Document 80p4erbOQdwqaaa2NjRYa7ed

~~~~p 3. Cancn Vol. 3, pp. 251-255. PerSamon Press 1967. Printed in Great Britain Benzene Leukaemia* ALESSANDRA FORNI and LUCIAN0 MORE0 Clinica del Lavoro "L. Devoto" University of Milan, Milan, ItaIy THE CHRONIC exposure to inhalation of benzene can produce toxic effects on the bone marrow, which result in hyporegenerative anaemias or pancytopenias. I n some cases the haemopathy can evolve into a leukaemia, mostly of the myeloblastic type [1-31. One such case is presented, which was followed up by cytogenetic studies from the phase of hyporegenerative anaemia to the leukaemic phase. Case report. Z.G. was a 38-year-old female, when first seen at the Clinica del Lavoro in December 1965. Nothing important in the family history. One normal pregnancy at the age of 26. The patient had been working from 1942 to 1964 as a finisher of electric cables. The cables were covered by a rubber muff, and were cleaned with solvents containing benzene; the solvent was poured on cotton-wool and this was rubbed on the external surface of the cable muff. From 1960 to 1964 the patient complained of weakness, headache, vertigo, irregular fever. I n September 1964 a severe anaemia was discovered (Hb 6 - 4 g/100 ml, RBC 2,lOO,OOO/mm3). She had two hospital admissions and improved under treatment with transfusions. From September to November 1965 she was admitted to a university hospital. The blood findings were: H b 4 - 5 g/100 ml, RBC 1,900,000/mm3, TVBC 4,80O/mm3, with normal diKerentia1 count, platelets 90,000/1nm3. No bone marrow could be obtained despite *Supported by PHS Grant No. OH-00263-01. several attempts. She was discharged with the diagnosis of benzene-induced hypoplastic anaemia. The case was reported to the Insurance Institute against Occupational Diseases and sent to the Clinica del Lavoro. On first admission (11/30/65-3/12/66) no important findings were observed by physical examination. The laboratory findings were: Hb 6.4 g/100 ml, RBC 2,OOOYOOO/mm3,WBC 3200/mm3 with neutrophils 7oy0, eosinophil l%, lymphocytes 27%, monocytes 2%; reticulocytes 1yo; platelets 120,00O/mm3, free protoporphyrin nine pg 41/100 ml RBC; plasma iron pg 149/100 ml; UIBC pg 110/100 ml plasma, h4CV p3 108; total serum bilirubin mg 0 7/100 ml; normal bleeding and coagulation times and thromboelastogram. At sternal puncture (2/2 1/66), the bone marrow was very poor with rare normoblasts and some cells of the myeloid series, mostly myeloblasts and neutrophil promyelocytes with abundant azurophilic granules. When seen in the out-patient clinic on 4/19/66, the spleen was palpable for the first time; TYBC 3000/mm3 with neutrophils 74%, lymphocytes 16%, monocytes 2%, metamyelo- cytes 4y0, myelocytes 3y0, myeloblasts 1%; normoblasts 2%. On second admission at the Clinica del Lavoro (5/25/66-7/30/66), the laboratory findings showed an accentuation of the anaemia, an increase of immature cells in the peripheral blood (10% of 3000 TYBC/mm3), low platelet count (48,OO0/inin3). The bone marrow at sternal puncture on 6/13/66, was rather scanty; 251 252 Date Hb \j-J3c Sf).elo- (g/100 ml) ;'mrn3 blasts o1r0 12/20,69 2;'21/66 4/19j66 6; 1/66' 6/13/66 ii28;66 9; 5/66 11/12/66 3/ 7/67 9 8.9 7.5 5 12 8 5 4.3 5000 2600 3500 3000 3000 10,000 10,000 10,000 0 0 1 10 7 16 17 18 Aies&dra F o k and Lucian0 dloreo LAP 10 168 101 108 90 80 Table 1. Sample 643 Distribution of chromosome counts 44-45 46 47 48-51 - Total -tetra- colIntS ploid PBlc 6 17 124 BM 3 3 10 3 6 z --150 37 PBlc 2 5 24 1 32 PB 2 1 3* 1 1 3 20 Bhf 2 1* 1 1 5 19 PB 2 1 3 PB 4 2* 1 1 1 18 PB 1 2" 26 1 2 32 PB 2 I* 25 28 LAP: Lcucocy:e alkaline phosphatase. B M : Boae marrow, direct preparation. * Pseudodiploid cells. PBlc: peripheral blood, lymphocyte culture. PB: peripheral blood, direct preparation. ! the differential count showed an increase of cells of tlie myeloid series, some of which with atypical features; the rare