Late-Appearing Philadelphia Chromosome in Acute Lymphoblastic Leukemia Mario Sessarego, Raffaella Defferrari, Anna Dejana, and Emanuele Salvidio
ABSTRACT: We describe the cytogenetic analysis of a patient affected by a common acute lymphoblastic leukemia, L2 type. At diagnosis and first relapse, the karyotype was normal, whereas at the second relapse more than 60% of the examined cells showed a Philadelphia chromosome, without any change in the morphological and immunophenotypical picture. This case confirms the observation that leukemic cells are susceptible to developing a Ph. considered a primary chromosomal abnormality, during the course of the disease.
INTRODUCTION About 2 0 - 2 5 % of adult patients affected by acute l y m p h o b l a s t i c leukemia (ALL) show a P h i l a d e l p h i a chromosome (Ph) at diagnosis: the t(9;22)(q34;q11) is the most c o m m o n structural rearrangement associated with ALL [1]. Morphologically the Ph + ALL, according to the French-American-British (FAB) classification [2], are L2 and rarely L1; most cases have an i m m u n o p h e n o t y p e compatible with B-cell differentiation. Clinically, P h + ALL adult patients show a WBC count higher than that of patients without a Ph and an average survival shorter than that of patients with normal karyotypes [3, 4]. Cytogenetically, the Ph found in ALL and in chronic myelogenous leukemia (CML) are indistinguishable [5]. Recently, molecular studies proved that at least some Ph + ALL patients show a bcr rearrangement different from the one found in CML: the c-abl oncogene translocates from the 9q34 band, where it is normally located, into the 5' region (centromeric} of the breakpoint cluster region (BCR) gene m a p p e d to the 2 2 q l l band. The chimeric transcript mRNA encodes a new 190-Kd protein kinase, p190, distinct from the p210 of CML [6-9]. Usually, the Ph, both in CML and ALL, is present at diagnosis: in CML it persists in all hematological stages, whereas in ALL it can d i s a p p e a r temporarily during complete remission [10]. We describe a case of ALL (L2 type) with a c o m m o n i m m u n o p h e n o t y p e and normal karyotype at diagnosis and during the first relapse and showing a Ph in more than 60% of the cells during the second relapse, without any morphological and i m m u n o phenotypical changes. From the Cattedra di Clinica Medico R (M. S., A. D.. E. S.), and BiologiaGenerale (R. D.), Universit~ di Genoa. Italy. Address reprint requests to: Mario Sessarego, M.D., ISMI-Cattedra Clinica Medica R, Viale Benedetto XV, 6, 16132 Genoa, Italy. Received June 8, 1989; accepted November 27, 1989.
35 © 1990 Elsevier Science Publishing Co., Inc.
Cancer Genet Cytogenet 48:35-38 (1990)
655 Avenue of the Americas. New York. NY 10010
0165-4608/90/$03.50
36
M. Sessarego et al.
A Figure 1 Partial Q-banded karyotypes showing chromosomes 9 and 22. (A) At diagnosis. (B) The t(9;22)(q34;q11) observed at the second relapse.
B 9
22
CASE REPORT A 45-year-old woman was admitted to our hospital in September 1987 with asthenia, slight persistent fever, and a hemorrhagic syndrome. Physical examination showed moderate splenomegaly. Blood examination revealed: white blood cell (WBC) count 98.000//~L with 85% m y e l o p e r o x i d a s e negative and periodic acid-Schiff (PAS)-positive blasts. The nuclear chromatin was finely dispersed; nuclear clefting and one or two nucleoli were present; the cytoplasm was basophilic and relatively abundant. Hematocrit was 26.8%, hemoglobin was 8.7 g/dl, and platelet count was 7,000/~L. Surface marker analysis of the peripheral blood (PB) cells showed the following: B1, 63%; B4, 68%; T3, 8% c o m m o n ALL antigen (CALLA), 66%; My4, less than 1%; My7, less than 1; My9, 5%; M1, 1%; SIg, less than 1%. The serum lactic dehydrogenase (LDH) was 1,373 U/L (normal value 125-250 U/L). The bone marrow (BM) aspirate showed 95% blasts with L2 morphology. The patient was treated with vincristine, daunoblastin, and p r e d n i s o n e combination weekly. A complete remission was achieved after 4 weeks. The patient received central nervous system prophylaxis (methotrexate and cytosine arabinoside intrathecally) and consolidation therapy with L-asparaginase and prednisone. Then 6-mercaptopurine, methotrexate, and cytosine arabinoside were a d m i n i s t e r e d as maintenance therapy. In May 1988 first relapse occurred: PB showed 40% blasts and the BM aspirate showed 80%. Blast cells were present in the liquor sample, as well. The patient was started on vincristine, cytosine arabinoside, amsacrine, and d e x a m e t h a s o n e in a d d i t i o n to the intrathecal methotrexate and prednisone. After two c h e m o t h e r a p e u t i c courses, a complete remission was obtained. In October 1988, a second relapse occurred with a high blast cell count both in the PB and BM. Morphologically, the blasts showed no differences as c o m p a r e d with that at diagnosis. Surface markers were unchanged. The patient died in November 1988.
