several important questions. If proteolysis of the cell periphery is responsible for some aspects of the common surface characteristics and dynamics dis played both by tumor cells and by normal cells (dur ing mitosis), we might profitably investigate the cellu lar mechanisms involved in regulating the release of proteases, particularly in normal cells. It also remains to be shown precisely how proteolysis of components on the external face of the plasma membrane might alter the trans-membrane linkage of membrane com ponents to regulatory elements on the inside of the membrane. Finally, more information is needed on the range of surface alterations that can result from dislocation of linkages between integral membrane components and membrane-associated cytoskeletal elements. In this brief outline we have attempted to summa rize some of the current research on the dynamics of cell-surface organization and indicate how perturba tion of the control mechanisms responsible for regu lating the mobility and distribution of surface compo nents might be of consequence in determining some of the properties of tumor cells. One potentially impor tant implication that emerges from this work is that expression of certain surface alterations need not in volve acquisition of uniquely "tumor-specific" surface components but could equally well arise simply from topographic rearrangement of existing surface com ponents also present on normal cells. Also implicit in this proposal is the concept that the topographic "pat terns" of cell-surface molecules found on different cells could act as a "code" by which normal cells rec ognize each other and regulate their growth and so cial behavior and that alterations in such topographic patterns, if sustained, could result in the spectrum of aberrant behavioral characteristics displayed by can cer cells. Although a high degree of speculation must currently surround the interpretation of informa tion on this subject, sufficient experimental data are already available to support a functional relation between the topography and dynamics of surface macromolecules and the control of cell-surface prop erties. It is therefore anticipated that future research on the mechanisms regulating the dynamics of cellsurface organization will yield important insights in to how the cell surface influences the growth and so cial behavior of cells, and it is expected this informa tion will be of relevance to both morphogenesis and cancer. References 22. Robbins JC. Nicolson GL: Surfaces of normal and transformed cells. Biology of Tumors: Surfaces. Immunology, and Comparative Patholo gy, Vol 4. Cancer A Comprehensive Treatise. Edited by FF Becker. New York. Plenum Press, 1975, pp 3-54 23. Nicolson GL: Trans-membrane control of the receptors on normal and tumor cells. II. Surface changes associated with transformation and ma lignancy. Biochim Biophys Acta 458:1-71, 1976 24. Posie G. Weiss L: Some considerations on ceil surface alterations in malignancy, Fundamental Aspects of Metastasis. Edited by L Weiss. Amsterdam. North-Holland Publishing Company, 1976, pp 25-47 25. New Horizons for Tissue Culture in Cancer Research. J Natl Cancer Inst 53:1429-1519. 1974 26. Hakomori S-I: Glycolipids of tumor cell membrane. Adv Cancer Res 18:265-315. 1973 > 27. Brady RO. Fishman PH: Biosynthesis of glycolipids in virus-trans formed cells. Biochim Biophys Acta 355:121-148, 1974 28. Hynes RO: Role of surface alterations in ceil transformation: the im portance of proteases and surface proteins. Cell 1:147-156, 1974 29. Yamada KM, Weston JA: Isolation of a major cell surface glycoprotein from fibroblasts. Proc Natl Acad Sci USA 71:3492*3496, 1974 30. Warren L, Fuhrer JP, Buck CA: Surface glycoproteins of cells before and after transformation by oncogenic viruses. Fed Proc 32:80*85,1973 31. Cold Spring Harbor Conferences on Cell Proliferation. Vol 2. Proteases and Biological Control. Edited by E Reich, DB Rifkin, E Shaw. New York, Cold Spring Harbor Laboratory, 1975 32. Holley RW: Control of growth of mammalian cells in cell culture. Nature 258:487-490, 1975 33. Hatanaka M: Transport of sugars in tumor cell membranes. Biochim Biophys Acta 355:77*104, 1974 34. Seifert W. Rudland PS: Cyclic nucleotides and growth control in cul tured mouse cells: correlation of changes in intracellular 3r:5' cGMP concentration with a specific phase of the cell cycle. Proc Natl Acad Sci USA 71:4920*4924, 1974 35. Pastan I, Johnson GS: Cyclic AMP and the transformation of fibro blasts. Adv Cancer Res 19:303*329, 1974 36. Edidin M, Weiss A: Restriction of antigen mobility in the plasma mem branes of some cultured fibroblasts, Cold Spring Harbor Conferences on Cell Proliferation. Vol 1. Control of Proliferation in Animal Cells. Edited by B Clarkson. R Baserga. New York. Cold Spring Harbor Laboratory, 1974, pp 213-220 37. Baldwin RW: Immunological aspects of chemical carcinogenesis. Adv Cancer Rea 18:1-75, 1973 38. Coggin JH Jr, Anderson NG: Cancer, differentiation and embryonic antigens' some central problems. Adv Cancer Res 19:105-165, 1974 39. Pardee AB: The cell surface and fibroblast proliferation: some current research trends. Biochim Biophys Acta 417:153*172, 1975 40. Klein G: Immunological surveillance against neoplasia. Harvey Lect 69:71*102, 1975 41. Hyman R: Genetic alterations in tumor cells resulting in their escape from the immune response. Cancer Chemother Rep 58:431-439. 1974 42. Old U, Slockert E, Boyse EA, et al: Antigenic modulation: loss of TL antigen from cells exposed to TL antibody: study of the phenomenon in vitro. J Exp Med 127:523*539. 1968 43. Stackpole CW, Jacobson JB, Lardii MP: Antigenic modulation In vitro. I. Fate of thymus-leukemia (TL) antigen-antibody complexes following modulation of TL antigenicity from the surfaces of mouse leukemia cells and thymocytes. J Exp Med 140:939-953, 1974 44. Hcllstrdm KE, Hellstrdm I: Immunity to neuroblastomas and melano* mas. Ann Rev Med 23:19-38, 1972 45. Shin S-I. Freedman VH, Risser R, ct al: Tumorigenicity of virus-trans formed cells in nude mice is correlated specifically with anchorage in dependent growth in vitro. Proc Natl Acad Sa .USA 72:4435-4439, 1975 46. Cuatrecasas P. Membrane receptors. Ann Rev Biochem 43:169-214, 1974 47. Boyse EA: Immuno-genetics in the study ofcell surfaces: Some implica tions for morphogenesis and cancer. Current Research in Oncology. Edited by CB Anfinsen, M Potter, AN Schechter. New York, Academ ic Press, 1972, pp 57-94 48. Nicolson GL, Winkelhake JL: Organ specificity of blood-borne tumour metastasis determined by cell adhesion? Nature 255:230-232, 1975 49. Nicolson GL, Winkelhake JL, Nussey AC: An approach to studying the cellular properties associated with metastasis: some in vitro proper ties of tumor variants selected in vivo for enhanced metastasis. Funda mental Aspects of Metastasis. Edited by L Weiss. Amsterdam, North- Holland Publishing Company, 1976, pp 291-303 50. Fidler IJ. Nicolson GL: Organ selectivity for implantation, survival and growth of B16 melanoma variant tumor lines. J Natl Cancer Inst (in press) AP00024442