A Case of Acute Eosinophilic Granulocytic Leukemia with PML

Home , Granulocytosis, Polycythemia vera

CORRESPONDENCE

CASE REPORT

A case of acute eosinophilic granulocytic leukemia with PML-RAR alpha fusion gene expression and response to all-trans retinoic acid R-Q Yu1, W Huang2, S-J Chen2, S-D Jiang1 and Z Chen2

1Division of Hematology, Department of Internal Medicine, Shanghai Chang-Zheng Hospital; and 2Laboratory of Molecular Biology, Shanghai Institute of Hematology, China

A typical case of eosinophilic granulocytic leukemia with PML- RAR alpha fusion gene expression is reported. The patient achieved complete remission after oral administration of all- trans retinoic acid without any exposure to cytotoxic agents. The facts strongly suggest that the genetic event occurred at the level of pluripotent stem cells. Keywords: leukemia; eosinophilic; PML-RAR alpha; retinoic acid

Introduction

It has been well demonstrated that the presence of a fusion gene, PML-RAR alpha, resulting from the reciprocal translocation of human chromosome 15 and 17, t(15;17)(q22:q21) is a specific molecular marker of acute promyelocytic leukemia, and plays an important role in the pathogenesis of that disease.1–4 Until now PML-RAR alpha fusion gene has not been found in other malignant cells. Recently, we saw a typi- Figure 1 Bone marrow smear showing coarse refractile eosino- cal case of acute eosinophilic granulocytic leukemia with philic granules in leukemic cells. PML-RAR alpha fusion gene expression that achieved complete remission after differentiation therapy with all-trans larity with a G/E ratio of 14.1:1. The differential count showed retinoic acid (ATRA). We would like to present it for further 5.1% myeloblasts, 1.0% neutrophilic promyelocytes, 14.5% attention. eosinophilic promyelocytes, 12.5% eosinophilic myelocytes, 10.0% eosinophilic metamyelocytes, 14.0% eosinophilic band-formed granulocytes and 26.0% eosinophilic-segmented Case history granulocytes. The eosinophilic granules seen on the smear were coarse, refractile, orange-red with slight blue staining Lee XX, a 66-year-old female, was admitted to hospital in and unevenly distributed (Figure 1). In some myeloblasts and December 1991 with complaints of dizziness and chest dis- tress for 2 weeks. On physical examination the patient revealed lassitude and pallor. The superficial lymph nodes were not enlarged. There was definite tenderness on slight pressure on the lower part of the sternum. The liver and spleen were not palpable under the costal margin. No petechia and ecchymosis could be found over the surface of the body. B-ultrasonic examination showed the size of the liver and spleen was normal. The analytical test of peripheral blood showed hemoglobin 108 g/l, leukocyte count 64.3 × 109/l, platelet count 192 × 109/l. Cell morphology in peripheral blood films showed 1% eosinophilic metamyelocyte, 13% eosinophilic band-formed granulocytes and 69% eosinophilic-segmented granulocytes. On bone marrow smear there was pronounced hypercellu-

Correspondence: R-Q Yu, Division of Hematology, Department of Internal Medicine, Shanghai Chang-Zheng Hospital, 415 Fong-Yang Figure 2 Electrophoresis showing the speciﬁc product of RT-PCR.