Cytogenetic Investigations Karyotype analysis was performed on BM and PB cells without mitogen stimulation at diagnosis and at the first and second relapses. The buffy coat layer was removed by centrifugation and 1 x 106 cells/ml were cultured for 24 and 48 hours. Chromosomes were analyzed on Q-banded preparations. At diagnosis, 21 metaphases were examined, and at first relapse 38 were examined: no clonal abnormalities were detected. The third cytogenetic study was performed on October 1988 at the second relapse: 15 of the 24 examined metaphases showed a Ph owing to the standard translocation (9;22)(q34;q11) (Fig. 1). P h + cells were found both in PB and BM cultures. The other nine metaphases were normal.
Late-Appearing Ph Chromosome in ALL
37
DISCUSSION All examined mitoses from the first and second cytogenetic studies showed a normal diploid karyotype. The last study, performed during the second relapse, showed a Ph in more than 60% of the cells. The PB and BM samples from the three analyses showed more than 80% lymphoblasts. Therefore Ph + cells may actually have been absent or present in only a very small percentage in the first two studies. A possible mosaicism can be excluded at the 95% confidence level for the P h + clone constituted 14% of the cells in the first study and 8% in the second [11]. The late appearance of a Ph subsequent to diagnosis has been described in chronic myelogenous leukemia (CML) patients [12], although it is a rare event. In our case, a typical c o m m o n ALL, the Ph + appeared during the second relapse, 5 months after the second cytogenetic study, whereas the morphological picture of blast cells and the i m m u n o p h e n o t y p e were unchanged. We do not know whether the bcr/c-abl rearrangement was present in the P h - cells observed in the first stages of the disease, as described for some P h - CML cases [13]. To the best of our knowledge, this is the second example of a late-developing Ph in ALL. Miller and co-workers [141 reported a case of childhood T-cell ALL with a 9p - chromosome at diagnosis showing 6 q - and a Ph as additional changes during relapse of the disease. The present article suggests the value of performing karyotype analysis in ALL, not only at diagnosis but also during evolution of the disease. It would also be useful to determine possible bcr rearrangements in P h - ALL, as in P h - CML. This work was supported by Grant No. 88.00492.44 from the CNR Progetto Finalizzato Oncologia and by the Associazione Italiana per la Ricerca sul Cancro.