cells of the erythroid series were mostly of basophilic type. During further observation as an out-patient, a progressive enlargement of liver and spleen was noted. The anaemia was always severe, the WBC counts varied between 6GOO and 24,00O/mni3 with 15-1 8'$6 myeloblasts. During the third admission at the Clinica del Lavoro (11/7,166-11;25j%6) and the clinical check-ups in the nes; months, the patient complained of fever and showed a progressive spleen edargement. The H b and RBC values decreased progressively, despite the treztrnent with transfusions; the IL'BC counts were around 1O:OOO/mm3 with 18-200.; myeloblasts. From 3/3/67 to 3 ( 3 O j G i the patient was admitted for the fourth time at the Clinica del Lavoro in a very Focr condition. She presented mycosis of the oral cavity, hyperpyrexia and bloody s p t u m . The chest X-ray shon.ed numerous infiltrates, which were interpreted as leukaenic infiltrates. The patient Ivishcd to be discharged despite her severe conditio:i and died at home on -April 7, 1967. Xo autopsy was performed. During the d o l e course of her illness, the patient was treatcd only Ivith transfusions, corticosteroids, antibiotics and vitamins. dla!el-icl find mefAods. From Dcccmbcr 19% to Slarcli 1367 sei.eral cytogcnctic studies were performed, at first on pcriphcrnl blood lymphocyte cultures, thcn on direct 1:rcpzrLtions of bone marrow and peripheral blood (Table 1). The 1ymphoc)ws fiom pcriphcral blood lvere cultured in i h r o fcr 72 lir in the presence of ph~tohaemoagglutiI?inaccording to a slight rncdification of the mcthocl of 1foori:ead r: GZ. [-IT]l.ic dircct prcpnrations of bone marrow and of peripheral blood \vih immature cells were performed according to a modification of the method of Punturieri [j]; the Colcemid treatment lasted 45 min for the bone marrow and 14 hr for the periplmd blood. Direct chromosome counts \cere performed. llost metaphases fi-om direct preparations of bone marrow and peripheral blood were als:, karyotyped. The leucocyte alkaline phosphatase \vas determined according to the histochemical score of Kaplow [GI. Chromorome studies. The results of chromosome counts are summarized in Table 1, together with some blood data at tlie time of study. The first study was performed on pcripheral blood lymphocytes. The preparations were very rich in transformed lyiiphocytes and mitoses, and the counts were performed on 150 elements. Of these cells, 4 showed c1iroma:id aberrations {gaps), 5 shoived unstable chromosome aberrations (acentric fragments, dicentric chromosomes) ivith a total of 8 caiculated breaks. One cell with .!G cliromosonies slion-ed an abnormal kaq-otype, doe to translocation of part of the long arm of one C and one G cI1ro~iosoIoi~n~a chromosome of the D group; the dc!etcd chromosome of the G group !cas si:nilar to thc Ph1 chronioso!ne. Three cclk had 47 chrornosomcs ; the cstra chromosome was a large suImietacentric not classifiable in one cell, an acrocentric of intermediate size betivccn those of t!ie D and those of the G group in anothcr cell; in one morc cell the extra cliromcjsomc \cas a submetacentric apprnxi:natzly of the size of pairs 17-18 2nd cjne chrornozomc of tlic G group had delcted long arms and \vas similar to Ph1. I n Felirur:r)* 13GG, \\.];en the paricnt :a:~s 3 C (to face p. 252) Y In a ture Ct mitosc peripl 52 hr chron: &err:. SomeS deletic dkalir In J count: blood. prepa mxrC out G chror C gro Three extra the G an ex chron c, D: one somes and c chroc the C 3rouF and some: one : most1 In pattc! cells \\.it11 were rc extra K anotl after cells; in p~ cells mat11 somc Cytogenetic Studies in a Case of Benzene Leukaemia 253 and leucopenic, still with no immature cells in tlie peripheral blood, one cell with 46 c]lromosomes presented an abnormally long ,crocentric chromosome in a direct preparation of bone marrow. Six out of 22 cells had 47 ,-l,romosomes. I n 4 analyzed cells with 