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Development of dysmyelopoietic syndrome in a hairy cell leukemia patient treated with chlorambucil: Cytogenetic and morphologic evaluation
Development of Dysmyelopoietic Syndrome in a Hairy Cell Leukemia Patient Treated with Chlorambucil: Cytogenetic and Morphologic Evaluation* Kathy S. Albain, Michelle M. Le Beau, James W. Vardiman, Harvey M. Golomb, and Janet D. Rowley
ABSTRACT: A splenectomized patient with hairy cell leukemia (HCL) who had received chemotherapy with a low-dose alkylating agent for 2 years developed dysmyelopoietic syndrome (DMPS), the first such case to be described. A portion of his marrow remained chronically involved with HCL, but the remainder evolved from being morphologically and karyotypically normal to involvement with DMPS, with cells having a bizarre karyotype. Three of four metaphase cells had consistent abnormalities, with a missing chromosome No. 5, structural rearrangements resulting in deletion of part of the long arm of chromosome No. 7, deletion of 2q, and a small ring chromosome; upon further clonal evolution, a translocation between chromosomes No. 12 and 15 and deletion of the long arm of chromosome No. 19 were seen. We therefore add HCL patients to the growing list of those at risk of developing a secondary hematologic malignant disease possibly due to cytotoxic therapy for their primary disorder. INTRODUCTION The occurrence of n o n r a n d o m , clonal chromosome abnormalities in chronic lymphoproliferative disorders, i n c l u d i n g hairy cell leukemia (HCL), has been well described [1-3]. That patients undergoing cytotoxic chemotherapy are at an increased risk for developing secondary acute n o n l y m p h o c y t i c leukemia (ANLL), of which dysmyelopoietic syndrome (DMPS) is an early stage, is being recognized with increasing frequency [4-15]. Secondary DMPS/ANLL has been described in patients with other hematologic malignant diseases such as Hodgkin's [12,14] and nonHodgkin's lymphomas [4], multiple myeloma [15], chronic lymphocytic leukemia [1], and polycythemia vera [10]; in those with solid tumors such as breast, lung, and ovarian carcinoma; as well as in patients with various n o n m a l i g n a n t conditions who have been treated with i m m u n o s u p p r e s s i v e agents [13]. We and others have
From the Departments of Medicineand Pathology, Universityof Chicago, Chicago, Illinois. *Presented at the AmericanAssociationfor Cancer Research Meetingin St. Louis, Missouri,April 1982. Address requests for reprints ta Dr. Kathy Albain, Box 420, 950 East 59th Street, Chicago, Illinois 60637. Received April 9, 1982; accepted June 1, 1982.
107 © ElsevierSciencePublishingCo., Inc.. 1983 52 VanderbiltAve., New York, NY 1 0 9 1 7
Cancer Genetics and Cytogenetics8, 107-115 (1983) 0165-4608/83/020107-09503.00
BC
9/29/81
48 h r e
48 hr e
24 h r
24 h r 24 h r 24 h r 7 days, P W M c
Cultures
2
1
14 1 1 19
No. of ceils analyzed
46,XY 46,XY 46,XY 46,XY/2 SCA d 46,XY, - 14,b(1)(cen), + r, + 2ace/ 46,XY, ÷ 5 D M s 4 7 , X Y , - 5 , - 1 2 , - 15,del(2)(q33),del(7)(q22q32), del(19)(q12), + del(19)(q12, + t(12;15)(12q;15q), + 2~ 49,XY, - 5,del(2)(q33),del(7)(q22q32), ÷ mar, + 3r, + 1 DMs f 4 6 , X Y / 4 5 , X Y , - 5 , - 1 2 , - 15,del(2)(33),del(7)(q22q32); del(19)(q12), + t(12;15)(12q;15q), + r f
Chromosome complement
dSCA, Single-cell abnormality. eCultured for 48 hr and synchronized with methotrexate and thymidine. fDescription of structural rearrangements: del(2)(pter-~q33:),del(7)(P ter~q22::q32-->qter)'del(19)(pter~q12:)'t(12;15)(12qter~cen--~15qter)'
~PWM, Pokeweed mitogen.
~PB, Peripheral blood; BC, bone core. bThree subsequent samples of PB cultured for 2 4 4 8 hr provided no mitotic cells.
PB
PB BC BC PB
3/6/78 b 5/18/79 4/21/80 9/15/80
analyses
Source~
Chromosome
DATE
Table 1
DMPS in a HCL Patient Treated with Chlorambucil
109
Figure 1 This bone marrow biopsy specimen, obtained 1 year after the diagnosis of HCL was made but prior to chlorambucil therapy, shows a mononuclear cell infiltrate with the characteristic water-clear cytoplasm surrounding the nuclei of the leukemic hairy cells. (H & E, x 500.)
