Analysis for loss of heterozygosity on chromosome arm 13q by STR analysis or SNP sequencing can replace analysis of FLT3-ITD to detect patients with prognostically adverse AML.

Abstract

We aimed at developing novel assays for Loss of Heterozygosity (LOH) detection on 13q as a substitute for FLT3-ITD analysis to identify acute myeloid leukemia (AML) patients with high risk of shorter survival. To this aim, we first analyzed a selected cohort of 185 patients with (n = 138) or without (n = 47) FLT3-ITD by short tandem repeat (STR) analysis for 13q LOH. In 46 of 138 FLT3-ITD positive cases, a FLT3-ITD/FLT3wt ratio of ≥ 1 was measured indicating LOH. Applying analysis with a combination of five different STR markers, a threshold of an STR allelic ratio of >65% allows the identification of LOH in 40/46 (87%) samples. Survival analysis revealed significantly inferior outcome in patients with LOH as detected by STR analysis (event free survival (EFS): no LOH: 13.3 months versus LOH: 4.5 months, p < 0.001; overall survival (OS): no LOH: 35.8 months versus LOH: 9.7 months, p = 0.001). In multivariate Cox regression analysis, 13q LOH was found to be an independent adverse parameter for EFS and OS (p = 0.003 and p < 0.001, respectively) besides age and white blood cell count. Thus, the prognostic impact of 13q LOH is nearly identical to the one of FLT3-ITD/FLT3wt load of ≥ 1 and thus is a feasible alternative to identify respective patients with high risk AML. In a second approach, a cohort of 91 patients was subjected to a proof-of-principle study of applying single nucleotide polymorphism analysis by next generation amplicon sequencing for detection of LOH. 53/91 cases had LOH and 50 were identified with this new method resulting in a positive detection rate of 94.3%.