KMT2A-PTD

We characterize the KMT2A fusions present in myeloid malignant samples. We also describe the abundance of KMT2A PTDs in both healthy donor and myeloid samples, with myeloid cases showing significantly higher PTD read counts. KMT2A PTD read count >2000 is present only in malignant samples but not in healthy donors.

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Given the results obtained with menin inhibitors in KMT2A-rearranged and NPM1-mutated AML, our findings open an opportunity for exploiting a therapeutic vulnerability in all HOX-AML including KMT2A-PTD AML or AML with high MEN1 expression. Since HOX-AML highly express genes according to the HOX differentiation profile, stage-specific surface proteins coded by these genes would be promising targets.

Patient B: 70/F, NPM1m, ECOG=1, 125 mg QD, Arm B, 1 prior line of treatment with decitabine and an investigational agent. BMF-219 is generally well tolerated with no DLT observed (and able to be taken with and without CYP3A4 inhibitors) with no pts discontinuing therapy due to toxicity. BMF-219 dose escalation is ongoing and approaching target exposure. BMF-219 demonstrates early signs of clinical activity in different genomic subgroups.

Therefore, some KMT2A-PTD variants could be potentially implemented in AML risk stratification. However, prospective studies and larger patient numbers are needed.

The NMAv2 has high specificity, sensitivity, accuracy and reproducibility (inter-lab and intra-lab) with sequencing results generated within 48 hours of assay initiation. As such, the assay is well suited for use to rapidly categorize the genomic alteration of AML to support clinical care in patients with active myeloid malignancies, including those patients potentially enrolled in the myeloMATCH program. Table 1: Results of validation experiment results for the NMAv2 Figure 1: Harmonization results for the NMAv2 between MoCha and MO labs, Right Panel = Indel; Left panel = SNV

Synthetic upsampling (SMOTE) for the training dataset (n=1369) counteracts bias towards the majority classes and increases performance for the random forest (sensitivity=0.9327; precision=0.9327; specificity=0.9965; F1=0.93; accuracy=0.9933), XGboost (sensitivity=0.9404; precision=0.9404; specificity=0.9969; F1=0.94; accuracy=0.9940) and linear SVM (sensitivity=0.9615; precision=0.9615; specificity=0.9980; F1=0.96; accuracy=0.9962) models. Conjointly, these models demonstrate the utility and effectiveness of a machine learning approach for classifying pAML samples from transcriptome sequencing data, which may have broad clinical and research utility, especially for fusion negative subtypes.

Outlier isoforms were also effective targeted RNA markers for PAX5 intragenic amplifications (B-ALL), KMT2A-PTD (myeloid malignancies), and rare NOTCH1 intragenic deletions (T-ALL). These findings support the use of outlier isoform analysis as a robust strategy for detecting clinically significant DNA events.

After analyzing demographic and genetic features, complementary genetic analyses, including KMT2A-PTD, were suggested for accurate IPSS-M categorization of patients with ASXL1, TET2, STAG2, RUNX1, SF3B1, SRSF2, DNMT3A, U2AF1, and BCOR mutations and those classified as MDS, not otherwise specified with single lineage dysplasia/multi-lineage dysplasia based on the 2022 ICC. This study confirmed that the IPSS-M can better risk-stratified MDS patients for optimized therapeutic decision-making.

The findings indicated that KMT2A-PTD had an adverse impact on the prognosis of patients with AML in the total population, and the conclusion can also be applied to some subgroups including karyotypically normal AML and old AML patients. KMT2A-PTD may be a promising genetic biomarker in patients with AML in the future.

Moreover, WT1-H patients had a significantly lower RFS rate than WT1-L patients within both FAB-M5 and KMT2A rearrangement subgroups (P = 0.010 and 0.028), whereas WT1 had no impact on RFS within other subgroups mentioned above (all P > 0.05). Therefore, high WT1 expression at diagnosis independently predicted induction chemotherapy failure in AML patients with non-favorable cytogenetic risk, and it was related to relapse just within FAB-M5 and KMT2A rearrangement subgroups.

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Our study showed that for the Non-KMT2A-MLLT3 group, the EVI1-high group had shorter OS and DFS than the EVI1-low group. High EVI1 expression showed an adverse effect in AML with KMT2A rearrangement in different risk stratification subtypes. For the EVI1-high patients with non-KMT2A-MLLT3 rearrangement, other novel regimens towards relapse should be taken into consideration.