Road, Shanghai 200003, PR China Lane 1, the patient; lane 2, positive specimen from patient with M3; Received 25 September 1996; accepted 11 December 1996 lane 3, normal subject; lane 4, blank control. Correspondence 610 eosinophilic promyelocytes the nuclei were twisted and twisted and folded nuclei. Auer rods could be found in their folded with a fine nuclear chromatin pattern. Auer rods could cytoplasm. On the basis of the above evidence, the diagnosis be found in their cytoplasm. Mitosis of the eosinophils could of acute eosinophilic granulocytic leukemia could be estab- easily be seen. The proliferation of erythrocytic series was lished, and according to the typically morphological feature depressed. Part of the erythroblasts underwent megaloblast- of the eosinophilic granules in the cytoplasm, acute promyelo- like transformation. Within the area of 2 × 3 cm of bone mar- cytic leukemia could also be ruled out. row smear, 65 megakaryocytes could be found; most of them PML-RAR alpha is a fusion gene resulting from reciprocal were granular and platelet-producing. There was an abundant translocation of human chromosomes by juxtaposing the PML amount of stainable iron in the histiocytes of the bone gene on chromosome 15 and the RAR alpha gene on chromo- marrow. some 17. It plays a key role in the pathogenesis of acute pro- Three days after admission ATRA therapy started 30 mg myelocytic leukemia and is also a specific molecular marker orally every 6 h. Ten days later the leukocyte count of periph- which has not been found in other malignancies. eral blood dropped from 64.3 × 109/l to 44.5 × 109/l, the plate- Here, interestingly enough, PML-RAR alpha fusion gene let count rose from 192 × 109/l to 332 × 109/l. Eosinophilic was unequivocally detected from both peripheral blood and granulocytes in the differential count decreased from 83 to bone marrow in a typical case of acute eosinophilic granulo- 64%. Twenty days after treatment tenderness of the sternum cytic leukemia that achieved complete remission after oral disappeared. Then, there was a gradual but steady decrease administration of ATRA without any exposure to cytotoxic in the leukocyte count and percentage of eosinophils in the agents. Owing to the blank control and normal control repeat- peripheral blood. Seventy days after treatment, bone marrow edly being negative, the possibility of contamination could be examination showed the cellularity had returned to normal excluded. In addition, the detection of RAR alpha-PML tran- with a G/E ratio of 3.52:1, eosinophilic promyelocytes script indicated that the abnormality of chromosomes was a decreased from 14.5 to 0.5%, eosinophilic myelocytes and reciprocal or a balanced translocation. Recently, Takatsuki et metamyelocytes fell from 12.5 and 10.0% to 1.5%, eosino- al6 found that PML-RAR alpha presented not only in CFU-GM, philic band-formed granulocytes decreased from 14.0 to but also in BFU-E. Altogether, these facts strongly suggest that 2.5%, and eosinophilic-segmented granulocytes from 26.0 to the formation of PML-RAR alpha fusion gene occurs at the 15%. Auer rods could no longer be found. One hundred and developmental stage prior to the progenitor cell for promyelo- three days after continuous oral administration of ATRA, the cyte, namely at the level of pluripotent stem cells.7 In the peripheral blood and bone marrow achieved complete main, transformed cells developed into an acute promyelo- remission. Next, three intensive courses of DAH protocol cytic leukemia, but in certain situations they could also (daunorubicin, cytosine arabinoside, harringtonine) were advance toward acute eosinophilic leukemia, although the given for consolidation. precise mechanism of distinct differentiation remains to be In June 1995, the patient was quite well with a hemoglobin elucidated. concentration of 121 g/l, leukocyte count of 4.4 × 109/l and The dosage of ATRA given to the patient was 30 mg every platelet count of 200 × 109/l. The differential count of leuko- 6 h. It corresponded to 68 mg/m2 and was much higher than cytes in the periperhal blood was normal with 1.0% eosino- the usual dose. The reason for doing so was that at the begin- philic-segmented granulocytes. Bone marrow examination ning we were really not certain if ATRA would work in that showed normal cellularity with a G/E ratio of 3.51:1. The particular case. To our surprise, that patient not only nucleated cells in all series were proportionally and morpho- responded but did not even show hyperleukocytosis at day logically normal. 10–20 after ATRA treatment. Individual discrepancy might Cytogenic study showed 46,XX. However, owing to the play a role in that situation. Another important possibility was unsatisfactory preparation of chromosome, no numerical and that most of the abnormal cells in the bone marrow and per- structural abnormality of chromosome could be found with G ipheral blood were relatively well differentiated, therefore banding analysis. they might not be prone to further division. Total RNAs were extracted from the peripheral blood and bone marrow using a one-step guanidine-thiocyanate-phenol- chloroform method. References RT-PCR was performed according to the procedures in our previous paper.5 From both peripheral blood and bone mar- 1 Kakizuka A, Miller WH Jr, Umesona K, Warrell RP Jr, Framkel SR, row samples, an L-type PML-RAR alpha transcript could be Murty VVVS, Dmitrovsky E, Evans RM. Chromosomal translo- definitely detected, which is shown in Figure 2. In the mean- cation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 1991; while a reverse juxtaposing product of translocation, RAR 66: 663–674. alpha-PML fusion gene could also be found. 2 de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a func- Discussion tionally altered RAR. Cell 1991; 66: 675–684. 3 Chen SJ, Chen Z, Chen A, Tong JH, Dong S, Wang Z, Waxman S, Zelent A. Occurrence of distinct PML-RAR alpha fusion gene The patient experienced typical manifestation of acute leuke- isoforms in patients with acute promyelocytic leukemia detected mia with rather sudden onset. There was a marked increase by reverse transcriptase-polymerase chain reaction. Oncogene of eosinophilic granulocytes in addition to the emergence of 1992; 7: 1223–1232. immature eosinophils in the peripheral blood. The bone mar- 4 Geng JP, Tong JH, Dong S, Wang ZY, Chen SJ, Chen Z, Zelent A, row was hypercellular with a pronounced augmentation of Berger R, Larsen CJ. Localization of the chromosome 15 break- eosinophilic granulocytes of all stages. The eosinophilic gran- points and expression of multiple PML-RAR alpha transcripts in acute promyelocytic leukemia: a study of 28 Chinese patients. ules seen on the smear were typical in morphology. The per- Leukemia 1993; 7: 20–26. centage of myeloblasts was higher than 5%. Some of the mye- 5 Huang W, Sun GL, Li XS, Cao Q, Lu Y, Jang GS, Zhang FQ, Chai loblasts and eosinophilic promyelocytes appeared with JR, Wang ZY, Waxman S, Chen Z, Chen SJ. Acute promyelocytic Correspondence 611 leukemia: clinical relevance of two major PML-RAR isoforms and formation at the level of pluripotent stem cell in acute promyelo- detection of minimal residual disease by retrotranscriptase-poly- cytic leukemia. Blood 1994; 84: 49a. merase chain reaction to predict relapse. Blood 1993; 82: 7 Bayby GC. Hematopoiesine. In: Annopoulas GS, Armur W, Maj- 1264–1269. erus PW, Varmus H (eds). The Molecular Basis of Blood Diseases, 6 Takatsuki H, Umemura T, Yufu Y, Nishimura J, Nawata H. Trans- 2nd edn, WB Saunders Co: Philadelphia, 1994, pp 71–103. Correspondence 612 CASE REPORT Translocation (2;8)(p12;q24) in blastic transformation of atypical chronic myeloid leukemia SK Ma1,KLAu2, TSK Wan1 and LC Chan1