described consistent chromosome abnormalities, particularly those involving deletions of chromosomes No. 5 and/or 7, in secondary, treatment-induced DMPS or ANLL [16-19]. We now report for the first time the development of secondary DMPS associated with a cytogenetic aberration in a HCL patient following chronic treatment with chlorambucil. REPORT OF A CASE
HCL was diagnosed on bone marrow examination in 12/77 in a 63-year-old white male patient with a calf abscess, splenomegaly, and a white blood cell (WBC) count of 1,900/mm 3. The findings of the remainder of his physical examination and of laboratory tests were normal. Because of progressive thrombocytopenia, with platelet counts decreasing from 130,000/ram s to 23,O00/mm 3, a splenectomy was done in 1/78; liver involvement with hairy cells was documented by a biopsy at that time. Cytogenetic analysis in 3/78 revealed a normal 46,XY karyotype (Table 1). The patients's platelet count gradually rose, and he did well without treatment until 11/78, when recurrent cellulitis developed. A bone marrow aspirate and biopsy showed approximately 80% hairy cells (Fig. 1). Although reduced in quantity, the erythroid, granulocytic, and megakaryocytic elements were all morphologically unremarkable. The patient was followed closely without therapy until 4/79, when his WBC count rose to 27,000/mm 3 with 80% hairy cells. The hematocrit was 32% and the platelet count 109,000/mm 3. The patient then began taking chlorambucil, 4 rag/ day. Between 5/79 and 7/79, he was hospitalized twice because of cellulitis and a fever and was given transfusions. His WBC count dropped to 1,500/mm 3, and the
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Figure 2 A peripheral blood smear obtained approximately 26 months after the initiation of chlorambucil treatment revealed macrocytosis and anisopoikilocytosis of red blood ceils, occasional giant platelets, acquired Pelger-Huet changes, and hypogranulation of neutrophils. (Wright's stain, × 1000.)
chlorambucil was stopped for 2 months; it was restarted at 2 mg q.o.d, in 9/79. By 11/79, his WBC count was 3,100/mm 3 with no hairy cells, his hematocrit was 40%, and his platelet count was 300,000/ram 3. In 4/80, a bone core biopsy showed a return of normal hematopoietic elements with 60-70% hairy cells. In 9/80, after more than 1 year of chlorambucil therapy, chromosome studies again showed a normal 46, XY karyotype except for two single-cell abnormalities (Table 1). The patient remained on chlorambucil until 6/81 (total, 24 months); at that time, his WBC count had dropped to 1,900/ram 3, his hematocrit was 32.9%, and his platelet count was 116,000/mm 3. A peripheral blood smear showed fewer than 1% hairy cells. However, new findings in the peripheral smear included poorly granulated polymorphonuclear and pseudo-Pelger-Huet forms, nucleated red blood cells, marked macrocytosis and anisopoikilocytosis, spiculated red blood cells, and giant platelets (Fig. 2). A repeat bone core biopsy showed 40% cellularity (decreased from the previous level of 60-70%) with approximately 70% hairy cells. A shift toward granulocytic immaturity and mild dyserythropoiesis was noted. Megakaryocytes were slightly increased in number and occurred in clusters. Chlorambucil was discontinued because of the decrease in cellularity. While on vacation 1 month later (7/81), the patient was hospitalized for 3 weeks with a fever, a left lower lobe infiltrate, an an infected anal fissure. The WBC count was 1,500/mm 3 with 50% polymorphonucleocytes, the platelet count was 11,000/ram 3, and the hemoglobin level 8.4 mg%. He required multiple transfusions. On returning to our clinic in 8/81, he complained only of low-grade fever, malaise, and perirectal pain. On physical examination, he appeared chronically ill, with
DMPS in a HCL Patient Treated with Chlorambucil
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normal vital signs and patchy, scaling erythroderma. There was no adenopathy. The lungs were clear except for left basilar dry rales. The abdomen was nontender and without hepatomegaly. An open anal fissure, not grossly infected, was present. Routine laboratory investigation gave normal results except for a WBC count of 2,300/mm 3 with one blast and 15% polymorphonucleocytes, some of which showed Pelger-Huet changes. The hemoglobin level was 11.1 mg% (posttransfusion), and the platelet count was 30,000/mm 3. The chest x-ray showed a persistent infiltrate in the left lower lobe. The peripheral blood finding of granulocytic dysplasia and a repeat bone marrow biopsy specimen were diagnosed as consistent with DMPS and residual HCL, In the bone marrow biopsy specimen, typical megakaryocytic hyperplasia, focal clusters of myeloblasts, and poor myeloid maturation were noted (Fig. 3). Cytogenetic analysis of the peripheral blood and bone marrow showed complex karyotypic abnormalities (Fig. 4 and Table 1). Both cells obtained from the bone core biapsy were hyperdiploid and showed aberrations similar to those in one of two mitotic cells from the unstimulated peripheral blood; the other peripheral blood cell had a normal karyotype. All three abnormal cells were missing a chromosome No. 5 and had an interstitial deletion of the long arm of one chromosome No. 7 as well as a deletion of the long arm of chromosome No. 2. One cell from the bane core specimen and from the peripheral blood also had a translocation involving chromosomes No. 12 and 15 and a deletion of No. 19(q12). Two and three ringlike structures were present in the bone core cells; the latter cell also had one marker and a double-minute chromosome. The aberrant cell from the peripheral blood had one ringlike structure. The patient was followed in the outpatient clinic for 1 month, until he developed spiking fevers, pseudomonas bacteremia, and right epididymitis.
