The BCR/ABL fusion gene originating from a t(9;22)(q34;q11.2) chromosomal translocation is the molecular marker of chronic myeloid leukemia (CML). In the vast majority of CML cases the breakpoint on chromosome 22 falls in the so-called major breakpoint cluster region of the BCR gene (M-bcr), leading to a hybrid BCR/ABL mRNA with a b2a2 and/or b3a2 junction, which encodes a p210 fusion protein involved in the mechanism underlying the chronic phase of CML. To date, only a few cases of CML with the breakpoint in the minor (m-bcr) and micro (μ-bcr) bcr region have been described, and were associated with peculiar CML phenotypes. Indeed, the m-bcr breakpoint has been associated with classical CML with a prominent monocytic component, while the μ-bcr breakpoint which gives origin to the e19a2 transcript corresponding to the p230 fusion protein, has been associated with a mild CML phenotype. Pane et al1 proposed classifying these latter cases as neutrophilic-chronic myeloid leukemia (CML-N), a rare disease characterized by moderate and persistent neutrophilia without precursors in the peripheral smear, absent or minimal splenomegaly, normal or raised leukocyte alkaline phosphatase (LAP) score, and a benign clinical course.
However, as more patients are being described, the correlation between the e19a2 genotype and CML-N, has become less clear. To date, an e19a2 transcript has been observed in nine patients with CML in the chronic phase,1,2,3,4,5,6 but also in four with CML rapidly evolving to the blastic phase,7,8,9,10 and in one with acute myeloid leukemia (AML).11 Moreover, patients in the chronic phase also presented characteristics of the classical type, such as pronounced leukocytosis and splenomegaly.5,6
We describe a case of CML with an e19a2 junction, whose phenotype shares some similarities with five of the previously published cases, such as low WBC count, no anemia, high platelet count and no splenomegaly. But, unlike the other cases, additional chromosomal abnormalities were present in the chronic phase.
A 63-year-old man was admitted to the Surgical Department in May 1999 for an intestinal subocclusion, due to a chronic appendicitis with adhesions. A hematological consultation was requested because a blood work-up disclosed a neutrophilic leukocytosis (WBC 23020/μl, N 70%), and the patient had a 3-year history of increasing thrombocytosis (413000/μl in 1997, 620000/μl in 1998 and 1209000/μl in 1999). Enlarged lymph nodes or hepatosplenomegaly were absent, and abdominal sonography findings were normal. At 1 month post-surgery, the patient was in excellent general condition and completely asymptomatic. Blood findings were: WBC 16500/μl with N 69%, Ly 20%, M 2%, E 5%, B 3%; Hb 12.8 g%, MCV 83, and Plts 784000/μl. The peripheral blood smear showed 73% segmented neutrophilic granulocytes and band cells, 3% promyelocytes, 5% myelocytes, 3% metamyelocytes, 1% myeloid blasts, 3% eosinophils, 3% basophils, 6% lymphocytes, and 3% monocytes. The LAP score was 11 (normal 10–100). Vitamin B12 (1363 pg/ml) and uric acid levels were high, while serum lactate dehydrogenase and plasma iron and ferritin concentrations were in the normal range.
CML was diagnosed, and the patient was placed on the α-interferon plus cytarabine therapeutic schedule. After 3 months, this treatment was suspended because the patient experienced loss of consciousness which was diagnosed as epilepsy, a rare side-effect of α-interferon. At this time, no hematological response had been obtained. The disease is now well controlled by hydroxyurea and the patient, still in good conditions, is on the waiting list for admission to an experimental trial employing inhibitors of tyrosine kinase activity.
The bone marrow aspirate was remarkably hypercellular due to a proliferation of all neutrophilic precursors, with dysmorphisms and eosinophils hyperplasia. Megakaryocytes were markedly increased, also in clusters, and showed dysmorphic characteristics (small in size, with hypolobulated nuclei and micromegakaryocytes).
The bone marrow biopsy showed the same pattern of hypercellularity with increased numbers of myeloid cells (also shown by the anti-myeloperoxidase antibody; M/E = 17/1), normal erythropoiesis (confirmed by the anti-glycophorin A antibody), increased megakaryocytes (confirmed by the anti-CD61 antibody), and decreased fat component. 2% of the cells were monocytes, as shown by the anti-CD68 antibody, while less than 5% were immature, CD34-positive cells; reticulin was normal.