Departments of Pathology, 1Queen Mary Hospital and 2Princess Margaret Hospital, Hong Kong

smears using labeled avidin-biotin technique (Dako, Carpin- Keywords: cytogentics; t(2;8); atypical CML teria, CA, USA) showed that the blasts were positive for HLA- DR and myeloid-associated antigen CD13 but negative for a panel of B and T cell markers. A diagnosis of myeloblastic Translocation (8;14)(q24;q32), t(8;22)(q24;q11) and transformation of atypical CML3 was made. Cytogenetic t(2;8)(p12;q24) are considered highly consistent and specific analysis of bone marrow cells showed: rearrangements in Burkitt’s lymphoma and high grade B cell 45,X,−Y,t(2;8)(p12;q24)[6] (Figure 2). Southern blot analysis lymphoma.1,2 Here, we report a case of atypical chronic on DNA restricted with BamHI and BglII and hybridized to a myeloid leukemia (CML) in blastic transformation in which large M-BCR probe revealed a germline configuration of the cytogenetic analysis showed the clonal abnormality of BCR gene. Chemotherapy was started but, unfortunately, the t(2;8)(p12;q24). patient ran a rapid downhill course and a computed tomog- A 51-year-old man was admitted for fever and headache. ram of brain showed multiple contrast-enhanced cerebral Physical examination showed pallor and massive hepato- lesions. He succumbed on the 3rd day of hospitalization and splenomegaly. Complete blood counts revealed: hemoglobin a post-mortem examination was not performed. 9 6.7 g/dl, leucocyte count 451 × 10 /l (differential: blasts 23%, Our case confirms that, at the cytogenetic level, the translo- promyelocytes 20%, myelocytes 16%, neutrophils 33%, lym- cations associated with Burkitt’s lymphoma can be present in phocytes 2% and monocytes 6%) and platelet count non-B lineage hematologic malignancies. The clinical and 9 89 × 10 /l. Dysplastic changes were noted in neutrophils and hematologic findings in our case are in keeping with a diag- monocytes (Figure 1). There was no basophilia. The marrow nosis of atypical CML in myeloblastic transformation. Atypical was markedly hypercellular with increased blasts of myeloid CML, which is classified by the French–American–British morphology accounting for 37% of all nucleated cells. Granu- Group as one of the chronic myleoid leukemias,3 has features locytic maturation was evident but dysplastic. Megakaryo- of a myelodysplastic syndrome as well as a myeloproliferative cytes were adequately present though erythropoiesis was sev- disorder. These cases lack the Ph chromosome and the BCR erely depressed. No ringed sideroblasts were found. gene is not rearranged. In a series of 10 such patients studied, Cytochemically, the blast cells were positive for myeloperoxi- five (50%) had clonal cytogenetic abnormality either on pres- dase and Sudan black B. Immunophenotyping on marrow entation or follow-up.4 Loss of the Y chromosome, which was noted in our patient in addition to t(2;8), is also a well docu- mented feature in acute myeloid leukemia both as the only karyotypic abnormality or as a secondary change.5 Another well recognized Burkitt translocation, t(8;14) (q24;q32), has also been reported in a case of CD8+ large granular lymphocyte leukemia,6 one of the mature T cell malignancies.7 Owing to the lack of material, it was not poss- ible to examine for rearrangements of c-myc and immuno- globulin ␬-light chain loci in our case with t(2;8)(p12;q24). It is important to correlate the findings from a larger series of patients with atypical CML to see if there is any consistent karyotypic abnormality in this recently defined entity.

