FLT3-ITD allelic ratio and HLF expression predict FLT3 inhibitor efficacy in adult AML

FLT3 internal tandem duplication (FLT3-ITD) is a frequent mutation in acute myeloid leukemia (AML) and remains a strong prognostic factor due to high rate of disease recurrence. Several FLT3-targeted agents have been developed, but determinants of variable responses to these agents remain understudied. Here, we investigated the role FLT3-ITD allelic ratio (ITD-AR), ITD length, and associated gene expression signatures on FLT3 inhibitor response in adult AML. We performed fragment analysis, ex vivo drug testing, and next generation sequencing (RNA, exome) to 119 samples from 87 AML patients and 13 healthy bone marrow controls. We found that ex vivo response to FLT3 inhibitors is significantly associated with ITD-AR, but not with ITD length. Interestingly, we found that the HLF gene is overexpressed in FLT3-ITD+ AML and associated with ITD-AR. The retrospective analysis of AML patients treated with FLT3 inhibitor sorafenib showed that patients with high HLF expression and ITD-AR had better clinical response to therapy compared to those with low ITD-AR and HLF expression. Thus, our findings suggest that FLT3 ITD-AR together with increased HLF expression play a role in variable FLT3 inhibitor responses observed in FLT3-ITD+ AML patients.

Biology and Clinical Implications of Complex Landscape of Cooperating Events in FLT3-ITD Acute Myeloid Leukemia

FLT3-ITD mutations are among the most common somatic mutations in acute myeloid leukemia (AML) and are important in prognostic determination as well as therapeutic allocation. Recent studies have demonstrated improved outcomes with the addition of FLT3 inhibitors and for some patients hematopoietic stem cell transplant (HSCT) in first remission (CR1). We have previously demonstrated that the outcome of FLT3-ITD patients can be quite heterogeneous based on the co-occurrence of a few specific risk stratifying mutations, including NPM1 and NUP98-NSD1. We sought to interrogate the complex landscape of cooperating events with FLT3-ITD AML and potential impacts on outcome in the context of contemporary therapies, including the FLT3 inhibitor sorafenib. Of the 1296 children and young adult patients with de novo AML enrolled on COG AAML1031, 229 had FLT3-ITD mutations and were included in this study. Patients with high allelic ratio (HAR; >0.4) FLT3-ITD were allocated to Arm C, received sorafenib in combination with chemotherapy and received HSCT in CR1. Those with low allelic ratio (LAR; £ 0.4) FLT3-ITD were treated on Arm A/B and received chemotherapy, no sorafenib, and did not receive HSCT in CR1 unless they had evidence of residual disease following induction I (MRD³ 0.1%) or a high-risk cytogenetic feature. FLT3-ITD status and allelic ratio were determined by PCR and all samples also underwent karyotyping, FISH, and next generation sequencing in 195 (85%) of cases for determination of comprehensive co-occurring mutational profile. Among the 229 FLT3-ITD positive patients, allelic ratio ranged from <0.1-20.8, with 96 (42%) patients classified as LAR and 133 (58%) patients as HAR. Among the cohort overall, the significant majority of 85% (n=195) harbored a cooperating genomic aberration. The most common co-occurring single gene mutations were: WT1 (31%, n=71), NPM1 (20%, n=46), NRAS (9.2%, n=21), FLT3-TKD (7%, n=16), CEBPA (6.5%, n=15), KMT2A-PTD (5.7%, n=13) (Figure 1A). KMT2A-PTD lesions were significantly more prevalent among FLT3-ITD vs non ITD patients, 5.7% vs. 0.65% (p<0.001). Normal karyotype was detected in 50% of patients. The most common recurring cytogenetic abnormalities were NUP98-NSD1/t(5;11) fusions (19.2%, n=44), trisomy 8 (10%, n=23), DEK-NUP214/t(6;9) fusions (7%, n=16), KMT2A rearrangements (3.9%, n=9)(Figure 1A). In contrast, the other high risk abnormalities (monosomy 5/del5q, monosomy 7) were absent or exceedingly rare, while the low risk lesions t(8;21) and inv(16) were also rare (3%, n=7 each). We have previously reported outcome of the more common and risk stratifying mutations with co-occurring NUP98-NSD1 resulting in dismal prognosis regardless of treatment arm, while outcome for those with WT1 was improved with Arm C treatment and approached that of other FLT3-ITD patients(Figure 1B). Evaluation of the FLT3-ITD/trisomy 8 patients demonstrated those treated on Arm C experienced poor outcomes with an EFS of 30% and was equivalent to 29% for those on Arm A/B (p=0.96, Figure 1C), with a corresponding OS of 40% vs. 34% (p=0.66) respectively. In contrast, evaluation of outcome of the KMT2A-PTD patients demonstrated those treated on Arm C had a favorable 5-year event-free survival (EFS) of 71% vs. 23% (p=0.05) for those on Arm A/B (Figure 1D), with a corresponding 5-year overall survival (OS) of 86% vs. 46% (p=0.15) respectively. Comprehensive sequencing demonstrated the FLT3-ITD samples identified co-occurring genetic mutations or cytogenetic abnormalities in the majority of cases. Although KMT2A-PTD is rarely reported in pediatric compared to adult AML, we found it was enriched in FLT3-ITD patients and this cohort experienced favorable outcomes when treated with transplant and sorafenib. Patients with dual FLT3-ITD/trisomy 8 had suboptimal outcomes similar to other poor risk co-occurring lesions and comparable regardless of AR or treatment arm. While there was some overlap with WT1 mutations in this cohort, further investigation into prognostic impact of this cooperating event is warranted. The prognostic implications FLT3-ITD mutations vary and we provide further data that the comprehensive cooperating mutational profile is critical to understanding the prognostic implications in specific patients, and may also impact response to FLT3 inhibitor therapy. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees.

