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HODGKIN'S DISEASE T E R M I N A T I N G I N A C U T E LYMPHOSARCOMA CELL LEUKEMIA A Metabolic Study c. PATRICBKURNSM, D," RUNEL. STJERNHOLRf, P H D , ~ AND ROBERITV . KELLERMEYERM, Dt A 40-year-old woman with Hodgkin's disease developed clinical signs of acute leukemia during the fourth year of her illness. She h a d been treated for Hodgkin's disease with supervoltage radiation therapy a n d extensive cytotoxic d r u g therapy. Metabolic studies of her abnormal leukocytes, using 14C-labeled glucose a n d propionate, confirmed the clinical impression that this was a lyrnphosarcoma cell leukemia. T h e development of acute leukemia secondary to radiation and cytotoxic therapy was considered, but the possibility that the lymphosarcoma cell leukemia was a terminal transition of the Hodgkin's disease was also raised. These lymphosarcoma cells also h a d a more rapid r a t e of incorporation of thymidine-CsHH,into DNA a n d a higher r a t e of lipid synthesis from 1Glabeled acetate than those cells recovered from lyrnphosarcoma cell leukemias studied previously. Leukocytes from this patient and those from another with acute leukemia may exemplify a characteristic metabolic pattern typical of the acute form of lymphosarcoma cell leukemia. THE ASSOCIATION OF L Y M P H O S A R C O M A ~A~ND Only one substantiated case of acute lymphoreticulum cell sarcoma33 with leukemia is blastic leukemias with two additional cases a well-described clinical phenomenon; how- which may fall in that category have been re- ever, Hodgkin's disease has rarely been docu- ported.15- 31 I n 4 other cases, the cells of ori- mented as terminating in acute leukemia. I n gin were in doubt; they were referred to as the cases described, i t has been difficult to stem cell leukemia, or the type was unspecidecide from what cell line the blasts were fied.2,3, 22 derived, since morphological distinction be- This study summarizes the results of bio- tween the blasts of the major leukocyte lines chemical techniques which differentiate be- is uncertain. Most cases reported have been tween monocytic, myelocytic, and lymphocytic acute myelocytic,7. 12, 23, 24 myelomonocytic,l~ cells and which were applied to the leukocytes or monocytic5*17 with rare instances of eryth- of a patient with Hodgkin's disease who devel- roleukemia,eo 7824 histiomonocytic,S and Stern- oped acute leukemia. This patient's leukemic berg-Reed cell leukemia.19 Leukemias of lym- cells had the same metabolic behavior as those phocytic cell origin are even more unusual. associated with lymphosarcoma. This associa- From the Departments of Biochemistry and Medicine, Case Western Reserve University and Unibelsity Hospitals of Cleveland, Cleveland, Ohio. tion of Hodgkin's disease with acute lymphosarcoma cell leukemia has not been reported previously. Supported, in part, by U.S. Public Health Cervice Grants CA-10706 and No. 5 R01AM-07841-06 from the National Institutes of Health, and Grant T-485 from the American Cancer Society. * U.S. Public Health Fervice Trainee in Medicine (Hematology) PO1 AM-05420, and recipient 0 6 an American Cancer Society Clinical Fellowship (Cuya- CASE REPORT J.M., a 34-year-old Caucasian woman, was first admitted to University Hospitals of hoga County Unit, Ohio Division). t Professor of Biochemistry, Case Western Reserve University. t Associate Profesqor of Medicine and Pharmaco!ogv, and a John and Mary Markle Scholar i n Academic Cleveland in March 1966, with a non-pitting edema of the left leg of one-month duration. O n examination, there were a few palpable small anterior cervical nodes and an adnexal Medicine, Case Western Reserve University. Address for reprints: C. Patrick Burns, MD, 2f65 Adelbert Rd., University Hospitals of Cleveland, Cleve- land, Ohio 44106. Received for publication September 17, 1970. pelvic mass, but n o hepatosplenomegaly. T h e hematocrit was 370/,, white blood count (WBC) 8100/mm3 with 75y0 segmented neutrophils, 2% bands, 11% monocytes, 8% lym- 806 No. 4 H O D C I i l N ' S DISEASE AND ACUTEL E U K E M I A * B u m s ct (11. 