REFERENCES 1. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Chromosomal abnormalities in acute lymphoblastic leukemia. Cancer Genet Cytogenet 4:101-110. 2. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (FAB Cooperative Group) (1976): Proposal for the classification of the acute leukaemias. Br J Haematol 33:451-458. 3. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Clinical significance of chromosomal abnormalities in acute lymphoblastic leukemia. Cancer Genet Cytogenet 4:11-137. 4. Bloomfield CD, Goldman AI, Alimena G, Berger R, Borgstrom GH, Brandt L, Catovsky D, de la Chapelle A, Dewald GW, Garson OM, Garwicz S, Golomb HM, Hossfeld DK, Lawler SD, Mitelman F, Nilsson P, Pierre RV, Philip P, Prigogina E, Rowley JD, Sakurai M, Sandberg AA, Secker Walker LM, Tricot G, Van Den Berghe H, Van Orshoven A, Vuopio P, WhangPeng J (1986): Chromosomal abnormalities identify high-risk and low-risk patients with acute lymphoblastic leukemia. Blood 67:415-420. 5. Helm S, Mitelman F (1987): Cancer Cytogenetics. Alan R. Liss, New York, pp 153-155. 6. Kurzrock R, Shtalrid M, Romero P, Kloetzer W, Talpaz M, Trujillo I, Blick M, Beran M, Gutterman J (1987): A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia. Nature 325:631-635. 7. Chan LC, Karhi KK, Rayter SI, Heisterkamp N, Eridani S, Powles R, Lawler SD, Groffen l, Foulkes JG, Greaves MF, Wiedemann LM (1987): A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 325:635-637. 8. Fainstein E, Marcelle C, Rosner A, Canaani E, (;ale RP, Dreazen O, Smith SD, Croce CM (1987): A new fused transcript in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 330:386-388. 9. Kurzrock R, Gutterman J, Talpaz M (1988): The molecular genetics of Philadelphia chromosome positive leukemias. N Engl J 'Med 319;990--998. 10. Bloomfield CD, Peterson LC, Yunis 1J, Brunning RD (1977): The Philadelphia chromosome
38
M. S e s s a r e g o et al.
(Phl) in adults presenting with acute leukaemia: A comparison of Ph + and Ph Br J Haematol 36:347-357.
patients.
11. Hook E (1977): Exclusion of chromosomal mosaicism: Tables of 90%, 95% and 99% (:onfidence limits and comments on use. Am I Hum Genet 29:94-97. 12. Lisker R, Casas L, Mutchinick O, Lopez-Ariza B, Labardini I (1982): Patient with chronic myelogenous leukemia and late-appearing Philadelphia chromosome. Cancer (,enet (]ytogenet 6:275-277. 13. Ganesan CM, Rassool F, Guo AP, Th'ng KH, Dowding C, Hibbin JA, Young BD, White H. Kumuran TO, Galton DAG, Goldman JM (19861: Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic Inyeloid leukemia. Blood 68:957-960. 14. Miller BA, Reid MM, Nell M, Lipton JM, Sallan SE, Nathan DG, Tantravahi R (1984): Tcell acute lymphoblastic leukaemia with late developing Philadelphia chromosome. Br l Haematol 56:139-146.
ABSTRACT: We describe the cytogenetic analysis of a patient affected by a common acute lymphoblastic leukemia, L2 type. At diagnosis and first relapse, the karyotype was normal, whereas at the second relapse more than 60% of the examined cells showed a Philadelphia chromosome, without any change in the morphological and immunophenotypical picture. This case confirms the observation that leukemic cells are susceptible to developing a Ph. considered a primary chromosomal abnormality, during the course of the disease.
INTRODUCTION About 2 0 - 2 5 % of adult patients affected by acute l y m p h o b l a s t i c leukemia (ALL) show a P h i l a d e l p h i a chromosome (Ph) at diagnosis: the t(9;22)(q34;q11) is the most c o m m o n structural rearrangement associated with ALL [1]. Morphologically the Ph + ALL, according to the French-American-British (FAB) classification [2], are L2 and rarely L1; most cases have an i m m u n o p h e n o t y p e compatible with B-cell differentiation. Clinically, P h + ALL adult patients show a WBC count higher than that of patients without a Ph and an average survival shorter than that of patients with normal karyotypes [3, 4]. Cytogenetically, the Ph found in ALL and in chronic myelogenous leukemia (CML) are indistinguishable [5]. Recently, molecular studies proved that at least some Ph + ALL patients show a bcr rearrangement different from the one found in CML: the c-abl oncogene translocates from the 9q34 band, where it is normally located, into the 5' region (centromeric} of the breakpoint cluster region (BCR) gene m a p p e d to the 2 2 q l l band. The chimeric transcript mRNA encodes a new 190-Kd protein kinase, p190, distinct from the p210 of CML [6-9]. Usually, the Ph, both in CML and ALL, is present at diagnosis: in CML it persists in all hematological stages, whereas in ALL it can d i s a p p e a r temporarily during complete remission [10]. We describe a case of ALL (L2 type) with a c o m m o n i m m u n o p h e n o t y p e and normal karyotype at diagnosis and during the first relapse and showing a Ph in more than 60% of the cells during the second relapse, without any morphological and i m m u n o phenotypical changes. From the Cattedra di Clinica Medico R (M. S., A. D.. E. S.), and BiologiaGenerale (R. D.), Universit~ di Genoa. Italy. Address reprint requests to: Mario Sessarego, M.D., ISMI-Cattedra Clinica Medica R, Viale Benedetto XV, 6, 16132 Genoa, Italy. Received June 8, 1989; accepted November 27, 1989.
