Essential Thrombocythemia with the philadelphia Chromosome and BCR-ABL Gene Rearrangement An Entity Distinct from Chronic Myeloid Leukemia and Philadelphia ChromosomeNegative Essential Thrombocythemia D. P. LeBrun, P. H. Pinkerton, B. L. Sheridan, Jeanne Chen-Lai, I. D. Dub6, and P. A. Poldre

ABSTRACT: A 64-year-old woman presented with a platelet count of 3,225 x 109/L. Bone morrow morphology showed massive megakaryocytic hyperplasia; cytogenetic studies showed the presence of the Philadelphia chromosome (Ph). The presence of a rearrangement involving the major breakpoint cluster region (mbcr) an chromosome 22 was confirmed by Southern blotting techniques. A diagnosis af Ph positive essential thrombocythemia (ET) was made. Such cases constitute less than 5% of patients with ET and it has been proposed that they be considered examples of chronic myelogenous leukemia (CML) because of a shared propensity to progress to blast crisis, An argument is presented for retaining Ph positive ET as an entity separate from Ph negative ET and Ph positive CML. INTRODUCTION Essential thrombocythemia {ET} is a clonal myeloproliferative disorder characterized primarily by a very high platelet count {over 1,000 x 109/L). Additional criteria have been proposed by the Polycythemia Vera Study Group in an attempt to exclude other conditions that may be associated with marked thrombocytosis, especially other myeloproliferative syndromes sharing clinical and laboratory findings with ET [1]. Accordingly, the presence of the Philadelphia chromosome {Ph} in a case with clinical and laboratory features highly suggestive of ET is considered diagnostic of chronic myelogenous leukemia {CML}, with a high risk of progression to blast crisis and death over a few years [2]. We present the case of a 64-year-old w o m a n with ET in w h o m the Ph was detected by classic cytogenetic techniques and confirmed by the demonFromthe Departmentsof LaboratoryHaematology(D. P. L., P. H. P., B. L. S., J. C.-L.,I. D. D.) and Medicine {P.A. P.},SunnybrookHealth ScienceCentre,Toronto,Ontario;and Universityof TorontoTeachingHospitals, Cancer CytogeneticsProgram {P. H. P., J. C.-L., I. D. D.}, Department of Pathology, Universityof Toronto, Toronto, Ontario. Address reprint requests to: P. H. Pinkerton, M.D., Deportment of Laboratory Haernatology, Sunnybrook Medical Centre, 2075 Bayview Avenue, Toronto, Ontario. M4N 3M5. Canada Received August 13, 1990; accepted October 8, 1990. 21 © 1991 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010

Cancer Genet Cytogenet54:21-25 (1991} 0165-4608/91/$03.50

22

D.P. LeBrun et al.

Figure 1 Bonemarrow biopsy showing striking megakaryocytic hyperplasia with dysplastic cytological features

stration of a rearrangement in the major breakpoint cluster region (mbcr) of the BCR gene in chromosome 22 by Southern analysis. An argument is presented for retaining Ph positive ET as an entity distinct from CML and Ph negative ET.

Case Report A 64-year-old woman with severe proliferative diabetic retinopathy and hypertension was admitted for vitrectomy in November 1989 and was found on routine laboratory examination to have a platelet count of 3,225 × 109/L. On clinical examination, there were no ischemic or hemorrhagic manifestations and the spleen was not palpably enlarged. Hemoglobin was 124 g/L and the white blood count was 38.6 × 109/L with 4% basophils, 2% eosinophils, and occasional immature granulocytic forms. Platelet aggregation in response to collagen and epinephrine was greatly reduced and the LAP score was normal. Bone marrow aspirate and biopsy showed marked megakaryocytic hyperplasia with large and dysplastic megakaryocytes occurring in clusters (Fig. 1). Granulopoiesis showed a left shift and occasional dysplastic forms, and erythropoiesis was reduced. Reticulin was not increased and no stainable iron was present. Cytogenetic studies on bone marrow using G-banding (GTG) showed the presence of a Ph [t(9;22)(q34;q11)] in all of 24 cells analyzed (Fig. 2), and BCR rearrangement was confirmed using standard Southern blotting techniques (Fig. 3). A diagnosis of Ph positive essential thrombocythemia was made. Treatment with hydroxyurea resulted in a prompt decrease in total white cell count, without significant effect on the platelet count. Melphalan, 10 rag/day for 5 days, and two plateletpheresis procedures reduced her platelet count to 300 × 109/L. Platelet aggregation in response to collagen and