Figure 3 Hypercellularity, with prominent megakaryocytichyperplasia, and a moderate shift toward granulocytic immaturity were found in the bone marrow 26 months after the initiation of chlorambucil therapy. (H & E, × 600.)
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Figure 4 Partial karyotypes of Giemsa-stained and Q-banded metaphase cells obtained from bone core (a) and peripheral blood (b) samples. (a) The cell is missing one chromosome No. 5 and shows deletions of the long arms of chromosomes No. 2, and 7 [del(2)(q33),del(7)(q22q32)]. One marker chromosome, three rings, and one double-minute chromosome were also present. (b) This cell shows similar abnormalities; however, two additional rearrangements have occurred. There is a deletion of chromosome No. 19 [del(19)(q12)] and a translocation of the long arm of chromosomes No. 12 and 15 [+(12;15)(12q15q)]. A single ring is present. The abnormal chromosomes are identified (arrows). Despite intensive support with antibiotics and antifungal agents, his disease had a rapidly progressive d o w n h i l l course characterized by persistent high fever, severe p a n c y t o p e n i a requiring multiple transfusions and, ultimately, fatal hepatic failure. A bone marrow s p e c i m e n obtained at autopsy on 10/8/81 yielded no evaluable material for cytogenetic analysis. The autopsy confirmed residual HCL in the liver and bone marrow as well as DMPS i n both blood and bone marrow.
DISCUSSION Ours is the first HCL patient described who developed DMPS; this diagnosis was confirmed both morphologically and cytogenetically. This case serves to point out some classic features of HCL that have been reviewed more extensively elsewhere [21,22]. Our patient had an initial response to a splenectomy, but he later required treatment with chlorambucil w h e n the leukemic phase developed [20]. This therapy added two productive years to his life. His course of HCL was characterized by typical frequent infections, which eventually led to his death w h e n persistent DMPS developed in the portion of the marrow not involved with HCL. Patients with secondary DMPS who progress to ANLL usually remain in the dysmyelopoietic phase
DMPS in a HCL Patient Treated with Chlorambucil
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from 1 to 24 months, with a median of 6 months [5,6,19]. Thus, because the patient died 3 months after developing DMPS, it is not surprising that he showed no further morphologic change toward ANLL, although the clonal evolution described above might have predicted such a progression had he survived the infections [19]. The patient developed DMPS 26 months after the start of chlorambucil therapy. Chronic treatment with chlorambucil and other alkylating agents has been implicated as a cause of secondary DMPS/ANLL that may occur from 1 to 15 years after completion of treatment, or during treatment, of Hodgkin's and non-Hodgkin's lymphomas [2,4,5,14], chronic lymphocytic leukemia [11], multiple myeloma [15], polycythemia vera [10], and various nonmalignant disorders [11]. Whether some of the reported cases represent a new second malignancy rather than a treatment-induced malignancy is not clear because of the lack of careful morphologic and cytogenetic follow-up [8]. Many patients had completed treatment of their original malignancy and were considered to be in remission when DMPS/ANLL developed. Others, however, including our patient, were still being treated, with persistence of the primary malignancy at the onset of the secondary disorder [4]. There was no significant change in this patient's marrow involvement with HCL when he became dysmyelopoietic; rather, the residual normal hematopoietic elements assumed the classic DMPS changes described above. DMPS has been shown to be a morphologically distinct entity that develops de novo or secondarily to cytotoxic agents. It appears that the morphologic features are independent of the original disease and the treatment used. DMPS, as defined by the French-American-British (FAB) Cooperative Group, includes refractory anemia with or without sideroblasts, as well as refractory anemia with excess blasts and chronic myelomonocytic leukemia. Other disorders included in this category have in the past been called preleukemia, subacute leukemia, or smoldering leukemia. Generally, the DMPS seen in patients treated with alkylating agents or radiotherapy is a process involving all three cell lines. As shown in Figures 2 and 3, our case meets the morphologic criteria for inclusion in this category [4,5,18,19]. We have previously described nonrandom, clonal chromosome aberrations, often with bizarre karyotypes, in patients with secondary DMPS and ANLL [18,19]. Loss af chromosome No. 5 and/or No. 7 has been the most consistent finding. As is true of the morphologic changes in DMPS, the karyotypic abnormality in these patients was different from that usually associated with their original disease, and there was no correlation with the type of treatment used initially. Furthermore, loss of chromosome No. 5 seems to be especially common in secondary DMPS/ANLL, although it is observed also