807 phocytes, and 47, eosinophils. Platelets on peripheral smear, urinalysis, uric acid, liver function studies, s:rum creatinine, chest x-ray, bone survey, and barium enema were normal. Intravenous pyelogram revealed a left hydro- nephrosis. Lymphangiograms showed enlarged nodes from inguinal to para-aortic area at L, level, above which no contrast media passed. During exploratory laparotomy, an external iliac node was excised and histologically revealed Hodgkin's disease (mixed type) with numerous Sternberg-Reed cells (Fig. 1). She was treated initially with 3700 R tumor dose to the abdominal lymph nodes. Subsequently, during the 45 months until her death, she received the following supervoltage therapy: 1700 R to the mandible (July 1966), 1000 R to the left orbit (July 1966), 2700 R to the abdominal lymph nodes (April 1967), 2'700 R to the left orbit (April 1967), 2650 R to the left neck (September 1967), 1450 R to the abdominal lymph nodes (August 1969),and 1929 R to each ureter (November 1969). Other therapy included 30 mg of intiavenous nitro- gen mustard (January 1967), chlorambucil 6-12 mg a day (May-August 196'7), 5-fluorouracil 1.5 g intrapleurally (September 1967), vinblastine 10-20 mg weekly (September- November 1967), procarbazine hydrochloride (Natulan, Roche) up to 150 mg daily (April- May 1968), and prednisone 10-30 mg daily (November 1967-November 1969), then 60 mg daily (November 1969 until death one month later). Each iorm of therapy was continued until no further beneficial response was evident. Complications of her disease included recurrent lymphedema of the legs, pulmonary infiltrates thought to be Hodgkin's disease (January 1967), splenomegaly (August 1967), unilateral exophthalmos (July 1966 and April FIG. 1. Lymph node showing Sternberg-Reed cell (H and E, x430). 808 CANCERA,fwil 1971 VOl. 27 1967), pleural effusion containing tumor cells (August 1967), herpes zoster of the trunk (October 1967), pulmonary embolus (November 1967), and anuria due to ureteral compression and invasion (August 1969 and October 1969). I n October 1969, she was admitted with a recurrence of anuria. A left ureteral catheter was inserted after ureterograms showed invasion of the right ureter and extrinsic compression of the left. It was during this admission that the leukemia became evident (Table 1 ) . WBC, which was normal on admission, increased to a peak of 82800/mm3 on the 30th hospital day (November 24, 1969), a t which time 91% of the leukocytes were of a n abnormal cell type with sparse cytoplasm, coarsely reticular nuclear chromatin, and notched nucleus. Although a few were blast cells with a single (rarely two) nucleolus rimmed with dense basichromatin, most were thought to be lymphosarcoma cells of intermediate maturity (Fig. 2). Other results included a reticulocyte count of 2.1%, serum creatinine 8.9 mg/lOO ml, serum lysozyme 23 pg/ml (spectrophotometric method using cell wall suspension of Micrococcus lysotieikticus-normal 5 to 7 pg/ml), uric acid 16.2 mg/100 ml, and a chest x-ray that revealed n o infiltrates. A sternal bone marrow aspiration produced only peripheral blood. Subsequent therapy included an increase i n the prednisone dose to 60 mg a day and 5 units of packed cells (approximately 200 ml cells per unit). Radiation therapy to the ureteral areas bilaterally and the indwelling left ureteral catheter relieved the azotemia temporarily, but her uremia progressed during the last 2 weeks of life and she died approximately 6 weeks after admission. There was no clinical improvement of the leukemic