Figure 1 Peripheral blood smear showing circulating blasts, immature granulocytes, two dysplastic neutrophils – cytoplasmic hypogranulation (thick arrow) and abnormal nuclear segmentation (thin arrow), and a dysplastic monocyte (arrowhead). Wright–Giemsa × 1000.

Correspondence: SK Ma, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong Figure 2 Partial karyotype showing t(2;8)(p12;q24) in the present Received 3 December 1996; accepted 5 December 1996 case. G-banding with trypsin/Giemsa. Correspondence 613 Acknowledgement chronic myeloid, and chronic myelomonocytic leukaemia. Pro- posal by the French–American–British Co-operative Group. Br J We thank Mr CKC So for his excellent technical assistance. Haematol 1994; 87: 746–754. 4 Oscier DG. Atypical chronic myeloid leukaemia, a distinct clinical entity related to the myelodysplastic syndrome? Br J Haematol 1996; 92: 582–586. References 5 Holmes RI, Keating MJ, Cork A, Trujillo JM, McCredie KB, Freire- ich EJ. Loss of the Y chromosome in acute myelogenous leukemia: 1 Dalla-Favera R, Martinotti S, Gallo RC, Erikson J, Croce CM. Trans- a report of 13 patients. Cancer Genet Cytogenet 1985; 17: 269– location and rearrangements of the c-myc oncogene locus in 278. human undifferentiated B-cell lymphomas. Science 1983; 219: 6 Brito-Babapulle V, Matutes E, Foroni L, Pomfret M, Catovsky D. 963–967. A t(8;14)(q24;q32) in a T-lymphoma/leukemia of CD8+ large 2 Croce CM, Nowell PC. Molecular basis of human B cell neoplasia. granular lymphocytes. Leukemia 1987; 1: 789–794. Blood 1985; 65: 1–7. 7 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, 3 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C. Proposals for the classiﬁcation of chronic Gralnick H, Sultan C, Cox C. The chronic myeloid leukaemias: (mature) B and T lymphoid leukaemias. J Clin Pathol 1989; 42: guidelines for distinguishing chronic granulocytic, atypical 567–584. Correspondence 614 CASE REPORT An extremely delayed cytogenetic response to interferon-␣ in a patient with chronic myeloid leukaemia AJ Whiteway1, CDL Reid1 and NCP Cross2

1Northwick Park Hospital, Watford Road, Harrow, Middlesex and 3LRF Centre for Adult Leukaemia, Hammersmith Hospital, London, UK