in the primary form. Whether patients with the latter have been exposed to mutagens or share other features with the treated patients awaits further clarification [16]. There is no evidence in our experience or in the literature that HCL, a lymphoproliferative disease, or other chronic lymphopraliferative disorders evolve naturally into DMPS. In this case, despite the limited number of mitotic cells available for analysis, the clonal chromosome changes observed ( - 5 and 7 q - ) provide further evidence for the development of DMPS, and an additional argument supporting the view that this is not part of the natural evolution of HCL. Various specific chromosome abnormalities have been described in many hematologic malignant diseases [2]. However, it has been difficult to demonstrate unique, nonrandom chromosome changes in chronic lymphoproliferative disorders because of the very low mitotic index of the cells [1]. The most consistent clonal chromosome abnormalities described to date in HCL have been an extra chromosome No. 12 or a missing Y chromosome, but these are not unique to HCL [3]. Lele et al. [23] recently reported cytogenetic studies in four patients with HCL; none had clonal abnormalities or a missing chromosome No. 5 or 7q as a nonclonal abnormality. Our patient had a
114
K . S . A l b a i n et al. normal karyotype both initially and 1 year after the start of c h l o r a m b u c i l therapy. From this n o r m a l karyotype, bizarre clonal changes evolved, i n c l u d i n g loss of chromosome No. 5 in all three abnormal cells examined. Loss of c h r o m o s o m e No. 5 has not been described in patients w i t h HCL [3]. Thus, the d e v e l o p m e n t of a k a r y o t y p i c abnormality that is shared with other patients who have secondary DMPS/ANLL provides further evidence that the DMPS was t r e a t m e n t - i n d u c e d and did not evolve from the HCL. An alternative theory m a y e x p l a i n the d e v e l o p m e n t of DMPS in this case. It is not k n o w n w h a t hairy cells are; both m o n o c y t i c and l y m p h o c y t i c origins have been postulated. In m a n y HCL patients, there is evidence of platelet abnormalities [24], u n e x p l a i n e d megaloblastic changes, and decreased granulocytic and m o n o c y t i c reserves, even w h e n the m a r r o w is not totally r e p l a c e d by hairy cells [25,26]. This suggests that HCL is perhaps a stem cell disorder. The abnormal stem cell might be easily d a m a g e d by cytotoxic agents. This theory might thus account for the progressive morphologic and cytogenetic abnormalities observed in the non-hairy cell population of our patient's marrow during long-term t h e r a p y with chlorambucil. Thus, p a n c y t o p e n i a in HCL patients can be due not o n l y ' t o splenic sequestration, to an increase in marrow i n v o l v e m e n t by hairy cells, or to cytotoxic treatment, but also to the d e v e l o p m e n t of DMPS secondary to chronic c h l o r a m b u c i l therapy. For this reason, we cannot e m p h a s i z e sufficiently the i m p o r t a n c e of sequential morphologic and cytogenetic evaluations in such patients. Morphologic analysis confirmed the presence of DMPS in a m a r r o w also chronically i n v o l v e d w i t h HCL. Cytogenetic data in this case show the classic c h r o m o s o m a l a b n o r m a l i t y seen in secondary or m u t a g e n - e x p o s e d DMPS/ANLL. Therefore, on the basis of these analyses, we propose that patients w i t h HCL be a d d e d to the growing list of those with malignant and n o n m a l i g n a n t conditions who are at risk of d e v e l o p i n g a secondary hematologic m a l i g n a n t disease related to cytotoxic therapy. Supported by USPHS Grant CA-19266-04 and the Bellman Research Fund.
REFERENCES 1. Whang-Peng J, Knutsen T (1980): Lymphocytic leukemias, acute and chronic. Clin Haematol 9(1):87-127. 2. Rowley JD (1980): Chromosome abnormalities in cancer. Cancer Genet Cytogenet 2: 175-198. 3. Golomb HM, Lindgren V, Rowley JD (1978): Hairy cell leukemia: An analysis of the chromosomes of 26 patients. Virchows Arch [Cell Pathol] 29:113-120. 4. Vardiman JW, Golomb HM, Rowley JD, Variakojis D (1978): Acute nonlymphocytic leukemia in malignant lymphoma: A morphologic study. Cancer 42:229-242. 5. Foucar K, McKenna RW, Bloomfield CD, Bowers TK, Brunning RD (1979): Therapy-related leukemia: A panmyelosis. Cancer 43:1285-1296. 6. Saarni MI, Linman TW (1973): Preleukemia: Hematologic syndrome preceding acute leu~ kemia. Am J Med 55:38-40. 7. Steigbigel RT, Kim H, Potolsky A, Schrier J (1974): Acute myeloproliferative disorder following long-term chlorambucil therapy. Arch Intern Med 134:728-731. 8. Sieber SM (1975): Cancer chemotherapeutic agents and carcinogenesis. Cancer Chemother Rep 59:915-918. 9. Anon (1977): Aklylating agent linked to occurrence of leukemias as second cancers (editorial). J Am Med Assoc 237:2697-2698. 10. Berk PD, Goldberg JD, Silverstein MN, Weinfeld A, Donovan PB, Ellis JT, Landau SA, Laszlo J, Najean Y, Pisciotta AV, Wasserman LR (1981): Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. New Engl J Med 304(8):441-447.
