A diagnostic challenge of KIT p.V559D and BRAF p.G469A mutations in a paragastric mass.

A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings.

Acquired resistance to DZNep-mediated apoptosis is associated with copy number gains of AHCY in a B-cell lymphoma model.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application. METHODS: To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis. RESULTS: Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium. CONCLUSIONS: This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep.

Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

Comprehensive Metaboproteomics of Burkitt's and Diffuse Large B-Cell Lymphoma Cell Lines and Primary Tumor Tissues Reveals Distinct Differences in Pyruvate Content and Metabolism.

Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are pathologically and clinically distinct subtypes of aggressive non-Hodgkin B-cell lymphoma. To learn more about their biology, we employed metabolomic and proteomic methods to study both established cell lines as well as cryopreserved and formalin-fixed paraffin-embedded (FFPE) tissue sections of BL and DLBCL. Strikingly, NMR analyses revealed DLBCL cell lines to produce and secrete significantly (padj = 1.72 × 10-22) more pyruvic acid than BL cell lines. This finding could be reproduced by targeted GC/MS analyses of cryopreserved tissue sections of BL and DLBCL cases. Enrichment analysis of an overlapping set of N = 2315 proteins, that had been quantified by nanoLC-SWATH-MS in BL and DLBCL cultured cells and cryosections, supported the observed difference in pyruvic acid content, as glycolysis and pyruvate metabolism were downregulated, while one-carbon metabolism was upregulated in BL compared to DLBCL. Furthermore, 92.1% of the overlapping significant proteins showed the same direction of regulation in cryopreserved and FFPE material. Proteome data are available via ProteomeXchange with identifier PXD004936.

Renal Extramedullary Hematopoiesis in Mast Cell Leukemia with Bone Marrow Fibrosis.

Systemic mastocytosis is defined by the clonal proliferation of abnormal mast cells. The clinical course can range from indolent forms with normal life expectancy to advanced mast cell leukemia with dismal prognosis. An association with other diseases, including myeloproliferative neoplasia, has been described. We present a case of a 75-year patient with a history of cutaneous mastocytosis who was diagnosed with mast cell leukemia more than 9 years ago and did not receive treatment. The patient presented to our clinic with acute kidney failure because of renal extramedullary hematopoiesis. Bone marrow histopathology revealed extensive fibrosis and 50% infiltration by mast cells with a c-KIT D816V mutation. No mutations supporting primary myelofibrosis were identified. Treatment with midostaurin was started, and the patient was discharged after improvement of renal function. Here, we discuss diagnostic challenges between different forms of mast cell leukemia and overlaps with other hematological malignancies.

High Concordance of Different Assays in the Determination of Homologous Recombination Deficiency-Associated Genomic Instability in Ovarian Cancer.

PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.

Response prediction in patients with gastric and esophagogastric adenocarcinoma under neoadjuvant chemotherapy using targeted gene expression analysis and next-generation sequencing in pre-therapeutic biopsies.

OBJECTIVES: Perioperative chemo-(radio-) therapy is the accepted standard in European patients with locally advanced adenocarcinoma of the esophagogastric junction or stomach (AEG/AS). However, 30-85% of patients do not respond to this treatment. The aim of our study was the identification of predictive biomarkers in pre-therapeutic endoscopic tumor biopsies from patients with histopathologic response (Becker-1) versus non-response (Becker-2/3) to preoperative chemotherapy. METHODS: Formalin-fixed paraffin-embedded biopsies from 36 Caucasian patients (Becker-1 n = 11, Becker-2 n = 7, Becker-3 n = 18) with AEG/AS, taken prior to neoadjuvant chemotherapy were selected. For RNA expression analysis, we employed the NanoString nCounter System. To identify genomic alterations like single nucleotide variants (SNV), copy number variation (CNV) and fusion events, we used Illumina TST170 gene panel. For HER2 and FGFR2 protein expression, immunostaining was performed. Furthermore, we analyzed the microsatellite instability (MSI) and Epstein-Barr virus (EBV) infection status by EBER in situ hybridization. RESULTS: Heat map and principal component analyses showed no clustering by means of gene expression according to regression grade. Concerning two recently proposed predictive markers, our data showed equal distribution for MSI (Becker-1: 2; Becker-2: 1; Becker-3: 3; out of 29 tested) and EBV infection was rare (1/32). We could not reveal discriminating target genes concerning SNV, but found a higher mutational burden in non-responders versus responders and fusion (in 6/14) and CNV events (in 5/14) exclusively in Becker-3. CONCLUSIONS: Although we could not identify discriminating target genes, our data suggest that molecular alterations are in general more prevalent in patients with AEG/AS belonging to the non-responding Becker group 3.

Integrative genomic analysis focused on cell cycle genes for MYC-driven aggressive mature B-cell lymphoma.

MYC is a transcriptional factor that regulates growth and proliferation through cell cycle pathways. MYC alterations, in particular MYC rearrangements, are important in assessing the prognosis of aggressive B-cell lymphoma. In this study, we focused on the impact of nine major cell cycle genes for MYC-driven aggressive mature B-cell lymphoma and analyzed the mutational status using targeted next generation sequencing. Our 40 cases of aggressive mature B-cell lymphomas included 5 Burkitt lymphomas, 17 high-grade B-cell lymphomas and 18 diffuse large B-cell lymphomas with MYC breaks in 100%, 88% and 11%, respectively. Our data allowed a molecular classification into four categories partially independent from the histopathological diagnosis but correlating with the Ki-67 labelling index: (I) harboring TP53 and CDKN2A mutations, being highly proliferative, (II) with MYC rearrangement associated with MYC and/or ID3 mutations, being highly proliferative, (III) with MYC rearrangement combined with additional molecular changes, being highly proliferative, and (IV) with a diverse pattern of molecular alterations, being less proliferative. Taken together, we found that mutations of TP53, CDKN2A, MYC and ID3 are associated with highly proliferative B-cell lymphomas that could profit from novel therapeutic strategies.

DZNep-mediated apoptosis in B-cell lymphoma is independent of the lymphoma type, EZH2 mutation status and MYC, BCL2 or BCL6 translocations.

Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin-A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis.