Analysis For Loss Of 13q Heterozygosity Using STR Or SNP Analysis Can Replace Analysis Of FLT3-ITD To Detect Prognostically Adverse AML

FLT3-ITD has been established as an adverse prognostic factor in acute myeloid leukemia (AML). However, it has been repeatedly shown that not the presence of FLT3-ITD by itself confers the unfavorable effect, but the excess load of the FLT3-ITD in comparison to the FLT3 wild-type (wt) allele, which occurs by loss of heterozygosity (LOH) due to somatic isodisomy of chromosome 13q.

As now analysis of FLT3-ITD in many countries is restricted by patent claims we aimed at developing a novel assay for LOH detection on 13q as a substitute for FLT3-ITD to identify patients (pts) with a higher risk for shorter survival.

1) First an intensively treated AML cohort with normal karyotype (NK) (n=453) was selected in which the FLT3-ITD/wt ratio was assessed by a standard procedure (fragment length analysis by gene scan). 2) In a second step, a subcohort (cohort 2) of 233/453 pts was analyzed for LOH in 13q by fragment analysis of polymorphic single tandem repeat (STR) markers flanking the FLT3 gene as previously described (Whitman et al., JCO 2001). 3) A further subset of 91 pts from cohort 2 was subjected to a proof-of-principle study of applying next generation amplicon sequencing (NGS) for detection of LOH using a combination of Access Arrays (Fluidigm, South San Francisco, CA) and the 250 bp paired-end read chemistry on a MiSeq instrument (Illumina, San Diego, CA). In total, 41 amplicons were designed covering 67 different known SNPs (single nucleotide polymorphisms), with 43 SNPs flanking the FLT3 gene in the telomeric and the centromeric direction (genomic position: chr13:26,755,356-38,231,018), 22 SNPs being located in FLT3 intronic regions and one SNP each in exons 2 and 6. The achieved median coverage per SNP was 6,143 reads (range 58 - 30,311).

1) In the control cohort of 453 NK-AML (FLT3-ITD+: n=162, 35.8%) the adverse impact of high FLT3-ITD burden was reproduced. According to FLT3-ITD/FLT3wt a ratio of <0.5 was detected in n=60 (13.2%), between 0.5 and <1 in 69 (15.2%) and ≥1 in 33 (7.3%) cases. Survival in patients (pts) with a FLT3-ITD load <0.5 was not different from those with FLT3-wt (54% 3-year survival for both, p=n.s.). In contrast, the outcome in pts with a load of >1 (LOH) was significantly shorter as compared to all others (EFS: 3.6 vs 14.4 months, p<0.001, respectively). These results clearly show that 13q-LOH outperforms the presence of FLT3-ITD per se as a prognostic marker. 2) Therefore, we next performed the STR analysis in a subcohort of 233 pts (FLT3-ITD+ load >1: n=83, FLT3-ITD+ load ≤1: n=105, FLT3wt: n=45). The median mutation load was 0.80 (range: 0.03-181.7). In all patients at least one informative STR marker was identified: 1: n=6 pts, 2: n=23 pts, 3: n=60 pts, 4: n=77 pts, 5: n=67 pts). STR allele ratios were similar for FLT3wt and FLT3-ITD with burden of ≤1. A threshold of a STR aIlelic ratio ≥70% allows identification of 81/83 samples with LOH (positive detection: 97.6%). Median EFS was 4.7 months in the 13q LOH group compared to 14.4 months in all others (p=0.001), clearly demonstrating that STR analysis using five 13q-specific markers can be used as a powerful tool to generate prognostic data. 3) As the gene scan procedure was time consuming, we next tested whether an NGS-based high-throughput SNP analysis can detect the LOH cases with the same precision. In total, 110 different SNPs were detected in this cohort (43 more than the anticipated 67, as many pts had additional rare SNPs). Complete homozygous SNP constellations were regarded as not informative. A median of 67 SNPs/pts (range: 44-80) were detected with a median informative allele constellation of 51/pts (range: 27-70). The allelic SNP load was calculated from the mean allelic load of all informative SNPs and then correlated to the FLT3-ITD load as calculated by gene scan. Across the total subset of 91 pts a very good correlation was detected (Spearman, R=0.807, p<0.001). This was even better when only the cases with LOH, as assessed by the other two methods, were regarded (n=48, R=0.916, p<0.001). In total, 47/48 of the LOH pts were identified by this method showing a positive detection rate of 97.9%.

1) Not the presence of FLT3-ITD per se but a high FLT3-ITD load due to LOH of 13q has prognostically adverse impact. 2) Assessment of LOH can be done either by STR analysis or even more accessible by quantification of SNP allelic ratios using NGS amplicon deep-sequencing with a positive detection rate of 97.9%.

Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Weber:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Alpermann: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.