state with prednisone therapy. N o autopsy was performed. METHODS On two occasions after the onset of the acute leukemia-I0 days and again 1 day before death--15 ml of whole blood were collected in heparinized tubes. T h e red blood cells were sedimented with 57& dextran. After 40 minutes, the leukocyte-rich supernatant was removed and processed as described previously.29 Separation of the cells with a column was not necessary. T h e cells were washed and suspended in 20% serum and Eagle's medium with the final suspension being made up of 95% lymphocytes or lymphosarcoma cells. Cell counts were clone with Unopette (Becton, Dickinson and Co.) and a Spencer Hemocytometer. Radioactive compounds were obtained from New England Nuclear Corporation. Leukocyte incubations with radioactive substrates and isolation of the products by sequential extractions, including ion-exchange and partition chromatography, have been described previously.26.30 T h e cells were incubated with 5 pc of thymidine-OH, for estimation of DNA turnover, 5 pc of uridine-5-3H for RNA turnover, 2 pc of mixed tritiated amino acids for protein synthesis, 1 pc glucose-U-lC for glu- TABLE1. Hematologic Studies on Patient J.AI. Lymphosarcoma Cells Mature Intermediate Blasts 0 16 0 12 - 0 4- 61 72 28 17 22 N o ,i -HODGKINDI'SEASAEND ACUTELEUKEMIA Biirris et (11. cose utilization and glycogen turnover, 2.5 pc acetate-2-14Cfor acetate utilization, and 2 pc propionate-3-*4C for propionate utilization. T h e incubation times for glucose, propionate, and acetate were 3 to 5 hours. T h e phytohemagglutinin (PHA), Difco, for stimulation of RNA turnover was used at a final concentration of 25 pg/ml. All equipment in contact with the intact cells was plastic or siliconecoated glass except the final incubation tubes for DNA, RNA, and amino acids, which were glass. At the time of the first study, the patient was receiving predriisone 15 mg per day; this had been increased to GO mg per day during tlie second study. 809 RESULTS Glucose utilkation by the patient's leukcmic cells was 0.52 pmoles per hour per los cells. T h e glycogen turnover value was 0.03 pmoles per hour per 108 cells. T h e results of propionate and acetate-labeling experiments are shown in Table 2. Propionate utilization was 17 nmoles per hour per 10s cells. Lipid synthesis. reflected by the incorporation of acetate-2-14C,was 8.9 nmoles per hour per los cells. T h e results of nucleic acid and protein-labeling experiments are shown in Table 3. Thymidine incorporation into DNA was 265 picomoles per hour per 108 cells. Uridine utili7ation reflecting RNA synthesis was 11.5 and 0.58 picomoles per hour per 10s cells after 4 and 2 1 hours' incubation, respectively. T h e uritline incorporation was stimulated only twofold with PHA. RNA tiirnover and protein synthesis, as determined after a 21-hour incubation period with radioactive precursors, were similar to normal lymphocytes. Metabolic studies on the abnormal leuko- cyte population of the patient were repeated one day prior to death, and the results were similar to those given above (Tables 2, 3). FIG.2. Peripheral blood smear after deielopment of leuhemia. U p p e r Irfl-blast cell with wveral nuclcoli. Upper ri~Izt--l~mpliosarcomacell with cleft and 1111cleolu5. Otliei cell h a s c\toplasnic tail. Loruer-cliimp of lymphosaicoma cells of intcrmetliate maturity (Tet i achrome stain, x 1000). Results of tlie metabolic studies of the leukocytes of patient J.M. were similar to those obtained from another patient (J.B.) with acute leukemia (Tables 2, 3 ) . Prior to therapy, J.R. had a leukocyte count of 94,000/mm3 h i t h (345% of the cells being immature with fine nuclear chromatin and a single large nucleolus. T h e abnormal leukocytes appcared to be lymphocytic, and some of the more mature-appearing cells had clefts in their