Expression of TCF3 target genes defines a subclass of diffuse large B-cell lymphoma characterized by up-regulation of MYC target genes and poor clinical outcome following R-CHOP therapy.

TCF3 is a lymphopoietic transcription factor that acquires somatic driver mutations in diffuse large B-cell lymphoma (DLBCL). Hypothesizing that expression patterns of TCF3-regulated genes can inform clinical management, we found that unsupervised clustering analysis with 15 TCF3-regulated genes and eight additional ones resolved local DLBCL cases into two main clusters, denoted Groups A and B, of which Group A manifested inferior overall survival (OS, p = 0.0005). We trained a machine learning model to classify samples into the Groups based on expression of the 23 transcripts in an independent validation cohort of 569 R-CHOP-treated DLBCL cases. Group A overlapped with the ABC cell-of-origin subgroup but its prognostic power was superior. GSEA analysis demonstrated asymmetric expression of 30 gene sets between the Groups, pointing to biological differences. We present, validate and make available a novel method to assign DLBCL cases into biologically-distinct groups with divergent OS following R-CHOP therapy.

Evidence-based review of genomic aberrations in diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS): Report from the cancer genomics consortium lymphoma working group.

Diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS) is the most common type of non-Hodgkin lymphoma (NHL). The 2016 World Health Organization (WHO) classification defined DLBCL, NOS and its subtypes based on clinical findings, morphology, immunophenotype, and genetics. However, even within the WHO subtypes, it is clear that additional clinical and genetic heterogeneity exists. Significant efforts have been focused on utilizing advanced genomic technologies to further subclassify DLBCL, NOS into clinically relevant subtypes. These efforts have led to the implementation of novel algorithms to support optimal risk-oriented therapy and improvement in the overall survival of DLBCL patients. We gathered an international group of experts to review the current literature on DLBCL, NOS, with respect to genomic aberrations and the role they may play in the diagnosis, prognosis and therapeutic decisions. We comprehensively surveyed clinical laboratory directors/professionals about their genetic testing practices for DLBCL, NOS. The survey results indicated that a variety of diagnostic approaches were being utilized and that there was an overwhelming interest in further standardization of routine genetic testing along with the incorporation of new genetic testing modalities to help guide a precision medicine approach. Additionally, we present a comprehensive literature summary on the most clinically relevant genomic aberrations in DLBCL, NOS. Based upon the survey results and literature review, we propose a standardized, tiered testing approach which will help laboratories optimize genomic testing in order to provide the maximum information to guide patient care.

Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Objective quantification of BCL2 protein by multiplex immunofluorescence in routine biopsy samples of diffuse large B-cell lymphoma demonstrates associations with survival and BCL2 gene alterations.

Up-regulation of BCL2 in cases of diffuse large B-cell lymphoma (DLBCL) can confer treatment resistance. Quantitative immunofluorescence (QIF) histology allows objective quantification of protein-based biomarkers. We investigated the utility of QIF for evaluating BCL2 as a biomarker in DLBCL by quantifying BCL2 selectively in CD20-expressing lymphoma cells in biopsy samples from 116 cases of DLBCL in two cohorts one of which consisted of relapsed/refractory cases from a clinical trial. BCL2 protein by QIF correlated with BCL2 mRNA abundance and was associated with both translocation and copy number gain of the BCL2 gene. Elevated BCL2 protein expression by QIF, but not immunohistochemistry or mRNA quantification, was associated with inferior overall and relapse-free survival in the relapsed/refractory cohort. QIF is an effective means of quantifying BCL2 protein objectively in routine cancer biopsy specimens and shows promise for identifying relapsed/refractory DLBCL patients at risk of inferior outcomes after salvage therapy.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

High-grade B-cell lymphoma with MYC and BCL2 rearrangements arising in a composite lymphoma.