Prognostic significance of FLT3-ITD length in AML patients treated with intensive regimens

FLT3-ITD mutations are detected in approximately 25% of newly diagnosed adult acute myeloid leukemia (AML) patients and confer an adverse prognosis. The FLT3-ITD allelic ratio has clear prognostic value. Nevertheless, there are numerous manuscripts with contradictory results regarding the prognostic relevance of the length and insertion site (IS) of the FLT3-ITD fragment. We aimed to assess the prognostic impact of these variables on the complete remission (CR) rates, overall survival (OS) and relapse-free survival (RFS) of AML patients with FLT3-ITDmutations. We studied the FLT3-ITD length of 362 adult AML patients included in the PETHEMA AML registry. We tried to validate the thresholds of ITD length previously published (i.e., 39 bp and 70 bp) in intensively treated AML patients (n = 161). We also analyzed the mutational profile of 118 FLT3-ITD AML patients with an NGS panel of 39 genes and correlated mutational status with the length and IS of ITD. The AUC of the ROC curve of the ITD length for OS prediction was 0.504, and no differences were found when applying any of the thresholds for OS, RFS or CR rate. Only four out of 106 patients had ITD IS in the TKD1 domain. Our results, alongside previous publications, confirm that FLT3-ITD length lacks prognostic value and clinical applicability.

Whole genome sequencing identifies allelic ratio distortion in sperm involving genes related to spermatogenesis in a swine model

Transmission Ratio Distortion (TRD), the uneven transmission of an allele from a parent to its offspring, can be caused by allelic differences affecting gametogenesis, fertilization or embryogenesis. However, TRD remains vaguely studied at a genomic scale. We sequenced the diploid and haploid genomes of three boars from leukocytes and spermatozoa at 50x to shed light into the genetic basis of spermatogenesis-caused Allelic Ratio Distortion (ARD). We first developed a Binomial model to identify ARD by simultaneously analysing all three males. This led to the identification of 55 ARD SNPs, most of which were animal-specific. We then evaluated ARD individually within each pig by a Fisher’s exact test and identified two shared genes (TOP3A and UNC5B) and four shared genomic regions harbouring distinct ARD SNPs in the three boars. The shared genomic regions contained candidate genes with functions related to spermatogenesis including AK7, ARID4B, BDKRB2, GSK3B, NID1, NSMCE1, PALB2, VRK1 and ZC3H13. Using the Fisher’s test, we also identified 378 genes containing variants with protein damaging potential in at least one boar, a high proportion of which, including FAM120B, TDRD15, JAM2 or AOX4 among others, are associated to spermatogenesis. Overall, our results show that sperm is subjected to ARD with variants associated to a wide variety of genes involved in different stages of spermatogenesis.