Wang, Bangchen

Diffuse large B-cell lymphoma (DLBCL), the most common type of lymphoma, arises from various pathogenic mechanisms including gene translocations and fusions. Detection of gene rearrangements using fluorescence in situ hybridization (FISH) is a standard practice for DLBCL diagnosis and guides treatment decisions. High-throughput chromosome conformation capture (Hi-C) DNA sequencing is a next-generation sequencing-based technology to identify genome-wide rearrangements using a single assay. In this study, Hi-C sequencing was performed using FFPE tissues from 159 patients with DLBCL, and identified 746 cancer genes at or proximal to the rearrangement breakpoints with a total of 1903 occurrences. Focusing on clinically important rearrangements, Hi-C detected 102 rearrangements of MYC, BCL2, and/or BCL6 in 92 patients and revealed the fusion partners, including 25 rearrangements missed by FISH. Causes of FISH negative results included FISH-cryptic breakpoints, complex or faint FISH signals, low percentage of FISH positivity, and issues related to tissue fixation. Moreover, in 20 patients (12.6%), Hi-C sequencing detected 22 rearrangements characteristic of other lymphoma types, including CCND1 rearrangements that could lead to reclassification as mantle cell lymphoma. Survival analysis for genome-wide rearrangements using machine learning models identified MYC, PD-L1, CCND1, BCL2, NTRK1/PRCC, RRAS, and FANCE rearrangements with significant prognostic effects in the DLBCL cohort. In conclusion, Hi-C sequencing detects gene rearrangements crucial for diagnosis in an unbiased and molecular manner and showed high sensitivity and specificity in our study. These advantages of Hi-C sequencing offer help to improve the workflow of clinical pathology laboratories, diagnostic precision, and treatment of large B-cell lymphoma.

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), accounting for approximately 30% of all NHL cases. DLBCL diagnosis relies on histologic examination, immunophenotyping, molecular and fluorescence in situ hybridization (FISH) analysis to differentiate it from similar NHL subtypes with overlapping morphological features, including more high-grade lymphomas like double-hit lymphoma. Because FISH is performed in a panel of single- or dual-target test format and analyzed through microscopy and manual image analysis, assessment of multiple target biomarkers can be cumbersome and expensive. As additional biomarkers for diagnosis, subtyping, prognosis, and therapeutic decision-making become available, target panels will become larger, posing additional challenges to clinical laboratories. Patients and Methods In this study, Hi-C sequencing of FFPE samples was performed to identify all genomic rearrangements by a single sequencing analysis for an unselected DLBCL case series of 159 patients, and the results were compared to FISH for the detection of clinically important variants. Results Hi-C sequencing showed superior performance compared to FISH: standard FISH for DLBCL includes testing for MYC, BCL2, and BCL6. Between FISH and Hi-C, these biomarkers were detected 105 times, of which, FISH detected 80/105 (76%) and Hi-C detected 102/105 (97%). The cohort included 4 double-hit lymphomas by FISH but Hi-C sequencing detected five. Besides DLBCL diagnostic biomarkers (MYC, BCL2, BCL6), many additional biomarkers were detected by Hi-C sequencing including variants not tested by FISH because the clinical suspicion was ambiguous and the relevant FISH probes were not used, and those not tested because the biomarkers were not thought to be common and, hence, left out of the FISH panel. In total, Hi-C sequencing detected additional biomarkers in 42 (26%) cases, including (1) variants targeted by FISH (i.e. MYC, BCL2, BCL6) but negative, (2) classification biomarkers for other lymphomas (CCND1, MALT, IRF4), and (3) those not tested because the biomarkers are not common and not included in the FISH panel. These additional biomarkers were supportive of suspected diagnosis of DLBCL versus double-hit lymphoma (24 cases), prompted reconsideration of a different diagnosis that may have led to different treatments (6 cases, e.g. mantle cell lymphoma), or biomarkers such as PDL1, ALK, and ATM which have implications for targeted therapies and/or inclusion in clinical trials (5 cases). Conclusion The Hi-C FFPE sequencing technology has the potential to improve the workflow of pathology labs for detection of a wide range of structural variants informing precision diagnosis and treatment for lymphomas.

Abstract Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare and aggressive lymphoma with molecular heterogeneity not well characterize. In this study, we performed next-generation sequencing analysis for a large number of DNA and RNA samples from patients with PT-DLBCL. DNA sequencing analysis identified ≥ 3 chromosomes with copy number variations (CNVs) and microsatellite instability as prognostic biomarkers, rather than MYD88 mutations and genetic subtypes. Remarkably, targeted RNA-seq analysis in 195 patients revealed that TP53 mutations with a ≥ 40% variant allele frequency had significantly adverse prognostic impact, and that a 150-gene expression signature subdivided PT-DLBCL into two distinct clusters, termed as testicular lymphoma tumor (TLT) and microenvironment (ME) subtypes. The TLT subtype featured upregulation of genes involved in B-cell receptor signaling, cell cycle, DNA damage and repair, higher frequencies of CNVs and MYD88 mutations, elder ages, larger tumor sizes, and significantly poorer survival. Genomic microRNA profiling analysis identified significantly differentially expressed microRNAs between 113 PT-DLBCL and 180 systemic DLBCL patients, and further subdivided the PT-DLBCL cohort by microRNA signatures. The subcohort with upregulation of 16 microRNAs associated with PT-DLBCL and testicular tissue expression had significantly better survival. This study revealed characteristic genetic, gene expression, and microRNA profiles and heterogeneity in PT-DLBCL.