35 © 1990 Elsevier Science Publishing Co., Inc.
Cancer Genet Cytogenet 48:35-38 (1990)
655 Avenue of the Americas. New York. NY 10010
0165-4608/90/$03.50
36
M. Sessarego et al.
A Figure 1 Partial Q-banded karyotypes showing chromosomes 9 and 22. (A) At diagnosis. (B) The t(9;22)(q34;q11) observed at the second relapse.
B 9
22
CASE REPORT A 45-year-old woman was admitted to our hospital in September 1987 with asthenia, slight persistent fever, and a hemorrhagic syndrome. Physical examination showed moderate splenomegaly. Blood examination revealed: white blood cell (WBC) count 98.000//~L with 85% m y e l o p e r o x i d a s e negative and periodic acid-Schiff (PAS)-positive blasts. The nuclear chromatin was finely dispersed; nuclear clefting and one or two nucleoli were present; the cytoplasm was basophilic and relatively abundant. Hematocrit was 26.8%, hemoglobin was 8.7 g/dl, and platelet count was 7,000/~L. Surface marker analysis of the peripheral blood (PB) cells showed the following: B1, 63%; B4, 68%; T3, 8% c o m m o n ALL antigen (CALLA), 66%; My4, less than 1%; My7, less than 1; My9, 5%; M1, 1%; SIg, less than 1%. The serum lactic dehydrogenase (LDH) was 1,373 U/L (normal value 125-250 U/L). The bone marrow (BM) aspirate showed 95% blasts with L2 morphology. The patient was treated with vincristine, daunoblastin, and p r e d n i s o n e combination weekly. A complete remission was achieved after 4 weeks. The patient received central nervous system prophylaxis (methotrexate and cytosine arabinoside intrathecally) and consolidation therapy with L-asparaginase and prednisone. Then 6-mercaptopurine, methotrexate, and cytosine arabinoside were a d m i n i s t e r e d as maintenance therapy. In May 1988 first relapse occurred: PB showed 40% blasts and the BM aspirate showed 80%. Blast cells were present in the liquor sample, as well. The patient was started on vincristine, cytosine arabinoside, amsacrine, and d e x a m e t h a s o n e in a d d i t i o n to the intrathecal methotrexate and prednisone. After two c h e m o t h e r a p e u t i c courses, a complete remission was obtained. In October 1988, a second relapse occurred with a high blast cell count both in the PB and BM. Morphologically, the blasts showed no differences as c o m p a r e d with that at diagnosis. Surface markers were unchanged. The patient died in November 1988.
Cytogenetic Investigations Karyotype analysis was performed on BM and PB cells without mitogen stimulation at diagnosis and at the first and second relapses. The buffy coat layer was removed by centrifugation and 1 x 106 cells/ml were cultured for 24 and 48 hours. Chromosomes were analyzed on Q-banded preparations. At diagnosis, 21 metaphases were examined, and at first relapse 38 were examined: no clonal abnormalities were detected. The third cytogenetic study was performed on October 1988 at the second relapse: 15 of the 24 examined metaphases showed a Ph owing to the standard translocation (9;22)(q34;q11) (Fig. 1). P h + cells were found both in PB and BM cultures. The other nine metaphases were normal.
