Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms.

FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition.

Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants.

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia.

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models.

Genetic Testing in the Diagnosis and Biology of Myeloid Neoplasms (Excluding Acute Leukemias).

Objectives: The 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology reviewed the role of genetic testing in the diagnosis of hematopoietic neoplasms, including non-acute leukemia myeloid malignancies.

Methods: The workshop panel assigned 98 submitted cases to the category of non-acute leukemia myeloid neoplasms, of which 13 were selected for oral presentation.

Results: Data from both conventional karyotyping and genetic sequencing had important impact on diagnosis, classification, and prognostication.

Genome-Wide Copy Number Variation Detection Using NGS: Data Analysis and Interpretation.

Copy number variants (CNVs) and copy neutral loss of heterozygosity (CN-LOH) represent important types of genomic abnormalities in cancer. Genomic DNA microarray serves as the current gold standard method for detecting genome-wide CNVs and CN-LOH. However, as next-generation sequencing (NGS) is widely used to detect gene variants in clinical testing, the ability of NGS to detect CNVs and CN-LOH has also been demonstrated.

Molecular Fingerprinting of Anatomically and Temporally Distinct B-Cell Lymphoma Samples by Next-Generation Sequencing to Establish Clonal Relatedness.

Context.—: B-cell lymphomas exhibit balanced translocations that involve immunoglobulin loci and result from aberrant V(D)J recombination, class switch recombination, or somatic hypermutation. Although most of the breakpoints in the immunoglobulin loci occur in defined regions, those in the partner genes vary; therefore, it is unlikely that 2 independent clones would share identical breakpoints in both partners.

Similar expression profiles in CD34 cells from chronic phase chronic myeloid leukemia patients with and without deep molecular responses to nilotinib.

The life expectancy of patients with chronic phase chronic myeloid leukemia on tyrosine kinase inhibitor therapy now approaches that of the general population. Approximately 60% of patients treated with second generation tyrosine kinase inhibitors achieve a deep molecular response, the prerequisite for a trial of treatment-free remission. Those patients unlikely to achieve deep molecular response may benefit from more intensive therapy up front.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

Aims: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS).

Detection and Quantification of Acute Myeloid Leukemia-Associated Fusion Transcripts.

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization.

Coexisting and cooperating mutations in NPM1-mutated acute myeloid leukemia.

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML.

Changes in peripheral blood lymphocytes in polycythemia vera and essential thrombocythemia patients treated with pegylated-interferon alpha and correlation with allelic burden.

Background: Pegylated-interferon alpha (PegINFα) treatment of patients with polycythemia vera (PV) and essential thrombocythemia (ET) has resulted in long-term clinical response, decreased allelic burden and restoration of polyclonal hematopoiesis. The mechanisms of the beneficial effects of PegINFα are not clear, but available evidence suggests direct suppression of -mutated clone, induction of dormant stem cells to proliferation, and augmentation of an immune effect against PV and ET clones.

Methods: We analyzed the phenotype and frequency of peripheral blood lymphocytes (PBL) from PegINFα treated patients and compared them to patients treated with hydroxyurea (HU).

Two Unrelated Burkitt Lymphomas Seven Years Apart in a Patient With X-Linked Lymphoproliferative Disease Type 1 (XLP1).

Objectives: We describe a rare case of a male child with X-linked lymphoproliferative disease type 1 (XLP1) who presented with Burkitt lymphoma (BL) when he was 6 years old, achieved a complete response to therapy, and developed a second BL after seven years.

Methods: Diagnostic H&E stained slides and ancillary studies were reviewed for both lymphomas. B-cell clonality by PCR and SNP array studies were performed on both specimens.

Targeted next-generation sequencing identifies a subset of idiopathic hypereosinophilic syndrome with features similar to chronic eosinophilic leukemia, not otherwise specified.

The distinction between chronic eosinophilic leukemia, not otherwise specified and idiopathic hypereosinophilic syndrome largely relies on clonality assessment. Prior to the advent of next-generation sequencing, clonality was usually determined by cytogenetic analysis. We applied targeted next-generation sequencing panels designed for myeloid neoplasms to bone marrow specimens from a cohort of idiopathic hypereosinophilic syndrome patients (n=51), and assessed the significance of mutations in conjunction with clinicopathological features.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy.

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel.

Detection of BCR-ABL1 mutations that confer tyrosine kinase inhibitor resistance using massively parallel, next generation sequencing.

Detection of BCR-ABL1 mutations that confer resistance to tyrosine kinase inhibitors is important for management of patients with t(9;22);BCR-ABL1-positive (Ph+) leukemias. Testing is often performed using Sanger sequencing (SS) which has relatively poor sensitivity. Given the widespread adoption of next generation sequencing (NGS), we sought to reevaluate the testing in the context of NGS methods.

A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.

Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves.

T-cell clonality assessment by next-generation sequencing improves detection sensitivity in mycosis fungoides.

Background: T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE).

Objective: We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF.

Molecular genetic biomarkers in myeloid malignancies.

Context: Recent studies using massively parallel sequencing technologies, so-called next-generation sequencing, have uncovered numerous recurrent, single-gene variants or mutations across the spectrum of myeloid malignancies.

Objectives: To review the recent advances in the understanding of the molecular basis of myeloid neoplasms, including their significance for diagnostic and prognostic purposes and the possible implications for the development of novel therapeutic strategies.

Data Sources: Literature review.

BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib.