ETV6-ABL1-positive “chronic myeloid leukemia”: clinical and molecular response to tyrosine kinase inhibition

While great progress has been achieved in the clinical management and molecular understanding of Ph+ chronic myeloid leukemia (CML), little is known about the optimal approach to monitor and treat patients with ETV6-ABL1 myeloproliferative neoplasms. Nine BCR-ABL1-negative CML patients with a variant ABL1 gene (9q34) rearrangement, involving fusion to ETV6 aka TEL (12p13) have been reported thus far.1–9 The ETV6-ABL1 fusion gene has also been identified in 3 patients with chronic myeloproliferative neoplasms other than “chronic myeloid leukemia” (cMPN) as well as 7 patients with BCR-ABL1 negative acute lymphoblastic leukemia and 4 patients with acute myeloid leukemia.10, 11 Of the 9 cases of ETV6-ABL1 chronic myeloid leukemia, only 2 were treated with a TKI in chronic phase4, 5 and only one of these reached a complete remission with a modest follow up of seven months (no molecular monitoring was performed).4 Among the other published ETV6-ABL1 reports, one patient was treated with a second generation TKI for a relapsed cMPN.12 We provide the first molecular documentation of sustained remission of ETV6-ABL1 chronic phase “chronic myeloid leukemia” (CML requires a BCR-ABL1 fusion according to the most recent 2008 WHO classification) to TKI. Molecular monitoring for the ETV6-ABL1 transcript is important given the fact that conventional karyotyping frequently fails to detect a cryptic translocation, i.e. the t(9;12). We provide evidence that treatment of ETV6-ABL1+ “chronic myeloid leukemia” with imatinib results in downregulation of C-MYC, BCL-XL, ID1 and NUP98, mediators of BCR-ABL1 transforming activity. Moreover, we found no associated mutations in UTX, ASXL1, EZH2, TET2 and IDH1/2 suggesting that ETV6-ABL1 “chronic myeloid leukemia” may be as tyrosine kinase focused as BCR-ABL1 driven disease.

The patient is a 36-year male who was found to have splenomegaly and a total white blood cell (WBC) count of 55×10/L (57% neutrophils, 6% lymphocytes, 1% monocytes, 3% eosinophils, 2% basophils, 7% metamyelocytes, 24% myelocytes). Lactate dehydrogenase was elevated at 653 IU/L. Bone marrow biopsy revealed myeloid hyperplasia suggestive of a myeloproliferative disorder. qRT-PCR for BCR-ABL1 translocation was negative. No BCR-ABL1 fusion signal was observed in interphase FISH analysis using BCR (22q11.2) and ASS-ABL1 (9q34) probes. Instead, 80% of interphase nuclei showed a variant signal pattern consisting of two signals for BCR and three signals for ASS-ABL1 consistent with rearrangement of ABL1 at 9q34 but not BCR at 22q11. Cytogenetic G-banding analysis and FISH showed t(9;12)(q34;p13) in an otherwise normal karyotype (Figure 1A and B). RT-PCR detected the ETV6-ABL1 translocation and the patient was diagnosed with ETV6-ABL1 CML-like disorder. Given the persistence of night sweats and fevers, and the persistent disease despite hydroxyurea at 1,000mg daily (WBC decreased to 7×10 cells/L after one month of hydroxyurea) imatinib mesylate, 400mg daily, was initiated. The patient tolerated imatinib and achieved a complete hematological remission after three months of treatment (WBC 5.6×10 cells/L). FISH testing after three months of imatinib revealed no evidence of rearrangement at the BCR or ETV6 loci (Figure 1B).

Figure 1.Diagnosis of ETV6-ABL1 positive CML. (A) G-banded karyotype showing t(9;12)(q34;p13) in the bone marrow sample from the patient with a clinical diagnosis of chronic myeloid leukemia. No additional cytogenetic abnormalities were observed (data not shown). (B) Bone marrow interphase cells showing rearrangements, as three signals were seen for ABL1 (orange) and ETV6 (green) genes by hybridization with BCR/ABL1 (upper left) and ETV6/AML1 (lower left) FISH probes. N.B. Only two signals were detected for the BCR (green, upper left) and AML1 (orange, lower left) loci; seven months after treatment with imatinib, no rearrangements were seen, as only two signals were generated by the ABL1 and ETV6 genes following hybridization with the BCR/ABL1 (upper right) and ETV6/AML1 (lower right) FISH probes.

To follow the patient’s response to therapy more sensitively, qRT-PCR was performed using primers for ETV6 and ABL1 (Online Supplementary Appendix). Quantification of the ETV6-ABL1 transcript level in peripheral blood cells one month prior to initiation of imatinib revealed 2,160×10 ETV6-ABL1 copies/μg RNA. After one month of treatment, ETV6-ABL1 transcript level dropped to 495×10 copies/μg RNA. The ETV6-ABL1 transcript became undetectable by seven months of treatment, indicating a complete and rapid molecular response. The patient’s molecular response closely mirrored normalization of the WBC count (Figure 2A). We also evaluated the expression of BCR-ABL1 target genes C-MYC, BCL-XL, ID1 and NUP98, pre- and post-imatinib treatment (Figure 2B). The expression of these genes closely mirrored ETV6-ABL1 expression: imatinib down-regulated their expression. The patient has continued to do well on imatinib 400mg/day with no evidence of ETV6-ABL1 transcript by qRT-PCR for the past five years.

Figure 2.Response to Imatinib in an ETV6-ABL1 positive chronic myeloid leukemia. (A) White blood cell count and peripheral blood qRT-PCR analysis of ETV6-ABL1 transcript levels pre-imatinib and throughout imatinib treatment. (B) Concomitant with decreases in ETV6-ABL1 transcript levels, decreases in C-MYC, ID1, BCL-XL, and NUP-98 transcripts were also seen. The patient remains in hema-tological remission with no identifiable ETV6-ABL1 transcripts in the peripheral blood after approximately five years of imatinib.

Somatic mutations in UTX, ASXL1, and TET2 have been reported in chronic myeloid leukemia and mutations in EZH2 and IDH1/2 in myeloid malignancies other than CML. We found no somatic alterations in these genes in the DNA extracted from whole blood prior to imatinib treatment, nor when the patient was in a molecular remission.

Our studies indicate that ETV6-ABL1 “chronic myeloid leukemia” can be sensitive to imatinib and there is significant overlap of molecular targets of ETV6-ABL1 with those of BCR-ABL1, suggesting that the ETV6-ABL1 fusion protein may trigger similar oncogenic cascades as BCR-ABL1. Finally, we were able to exclude mutations in any of the recently identified “myeloid” genes including UTX, ASXL1, EZH2, TET2 and IDH1/2 suggesting that the pathogenesis of ETV6-ABL1 “chronic myeloid leukemia” may be as tyrosine kinase focused as BCR-ABL1 driven disease.

Acknowledgments

The authors thank Masakatsu Hishizawa and Takashi Uchiyama from the Deptartment of Hematology/Oncology, Kyoto University Hospital, Japan for following the patient in Japan, Tony Deblasio for collecting and storing the samples, and Emily Dolezal for generating the database that facilitated our analysis.