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11. Auclerc G, Jacquillat C, Auclerc MF, Weil M, Bernard J (1979): Post-therapeutic acute leukemia. Cancer 44:2017-2025. 12. Coleman CN, Williams CJ, Flint A, Glatstein EJ, Rosenberg SA, Kaplan HS (1977): Hematologic neoplasms in patients treated for Hodgkin's disease. New Engl J Med 297(23):12491252. 13. Totternia G, Mielot F, Subtil E, Parmentier C (1976): Acute myeloblastic leukemia after immunodepressive therapy for primary nonmalignant disease. Blood Cells 2:67-80. 14. Kitahara M, Gosgriff T, Egre H (1980): Sideroblastic anemia as a preleukemic event in patients treated for Hodgkin's disease. Ann Intern Med 92:625-627. 15. Bergsagel DE, Bailey AJ, Langley GR, MacDonald R, White DF, Miller AB (1979): The chemotherapy of plasma cell myeloma and the incidence of acute leukemia. New Engl J Med 301:743-748. 16. Mitelman F, Nilsson PG, Brandt L, Alimena G, Montuoro A, Dallapiccolo B (1979): Chromosomes, leukaemia and occupational exposure to leukaemogenic agents. Lancet 2:11951196. 17. Rowley JD; Golomb HM, Vardiman J (1977): Nonrandom chromosomal abnormalities in patients treated for Hodgkin's disease and non-Hodgkin's lymphomas. Blood 50:759-770. 18. Streuli RA, Testa JR, Vardiman JW, Mintz U, Golomb HM, Rowley JD (1980): Dysmyelopoietic syndrome: Sequential clinical and cytogenic studies. Blood 55(4):636-644. 19. Rowley JD, Golomb HM, Vardiman JW (1981): Nonrandom chromosome abnormalities in acute leukemia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 58(4):759-767. 20. Golomb HM (1981): Progress report on chlorambucil therapy in postsplenectomy patients with progressive hairy cell leukemia. Blood 57(3):464-467. 21. Catovsky D (1977): Hairy cell leukemia and prolymphocytic leukemia. Clin Haematol 6:245-268. 22. Golomb HM (1978): Hairy cell leukemia: An unusual lymphoproliferative disease--A study of 24 patients. Cancer 42:946-956. 23. Lele KP, Filippa DS, Chaganti RSK (1981): Cytogenetic studies of hairy cell leukemia. Cancer Genet Cytogen 4:325-330. 24. Sweet DL, Golomb HM (1979): Correction of platelet aggregation defect after splenectomy in HCL (letter). J Am Med Assoc 241(10):1084. 25. Yam LT, Chaudhry AA, Janckila AJ (1977): Impaired marrow granulocyte reserve and leukocyte mobilization in leukemic reticuloendotheliosis. Ann Intern Med 87:444-445. 26. Seshadri RS, Brown E, Zipursky A (1976): Leukemic reticuloendotheliosis: A failure of monocyte production. New Engl J Med 295(4):181-184.
ABSTRACT: A splenectomized patient with hairy cell leukemia (HCL) who had received chemotherapy with a low-dose alkylating agent for 2 years developed dysmyelopoietic syndrome (DMPS), the first such case to be described. A portion of his marrow remained chronically involved with HCL, but the remainder evolved from being morphologically and karyotypically normal to involvement with DMPS, with cells having a bizarre karyotype. Three of four metaphase cells had consistent abnormalities, with a missing chromosome No. 5, structural rearrangements resulting in deletion of part of the long arm of chromosome No. 7, deletion of 2q, and a small ring chromosome; upon further clonal evolution, a translocation between chromosomes No. 12 and 15 and deletion of the long arm of chromosome No. 19 were seen. We therefore add HCL patients to the growing list of those at risk of developing a secondary hematologic malignant disease possibly due to cytotoxic therapy for their primary disorder. INTRODUCTION The occurrence of n o n r a n d o m , clonal chromosome abnormalities in chronic lymphoproliferative disorders, i n c l u d i n g hairy cell leukemia (HCL), has been well described [1-3]. That patients undergoing cytotoxic chemotherapy are at an increased risk for developing secondary acute n o n l y m p h o c y t i c leukemia (ANLL), of which dysmyelopoietic syndrome (DMPS) is an early stage, is being recognized with increasing frequency [4-15]. Secondary DMPS/ANLL has been described in patients with other hematologic malignant diseases such as Hodgkin's [12,14] and nonHodgkin's lymphomas [4], multiple myeloma [15], chronic lymphocytic leukemia [1], and polycythemia vera [10]; in those with solid tumors such as breast, lung, and ovarian carcinoma; as well as in patients with various n o n m a l i g n a n t conditions who have been treated with i m m u n o s u p p r e s s i v e agents [13]. We and others have
From the Departments of Medicineand Pathology, Universityof Chicago, Chicago, Illinois. *Presented at the AmericanAssociationfor Cancer Research Meetingin St. Louis, Missouri,April 1982. Address requests for reprints ta Dr. Kathy Albain, Box 420, 950 East 59th Street, Chicago, Illinois 60637. Received April 9, 1982; accepted June 1, 1982.
107 © ElsevierSciencePublishingCo., Inc.. 1983 52 VanderbiltAve., New York, NY 1 0 9 1 7
Cancer Genetics and Cytogenetics8, 107-115 (1983) 0165-4608/83/020107-09503.00
BC
9/29/81
48 h r e
48 hr e
24 h r
24 h r 24 h r 24 h r 7 days, P W M c
Cultures
2
1
14 1 1 19
No. of ceils analyzed
46,XY 46,XY 46,XY 46,XY/2 SCA d 46,XY, - 14,b(1)(cen), + r, + 2ace/ 46,XY, ÷ 5 D M s 4 7 , X Y , - 5 , - 1 2 , - 15,del(2)(q33),del(7)(q22q32), del(19)(q12), + del(19)(q12, + t(12;15)(12q;15q), + 2~ 49,XY, - 5,del(2)(q33),del(7)(q22q32), ÷ mar, + 3r, + 1 DMs f 4 6 , X Y / 4 5 , X Y , - 5 , - 1 2 , - 15,del(2)(33),del(7)(q22q32); del(19)(q12), + t(12;15)(12q;15q), + r f
Chromosome complement
dSCA, Single-cell abnormality. eCultured for 48 hr and synchronized with methotrexate and thymidine. fDescription of structural rearrangements: del(2)(pter-~q33:),del(7)(P ter~q22::q32-->qter)'del(19)(pter~q12:)'t(12;15)(12qter~cen--~15qter)'
~PWM, Pokeweed mitogen.
~PB, Peripheral blood; BC, bone core. bThree subsequent samples of PB cultured for 2 4 4 8 hr provided no mitotic cells.
PB
PB BC BC PB
3/6/78 b 5/18/79 4/21/80 9/15/80
analyses
Source~
Chromosome
DATE
Table 1
DMPS in a HCL Patient Treated with Chlorambucil
109
Figure 1 This bone marrow biopsy specimen, obtained 1 year after the diagnosis of HCL was made but prior to chlorambucil therapy, shows a mononuclear cell infiltrate with the characteristic water-clear cytoplasm surrounding the nuclei of the leukemic hairy cells. (H & E, x 500.)
described consistent chromosome abnormalities, particularly those involving deletions of chromosomes No. 5 and/or 7, in secondary, treatment-induced DMPS or ANLL [16-19]. We now report for the first time the development of secondary DMPS associated with a cytogenetic aberration in a HCL patient following chronic treatment with chlorambucil. REPORT OF A CASE
HCL was diagnosed on bone marrow examination in 12/77 in a 63-year-old white male patient with a calf abscess, splenomegaly, and a white blood cell (WBC) count of 1,900/mm 3. The findings of the remainder of his physical examination and of laboratory tests were normal. Because of progressive thrombocytopenia, with platelet counts decreasing from 130,000/ram s to 23,O00/mm 3, a splenectomy was done in 1/78; liver involvement with hairy cells was documented by a biopsy at that time. Cytogenetic analysis in 3/78 revealed a normal 46,XY karyotype (Table 1). The patients's platelet count gradually rose, and he did well without treatment until 11/78, when recurrent cellulitis developed. A bone marrow aspirate and biopsy showed approximately 80% hairy cells (Fig. 1). Although reduced in quantity, the erythroid, granulocytic, and megakaryocytic elements were all morphologically unremarkable. The patient was followed closely without therapy until 4/79, when his WBC count rose to 27,000/mm 3 with 80% hairy cells. The hematocrit was 32% and the platelet count 109,000/mm 3. The patient then began taking chlorambucil, 4 rag/ day. Between 5/79 and 7/79, he was hospitalized twice because of cellulitis and a fever and was given transfusions. His WBC count dropped to 1,500/mm 3, and the
1 10
K.S. Albain et al.
Figure 2 A peripheral blood smear obtained approximately 26 months after the initiation of chlorambucil treatment revealed macrocytosis and anisopoikilocytosis of red blood ceils, occasional giant platelets, acquired Pelger-Huet changes, and hypogranulation of neutrophils. (Wright's stain, × 1000.)
chlorambucil was stopped for 2 months; it was restarted at 2 mg q.o.d, in 9/79. By 11/79, his WBC count was 3,100/mm 3 with no hairy cells, his hematocrit was 40%, and his platelet count was 300,000/ram 3. In 4/80, a bone core biopsy showed a return of normal hematopoietic elements with 60-70% hairy cells. In 9/80, after more than 1 year of chlorambucil therapy, chromosome studies again showed a normal 46, XY karyotype except for two single-cell abnormalities (Table 1). The patient remained on chlorambucil until 6/81 (total, 24 months); at that time, his WBC count had dropped to 1,900/ram 3, his hematocrit was 32.9%, and his platelet count was 116,000/mm 3. A peripheral blood smear showed fewer than 1% hairy cells. However, new findings in the peripheral smear included poorly granulated polymorphonuclear and pseudo-Pelger-Huet forms, nucleated red blood cells, marked macrocytosis and anisopoikilocytosis, spiculated red blood cells, and giant platelets (Fig. 2). A repeat bone core biopsy showed 40% cellularity (decreased from the previous level of 60-70%) with approximately 70% hairy cells. A shift toward granulocytic immaturity and mild dyserythropoiesis was noted. Megakaryocytes were slightly increased in number and occurred in clusters. Chlorambucil was discontinued because of the decrease in cellularity. While on vacation 1 month later (7/81), the patient was hospitalized for 3 weeks with a fever, a left lower lobe infiltrate, an an infected anal fissure. The WBC count was 1,500/mm 3 with 50% polymorphonucleocytes, the platelet count was 11,000/ram 3, and the hemoglobin level 8.4 mg%. He required multiple transfusions. On returning to our clinic in 8/81, he complained only of low-grade fever, malaise, and perirectal pain. On physical examination, he appeared chronically ill, with
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normal vital signs and patchy, scaling erythroderma. There was no adenopathy. The lungs were clear except for left basilar dry rales. The abdomen was nontender and without hepatomegaly. An open anal fissure, not grossly infected, was present. Routine laboratory investigation gave normal results except for a WBC count of 2,300/mm 3 with one blast and 15% polymorphonucleocytes, some of which showed Pelger-Huet changes. The hemoglobin level was 11.1 mg% (posttransfusion), and the platelet count was 30,000/mm 3. The chest x-ray showed a persistent infiltrate in the left lower lobe. The peripheral blood finding of granulocytic dysplasia and a repeat bone marrow biopsy specimen were diagnosed as consistent with DMPS and residual HCL, In the bone marrow biopsy specimen, typical megakaryocytic hyperplasia, focal clusters of myeloblasts, and poor myeloid maturation were noted (Fig. 3). Cytogenetic analysis of the peripheral blood and bone marrow showed complex karyotypic abnormalities (Fig. 4 and Table 1). Both cells obtained from the bone core biapsy were hyperdiploid and showed aberrations similar to those in one of two mitotic cells from the unstimulated peripheral blood; the other peripheral blood cell had a normal karyotype. All three abnormal cells were missing a chromosome No. 5 and had an interstitial deletion of the long arm of one chromosome No. 7 as well as a deletion of the long arm of chromosome No. 2. One cell from the bane core specimen and from the peripheral blood also had a translocation involving chromosomes No. 12 and 15 and a deletion of No. 19(q12). Two and three ringlike structures were present in the bone core cells; the latter cell also had one marker and a double-minute chromosome. The aberrant cell from the peripheral blood had one ringlike structure. The patient was followed in the outpatient clinic for 1 month, until he developed spiking fevers, pseudomonas bacteremia, and right epididymitis.
Figure 3 Hypercellularity, with prominent megakaryocytichyperplasia, and a moderate shift toward granulocytic immaturity were found in the bone marrow 26 months after the initiation of chlorambucil therapy. (H & E, × 600.)
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Figure 4 Partial karyotypes of Giemsa-stained and Q-banded metaphase cells obtained from bone core (a) and peripheral blood (b) samples. (a) The cell is missing one chromosome No. 5 and shows deletions of the long arms of chromosomes No. 2, and 7 [del(2)(q33),del(7)(q22q32)]. One marker chromosome, three rings, and one double-minute chromosome were also present. (b) This cell shows similar abnormalities; however, two additional rearrangements have occurred. There is a deletion of chromosome No. 19 [del(19)(q12)] and a translocation of the long arm of chromosomes No. 12 and 15 [+(12;15)(12q15q)]. A single ring is present. The abnormal chromosomes are identified (arrows). Despite intensive support with antibiotics and antifungal agents, his disease had a rapidly progressive d o w n h i l l course characterized by persistent high fever, severe p a n c y t o p e n i a requiring multiple transfusions and, ultimately, fatal hepatic failure. A bone marrow s p e c i m e n obtained at autopsy on 10/8/81 yielded no evaluable material for cytogenetic analysis. The autopsy confirmed residual HCL in the liver and bone marrow as well as DMPS i n both blood and bone marrow.
