Identification and Characterization of Three Novel Cytogenetically Cryptic EVI1 Rearrangements Identified in 27 AML Patients All Predicting An Unfavorable Outcome

Increased expression of EVI1 was reported to be associated with poor outcome in AML. The main mechanism of increased EVI1 expression is based on chromosomal rearrangements involving 3q26, where the EVI1 gene is located. The most frequently observed EVI1 rearrangements are inv(3)(q21q26) and t(3;3)(q21;q26). In addition, a variety of additional partner chromosomes and respective fusion partner genes were identified: 1q41 (DUSP10), 2p15–23, 3p25, 7q21 (CDK6), 7q34 (TCRB), 8q24, 12p13 (ETV6), or 21q22 (RUNX1). In addition, increased EVI1 expression has been reported in subsets of AML with normal karyotype or −7/7q-. Aim: We asked the question whether these AML subsets harbor cytogenetically cryptic EVI1 rearrangements. Patients and Methods: 606 AML and 377 MDS cases with normal karyotype or −7/7q- were screened using FISH technology with probes flanking breakpoints occurring in the EVI1 region (Kreatech, Amsterdam, The Netherlands). Results:EVI1 rearrangements were detected in twenty-seven patients with cytogenetically normal chromosomes 3. By further characterization using FISH analyses on metaphases three new and distinct EVI1 rearrangements were identified in these 27 cases. In detail, ten (2 MDS, 8 AML) cases demonstrated an inv(3)(p24q26), nine (5 MDS, 4 AML) cases showed a t(3;21)(q26;q11), and another eight (1 MDS, 7 AML) cases had a thus far not known der(7)t(3;7)(q26;q21). Moreover, EVI1 expression was measured by quantitative RT-PCR in 22/27 cases with material available (all values given as %EVI1/ABL1). In all investigated cases EVI1 expression was elevated. In 7 cases with inv(3)(p24q26) median EVI1 expression was 92.8 (range: 29.8–146.1), in 8 patients with t(3;21)(q26;q11) 104.9 (range: 41.4–176.3), and in 7 cases with der(7)t(3;7)(q26;q21) 101.8 (range: 4.4–210.4). For comparison, in 56 cases with inv(3)(q21q26)/t(3;3)(q21;q26) median EVI1 expression was 73.9 (range: 7.3–585.6), while EVI1 expression in normal bone marrow was 0.84 (range 0.75–1.28). We next aimed at investigating the novel partner genes deciphered here by using SNP array analyses (Cytogenetics Whole-Genome 2.7M array, Affymetrix, Santa Clara, CA). In 4 cases with der(7)t(3;7)(q26;q21) the high-resolution SNP microarrays revealed breakpoints in the CDK6 gene (breakpoints between 92,399,507 and 92,458,111; range: 59 kb) and centromeric of the EVI1 gene (breakpoints between 168,623,118 and 168,801,200; range: 178 kb). In 3 cases the EVI1-CDK6 rearrangements were confirmed by Sanger sequencing. The t(3;21)(q26;q11) was resolved as follows: in one case with t(3;21)(q26;q11) a duplication of the derivative chromosome 21 was present allowing the identification of breakpoints on chromosomes 3 and 21 by SNP microarray analysis. On chromosome 3 the breakpoint was located within the EVI1 gene (intron 2, breakpoint at 169,011,622) and on chromosome 21 within the NRIP1 gene (intron 3, breakpoint at 16,368,545). This rearrangement was confirmed by Sanger sequencing. In 7 additional cases with t(3;21)(q26;q11) an NRIP1-EVI1 fusion was detected by PCR. In 3 further cases the NRIP1 -EVI1 fusion was characterized on the DNA level by Sanger sequencing. Breakpoints in the EVI1 gene were located in intron 2 and 4 and in the NRIP1 gene in intron 1, 2 and 3, respectively. Finally, we performed an outcome analysis taking available clinical information into account. In cases with the novel EVI1 -rearrangements, median overall survival (OS) was 11.0 months and thus comparable to the median OS of AML with other described EVI1 -rearrangements (cohort: n=80 inv(3)(q21q26)/t(3;3)(q21;q26), n=24 other EVI1 -rearrangements). Conclusions: FISH screening with loci-specific probes for the detection of EVI1 rearrangements identifies a subgroup of patients with cryptic rearrangements and poor outcome, which cannot be detected by chromosome banding analysis. Thus, screening for EVI1 rearrangements enhances diagnostic accuracy and this method is particularly appropriate in AML with normal karyotype and AML with chromosome 7 abnormalities.

Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.