Characterization and Quantification Of Rare BCR-ABL1 Fusion Transcripts

In chronic myeloid leukemia (CML) treatment with tyrosine kinase inhibitors induces extremely good responses in most of the patients (pts). To control for treatment responses molecular monitoring by quantitative real time PCR has become a standard procedure. Many international efforts have been done to standardize PCR assays, an international conversion factor has been introduced which allows comparison of results between laboratories, and response criteria have been defined based on %BCR-ABL1/ABL1 values. A drawback of the current state is that standardization, so far, addressed only the most common BCR-ABL1 fusion transcripts e13a2 and e14a2. For rare fusion types response criteria have not been applied and biological and prognostic significance are widely unknown.

1) Analyze incidence and associations of rare BCR-ABL1 fusion types with other biological parameters. 2) Develop assays for rare BCR-ABL1 fusions to allow molecular monitoring and analysis of therapy response in CML patients with rare fusion types.

In 5,730 individual CML pts a detailed molecular characterization of the BCR-ABL1 rearrangement was performed between 8/2005 and 6/2013. Rare fusion transcripts were further characterized by Sanger sequencing, and quantification was performed by a LightCycler based real time assay using hybridization probes. Probes and reverse primers delevoped for M-bcr transcripts were used (Emig et al., Leukemia, 1999). Solely the forward primer was modified and adapted to the BCR breakpoint. Assays were optimized to obtain similar efficiencies and sensitivities (1:10,000) compared to the standard p210 assay.

In the entire cohort of 5,730 CML pts rare fusion types were detected in 112 cases (2.0%). In detail, of the rare types 49/112 (43.8%) had breakpoints in the m-bcr (minor breakpoint cluster region, corresponding to p190): e1a2 (n=46, 41.1%) and e1a3 (n=3, 2.7%). 26 cases (23.2%) had rare M-bcr types (major breakpoint cluster region, corresponding to p210): e13a3 (n=8), e14a3 (n=15), e13e14a3 (n=2) and one case with e14a3 and insertion of 45 nucleotides. 20 pts (17.9%) had a typical µ-bcr breakpoint with e19a2 (corresponding to p230), including one case with an additional alternative splicing event that leads to an e13a2 fusion with 19 bp insertion. Further very rare fusions were: e4a2 (n=2), e6a2 (n=5), e16a2 (n=1), e18a2 (n=2), and e8a2 (n=7) (e8a2 fusions had in frame additions or deletions of 39-70 bp). The male/female ratio in this cohort was 65/47 (1.4), and median age was 66.9 years (y) (range: 20-87y). In 77 of the 112 cases a sample at diagnosis was available. In the remaining 35 pts monitoring started later. Of the 61 cases with available cytogenetics 38 pts had t(9;22) sole and 23 (37.7%) had additional chromosomal aberrations (ACA): -7 (n=2), +8 (n=4), i(17q) (n=1), dup(17)(q23q25) (n=1), -Y (n=7), +der(22)t(9;22) (n=3), rare translocations (n=5). Thus, the frequency of ACA was significantly higher than has been described for CML in general (6.9%, Fabarius et al., Blood, 2011).

Of the 112 patients 714 individual samples were quantified (e1a2 and e1a3: n=237, e13a3 and e14a3: n=191, e19a2: n=144, e16a2: n=24, e18a2: n=21, e8a2: n=60, e6a2: n=28 e4a2: n=9). Regular monitoring at every 3 months was available in 76 pts who were treated with firstline imatinib (n=64), followed by second line nilotinib (n=4) or dasatinib (n=3); firstline dasatinib (n=7), followed by nilotinib (n=1), or firstline nilotinib (n=5). Response was evaluated after 1 year and subdivided as follows: 1) CMolR (complete molecular response): undetectable BCR-ABL1, 2) MMolR (major molecular response): ≤0.1% BCR-ABL1/ABL1, 3) no response: no or less that 1 log decrease, 4) suboptimal response: > 0.1% BCR-ABL1/ABL1. In 15 pts (19.7%) a CMolR and in 20 pts (26.3%) a MMolR was reached (in 1 case after switch from imatinib to nilotinib and in 1 from imatinib to dasatinib). However, there was a large number of cases with no response: n=19 (25.0%) or suboptimal response: n=22 (28.9%). In 55 of the 76 continuously monitored pts cytogenetics at time of diagnosis was available. In the poor responders slightly more cases had ACA (12/28; 42.9%) compared to the good responders (7/27; 25.9%) (n.s.).

Rare BCR-ABL1 fusion types 1) occur in 2% of all CML, 2) are associated with higher number of ACA, 3) have no or suboptimal response to TKI treatment in more than 50% of pts.

Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.