23

Philadelphia Chromosome BCR-ABL Rearrangement in ET

I I m

Figure 2 Partial karyotypes showing the t(9;22)(q34;q11) encountered in bone marrow cells from this patient, using G-banding. There was no other chromosomal abnormality. epinephrine improved. She underwent vitrectomy in February 1990 with no hemorrhagic complications. In the subsequent 4 months the platelet count gradually rose to 1,500 × 109/L. She died of pneumonia and septic cholangitis complicating cholelithiasis 9 months after diagnosis of ET. Discussion Cases of ET that are Ph negative have a favorable long-term prognosis with a low indidence of transformation to leukemia, myelofibrosis with myeloid metaplasia, or polycythemia rubra vera. The prognosis is worse when the Ph is present. Most patients die within 5-7 years, having developed the accelerated phase of CML or blast crisis [2]. It has been proposed that the diagnosis in this latter group should be regarded as CML [1]. However, important differences exist between Ph positive ET and typical CML. These include the predilection of Ph positive ET for females [2-5] and the frequent absence of splenomegaly [2, 3, 6]. The typical thrombotic or hemorrhagic events often seen in ET, with or without the Ph, are not common in CML. The results of conventional laboratory studies that readily distinquish ET from CML do not reliably predict the presence of the Ph in cases of ET, although peripheral basophilia and the appearance of megakaryocytes in the marrow are said to provide clues [2]. The leukocyte alkaline phosphatase is much less frequently low in Ph positive ET

24

D.P. LeBrun et el.

Bam HI

Bgl ]I

I

Hind ]]I

I

A

B

C

I1'

D

E

F

I

G

H

I

-5.4 ~

-4.5

Figure 3 Southern analysis using BCR probe 1. This probe is a 1.2Kb HindlII/BgllI fragment from the 3' end of the mbcr [8]. Lanes A, C, D, F, G, I contain control DNA. Patient DNA is in Lanes B, E, and H. Arrows indicate rearranged bands observed in patient DNA with all three enzymes. Lane B with BamH1, 13 and 5.3Kb; lane E with BglII, 4.8 and 2.6Kb; lane H with HindIII, 11 and 6.6Kb. Germline fragments are as follows: 3.4Kb with BamH1, 5.4Kb with BglII, and 4.5Kb with HindIII.

than in CML [2, 3, 6]. In addition, the m e d i a n time interval from diagnosis to blastic transformation is 3.5 years in CML and appears to be somewhat longer in Ph positive ET, although the n u m b e r of patients followed has been small [2-4]. Patients with Ph positive ET a p p e a r to have a longer interval from diagnosis to blastic transformation than those with CML, and at transformation the blast cells are more likely to show megakaryoblastic differentiation [2, 3, 4, 6]. As in patients with Ph negative CML, occasional cases of ET may be found that are Ph negative, but w h i c h show the classic major breakpoint cluster region rearrangement [71. The observation that the region involved in rearrangements of the BCR gene in this and other [5] cases of Ph positive ET is the same as that seen in CML strongly supports the notion that in both conditions the initiating or potentiating event is the same. The

P h i l a d e l p h i a Chromosome BCRoABL Rearrangement in ET

25

differences in clinical and laboratory findings may be due to the presence of other secondary mutations that modify the effect of the BCR/ABL fusion gene. Alternatively, the translocation that gives rise to the Ph may occur in cells at different stages of h e m o p o i e t i c differentiation. Because the role played by the BCR/ABL fusion gene in the manifestation of these conditions has yet to be completely elucidated, and in view of the clinical and laboratory features that still serve to distinquish the two entities, it seems premature to consider as CML those cases of ET w h i c h have the P h i l a d e l p h i a chromosome. We suggest that Ph positive ET with mbcr rearrangement should be c o n s i d e r e d an entity separate from either CML or Ph negative ET.

REFERENCES 1. Murphy S, Iland H, Rosenthal D, Laszlo J (19861: Essential thrombocythemia: an interim report from the Polythemia Vera Study Group. Semin Hematol 23:177-182. 2. Stoll DB, Peterson P, Exten R, Laszlo J, Pisciota AV, Ellis JT, White P, Vaidya K, Bozdech M, Murphy S (1988): Clinical presentation and natural history of patients with essential thrombocythemia and the Philadelphia chromosome. Am J Hematol 27:77-83. 3. Morris CM, Fitzgerald PH, Hollings PE, Archer SA, Rosman I, Beard MEJ, Heaton DC, Newhool< CT 11988): Essential thrombocythemia and the Philadelphia chromosome. Br J Hematol 70:13-19. 4. Sessarego M, Defferrari R, Dejana AM, Rebuttato AM, Fugazzo G, Salvidio E, Ajmar F/1989): Cytosenetic analysis in essential thrombocythemia at diagnosis and at transformation. A 12year study. Cancer Cenet Cytogenet 43:57-65. 5. Martiat P, Zfrah N, Rassool F, Morgan G, Giles F, Gow T, Goldman JM (1989): Molecular analysis of Philadelphia positive essential thrombocythemia. Leukemia 3:563-565. 6. Michiels JJ, Prins ME, Hagermeiier A, Brederoo P, van der Meulen T, Huub HDMV, Abels J (1987): Philadelphia chromosome-positive thrombocythemia and megakaryoblast leukemia. Am J Clin Path 88:645-652. 7. Tesch H, Zankovich R, Fouatrsch C, Diehl V (1990): Analysis of bcr rearrangements in primary (essentiall thrombocythemia. Leukemia Res 14:241-246. 8. Dube I, Dixon J, Beckett T, Grossman A, Weinstein M, Beun P, McKeithan T, Norman C, Pinkerton P (1989): Location of breakpoints within the major breakpoint cluster region (bcr) in 33 patients with bcr rearrangement-positive chronic myeloid leukemia (CML) with complex or absent Philadelphia chromosomes. Genes Chrom Cancer 1:106-111.