47 cllromosomes, the extra chromosome was siliiilar to one of the C group in one cell, to those of pair 16in one cell, to those of the F group in one cell and to those of the G group in one more cell. In April 1966, when for the first time immature cells appeared in the peripheral blood, no mitoses were seen in direct preparations of pcripheral blood. In lymphocytes cultured for 72 hr with PHA, 2 out of 32 cells (one with 47 chromosomes) showed unstable chromosome aberrations, and 2 metaphases with 4.6 chromo- somes had an abnormal karyotype due to deletions and translocations. The leucocyte alkaline phosphatase (LAP) score was low. In June 1966 the patient had still loiv blood counts with 10% myeloblasts in the peripheral 1)lood. The chromosome counts on direct preparations of peripheral blood and bone marrow showed a modal number of 47 in 22 out of 39 metaphases. Most cells with 47 chromosomes had an extra chromosome in the C group, which could not be classified further. Three cells with 48 chromosomes had an extra chromosome in the C group and one in the G group; another cell had an extra C and an extra D chromosome. One cell with 49 chromosomes had a n extra chromosome in the C, D, E groups, and one had 2 extra C and one extra G. Another cell with 49 chromosomes had 4 extra chromosomes in tlie C group and one missing of pair 2. One cell with 51 chromosomes had one missing chromosome in the C group, one extra chromosome in the D group, 3 extra chromosomes in the G group and 2 "minute". The cells with 46 chromosomes were pseudodiploid with an extra C and one missing chromosome in another group, mostly G. In further chromosome studies, the same pattern of group C trisomy was constant in cells with 47 chromosomes, escept for 2 cells with an extra G. Cells with 46 chromosomes wcre pseudodiploid due to the presence of an extra C and one missing chromosome of another group. Of interest was the appearance after July 1966 of occasional near-tetraploid cells; this fact coincided ivith the appearance in peripheral blood smears of some binucleatcd cclls of the myeloid series, both immature and mature. In occasional cells the estra chromosome could be classified as C 9, due to the presence of 3 chromosomes of the same length with evident secondary constriction in group C (Fig. 1). This was particularly clear in a metaphase with 94 chromosomes, in which 6 chromosomes of the C group showed the secondary constriction (Fig. 2). DISCUSSION In the case described here, a leukaemia occurred after a long-standing hyporegenerative anaemia from chronic exposure to benzene for occupational reasons. On the first and third chromosome studies, the patient's lymphocytes cultured in vitro with PHA showed a high rate of chromosome abnormalities. Chromosome changes in people with benzene haemopathy have been described before [7, 81. Also subjects who have recovered from benzene haemopathy [9] and subjects exposed to benzene without signs of toxicity [9, 101 show a high rate of chromosome aberrations both of the unstable and of the stable type in their lymphocytes. I t seems therefore that chroniosome changes in lymphocytes may be an indicator of exposure to the toxic agent, provided the exposure to radiation and to cytotoxic drugs may be ruled out. Of interest is the fact that in the present case manifold chromosome changes were present in the bone marrow before the onset of leukaemia. The cells with 47 chromosomes showed different karyotypes. Cells with abnormal karyotypes had been observed also in people with past benzene haemopathy, apparently recovered both from the clinical and the haematological point of view [9]. After the onset of leukaemia, the leukaemic cells both in the bone marrow and in the peripheral blood showed a stable abnormal karyotype with a C group trisomy. The extra chromosome could sometimes be identified as belonging to pair 9. The problem of the relationship of group