Introduction: Primary diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS), traditionally referred to as primary CNS lymphoma (PCNSL), is a rare type of primary large B-cell lymphoma of immune-privileged sites with significantly poorer prognosis than systemic DLBCL. Genetic profiles of PCNSL were widely studied, whereas the tumor microenvironment has not been well characterized. Patients and Methods: A series of molecular analyses were performed using DLBCL FFPE tissues collected by the International Lymphoma Consortium. First, multiplex fluorescent immunohistochemistry (mfIHC) using antibodies for CD20, PAX5, CD3, CD4, CD8, FOXP3, CD45RO, CD68, CD56, PD-1, PD-L1, PD-L2, and CTLA-4 was performed on PCNSL tissues from 121 immunocompetent patients and de novo systemic DLBCL tissues from 405 patients. Digital quantification results for 405 patients with EBV-negative systemic DLBCL or primary testicular (PT) DLBCL have been reported previously. The preliminary mfIHC results of PCNSL will be presented. Second, mfIHC for multiple B cell biomarkers, CD3, CD4, CD8, CD68, CD163, CD11c, and CD31 were performed for 10 PCNSL validation cases, 24 PT-DLBCL and 451 systemic DLBCL tissues (unpublished). Third, RNA samples were extracted and sequenced for the targeted 1408 cancer-associated genes, successfully in 142 PCNSL cases, including 102 cases evaluated by either of these two mfIHC panels and additional 40 PCNSL cases, Results: First, in the first large mfIHC cohort, PCNSLs compared with de novo systemic nodal or extranodal DLBCL had significantly higher mean/median CD56+ cell densities whereas lower T cell densities (consistent with the immune privilege), as well as lower CD45RO+, PD-1+, and FOXP3+ percentages in T cells whereas higher CD4¯CD8¯ double-negative percentages in T cells. Prognostic analysis in the PCNSL cohort showed that high CD56+ cell densities and high CD45RO+CD8+ T cell densities were associated with significantly poorer and better patient survival, respectively, which are opposite to their prognostic effects in systemic DLBCL as previously reported. Twelve patients had very high PD-L1 expression in B cells and significantly poorer survival in the PCNSL cohort. Second, as validation, the 10 PCNSL cases in the second mfIHC study showed significantly less T cells than systemic DLBCL and PT-DLBCL cases, and low CD8 T cell densities and having low percentage of CD8 T cells adjacent to tumor cells were associated with significantly poorer survival. Immunofluorescence landscapeand spatial analysisrevealed significant differences in immune privilege between PCNSL and PT-DLBCL. Third, gene expression profiling analysis by targeted RNA-seq identified 282 significantly differentially expressed genes between PCNSL and systemic DLBCL, referred to as the PCNSL signature. Unsupervised clustering analysis divided the PCNSL cohort into two clusters, which had no significant difference in MYD88 mutation frequencies analyzed at the mRNA level. Importantly, the cluster with higher expression of many neural glial genes in the PCNSL signature had significantly poorer survival, which remained significant in multivariate analysis adjusting for clinical factors and/or treatments, two PCNSL subcohorts (as training and validation cohorts), and methotrexate-treated patients. NCAM1 (Neural Cell Adhesion Molecule 1, encoding CD56) was among the upregulated PCNSL signature genes, explaining the poorer prognosis associated with higher CD56+ cell densities in PCNSL by the mfIHC analysis. Summary: Our studies represent the largest immunophenotypic and gene expression profiling studies currently in PCNSL, a rare type of primary DLBCL of immune-privileged sites. Multiplex immunofluorescence revealed the immune landscape in PCNSL in comparison with systemic and testicular DLBCL, and identified prognostic immune biomarkers. Targeted RNA-seq identified a PCNSL gene signature enriched with neuronal-glial genes and showing significant prognostic effect in PCNSL. These novel findings in the PCNSL microenvironment warrant further studies.

Abstract Prostate cancer remains the second leading cause of cancer death among American men. Radiotherapy is a potentially curative treatment for localized prostate cancer, and failure to control localized disease contributes to the majority of prostate cancer deaths. Neuroendocrine differentiation (NED) in prostate cancer, a process by which prostate adenocarcinoma cells transdifferentiate into neuroendocrine-like (NE-like) cells, is an emerging mechanism of resistance to cancer therapies and contributes to disease progression. NED also occurs in response to treatment to promote the development of treatment-induced neuroendocrine prostate cancer (NEPC), a highly aggressive and terminal stage disease. We previously demonstrated that by mimicking clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces prostate cancer cells to undergo NED in vitro and in vivo. Here, we performed transcriptomic analysis and confirmed that FIR-induced NE-like cells share some features of clinical NEPC, suggesting that FIR-induced NED represents a clinically relevant model. Furthermore, we demonstrated that protein arginine methyltransferase 5 (PRMT5), a master epigenetic regulator of the DNA damage response and a putative oncogene in prostate cancer, along with its cofactors pICln and MEP50, mediate FIR-induced NED. Knockdown of PRMT5, pICln, or MEP50 during FIR-induced NED and sensitized prostate cancer cells to radiation. Significantly, PRMT5 knockdown in prostate cancer xenograft tumors in mice during FIR prevented NED, enhanced tumor killing, significantly reduced and delayed tumor recurrence, and prolonged overall survival. Collectively, our results demonstrate that PRMT5 promotes FIR-induced NED and suggests that targeting PRMT5 may be a novel and effective radiosensitization approach for prostate cancer radiotherapy.