Late-Appearing Ph Chromosome in ALL
37
DISCUSSION All examined mitoses from the first and second cytogenetic studies showed a normal diploid karyotype. The last study, performed during the second relapse, showed a Ph in more than 60% of the cells. The PB and BM samples from the three analyses showed more than 80% lymphoblasts. Therefore Ph + cells may actually have been absent or present in only a very small percentage in the first two studies. A possible mosaicism can be excluded at the 95% confidence level for the P h + clone constituted 14% of the cells in the first study and 8% in the second [11]. The late appearance of a Ph subsequent to diagnosis has been described in chronic myelogenous leukemia (CML) patients [12], although it is a rare event. In our case, a typical c o m m o n ALL, the Ph + appeared during the second relapse, 5 months after the second cytogenetic study, whereas the morphological picture of blast cells and the i m m u n o p h e n o t y p e were unchanged. We do not know whether the bcr/c-abl rearrangement was present in the P h - cells observed in the first stages of the disease, as described for some P h - CML cases [13]. To the best of our knowledge, this is the second example of a late-developing Ph in ALL. Miller and co-workers [141 reported a case of childhood T-cell ALL with a 9p - chromosome at diagnosis showing 6 q - and a Ph as additional changes during relapse of the disease. The present article suggests the value of performing karyotype analysis in ALL, not only at diagnosis but also during evolution of the disease. It would also be useful to determine possible bcr rearrangements in P h - ALL, as in P h - CML. This work was supported by Grant No. 88.00492.44 from the CNR Progetto Finalizzato Oncologia and by the Associazione Italiana per la Ricerca sul Cancro.
REFERENCES 1. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Chromosomal abnormalities in acute lymphoblastic leukemia. Cancer Genet Cytogenet 4:101-110. 2. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (FAB Cooperative Group) (1976): Proposal for the classification of the acute leukaemias. Br J Haematol 33:451-458. 3. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Clinical significance of chromosomal abnormalities in acute lymphoblastic leukemia. Cancer Genet Cytogenet 4:11-137. 4. Bloomfield CD, Goldman AI, Alimena G, Berger R, Borgstrom GH, Brandt L, Catovsky D, de la Chapelle A, Dewald GW, Garson OM, Garwicz S, Golomb HM, Hossfeld DK, Lawler SD, Mitelman F, Nilsson P, Pierre RV, Philip P, Prigogina E, Rowley JD, Sakurai M, Sandberg AA, Secker Walker LM, Tricot G, Van Den Berghe H, Van Orshoven A, Vuopio P, WhangPeng J (1986): Chromosomal abnormalities identify high-risk and low-risk patients with acute lymphoblastic leukemia. Blood 67:415-420. 5. Helm S, Mitelman F (1987): Cancer Cytogenetics. Alan R. Liss, New York, pp 153-155. 6. Kurzrock R, Shtalrid M, Romero P, Kloetzer W, Talpaz M, Trujillo I, Blick M, Beran M, Gutterman J (1987): A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia. Nature 325:631-635. 7. Chan LC, Karhi KK, Rayter SI, Heisterkamp N, Eridani S, Powles R, Lawler SD, Groffen l, Foulkes JG, Greaves MF, Wiedemann LM (1987): A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 325:635-637. 8. Fainstein E, Marcelle C, Rosner A, Canaani E, (;ale RP, Dreazen O, Smith SD, Croce CM (1987): A new fused transcript in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 330:386-388. 9. Kurzrock R, Gutterman J, Talpaz M (1988): The molecular genetics of Philadelphia chromosome positive leukemias. N Engl J 'Med 319;990--998. 10. Bloomfield CD, Peterson LC, Yunis 1J, Brunning RD (1977): The Philadelphia chromosome
38
M. S e s s a r e g o et al.
(Phl) in adults presenting with acute leukaemia: A comparison of Ph + and Ph Br J Haematol 36:347-357.
patients.
11. Hook E (1977): Exclusion of chromosomal mosaicism: Tables of 90%, 95% and 99% (:onfidence limits and comments on use. Am I Hum Genet 29:94-97. 12. Lisker R, Casas L, Mutchinick O, Lopez-Ariza B, Labardini I (1982): Patient with chronic myelogenous leukemia and late-appearing Philadelphia chromosome. Cancer (,enet (]ytogenet 6:275-277. 13. Ganesan CM, Rassool F, Guo AP, Th'ng KH, Dowding C, Hibbin JA, Young BD, White H. Kumuran TO, Galton DAG, Goldman JM (19861: Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic Inyeloid leukemia. Blood 68:957-960. 14. Miller BA, Reid MM, Nell M, Lipton JM, Sallan SE, Nathan DG, Tantravahi R (1984): Tcell acute lymphoblastic leukaemia with late developing Philadelphia chromosome. Br l Haematol 56:139-146.
