Genetic Heterogeneity and Stratification of AML Samples with NPM1 Mutation Detected by the MyAML® NGS Test

H19Svetlana Gramatikova 1, Andrew Carson 1, Bradley Patay 1, Tim Stenzel1, and Jeffrey E. Miller1
1Invivoscribe, San Diego, USAAbstract

ELN Recommendations for AML Treatment Based on Genetics

Variant Allele Frequencies

Introduction: Molecular abnormalities in multiple genes cause acute myeloid leukemia (AML), a clinically andgenetically heterogeneous disease. A recent study has shown that up to one-third of AML patients would have had
their treatment altered using a knowledge bank of matched genomic − clinical data, as compared to the treatment
they received using current practice recommendations1. With a frequency of ~30%, nucleophosmin (NPM1)-mutatedAML is the largest genomic subgroup in this disease2,3. The key purpose of this study is characterization of thegenetic heterogeneity and stratification of NPM1-mutated AML samples.

5

European LeukemiaNet - Conventional Care Regimens
Consolidation therapy‡,§
Younger patients (18-60/65 y)Favorable-risk genetics
Intermediate-risk genetics

Methods: Using the MyAML® next generation sequencing (NGS) panel targeting 194 genes, we analyzed the genetic
profile of 22 AML samples with driver mutations in the NPM1 gene. The variants were annotated and interpreted
followi ng the Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology,
College of American Pathologists, and American College of Medical Genetics and Genomics .
Results: In all cases the NPM1 mutations co-occurred with mutations in the FLT3 tyrosine ki nase, DNMT3A
methyltransferase, IDH1 or IDH2 isocitrate dehydrogenase genes. All FLT3 mutations were located in the protei nkinase catalytic domain and most mutations were internal tandem duplications (ITD). Four samples i ncluded morethan one FLT3-ITD. Most of the DNMT3A mutations were detected at the mutational hotspot Arg882; all IDH1mutations were at the mutational hotspot Arg132; most IDH2 mutations were at mutational hotspot Arg140, and allSRSF2 mutations were found at mutational hotspot Pro95. Mutations in IDH2 were more frequent than in IDH1, andmutations in these two genes di d not co-occur in the same sample. FLT3-ITDs had the largest variability in allele
frequency, when compared to mutations in DNMT3A and IDH1/2. The NPM1mut/FLT3ITD/DNMT3Amut genotype,associated with poor prognosis in AML patients, was observed in 6 samples (~27%), close to a previ ously reported
frequency3. All samples i ncluded mutations in the epigenetic modifiers DNMT3A, IDH1/2, TET2 and WT1, which areinvolved in the DNA methylation/hydroxymethylation pathway6.

Mutational Profiles of AML Samples

AB

A. New agents targeting epigenetic processes.13.
B. Rydapt (midostaurin) is used for treatment ofAML patients who are FLT3 mutation-positive.

Allogeneic HCT from matched-related or unrelated donor
22-4 cycles of IDAC (1000-1500 mg/m IV over 3 h q12h, d1-3; or 1000-1500
2mg/m IV over 3 h d1-5 or 6), orHigh-dose therapy and autologous HCTAllogeneic HCT from matched-related or unrelated donor

Adverse-risk genetics
Older patients (> 60/65 y)Favorable-risk genetics

222-3 cycles of IDAC (500-1000 mg/m IV over 3 h q12h, d1-3; or 500-1000 mg/m
IV over 3 h d1-5 or 6)

Intermediate/adverse-risk genetics

Patients considered not candidates for intensive chemotherapy
Low-dose cytarabine**

No established value of intensive consolidation therapy; consider allogeneic HCT
in patients with low HCT-Comorbidity Index, or investigational therapy
Low-dose cytarabine (20 mg q12h, SC, d1-10, q4 wk; until progression); notrecommended in patients with adverse-risk genetics‡ Patients, at least those aged 18 to 60 y, with newly diagnosed AML and activating FLT3 mutations may beconsidered to receive additional therapy with midostaurin (administered after the chemotherapy). § Results fromassessment of MRD should be taken into account for selecting consolidation therapy. ** In some countries used
in a dosage of 20 mg/m2 SC once daily. HCT, hematopoietic cell transplantation

Effect of Mutations in Mediators of the DNA Methylation Pathway
IDH mutations lead to
production of 2hydroxyglutarate (2HG), which
inhibits TET2. TET2 mutations
disrupt interaction with WT1.
WT1 mutations cause failure
to properly direct TET2 totarget sites, either bydisruption of the interaction
itself or of DNA binding. aKG,alpha ketoglutarate6.

Risk Stratification of AML Samples Based on Genetics

Combinational Therapies: Commercial and Developmental Drugs

22-4 cycles of IDAC (1000-1500 mg/m IV over 3 h q12h, d1-3; or 1000-1500
2mg/m IV over 3 h d1-5 or 6)

Conclusions• The MyAML® NGS test allows a comprehensive, rapid, and cost-effective molecular profiling of AMLsamples.
• We confirmed the previously reported occurrences and statistically significant associations of driver
mutations in AML samples2,3,8,11. NPM1 mutations associate with mutations in the mediators of the DNAmethylation/hydroxymethylation pathway and FLT3. Aberrant DNA methylation is a crucial processduring tumorigenesis.
• The complexity of molecular interactions and their role in AML is a major challenge for clinicalapplications of genetic data. The classification “AML with NPM1 mutation” only partially contributes tothe risk stratification, based on the 2017 Recommendations by NCCN4 and ELN5, as well as onliterature analysis. This classification does not inform about currently available treatments and clinical
trials, which depend on the mutational profile of the AML sample obtained from sequencing of multiplegenes.
• Therapies that combine conventional treatment and drugs targeting driver mutations are a promisingstrategy for improving the disease outcome. A combination of DNA methyltransferase and FLT3
inhibitors have already been shown to be effective in AML patients9.
• NPM1-positive minimal residual disease (MRD) has recently emerged as a sole prognostic factor forrelapse, regardless of the presence of other gene alterations8. MRD monitoring allows precise andsensitive tracking of leukemia clones in order to apply personalized medicine7. Patients whose
leukemia evolves from a pre-leukemic clone (e.g. DNMT3A, TET2 mutations)10 may require therapy
that exceeds standard chemotherapy. Monitoring of the pre-leukemic clone for acquisition of driver
mutations (e.g. NPM1, FLT3ITD) during remission might prompt initiation of aggressive therapy, such as
allogenic hematopoietic stem cell transplantation12.

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AMP, November 16 –18 2017, Salt Lake City, USA