In chronic myeloid leukaemia (CML), treatment with interferon response unlikely and the drug may then be discontinued. The ␣ ′ alpha IFN- results in loss of the Ph chromosome in a signifi- clinical course of the patient reported here suggests that per- cant proportion of patients. Most cytogenetic responses occur severance with IFN-␣, in the face of early failure to impact on early at a median of 9 months after initiation of treatment and ′ failure to detect a cytogenetic response within a predetermined the Ph positive clone, may still be rewarded with complete period may be a reason for IFN-␣ withdrawal. We report a response after many years of treatment. patient in whom IFN-␣ dosage was initially severely limited by bone marrow suppression but in whom continuing treatment led to a first cytogenetic response only after 53 months. Case report Increasing Ph′ negativity over a further 2 years was associated with improving haematological tolerance which permitted IFN- ␣ dose escalation and complete cytogenetic remission was In September 1988 a 49-year-old Afro-Caribbean man achieved at 7 years after diagnosis. This remission has been presented with an acutely swollen right calf and a 6-week his- sustained and has thus followed the most delayed cytogenetic tory of weight loss and fevers. He was known to have treated response to IFN-␣ so far reported. hypertension and sickle cell trait. Investigations showed the Keywords: chronic myeloid leukaemia; Philadelphia chromosome; calf swelling to be a haematoma; he also had a right retinal interferon-␣ haemorrhage and splenomegaly palpable 4 cm below the costal margin. There were several subcutaneous nodules over both thighs and biopsy showed these were leukaemic Introduction infiltrates. Further investigation showed: haemoglobin 8.6 g/dl, white Chronic myeloid leukaemia (CML) is characterised in most blood cell count 230 × 109/l (blasts 9.6 × 109/l), platelets cases by the presence of the Philadelphia chromosome, a 118 × 109/l. Prothrombin time, kaolin cephalin clotting time, translocation between chromosomes 9 and 22. The resulting thrombin time and bleeding time were all within the normal fusion of bcr and abl genes results in production of a chimeric range. Bone marrow aspirate was dry and an adequate tre- 210 kDa protein with tyrosine kinase activity that may play a phine was not obtained. Cytogenetic analysis of peripheral part in the disordered stem cell growth of CML. The median blood cells showed 11/11 metaphases contained the Philadel- survival for this condition has lengthened from 3 years to more phia chromosome translocation, t(9;22)(q34;q11). 1 recent estimates of 5 to 6 years. Following initial leukopheresis busulphan was introduced Chemotherapy has been the mainstay of treatment for the 1,2 and continued to February 1989 at which time the splenomeg- disease. More recently it has become clear that interferon aly had resolved. A sibling bone marrow donor could not be ␣ alpha (IFN- ) is effective in the induction of both haematologi- found, so in April 1989 he was entered into the Medical 3 ␣ cal and cytogenetic remission. The mechanism of IFN- in Research Council CML 3 study, at which time his bone mar- CML is unclear but antiproliferative and immunomodulatory row aspirate was hypercellular with an M:E ratio of 7:1; meta- effects may be involved. It is likely that achievement of cyto- myelocytes 30%, myelocytes 16%, promyelocytes 9%, blasts genetic response equates with reduction in the leukaemic 1%. Cytogenetic examination of marrow cells showed 30/30 clone and that a complete cytogenetic response might there- to be Ph′ positive. Busulphan 4 mg daily was continued until fore be expected to give the best chance of long-term survival the white cell count was reduced to less than 20 × 109/l, at or even cure. The most sensitive index of the disease uses the which time (June 1989) human lymphoblastoid IFN-␣ polymerase chain reaction (PCR) to detect and quantitate bcr- (Wellferon; Glaxo Wellcome, UK) was started at a dose of 3 abl transcripts. Most patients who achieve complete cyto- megaU daily (indicated in Table 1). Subsequent dosing with genetic remission remain PCR positive but the levels of interferon was limited by thrombocytopenia such that he was residual bcr-abl transcripts may differ by as much as four 4,5 receiving as little as 10.5 megaU weekly when his platelet orders of magnitude. count reached a nadir of 37 × 109/l after 48 months of IFN-␣. ␣ Numerous studies have employed different IFN- dosage Despite minor toxicity including ’flu-like symptoms and some strategies and have reported a variable incidence of cyto- mood effects IFN-␣ was not stopped at any stage. genetic response, varying rapidity of response and differing Bone marrow aspirates were obtained at 6 monthly intervals effects on survival.3,6–11 However, despite indications that ␣ for morphological and cytogenetic assessment. Table 1 rec- IFN- may enhance survival even without a cytogenetic ords the course of his treatment and the first appearance of 6 ′ response, absence of an early reduction in Ph positive meta- Ph′ negative metaphases at 53 months from the start of IFN- phases has been thought to make an eventual complete ␣ therapy. This was followed by further reductions in Ph′ positive cells until a complete cytogenetic remission (CCyR) at 84 months from diagnosis (76 months from starting interferon). Correspondence: CDL Reid With the improvement in the cytogenetic findings there was Received 18 November 1996; accepted 10 January 1997 increasing haematological tolerance for IFN-␣ and he now Correspondence 615 Table 1 The pattern of interferon dose, white cell count and karyotype over time

Date White cell count Platelet count Interferon dose Bone marrow × 109/l × 109/l megaU/week karyotypea