Around 10% of patients with MDS lack NGS-detectable mutations by current methods. These patients have different risk categories according to classical classifications, and there is an significant percentage of patients with tMDS, which also present with worse prognosis. The biological pathways altered in this subset of patients needs to be understood.

We show that nanopore sequencing can accelerate and improve comprehensive analysis of genome-wide chromosomal and specific molecular aberrations in AML patients. Rapid classification according to WHO classification and ELN risk stratification is able to immediately impact on targeted and risk adapted treatment of AML patients. Due to the low coverage, detection of small subclones will not be possible by LC-WGS.

Targetable mutations are frequent in EM-AML and EM-site NGS is warranted for selecting potential targeted therapies for patients with EM-AML.

By multivariate analysis, CCSS by OGM only (not CBA), TP53 mutation and BM blasts independently predicted survival. This is the first and largest study reporting the value of combined SVP and NGS for MDS prognostication.

Moreover, in serial samples, complexity was associated with relapse and secondary transformation. Taken together, we provide approaches to integrate quantitative and allelic assessment of KMT2A-PTDs into targeted DNA NGS and demonstrate that secondary genetic events occur in KMT2A-PTD by multiple mechanisms that may be linked to myeloid disease progression by driving increased expression from the affected allele.

In a multivariable analysis, FLT3 non-ITD mutations were not an independent prognostic factor. However, patients with dual FLT3 non-ITD and KMT2A-PTD mutations showed a trend for inferior outcome, which points at a functional interaction in this subset of AML.

AEL is a heterogenous subgroups of AML characterized in common by upregulated JAK/STAT5 signaling. Gains/amplifications of JAK2/EPOR are frequent in TP53-mutated cases, particularly those with the PEL phenotype, and could be exploited as potential therapeutic targets using JAK2 inhibitors.

Survival was significantly longer in patients with molCR vs no molCR post-HSCT (OS: not reached vs. 30.4 months, p=0.004; relapse-free survival: not reached vs. 9.1 months, p<0.001), while other factors, such as cytogenetic risk, total body irradiation-based conditioning, RIC vs MAC, use of FLT3i pre-HSCT or post-HSCT had no significant impact on survival.Meningeal leukemia (n=5) or CNS chloroma (n=1) were observed as late events at a median of 16.3 months post-HSCT and occurred on Sorafenib (n=2), Gilteritinib (n=1) or decitabine-based salvage therapy after failure of FLT3i (n=3).  Patients with FLT3-mutated AML and active disease at the time of allogeneic HSCT, combined with failure to achieve MRD-negativity after allogeneic HSCT have a high risk of CNS-relapse and leukemic death. This was not abrogated by pre-emptive or salvage-therapy with FLT3i or HMA and prophylactic intrathecal therapy may be considered to avoid CNS relapse.

The assay for transposase-accessible chromatin sequencing of AMLs supported enhanced chromatin accessibilities around genomic regions, such as HOXB cluster genes, SCHIP1, and PRDM16, which were associated with DNA methylation changes in AMLs with FLT3-ITD and high PRDM16 expression. Our results suggest that DNA methylation levels at specific CpG sites are useful to support genetic alterations and gene expression patterns of patients with pediatric AML.

Although KMT2A -PTD patients with and without FLT3 -ITD had similar outcomes, the cohort who received HSCT in CR1 (most with FLT3 -ITD) experienced improved outcomes compared to the rest of the cohort, suggesting that intensification of therapy may benefit this group of patients overall. Further research into KMT2A -PTD in pediatric AML will guide future risk-adapted therapy and enhance understanding of biologic implications of this lesion, including whether altered KMT2A may serve as a therapeutic target as it may for KMT2A -r AML.

Finally, we proved that the dynamic changes in the KMT2A-PTD fusion ratio were consistent with the overall process of disease progression. In summary, we applied the UMI-based RNA panel to quantitatively detect KMT2A-PTD and elucidate its clinical relevance, which complemented the integrative network of various genetic alterations in AML.

In conclusion, our results emphasize that KMT2A-PTD should be considered as a potential adverse prognostic factor. However, as KMT2A-PTD-mutated patients are usually considered an intermediate risk group, upfront HSCT should be considered in first CR due to the high relapse rate observed in this subset of patients.

We showed that the negative impact of this mutation might be overcome by HSCT in younger patients. In elderly unfit patient, targeted therapies such as IDH inhibitors and venetoclax-based regimens might be of interest.