C trisomy and leukaemia or leukaemoid reactions is a n actual one. Several reports have been published of group C trisomy in acute myeloblastic leukaemia, blastic crisis of chronic myelogenous leukaemia and myeloproliferative disorders with leukaemoid reaction [ll, 121 (see review of the literature in [13]). As suggested by Sandberg [13], the development of a clone with C 9 trisomy might really play a role in the onset of leukaemia. The findings in our case seem to support this hypothesis. The occasional cells with 46 and 48 chromosomes with one missing or one extra chromosome in other groups (mostly D and G) and C trisomy, might be part of clones resulting from 254 Alessandra Forni`anaLucian0 More0 abnormal chromosome distribution during mitosis in the leukaemic cells. The development of the leukaemic phase was accompanied in our case by the findins of a high leukocyte alkaline phosphatase le\d. This fact does not seem to be related to the C 9 trisomy, since also cases with C trisomy and low granulocyte alkaline phosphatase have been described [lP]. The fact that in our case a high score of leucocyte alkaline phosphatase was first found at a time of an infectious complication might be of interest. As for the mechanism of induction of leukaemia by benzene, one can suggest the hypothesis that multiple chromosome aberrations might be produced by the toxic agent on bone marrow cells, resulting in cells with abnormal chromosome complement. If the abnormal cells are able to undergo mitotic division, different clones may develop, one of ndiich might e\*cntually become predominant due to selective advantage and become the leukaemic clone. This seems to have happened in the case described. I t is possible that a benign course of benzcne haemopathy might occur, when there is no formation of potentially leukaemic clones. O n the other side, the toxic action of benzene on the lymphocyte population might play a role in the development of leukaemia, by affecting the potentiality of the immune system. RESUME Un cas d'ane`mie due au benzine, icoluant uers la leucimie aigue myiloblastique, aprk une longue pkriode de latence, a ktt contro'le` par de multiples analyses cytoglnktiques. A u moment oir le patient prksentait une animie sivkre, une moelle aplastique, et une absence totale de leucocytes immatures dans le sang pe`riphirique, de nombreuses aberrations chromosomiques "stables" et "iiutables" furent observe`es dans les lymphocytes des culfures de sang. Quelques mois plus tard, toujours en l`absence de formes immatures dans le sang, de nombseuses cellules ci: 47 chromosomes furent mises en lvidence par I`examen direct de la moelle osseuse. Les cellules h&erdiploides apparteizaient ci des clones di@rents, le chromosomes suppltmentaire ressemblant soit d un chromosome du groupe C, soit 2 un chromosome des groupes F ou G. Au moment oir des mjkloblartes apparaissaient d a m le sang, toutes les cellules ci 47 chromosomes, tant par l'examen direct de la moelle que par la culture du sang, montraient le mtme caryotype, avec un chromosome supplPmentaii-e du groupe C. Quelques cellules avaient plus de 47 chromo- somes. Les auteurs itnettent l'hypoth2se que les cellules auec difirentes aberrations chromosomiques induites par le benzine peuvent &re ci l'origine de clones anormaux. L'un de ceux-ci pourrait devenir leuckmique et supplanter les autres par un processus dt sklection. SUMMARY A case of benzene anaemia evolving into acute mj.eloblastic leukaemia after a long latent period was folloxed up by multiple gtogenetic studies. At a tine when the patient shoced setrere anaemia, aplastic bone marrow, no immature leucocytes in the peripheral blood a high rate of "unstable" and "stable" chromosonze aberratiotrr w a f~ound in cultured peripheral blood lymphocytes. A f e w months later, still in the absence of immature leucocytes in the peripheral blood, numerous cells with 47 chromosomes