Sept 1988 230 118 0 11/11 Apr 1989 36 118 0 30/30; June 1989 10.7 98 21 — Dec 1989 9.5 100 15 30/30; + t(8;21) in 2 cells Oct 1990 6.3 106 24.5 30/30; + t(8;21) in 1 cell May 1991 7.1 87 24.5 30/30 Jan 1992 5.8 42 24.5 30/30 Aug 1992 3.1 37 14 30/30 Feb 1993 2.8 43 10.5 30/30 Jul 1993 5.4 37 10.5 — Nov 1993 3.1 51 10.5 28/30 Feb 1994 3.1 74 21 25/30 Sept 1994 3.2 92 21 27/30 Mar 1995 3 105 31.5 24/30 Oct 1995 2.8 99 42 0/50 June 1996 3.4 125 42 0/50 aNumber of Ph+ metaphases/total number of cells examined. bAnalysis of peripheral blood at diagnosis. tolerates 42 megaU weekly with neutrophil counts above was achieved at 6 weeks and for cytogenetic non-responders 1 × 109/l and platelets above 100 × 109/l. Early in the treat- further dose escalation occurred at 8 months. At 14 months, ment a clone of cells with an additional t(8;21)(q22;q22) was however, non-responders had the IFN-␣ dose reduced to 9 identified but this had disappeared after 24 months of IFN- megaU per week. They observed that 9% of first CyR did not ␣ therapy. occur until 14–24 months of IFN-␣ treatment and that the Analysis of peripheral blood by the polymerase chain reac- maximal CyR was not seen until 60 months in two out of 17 tion identified this patient as having b2a2 and b3a2 bcr-abl patients achieving CCyR. The authors speculated whether the RNA products. Semi-quantitative assay of the bcr-abl RNA12 CyR rate would have been better if the cytogenetic non- was performed and the results are tabulated in Table 2 which responders had continued to receive maximum tolerated demonstrates a fall in numbers of bcr-abl transcripts to levels doses of IFN-␣ – as occurred in our patient. Median survival compatible with a CCyR.4,5 in the IFN group was 72 months, significantly better than the chemotherapy group. The randomised German CML study9 compared chemo- Discussion therapy (hydroxyurea or busulphan) with IFN-␣, escalating from a starting dose of 5 megaU/m2/day. The IFN arm achie- IFN-␣ treatment produces a complete haematological ved a CyR rate of 18%, a CCyR of 7.2% and the longest response in 30–80% of cases and major cytogenetic responses reported interval to CCyR was 30 months. The interval to first (less than 35% metaphases Ph′ positive) in 10–40% of CyR is not reported. Although there was a trend for improved patients.1,2 In their non-randomized study the CALGB group7 survival in cytogenetic responders compared to non- used an IFN-␣ dose of 5 megaU/m2 daily and reported a first responders, this was not significant. In the MD Anderson cytogenetic response (CyR) at a median time of 9 months in study10 using 5 megaU/m2 IFN-␣ daily, the CyR rate was 58% 40% of evaluable cases. CCyR was obtained in 18% of cases and the CCyR rate 26% at a median of 16 months though with at a median time of 9 months. They found that in some a wide range of 3–70 months. Some of these patients also patients cytogenetic responses were slow to develop and 5% received IFN-gamma or hydroxyurea. The median survival of CyR were beyond 18 months of treatment, progressing to was 89 months and was significantly longer in those achieving CCyR at up to 24 months. The latest CyR occurred after 40 a CyR. This single centre study suggests that high CyR rates months of treatment. The randomised study of the Italian Co- may be achieved by consistent and aggressive dosing with operative Study Group8 compared chemotherapy with a IFN-␣ which might be difficult to maintain in multicentre stud- regime of escalating IFN-␣ dosage. A dose of 9 megaU daily ies. Factors associated with major cytogenetic response were the presence at diagnosis of good performance status, being asymptomatic, having a small spleen, high haemoglobin, low Table 2 Results of PCR assays for BCR-ABL white cell count and a low peripheral blast count. In contrast to most studies using comparatively high doses Date Bone marrow karyotype Bcr-abl/ Ph+cells/Cells examined Abl ratio % of IFN, one small study has shown that a low dosage of 6 megaU/m2/week resulted in a 26% CyR rate at a median inter- Mar 1995 24/30 5 val of 6 months with a CCyR rate of 7% and improved sur- Oct 1995 0/50 7 vival.11 The MRC trial (in which our patient was enrolled) gave May 1996 0/50 0.9 a mean weekly IFN-␣ dose