BACKGROUND: We report the first case of composite lymphoma consisting of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL) and high-grade B-cell lymphoma with MYC and BCL2 rearrangements within the same needle biopsy in which a clonal relationship between the FL and high-grade B-cell lymphoma components was demonstrated by molecular cytogenetics. CASE PRESENTATION: An 85-year-old man presented with masses in his neck and right groin. Cutting needle biopsy of the inguinal mass revealed the three lymphoma types which were morphologically, immunophenotypically and topographically distinct. Fluorescence in situ hybridization (FISH) identified an IGH-BCL2 rearrangement in both the FL and high-grade B-cell components while a MYC rearrangement was detected in the high-grade B-cell component alone. CONCLUSIONS: Our findings suggest that the high-grade lymphoma with MYC and BCL2 translocations evolved through transformation of the FL by a process that entailed acquisition of the MYC translocation. No clonal relationship between the FL and CLL/SLL components was evident since the IGH-BCL2 rearrangement was present in in the former but not the latter. This unique case of co-localized FL, CLL/SLL, and high-grade B-cell lymphoma contributes to our understanding of the clonal relationships that may exist between the components of composite lymphomas.

The CDK inhibitor AT7519M in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. A Phase II study of the Canadian Cancer Trials Group.

AT7519M is a small molecule inhibitor of cyclin-dependent kinases 1, 2, 4, 5, and 9 with in vitro activity against lymphoid malignancies. In two concurrent Phase II trials, we evaluated AT7519M in relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) using the recommended Phase II dosing of 27 mg/m2 twice weekly for 2 of every 3 weeks. Primary objective was objective response rate (ORR). Nineteen patients were accrued (7 CLL, 12 MCL). Four CLL patients achieved stable disease (SD). Two MCL patients achieved partial response (PR), and 6 had SD. One additional MCL patient with SD subsequently achieved PR 9 months after completion of AT7519M. Tumor lysis syndrome was not reported. In conclusion, AT7519M was safely administered to patients with relapsed/refractory CLL and MCL. In CLL, some patients had tumor reductions, but the ORR was low. In MCL, activity was noted with ORR of 27%.

HLA-DR(negative), CD34(negative) hypergranular acute myeloid leukemia with trisomy 6 and del(5)(q22q33): case report and review of the literature.

We report a unique pediatric case of hypergranular acute myeloid leukemia with myelodysplasia-related changes. The patient presented with moderate leukocytosis with neutrophilia with left-shift maturation and dysplasia, anemia, and multiple sclerotic bone lesions. The bone marrow was hypercellular with a predominance of myeloblast cells and/or abnormal promyelocytes with hypergranular cytoplasm. Flow cytometric immunophenotyping showed that the leukemic cells were positive for CD13, CD33, and myeloperoxidase, and negative for HLA-DR and CD34. Morphology and immunophenotyping were highly suggestive of acute promyelocytic leukemia. The classic t(15;17) or other RARα rearrangements were not detected by cytogenetic or molecular assays, ruling out acute promyelocytic leukemia. Standard cytogenetic analysis showed that the karyotype of the predominant clone was 47,XY,+6 with evidence of clonal evolution to 47,XY,+6,del(5)(q22q33). A literature and database review showed that trisomy 6 is a rare occurrence in hematological malignancies and, to our knowledge, has never been reported in association with del(5)(q22q33) in a child presenting with hypergranular acute myeloid leukemia with myelodysplasia-related changes. We present a current review of the literature and summarize the clinical features of 57 cases of trisomy 6 as the primary chromosomal abnormality in hematological disease.

Huntingtin phosphorylation on serine 421 is significantly reduced in the striatum and by polyglutamine expansion in vivo.

Huntington disease (HD) results from polyglutamine expansion in the huntingtin protein (htt). Despite the widespread tissue expression pattern of htt, neuronal loss is highly selective to medium spiny neurons of the striatum. Huntingtin is phosphorylated on serine-421 (S421) by the pro-survival signaling protein kinase Akt (PKB) and this has been previously shown to be protective against the toxicity of polyglutamine-expanded htt in cell culture. Using an antibody specific for htt phosphorylated on S421, we now demonstrate that htt phosphorylation is present at significant levels under normal physiological conditions in human and mouse brain. Furthermore, htt phosphorylation shows a regional distribution with the highest levels in the cerebellum, less in the cortex, and least in the striatum. In cell cultures and in YAC transgenic mice, the endogenous phosphorylation of polyglutamine-expanded htt is significantly reduced relative to wild-type htt. The presence and pattern of significant htt phosphorylation in the brain indicates that this dynamic post-translational modification is important for the regulation of htt and may contribute to the selective neurodegeneration seen in HD.