nuclei. T h e abnormal cells in tlie peripheral blood of this patient were thought to be lymphosarcoma cells. At autopsy, small and large lymphocytes were found invading most of the organs of the body. T h e morphology of these invasive cells was consistent with lymphosarcoma. TABLE2. _- Results of Propionate and Acetate-labeling Experiments on Cells of J.M. and J.B. Compared t o 3 Types of Leukemia J.M. 11/22/69 J.M. 12/2/69 J.B. LSCL* ALL CLL Propionate utilization nmoles/hr/lO* cells Lipid synthesis nmoles/hr/l08 cells 17 8.9 - 29 18-36 2-3 5-6 6.2 12.6 1.2-2.0 0.8-1.7 0.5-0.8 * As previously reported.27.28 ALL-acute lymphocytic leukemia; CLL-chronic lymphocytic leukemia. 81.0 CANCERA p i i l 1971 TABLE3. Results of Nucleic Acid and Protein-labeline ExDeriments DNA RNA RNA +RNA PHX pM/hr/108 pM/hr/l08 pM/hr/lOB pM/hr/l08 4 hrs.+ 4 hrs. 21 hrs. 21 hrs. Normal lymphocyte* 8.6+ 2.9 3.2h2.0 J.M. 11/22/69 265 11.5 J.M. 12/2/69 J.B. 129 10.0 60 - * Mean of 4 normals i l S.D. t Incubation time. pM = picomoles; dpm = disintegrations per minute. 1.6f0.3 0.58 - 21 10.4f3.6 1.25 - 17.8 Vol. 27 Amino acids dpm,fhr/lO8 21 hrs. 18200i~10000 20000 - DISCUSSION Lymphosarcoma cell leukemia (LSCL) has been identified within the larger group of lymphocytic leukemias.13.14,21 It can be divided into an acute and chronic form. Morpliological criteria are used to distinguish these from the corresponding acute and chronic lymphocytic leukemias. Biochemical evidence has substantiated the morphological similarities and differences between LSCL and other lymphocytic leukemias.27~28 T h e cells from patient J.M. had a low glucose utilization thereby defining their glycolytic rate as that characteristic of a lymphocyte (0.28-0.50 pmoles per hour per lo8 cells) .z7 This low rate which persists even when the lymphocyte is abnormal as i n acute lymphocytic leukemia or LSCL is only about 10% of that found in neutrophils, eosinophils, and monocytes.27 T h e low glycogen turnover value confirmed the identification of the cell as being of lymphocytic t y ~ e . ~ 7 Propionate utilization of the magnitude recorded for the cells of J.M. has been described only for cells from acute monocytic leukemia or LSCL.28 This result contrasts with the lower values typical of acute and chronic lymphocytic leukemia (Table 2).28 Together, the rates of glucose and propionate utilization identify the cells studied from J.M. as lymphosarcoma cells. Serum lysozyme values have been found useful in identifying various forms of leukemia. T h e Iysozyme levels are usually normal or low in the lymphocytic cell leukemias including LSCL.27 Values greater than 50 pg/ml are characteristic of the monocytic leukemias.27 Azotemia is a well-documented cause of elevation oE lysozyme,lo and this is the likely cause of the moderate elevation in T.M. T h e high thymidine utilization is similar to the values found in acute 1eukemias.z~Such high utilization suggests that the cell is undergoing mitosis. This high value supports the presumption that the abnormal cells are immature. T h e lack of proportional elevation i n uridine utilization and protein synthesis has been noted in acute leukemia, and this perhaps reflects the fact that transcription and translation are secondary events during mitosis. PHA added to the incubation mixtures stimulated RNA synthesis by a factor of 2 compared with the five to ten fold increase observed with normal lymphocytes (Table 3). Insensitivity to PHA has been reported to occur i n lymphocytes of patients with Hodgkin's disease; the degree of insensitivity seemed to correlate directly with the clinical activity of the disease.ll Impairment of stimulation is a characteristic of the lymphocytes of chronic lymphocytic leukemia.20 Lymphocytes obtained during acute viral infection also fail to respond normally to PHA.32 Lipid synthesis by the cells from J.M. was higher than that previously reported for LSCL.27 T h e fact that J.M.'s cells were divid- ing more rapidly may account for this increase, since more lipid for cell membrane synthesis would be required. T h e cells from the patients with LSCL reported earlier had a normal or only slightly elevated DNA turn0ver.