Using primers specific for the major (CA3-: 5′-TGTTGAC TGGCGTGATGTAGTTGCTTGG-3′; b2+: 5′-TTCAGAAGCTTCTC CCTGACAT-3′) as well as the minor breakpoint (JC-: 5′-GG AGTGTTTCTCCAGACTGTTG-3′: BAL+: 5′-GAACTCGCAACAGTC CTTCGAC-3′), RT-PCR analysis failed to show visible bands corresponding to b2a2, b3a2 and e1a2 transcripts; a faint band of approximately 900 bp was seen with the M-bcr primers, suggesting the presence of a downstream breakpoint consistent with an e19a2 transcript. This hypothesis was confirmed by RT-PCR using primers we specifically designed for the μ-breakpoint; C3-A: 5′-GAGAGAGA GGTCCAAGGTGC-3′; ABL-A: 5′-CAGACTGTTGACTGGCGTGA-3′; a 426 bp amplification band was present on both peripheral blood and bone marrow samples; hybridization of the amplified products with an oligo-probe spanning the e19a2 junction, and direct sequencing further corroborated the result (Figure 1).
Cytogenetic analysis showed a Ph-positive clone, and also revealed additional aberrations, including loss of the Y chromosome and pericentric inversion of chromosome 8, in all 25 metaphases analysed: 45,X,-Y,inv(8)(p21q21.2),t(9;22)(q34,q11).
In both metaphase and interphase, FISH analysis could detect a BCR/ABL rearrangement in the μ-breakpoint using probes for the M-bcr and the m-bcr, since the μ-region is located 3′ to the minor and the major sites.
Saglio et al2 first recognized the e19a2 BCR/ABL fusion transcript as the molecular alteration in two patients with CML. Subsequently, the same group proposed that patients with this rare variant of BCR/ABL junction be classified as having CML-N, an atypical form of CML characterized by a mild phenotype and a benign clinical course.1
While the presence of additional chromosomal abnormalities, such as extra-Ph, iso17q and +8, is typical of the blastic phase, additional cytogenetic changes in the chronic phase of CML are uncommon.12 Moreover, their occurrence at the time of diagnosis has been associated with shortened survival, independently of the aberration type.12 The presence in our patient of a loss of chromosome Y and the pericentric inversion of chromosome 8, in addition to the Philadelphia chromosome was suggestive of a worse prognosis. On the other hand, the presence of good prognostic indicators such as the absence of splenomegaly, a low number of circulating blasts, a low percentage of basophils and eosinophils along with less than 5% of marrow blasts, was suggestive of a benign clinical course of the disease. Interestingly, thrombocytosis was the main clinical feature of our patient, with only a moderate increase in leukocyte count, mimicking essential thrombocytemia (ET), a chronic myeloproliferative disease with a better prognosis and a different therapeutic approach compared to CML.
Thrombocytosis (platelet count >600000/μl) was also observed in nine of the 14 patients previously described (in one case, the platelet count is not documented); indeed in five of them the diagnosis of ET was considered before the cytogenetic analysis. Two cases3,4 had clinical features similar to our patient. The other three7,8,10 presented a rapid disease progression. Additional chromosomal abnormalities typical of blast crisis characterized two of them7,8 while the other10 presented the t(8;21), typical of AML. Due to the benign clinical course of the disease, a pre-existing silent phase could be advanced in the first two cases, but the rapid evolution in the latter one seems related to a different molecular alteration.
A newly reported case of accelerated phase CML, characterized by an e19a2 junction as a single molecular feature and severe disease progression9 and recent experimental findings concerning in vivo leukemogenic activity of the p230 protein compared to the other BCR/ABL forms,13 seem to invalidate the hypothesis that the e19a2 rearrangement is indicative of a good prognosis. In our opinion, the characterization of new cases and long-term evaluation of these patients are needed in order to establish the real clinical relevance of the e19a2 BCR/ABL fusion transcript.
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We thank Miss Rossana Trevisan for technical assistance, Mrs Patricia Segato for help in preparing the manuscript and Mr Pierantonio Gallo for artwork.
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Bertorelle, R., Bonaldi, L., Bianchini, E. et al. The e19a2 BCR/ABL fusion transcript with additional chromosomal aberrations on a new case of chronic myeloid leukemia (CML) of mild type. Leukemia 15, 2003–2004 (2001). https://doi.org/10.1038/sj.leu.2402292
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