DISCUSSION Ours is the first HCL patient described who developed DMPS; this diagnosis was confirmed both morphologically and cytogenetically. This case serves to point out some classic features of HCL that have been reviewed more extensively elsewhere [21,22]. Our patient had an initial response to a splenectomy, but he later required treatment with chlorambucil w h e n the leukemic phase developed [20]. This therapy added two productive years to his life. His course of HCL was characterized by typical frequent infections, which eventually led to his death w h e n persistent DMPS developed in the portion of the marrow not involved with HCL. Patients with secondary DMPS who progress to ANLL usually remain in the dysmyelopoietic phase
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from 1 to 24 months, with a median of 6 months [5,6,19]. Thus, because the patient died 3 months after developing DMPS, it is not surprising that he showed no further morphologic change toward ANLL, although the clonal evolution described above might have predicted such a progression had he survived the infections [19]. The patient developed DMPS 26 months after the start of chlorambucil therapy. Chronic treatment with chlorambucil and other alkylating agents has been implicated as a cause of secondary DMPS/ANLL that may occur from 1 to 15 years after completion of treatment, or during treatment, of Hodgkin's and non-Hodgkin's lymphomas [2,4,5,14], chronic lymphocytic leukemia [11], multiple myeloma [15], polycythemia vera [10], and various nonmalignant disorders [11]. Whether some of the reported cases represent a new second malignancy rather than a treatment-induced malignancy is not clear because of the lack of careful morphologic and cytogenetic follow-up [8]. Many patients had completed treatment of their original malignancy and were considered to be in remission when DMPS/ANLL developed. Others, however, including our patient, were still being treated, with persistence of the primary malignancy at the onset of the secondary disorder [4]. There was no significant change in this patient's marrow involvement with HCL when he became dysmyelopoietic; rather, the residual normal hematopoietic elements assumed the classic DMPS changes described above. DMPS has been shown to be a morphologically distinct entity that develops de novo or secondarily to cytotoxic agents. It appears that the morphologic features are independent of the original disease and the treatment used. DMPS, as defined by the French-American-British (FAB) Cooperative Group, includes refractory anemia with or without sideroblasts, as well as refractory anemia with excess blasts and chronic myelomonocytic leukemia. Other disorders included in this category have in the past been called preleukemia, subacute leukemia, or smoldering leukemia. Generally, the DMPS seen in patients treated with alkylating agents or radiotherapy is a process involving all three cell lines. As shown in Figures 2 and 3, our case meets the morphologic criteria for inclusion in this category [4,5,18,19]. We have previously described nonrandom, clonal chromosome aberrations, often with bizarre karyotypes, in patients with secondary DMPS and ANLL [18,19]. Loss af chromosome No. 5 and/or No. 7 has been the most consistent finding. As is true of the morphologic changes in DMPS, the karyotypic abnormality in these patients was different from that usually associated with their original disease, and there was no correlation with the type of treatment used initially. Furthermore, loss of chromosome No. 5 seems to be especially common in secondary DMPS/ANLL, although it is observed also in the primary form. Whether patients with the latter have been exposed to mutagens or share other features with the treated patients awaits further clarification [16]. There is no evidence in our experience or in the literature that HCL, a lymphoproliferative disease, or other chronic lymphopraliferative disorders evolve naturally into DMPS. In this case, despite the limited number of mitotic cells available for analysis, the clonal chromosome changes observed ( - 5 and 7 q - ) provide further evidence for the development of DMPS, and an additional argument supporting the view that this is not part of the natural evolution of HCL. Various specific chromosome abnormalities have been described in many hematologic malignant diseases [2]. However, it has been difficult to demonstrate unique, nonrandom chromosome changes in chronic lymphoproliferative disorders because of the very low mitotic index of the cells [1]. The most consistent clonal chromosome abnormalities described to date in HCL have been an extra chromosome No. 12 or a missing Y chromosome, but these are not unique to HCL [3]. Lele et al. [23] recently reported cytogenetic studies in four patients with HCL; none had clonal abnormalities or a missing chromosome No. 5 or 7q as a nonclonal abnormality. Our patient had a
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K . S . A l b a i n et al. normal karyotype both initially and 1 year after the start of c h l o r a m b u c i l therapy. From this n o r m a l karyotype, bizarre clonal changes evolved, i n c l u d i n g loss of chromosome No. 5 in all three abnormal cells examined. Loss of c h r o m o s o m e No. 5 has not been described in patients w i t h HCL [3]. Thus, the d e v e l o p m e n t of a k a r y o t y p i c abnormality that is shared with other patients who have secondary DMPS/ANLL provides further evidence that the DMPS was t r e a t m e n t - i n d u c e d and did not evolve from the HCL. An alternative theory m a y e x p l a i n the d e v e l o p m e n t of DMPS in this case. It is not k n o w n w h a t hairy cells are; both m o n o c y t i c and l y m p h o c y t i c origins have been postulated. In m a n y HCL patients, there is evidence of platelet abnormalities [24], u n e x p l a i n e d megaloblastic changes, and decreased granulocytic and m o n o c y t i c reserves, even w h e n the m a r r o w is not totally r e p l a c e d by hairy cells [25,26]. This suggests that HCL is perhaps a stem cell disorder. The abnormal stem cell might be easily d a m a g e d by cytotoxic agents. This theory might thus account for the progressive morphologic and cytogenetic abnormalities observed in the non-hairy cell population of our patient's marrow during long-term t h e r a p y with chlorambucil. Thus, p a n c y t o p e n i a in HCL patients can be due not o n l y ' t o splenic sequestration, to an increase in marrow i n v o l v e m e n t by hairy cells, or to cytotoxic treatment, but also to the d e v e l o p m e n t of DMPS secondary to chronic c h l o r a m b u c i l therapy. For this reason, we cannot e m p h a s i z e sufficiently the i m p o r t a n c e of sequential morphologic and cytogenetic evaluations in such patients. Morphologic analysis confirmed the presence of DMPS in a m a r r o w also chronically i n v o l v e d w i t h HCL. Cytogenetic data in this case show the classic c h r o m o s o m a l a b n o r m a l i t y seen in secondary or m u t a g e n - e x p o s e d DMPS/ANLL. Therefore, on the basis of these analyses, we propose that patients w i t h HCL be a d d e d to the growing list of those with malignant and n o n m a l i g n a n t conditions who are at risk of d e v e l o p i n g a secondary hematologic m a l i g n a n t disease related to cytotoxic therapy. Supported by USPHS Grant CA-19266-04 and the Bellman Research Fund.
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