A Cryptic Rearrangement Involving ABL1/ETV6 Fusion in a Patient with Philadelphia-Negative Chronic Myeloid Leukemia Joann C

Cancer Genetics and Cytogenetics 192 (2009) 36e39

Short communication Insertion (12;9)(p13;q34q34): a cryptic rearrangement involving ABL1/ETV6 fusion in a patient with Philadelphia-negative chronic myeloid leukemia JoAnn C. Kellya,*, Nasrin Shahbazia, Jay Scheerlea, Jennifer Jahna, Stephany Suchena, Nicole C. Christacosa, Philip N. Mowreya, Mary H. Wittb, Alden Hostetterb, Aurelia M. Meloni-Ehriga aCytogenetics Department, Quest Diagnostics Nichols Institute, 14225 Newbrook Dr., Chantilly, VA 20151 bCancer Center, Rockingham Memorial Hospital, Harrisonburg, VA 22801 Received 22 December 2008; received in revised form 12 February 2009; accepted 13 February 2009

Abstract We report a rare cryptic ins(12;9)(p13;q34q34), a chromosomal abnormality involving the ABL1 (9q34) and the ETV6 (alias TEL; 12p13) genes, detectable only by ﬂuorescence in situ hybridization (FISH), in a patient with Philadelphia-negative chronic myeloid leukemia (CML). Using reverse 40,6-diamidino-2-phenylindole banding on metaphase cells, FISH analysis with BCR/ABL dual- fusion and ETV6 break-apart probes showed that a third ABL signal was inserted into 12p, splitting the ETV6 signal into two adjacent signals. CML patients with an ABL1/ETV6 fusion historically have demonstrated a variable and sometimes transient response to treatment with imatinib mesylate, which was also the case in the present patient. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Here we describe the case of a 79-year-old man with atypical CML whose fluorescence in situ hybridization (FISH) The t(9;22)(q34;q11.2), or Philadelphia chromosome, is studies using the BCR/ABL1 dual-fusion probe demonstrated usually consistent with the diagnosis of chronic myeloid a rare cryptic rearrangement, ins(12;9)(p13;q34q34), leading leukemia (CML) and, less frequently, with the diagnosis to an ABL1/ETV6 fusion. The insertion is a variant of of acute leukemia. The t(9;22) generates the BCR/ABL1 t(9;12)(q34;p13) that has been reported in only few cases fusion gene encoding a protein with increased tyrosine of atypical CML [1].This case study emphasizes the impor- kinase activity and transforming properties [1]. Although tance of FISH in detecting cryptic and variant rearrange- the fusion is correlated with an initiating event BCR/ABL1 ments, particularly when an abnormality is associated with in the development of CML, the underlying genetic mech- a malignancy in which a specific therapy may be beneficial. anisms are less well understood. Some cytogenetic and molecular genetic changes have been associated with disease progression [2,3]. The atypical CMLs are a particular group of myeloprolif- 2. Patient, materials, and methods erative disorders (MPDs) recently redefined as unclassified MPDs that are not characterized by the t(9;22) BCR/ABL1 2.1. Case report fusion. Approximately a dozen patients have been reported The patient initially presented in 2005 for evaluation of with variant ABL1 (9q34) rearrangements involving 12p13 increased white blood cell count after several weeks with e (ETV6), rather than 22q11.2 (BCR) [4 12]. This transloca- decreased appetite, an unintentional weight loss of 7 tion results in an ABL1/ETV6 fusion. In various hematolog- pounds, and lower abdominal pain. His laboratory parame- ical disorders, ETV6 encodes for a transcriptional repressor ters showed a white blood cell count of 35.2 103/mL, and is known to fuse to ~20 different genes, most of which hemoglobin 14.1 g/dL, platelet count 176 103/mL, with e code for tyrosine kinases or transcription factors [13 15]. 72% neutrophils, 6% lymphocytes, 16% monocytes, and 6% eosinophils. Within 5 days, his white blood cell count 3 * Corresponding author. Tel.: (703) 802-7024; fax: (703) 802-7103. increased to 41.6 10 /mL; hemoglobin was 13.9 g/dL, 3 E-mail address: [email protected] (JoAnnC. Kelly). and the platelet count was 202 10 /mL. At that time, he