were detected in a direct preparation of bone marrow. The hyperdiploid cells belonged to di$erent clones, the extra chromosome being siniilar at times io one of the C groups, at times to one o f the F or G groups. When myeloblasts appeared in the peripheral blood, all cells with 47 chromosomes in direct preparations of bone marrow and peripheral blood showed the same kayogpe with an extra chromosome in the C group; occasional cells with more than 47 chromosomes were also obselred. The lypothesis is suggested that cells with dtffesent chromosome alterations induced by benzene may give origin to abnormal clones. One o f these might become the leukaemic clonc selective adcantage. ZUSAMMENFASSUNG E i n Fall con Benzolanamie, der nach h e r langcn Latenzperiode in eine akute MjeloblasierileuX-amie umschlug, wurde mit H i y e rahlreicher zyfogcrietischer Untersuchutigett verfolgt. <u einer z e d , ais der Palielit cine schuere Anamie, ein aplastisches hpnochenmark und keine unreifen Leukozjten im peripheren Blut hatte, zcurde eine hohe Rate von slabilen und unstabilen Cliromosomenalierrationen in h-ulturen con peri'heren Ljmphozjtetz Cytogenetic Studies in a X u e vf .Benzene Leukaemia gejiunden. Einige illonate spater, immer noch beim Fehlen unreifer Leukozyten im peripheren Blut, konnfen in einem direkten ITnochenmarkspriiparat zahlreiche <elLen mit 47 Chromosomen gejunden werden. Die hyperdiploiden <ellen gehorten uerschiedenen h`lonen an. Die Extrachromosomen waren einmal denen der C-Gruppe, wieder andere Male denen der I;- oder G-Giirppe ahnlich. Als AQeloblasten im peripheren Blut atlftauchten, peigfen alle <ellen mit 47 Chromosomen, die in direliten Praparaten von hfnochenmark und peripheren Blut gejiunden wurden, den gleichen Kerntyp mit einem Extrachromosom in der C-Gruppe. Geiegentlich wurden auch <ellen mit mehr als 47 ChroGosomen gefunden. Es wird die fibothese vorgeschlagen, daJ <ellen mit verschie denen Chromosornenveranderungen, die durch Benzol auqelost sitid, zum Ursprung abnormer Klonen werden konnen. Einer von diesen kiinnte der leukamische Klonus werden aufgrund von Seleklionsvorteilen. REFERENCES 1. E. P. CRONKITE, Evidence for radiation and chemicals as leukemogenic agents. Arch. environrn. Hlth 3, 297-303 (1961). 2. S. hkuGERI, R. COLOMBGI,. POLLINI, E. STROSSELaLnId R. CORSICOC,aratteristiche ed evoluzione dell'emopatia benzolica. Med. d. Lavoro 56, 554-560 (1965). 3. E. C. VIGLIAXI and G. SAITAB, enzene and leukemia. .?few Engl. 3. Med. 271, 872-876 (1964). 4. P. S. MOORHEAD,P. C . NOWELLW, . J. MELLMAND, . M. BATTIPSand D. A. HUKGERFORCDhr,omosome preparations of leukocytes cultured from human peripheral blood. Ex$ Cell Res. 20, 613-616 (1960). 5. E. PUNTURIERI, Metodo diretto per la preparazione cromosomica dal midollo umano. Haernatologica 49, 488-494 (1964). 6. L. S. KAPLOWA, histochemical procedure for localizing and evaluating leucocyte alkaline phosphatase activity in smears of blood and marrow. Blood 10, 1023-1029 (1955). 7. G. POLLINI and R. COLOMBI, I1 danno cromosomico midollare nell'anemia aplastica benzolica. Med. d. Lauoro 55, 241-255 (1964). 8. G. POLLIXI and R. COLOAEIH1,danno cromosomico dei linfociti nell'emopatia benzenica. Med. d. Lavoro 55, 641-654 (1964). 9. A. FORNIC, hromosome changes due to chronic exposure to benzene. Proc. X V Int. Congr. Occupational Hlth, 1111 pp. 437-439. Vienna (1966). 10. I. M. TOUGH and W. M. COURT BROWN, Chromosomeaberrations and exposure to ambient benzene. Lancet i, 684-684 (1965). 11. A, A. SAXDBERG, T. ISHIHARA and L. H. CROSSWHITEG, r o u p 4 Trisomy in myeloid metaplasia with possible leukemia. Blood 24, 716-725 (1964). 12. A. WINKELSTERIN. ,S. SPARKEaSnd C. G. CRADDOCTKri,somy of group C in a myeloproliferative disorder. Report of case. Blood 27, 722-733 (1966). 13. A. A. SAKDBERGTh, e chromosomes and causation of human cancer and leukaemia. Cancer Res. 26, part I, 2064-2081 (1966). 255