A Distinct Pattern of Additional Aberrations and Novel Mechanisms of MLLRearrangements Are Detectable in AML with 11q23 Aberrations Using SNP Arrays
Authors: Haferlach Claudia , Kohlmann Alexander , Rauhut Sonja , Dicker Frank , Kern Wolfgang , Schnittger Susanne , Haferlach Torsten 
Published: April 2020

Abstract
Chromosomal rearrangements involving the MLL gene occur in 3–5% of adult AML. More than 50 different partner genes have been described in acute leukemia with 11q23-abnormalities. Although MLL-rearrangements per se have a high leukemic potential, additional genetic aberrations occur. This study was intended to decipher MLLrearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP 6.0 microarray analyses were performed in 47 newly diagnosed AML with 11q23 aberrations. First, as a proof of principle, all gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included recurring gains of whole chromosomes; 4 (n=3), 8 (n=7), and 19 (n=2). In addition, the following unbalanced abnormalities were detected: gain of 1q31.3 to 1q43 (n=5) and a gain of 3q (n=2). In 40/47 cases the following partner genes had been identified based on the translocation observed in chromosome banding analysis and RTPCR: AF9 (n=27), AF6 (n=4), AF10 (n=3), ELL (n=2), AF4 (n=1), AF17 (n=1), ENL (n=1), SEPT5 (n=1). In 4/47 cases results from chromosome banding analysis suggested partner genes to be located at 11q13 (n=1), 10p11 (n=1), and 19p13 (n=2). In 3/47 cases the MLL rearrangement was cryptic and only suspected by FISH analysis. Two of those (#1, #2) showed a del(11)(q23q25) in chromosome banding analyses and FISH analyses demonstrated a loss of the 3′ flanking MLL probe. In the remaining case (#3) cytogenetics showed an i(21)(q10). FISH analysis on metaphase spreads identified an additional copy of the 5′ flanking MLL probe which localized on 6q27. SNP analyses were able to resolve all three cases: #1) The deletion was fine-mapped by SNP microarray data and ranged from physical map position 117,859,541 to 11qter including exons 10 to 28 of the MLL gene. In addition, SNP microarray data revealed a gained segment on 6q ranging from physical map position 167,977,103 to 6qter including exons 2 to 28 of AF6. #2) In this case the 11q deletion spans from physical map position 117,859,541 to 121,033,713 including exons 10 to 28 of the MLL gene. SNP microarray data revealed a gained segment on 6q ranging from physical map position 168,036,784 to 168,457,799 including exons 9 to 28 of AF6. #3) Corresponding to FISH analysis SNP microarray data revealed a gained segment on 11q ranging from physical map position 117,760,488 to 117,859,673, including exons 1 to 9 of MLL. Moreover, on chromosome 6 a small deletion of 177 kb was detected, starting at physical map position 167,804,673 towards 167,982,457. This deletion included exon 1 of AF6 and a small adjacent centromeric region. In all 3 cases, subsequent RT-PCR analyses confirmed the predicted MLL-AF6 fusion. Analyzing the MLL gene further in the remaining cases revealed copy number changes in 2 cases showing gains of 11q starting from exon 12 of the MLL gene to 11qter (physical map position 117,863,291 to 11qter and 117,862,916 to 11qter). These were due to an extra copy of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3), respectively. In two additional cases very small deletions within MLL with a size of 4.831 kb including exons 10 and 11 (physical map position 117,859,541 to 117,864,372) and 1.699 kb including exons 10 and 11 (physical map position 117,859,541 to 117,861,240) were observed (MLL-AF6- and MLL-AF4-rearrangement). With respect to the various MLL partner genes, deletions starting in the partner genes were observed in 2 cases with MLL-AF9 rearrangement (size: 8 MB and 6.1 MB, physical map position 20,334,335 to 28,350,412 and 20,342,604 to 26,451,390). The region deleted in both cases spanned 37 genes, including several genes of the interferon alpha family and the tumor suppressor candidate TUSC1. Copy number gains were observed in the region of the partner genes in both cases with a doubling of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3). In conclusion, using high resolution SNP arrays we identified three novel mechanisms leading to MLL-AF6 fusions which are cytogenetically cryptic and associated with atypical FISH signal constellations. Furthermore, a distinct pattern of additional aberrations was observed showing trisomies of chromosomes 4, 8 and 19. SNP microarray data also revealed a small deletion on the short arm of chromosome 9 as a recurrent additional genetic change in AML with MLL-AF9-rearrangements.