~7 T h e other patient with LSCL (J.B.), like J.M., had a markedly elevated DNA turnover and lipid synthesis (Tables 2, 3). From the results obtained with the cells from these two patients, a metabolic pattern may be emerging that is characteristic of LSCL. T h e high turnover of DNA may correlate with an acute clinical phase of the disease. T h e relationship of the terminal leukemia No. 4 -HODGKINDI'SSEASAEND ACUTELEUKEMIA Burns et al. 811 to Hodgkin's disease in this patient is speculative. When one disease develops in the setting of another, i t is difficult t o rule out coincident e. However, the appearance of the leukemia a t the time of inexorable progression of the Hotlgkin's disease suggests a closer relationship. T h e similarity of Hodgkin's disease antl lpmphosarcoma before bone marrow in- volvement occurs and the rarity of reported cases of lymphoproliferative disorders after radiation exposurelp 4 strengthen the possibility. Although the relationship of acute leukemia to Hotlgkin's disease is as yet uncertain, this study reports a n association of the 2 diseases and documents the diagnosis of LSCL with both morphological and metabolic data. REFERENCES 1. Brill, A. B., Tomonaga, M., and Heyssel, R. hf.: Leukemia in man following exposure to ionizing radiation. A summary of the findings in Hiroshima and Nagasaki, and a conipai-ison with other human expcrience. A n n . Z n t e m . M e d . 56:59&609, 1962. 2. Cavallero, G., and Bo, G.: Associazione di leucosi acuta indifferenziata o di granulomatosi maligna tli Hotlgkin in un bambino. Pathologica 58:lll-120, 1966. 3. Cohrn, H., Rubin, S., and Eisrmann, G . : Hotlgkin's disease (familial) associatcd with multiple malignant neoplasms. 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E., and Finch, S. C.: Urinary muraniidase and renal disease. Correlation with I.enal liistology and implication for the mechanism of cnzymuria. New Eng. J. Med. 279:506-512, 1968. 11. Hcrsh, E. M., and Oppenheimer, J. J.: Impaired in vitro lymphocyte transformation in Hodgkin's disease. Nerir Eng. J. Med. 273:1006-1012, 1965. 12. Hollard, D., Morel, P., and Revol., L.: Maladies de Hodgkin terminecs en leucose aigue. Lyon M e d . 215:1373-1377, 1966. 13. Isaacs, R.: Lymphosarcoma cell leukemia. A n n . Intern. iMed. 115557-662, 1937. 14. Kundrat: Ueber lympho-sarkomatosis. W i e n . Klin. Wschr. 6:211-213, 234-239, 1893. 15. Lacher, M.J., and Sussman, L. N.: Leukemia and Hodgkin's disease. Ann. Intern. M e d . 59:369-378, 1963. 16. Newman, D. R., Maldonado, J. E., Harrison, E. G., Kiely, J. M., and Linman, J. W.: Myelomonocytic leukemia in Hodgkin's disease. Cancer 25: 128-134, 1970. 17. Razis, D. V.,Diamond, H. D., and Craver, L. 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H.: Carbohydrate metabolism in Icukocytes. XII. Metabolism of the human lymphocyte. J . Reticuloendothel. SOC. 6:590-605, 1969. 30. Stjernholm, R. L., Wheelock, E. F., and van den Noort, S.: A lymphotoxic factor in the sera from patients with multiple sclerosis. J. Reticuloendothel. SOC. 8:334-341, 1970. 31. Watson, C. J.: Handbook of Hematology, vol. 4, H. Downey, Ed. New York, Paul B. Hoeber Inc., 1938. 32. Willems, F. T . C., Melnich, J. L., and Rawls, W. E.: Viral inhibition of the phytohemagglutinin response of human lymphocytes and application to viral hepatitis. Proc. SOC.Exp. Biol. M e d . 130:652-661, 1969. 33. Zeffren. 1. L.. and Ultmann. 1. E.: Reticulum cell sarcoma ierminating in acute "leukemia. Blood 15:277-284, 1960.