0165-4608/09/$ e see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.02.012 J.C. Kelly et al. / Cancer Genetics and Cytogenetics 192 (2009) 36e39 37 had a marked left shift, with many promyelocytes and band metaphases analyzed. FISH studies were not performed in forms, significant eosinophilia, occasional basophils, and 2005. In 2006, three consecutive FISH studies were per- nucleated red blood cells. The patient appeared to have formed, which were instrumental in detecting and charac- a myeloproliferative disorder, likely chronic myeloid terizing a rare cryptic ins(12;9)(p13;q34q34). leukemia (CML). Polymerase chain reaction analysis was A FISH study using the BCR/ABL dual-fusion probe negative for the Philadelphia chromosome, thus excluding demonstrated two green signals for the BCR gene and three Philadelphia chromosome-positive CML. orange signals for the ABL1 gene in 92% of interphase In 2006, the patient experienced abdominal discomfort, cells and in the majority of metaphase cells analyzed. respiratory problems, chills and possible fever, as well as Additional FISH studies were performed with the 9q34 generalized malaise and fatigue, and falling on one occa- locus-specific ASS probe, proximal to the ABL1 region, sion. He also had an elevated white blood count of 205 mainly to determine whether the three ABL1 signals were 103/mL, a hematocrit of 36%, and a low platelet count of consistent with three intact copies of ABL1 or were the 89 103/mL. His peripheral blood smear showed predom- result of a rearrangement of this region that split apart inance of myeloid blasts. Neither lymphadenopathy nor one of the ABL1 signals. Two ASS aqua signals were hepatosplenomegaly was noted. After the FISH analysis, observed in all interphase cells examined. FISH on meta- which confirmed the presence of the ABL1/ETV6 rearrange- phase cells showed that both ASS signals still hybridized ment, the patient was placed on a therapeutic regimen that to the 9q34 region. included imatinib mesylate. Our patient demonstrated Translocations involving ABL1 and genes other than a transient response to imatinib, similar to what has been BCR have been reported in atypical CML. One of these indicated in the literature [11]. He returned to the hospital translocation is the t(9;12)(q34;p13) [4,5]. Using reverse- after 1 month with respiratory failure and a white blood cell 40,6-diamidino-2-phenylindole banding on metaphase cells, count of 205 103/mL with 39% blasts. He died within we confirmed that the third ABL1 signal was located on 12p hours of arrival. (Fig. 1). Furthermore, FISH analysis using the ETV6 (12p13) break-apart probe demonstrated two greeneorange 2.2. Chromosome analysis fusion signals for the ETV6 gene in all metaphase cells examined (Fig. 2). On interphase cells, however, a split The G-banded chromosome analysis was performed on ETV6 signal was observed on the abnormal 12p, with the the peripheral blood sample using standard cytogenetic 30 and the 50 regions remaining in constant vicinity of each protocols. Twenty metaphases were analyzed and karyo- other (Fig. 3). Because the 30 and 50 regions of ETV6 were grams were prepared using a computer-assisted karyotyping so close, no splitting was evident on metaphase cells system (CytoVision; Applied Imaging, San Jose, CA). The (Fig. 2). karyotypes were described according to ISCN 2005 [16].

2.3. FISH The FISH slides were processed according to the manu- facturer’s guidelines. Analyses were performed on both interphase and metaphase cells. The LSI BCR/ABL dual- color, dual-fusion translocation probe was used to identify any t(9;22)(q34;q11.2) present; the LSI TEL (ETV6) dual-color, break-apart probe was used to identify any rearrangement involving the ETV6 gene; and the LSI 9q34 SpectrumAqua probe to determine the number and location of ASS signals. All probes were purchased from Abbott Molecular, Des Plaines, IL. All FISH analyses were performed using a BX51/BX52 Olympus fluorescence microscope (Exfo America; Olympus, Richardson, TX). The images were captured using a charge-coupled device camera and the Isis imaging system (MetaSystems, Watertown, MA; Altlussheim, Germany). Fig. 1. Metaphase fluorescence in situ hybridization (FISH) with the BCR/ABL1 dual-fusion, dual-color probe shows two BCR signals (green) and three ABL1 signals (orange). The third ABL1 signal was determined 3. Results to be located on 12p. Therefore, two of the ABL1 signals (small orange) are derived from splitting of the probe signal with one located on 9q34 Conventional chromosome analyses performed in 2005 and the other on 12p13; and one normal ABL1/ASS signal (large orange) and 2006 yielded apparently normal results in all 20 on 9q34. 38 J.C. Kelly et al. / Cancer Genetics and Cytogenetics 192 (2009) 36e39 are highly suggestive of CML, to exclude the presence of the BCR/ABL1 fusion and then also to detect abnormalities involving either ABL1 or BCR that are more suggestive of an atypical CML. Our case is an example of a cryptic rearrangement associated with atypical CML. The conventional chromosome studies performed in this patient yielded an apparently normal result. The FISH studies, however, were instrumental in detecting and characterizing a cryptic insertion of the ABL1 region into 12p13, splitting the ETV6 region apart. The FISH study on interphase cells revealed a consistent splitting of the ETV6 fusion signal, with the 30 and the 50 regions remaining in constant vicinity of each other. At the metaphase level, there was no significant separation Fig. 2. Metaphase FISH with the break-apart ETV6 (12p13) and the ASS of the ETV6 fusion. The FISH result was therefore inter- (9q34) probes shows two apparently intact ETV6 signals (fusion) on preted as a small insertion of ABL1 into ETV6, rather than 12p13. a simple split. The ETV6/ABL1 fusion gives rise to a cyto- plasmic protein with elevated tyrosine kinase activity, 4. Discussion which can transform cell lines from factor dependent to factor independent [7,20] and to induce myeloproliferative The translocation (9;22)(q34;q11.2) is generally consis- disease in mice [21]. A recent report described a patient tent with CML and, at a lower frequency, with acute with ETV6/ABL1-positive acute leukemia who responded leukemia [17]. Other abnormalities have been associated transiently to imatinib [11], a tyrosine kinase inhibitor of with atypical CML, a chronic myeloproliferative disorder BCR/ABL1 and ABL1 [22]. mimicking typical CML but lacking the t(9;22) or the This case illustrates the importance of FISH in detecting BCR/ABL1 fusion [18]. These atypical CML cases exhibit cryptic and variant rearrangements, particularly when the translocations involving oncogenes including, but not abnormality in question is associated with a malignancy limited to, ABL1, PDGFR, and ETV6 [19].The in which a specific therapy may be beneficial. Our patient t(9;12)(q34;p13) is a recent finding and has been reported demonstrated a transient response to imatinib mesylate. only in a few cases [4,6,7,19,20], although some cases Studies of additional similar cases are warranted to better might have been missed, given the possibly cryptic nature understand the biology and clinical course of these atypical of the rearrangement. Fluorescence in situ hybridization CMLs. should be performed when myeloproliferative disorders

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