of 22.6 megaU and resulted in a Sept 1996 — 0.54 CyR rate of 22% (half of which were major or complete). Earl- iest CyR were seen at a median of 32 weeks and the longest Correspondence 616 treatment interval to response (other than our patient) was 108 References weeks.6 There was no significant difference between the median received doses for non-responders and those with any level of CyR. Survival in the responders was significantly bet- 1 Goldman JM. Management of chronic myeloid leukaemia. Blood Rev 1986; 1: 21–29. ter than non-responders, who in turn had a better survival than ␣ 2 Kumar L, Gulati S. Alpha-interferon in chronic myelogenous leu- patients that did not receive IFN- . kaemia. Lancet 1995; 346: 984–985. This patient had several of the presenting characteristics 3 Talpaz M et al. Leukocyte interferon-induced myeloid cytoreduc- (marked splenomegaly, anaemia, high white cell count, blasts tion in chronic myelogenous leukaemia. Blood 1983; 62: 689– Ͼ4%) that have been associated with a poor prognosis for 692. IFN-␣-treated patients.10 Despite this IFN-␣ rapidly achieved 4 Hochhaus A et al. Variable numbers of BCR-ABL transcripts persist in CML patients who achieve complete cytogenetic remission with haematological control of his disease, accompanied by con- ␣ siderable myelotoxicity which was dose-limiting. This toxicity interferon- . Br J Haematol 1995; 91: 126–131. 5 Hochhaus A et al. Quantification of residual disease in chronic together with persistence of 100% Ph′ positive metaphases for ␣ myelogenous leukaemia patients on interferon-a therapy by com- more than 4 years mght have led us to withdrawn IFN- . petitive polymerase chain reaction. Blood 1996; 87: 1549–1555. However, because of evidence that there may be a survival 6 Allan NC et al. UK MRC randomised, multicentre trial of IFN-␣n1 advantage even in non-cytogenetic responders,6 IFN-␣ treat- for CML: improved survival irrespective of cytogenetic response. ment was continued, leading to CyR at 53 months and event- Lancet 1995; 345: 1392–1397. ual development of a CCyR after 6 years 4 months of treat- 7 Ozer H et al. Prolonged subcutaneous administration of recombi- ␣ ment (over 7 years from diagnosis). It is notable that the nant 2b interferon in patients with previously untreated Philadel- additional translocation t(8;21)(q22;q22) that appeared in phia chromosome-positive chronic-phase chronic myelogenous leukaemia: effect on remission duration and survival: Cancer and 2/30 metaphases 15 months after diagnosis disappeared after ␣ ′ Leukaemia Group B Study 8583. Blood 1993; 82: 2975–2984. 1 year of IFN- treatment even though Ph positive chromo- 8 Italian Cooperative Study Group on CML. Interferon ␣-2a as com- some numbers were unaffected. pared with conventional chemotherapy for the treatment of The clinical history of this patient with CML suggests that chronic myeloid leukaemia. New Engl J Med 1994; 330: 820–825. IFN-␣ should be continued at maximally tolerated doses even 9 Hehlmann R et al. Randomised comparison of interferon-␣ with in the absence of an early cytogenetic response. At least one busulphan and hydroxyurea in chronic myelogenous leukaemia. study6 supports a role for this drug in prolonging survival that Blood 1994; 84: 4064–4077. 10 Kantarjian HM et al. Prolonged survival in chronic myelogenous may be independent of cytogenetic response. If a minority of ␣ ′ leukaemia after cytogenetic response to interferon- therapy. Ann patients can achieve depletion of the Ph positive clone after Intern Med 1995; 122: 254–261. very prolonged treatment this would be an additional reason 11 Schofield JR et al. Low doses of IFN-␣ are as effective as higher to persevere with therapy. It was notable that the increased doses in inducing remissions and prolonging survival in CML. Ann haematological tolerance to IFN-␣ accompanied the depletion Intern Med 1994; 121: 736–744. of the Ph′ positive clone but it remains unclear whether the 12 Cross N et al. Competitive PCR to estimate the number of bcr-abl subsequent dose escalation was responsible for the attainment transcripts in CML after bone marrow transplantation. Blood 1993; of complete cytogenetic remission. 82: 1929–1936. Correspondence 617 LETTER TO THE EDITOR

Association between b3a2 BCR/ABL fusion and chronic myeloid leukemia with thrombocythemic onset: fortuitous or real?

Cervantes et al1 recently reported six cases of chronic myeloid could be associated with a given BCR/ABL fusion. These con- leukemia with a thrombocythemic onset. These cases flicting findings underline the problems of ascribing clinical resembled essential thrombocythemia on presentation, with characteristics to particular BCR/ABL fusion junctions, a sub- platelet counts Ͼ1000 × 109/l, and moderate increases in ject that has received much current attention.7 white cell counts (Ͻ20 × 109/l). With molecular analysis, five Recently, it has been shown that minute amounts of of six cases showed a b3a2 BCR/ABL fusion, and only one BCR/ABL can be detected in normal individuals who do not showed a b2a2 fusion. Cervantes et al regarded these cases have leukemia.8 Therefore, it appears that while BCR/ABL as another form of CML, and suggested that there was an may confer a proliferative advantage on a pluripotential stem association between b3a2 fusion and CML with thrombo- cell, additional genetic lesions may be needed for the devel- cythemia. opment of CML. Indeed, the acquisition of additional genetic CML in chronic phase may present with hyperplasia of any lesions also underlies the development of the accelerated and or all of the erythroid, granulocytic and megakaryocytic lin- blastic phases of CML. As the malignant phenotypes in leuke- eages, thereby mimicking other myeloproliferative disorders. mia are largely determined by genetic alterations,9 the For this reason, the diagnosis of essential thrombocythemia mutation(s) giving rise to a predominant granulocytic hyper- (ET) requires the exclusion of the Philadelphia (Ph) translo- plasia and a predominant megakaryocytic hyperplasia may be cation t(9;22)(q34;q11),2 as ET with t(9;22) has a clinical different, although in both forms BCR/ABL fusion is involved course similar to that of CML, and should receive similar treat- as the initiating event. Therefore, instead of simply regarding ment. thrombocythemia with BCR/ABL as another facet of CML, We have encountered five patients with apparent ET further investigations of these cases may disclose putative gen- (platelet counts 1100, 2400, 2460, 2700 and 3200 × 109/l) etic alterations that may contribute to our understanding of who were found to be BCR/ABL positive (three of them had the progression of the BCR/ABL clone, as well as factors con- been reported previously).3 Karyotypic analysis showed that trolling megakaryocytic differentiation. three cases had 100% Ph-positive metaphases on presen- Nevertheless, it is imperative that discussions on the under- tation, one case had 66% Ph-positive and 33% normal meta- lying genetic lesions in BCR/ABL-positive thrombocythemia phases, and one case had normal metaphases only. On mol- should not distract us from the important issue, as pointed ecular analysis by reverse transcription polymerase chain out by Cervantes et al and many other groups,10–14 that these reaction, three patients showed a b3a2 BCR/ABL fusion, while patients have a disease with a clinical course very similar to two showed a b2a2 fusion. It is noteworthy that one case was CML. Therefore, they should receive treatment strategies karyotypically normal, underlying the importance of molecu- identical to those of CML. lar investigations for BCR/ABL in thrombocythemia. Secondly, one case had 33% normal metaphases on presentation, an YL Kwong University Department of Medicine, unusual finding with reference to CML in chronic phase. Professorial Block Although the number of cases of thrombocythemia with Queen Mary Hospital BCR/ABL reported were small and a definitive conclusion can- Pokfulam Road not be drawn, it may be, contrary to the suggestion of Cerv- Hong Kong antes et al, that the association between b3a2 BCR/ABL fusion and thrombocythemia is fortuitous. As b3a2 and b2a2 fusions were more or less equally as frequent in CML,4 and most of these cases presented with granulocytic hyperplasia instead of References simply high platelet counts, it is unlikely that b3a2 per se can explain the thrombocythemia. Furthermore, b3a2 is not the 1 Cervantes F, Colomer D, Vives-Corrons JL, Rozman C, Montserrat E. Chronic myeloid leukemia of thrombocythemic onset: a CML only BCR/ABL junction thought to be associated with throm- subtype with distinct hematological and molecular features? Leu- bocythemia. Yamagata et al5 reported a CML patient with kemia 1996; 10: 1241–1243. thrombocythemia and minimal granulocytosis (1400 × 109/l 2 Murphy S, Iland H, Rosenthal DS, Laszio J. Essential thrombocy- and 17 × 109/l respectively), in whom a c3a2 BCR/ABL fusion themia: an interim report from the polycythemia vera study group. was demonstrated, with the BCR breakpoint located in ␮-bcr, Semin Hematol 1986; 23: 177–182. the most 3′ breakpoint cluster region.6 The authors postulated 3 Kwong YL, Chiu EKW, Liang RHS, Chan V, Chan TK. Essential thrombocythemia with BCR/ABL rearrangement. Cancer Genet that c3a2 might be related to thrombocytosis. On the other Cytogenet 1996; 89: 74–76. hand, a similar c3a2 fusion has also been found to be associa- 4 Nichols J, Dimer SD. Transcription factors, translocations, and leu- ted with ‘neutrophilic’ CML that was characterized by a pre- kemia. Blood 1992; 80: 2953–2963. dominant neutrophilia, but without an elevated platelet 5 Yamagata T, Mitani K, Kanada Y, Yazaki Y, Hirai H. Elevated count.6 Therefore, it appears that different disease phenotypes platelet count features the variant type of BCR/ABL junction in chronic myelogenous leukaemia. Br J Haematol 1996; 94: 370– 372. 6 Pane F, Frigeri F, Sindona M, Luciano L, Ferrara F, Cimino R, Correspondence: YL Kwong Meloni G, Saglio G, Salvatore F, Rotoli B. Neutrophilic chronic Received 5 November 1996; accepted 20 December 1996 myeloid leukemia: a distinct disease with a specific molecular Correspondence 618 marker (BCR/ABL with C3/A2 junction). Blood 1996; 88: 2410– 11 Morris CM, Fitzgerald PH, Hollings PE, Archer SA, Rosman I, 2414. Beard MJ. Essential thrombocythemia and the Philadelphia chro- 7 Melo JV. The diversity of BCR-ABL fusion proteins and their mosome. Br J Haematol 1988; 70: 13–19. relationship to leukemia phenotype. 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