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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.

Despite considerable treatment advances, multiple myeloma (MM) remains an incurable hematological cancer due to treatment resistance. A systematic literature search was conducted to identify determinants for clonal evolution driving relapse and drug resistance in MM. A total of 631 non-duplicate publications were screened of which 28 articles were included for data extraction. Genetic alterations, mutational signatures, evolutionary trajectories, and non-genetic determinants were identified as key topics to characterize clonal evolution in relapsed MM. A variety of factors led to clonal diversification and increased tumor mutation burden, such as MAPK-Ras mutations and incremental changes related to chromosomal bands 1 and 17, while mutational signature analyses revealed that APOBEC activity and melphalan treatment leave a distinct impact on the clonal composition in MM genomes. To capture and dissect tumor heterogeneity, our review suggests combining methods or using technical approaches with high resolution to assess the impact of clonal evolution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells due to constitutive B-cell receptor (BCR) signaling, leading to apoptosis resistance and increased proliferation. This study evaluates the effects of the Bruton Tyrosine Kinase (BTK) inhibitor ibrutinib on the molecular composition, clonality, and kinetics of B cells during treatment in CLL patients. Employing a multi-omics approach of up to 3.2 years of follow-up, we analyzed data from 24 CLL patients, specifically focusing on nine patients treated with ibrutinib monotherapy. In this study, clonal stability was observed within the ibrutinib-treated group following an effective initial clinical response, where clonotype frequencies of residual CLL cells remained high and stable, ranging from 74.9% at 1.5 years to 87.7% at approximately 3 years. In contrast, patients treated with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax exhibited substantial reductions in clonal frequencies, approaching molecular eradication. Deep whole-exome sequencing revealed minimal genomic progression in the ibrutinib group, maintaining somatic drivers and variant allele frequencies (VAF) above 0.2 throughout treatment. At the single-cell level, the NF-κB pathway inhibition and apoptotic signals were detected or even augmented during treatment in ibrutinib-treated patients. These findings may corroborate the role of ibrutinib in stabilizing the genomic landscape of CLL cells, preventing significant genomic evolution despite maintaining a high clonal burden within the residual B-cell compartment.

Multiple myeloma (MM) is a haematological malignancy with abnormal proliferation of plasma cells in the bone marrow (BM), and MM patients with highly proliferative plasma cells have reduced overall survival. Circular RNAs (circRNAs) are endogenous, non-coding molecules that are promising biomarkers in cancer. Here, we present the largest study of circRNAs in MM to date and explore the prognostic potential of circRNAs and the link between proliferation and circRNA expression in MM. We performed deep total RNA sequencing (RNA-seq) on two cohorts: one cohort consisting of 45 whole BM MM patient samples and 13 healthy controls (HCs), and another cohort consisting of 43 CD138-purified plasma cell MM patient samples. We found that circRNAs are globally upregulated in the whole BM of MM patients compared to HCs. In whole BM, low proliferation and high circRNA levels were associated with a poor prognosis, while in purified plasma cells, low proliferation and high circRNA levels were associated with a favourable prognosis. Individual circRNAs from purified plasma cells were found to be significantly associated with MM patient outcomes and provide additional prognostic value to the proliferative indexes. Together, our findings emphasise the potential of circRNAs as prognostic biomarkers in MM., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Deficient (d) DNA mismatch repair (MMR) is a biomarker predictive of better response to PD-1 blockade immunotherapy in solid tumors. dMMR can be caused by mutations in MMR genes or by protein inactivation, which can be detected by sequencing and immunohistochemistry, respectively. To investigate the role of dMMR in diffuse large B-cell lymphoma (DLBCL), MMR gene mutations and expression of MSH6, MSH2, MLH1, and PMS2 proteins were evaluated by targeted next-generation sequencing and immunohistochemistry in a large cohort of DLBCL patients treated with standard chemoimmunotherapy, and correlated with the tumor immune microenvironment characteristics quantified by fluorescent multiplex immunohistochemistry and gene-expression profiling. The results showed that genetic dMMR was infrequent in DLBCL and was significantly associated with increased cancer gene mutations and favorable immune microenvironment, but not prognostic impact. Phenotypic dMMR was also infrequent, and MMR proteins were commonly expressed in DLBCL. However, intratumor heterogeneity existed, and increased DLBCL cells with phenotypic dMMR correlated with significantly increased T cells and PD-1 + T cells, higher average nearest neighbor distance between T cells and PAX5 + cells, upregulated immune gene signatures, LE4 and LE7 ecotypes and their underlying Ecotyper-defined cell states, suggesting the possibility that increased T cells targeted only tumor cell subsets with dMMR. Only in patients with MYC¯ DLBCL, high MSH6/PMS2 expression showed significant adverse prognostic effects. This study shows the immunologic and prognostic effects of genetic/phenotypic dMMR in DLBCL, and raises a question on whether DLBCL-infiltrating PD-1 + T cells target only tumor subclones, relevant for the efficacy of PD-1 blockade immunotherapy in DLBCL., Competing Interests: D.O., Q.A., and H.N. are employees of NeoGenomics Laboratories, Inc. The NeoGenomics Laboratories, Inc. had no role in the design of the study, in the writing of the manuscript, or in the decision to publish the results., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3-5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27-54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6 , in addition to BTK , are essential for the response to platinum-based drugs in DLBCL.

Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance., Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution., Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell-like/activated B-cell-like classification and TIL-T frequency. The identified TIL-B-high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts., Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications., (©2023 American Association for Cancer Research.)

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is an extremely rare B-cell lymphoma. The disease is typically indolent and treatment with splenectomy usually results in durable remissions. Here, we present a case of an extremely aggressive course of SDRPL with transformation to diffuse large B-cell lymphoma and multiple relapses immediately following cessation of immunochemotherapy. We provide results from whole-exome sequencing from debut of SDRPL and from following transformed stages and identified a novel somatic mutation in RB1 as the possible driver of this aggressive disease, which has not been reported earlier in SDRPL., Competing Interests: All authors declare no conflict of interest., (Copyright 2023, Christensen et al.)

Background and Aims: The treatment of locally advanced rectal cancer often consists of neoadjuvant chemoradiotherapy followed by surgery. However, approximately 15% of patients show no response to this neoadjuvant chemoradiotherapy. This systematic review aimed to identify biomarkers of innate radioresistant rectal cancer., Method: Through a systematic literature search, 125 papers were included and analyzed using ROBINS-I, a Cochrane risk of bias tool for non-randomized studies of interventions. Both statistically significant and nonsignificant biomarkers were identified. Biomarkers mentioned more than once in the results or biomarkers with a low or moderate risk of bias were included as the final results., Results: Thirteen unique biomarkers, three genetic signatures, one specific pathway, and two combinations of two or four biomarkers were identified. In particular, the connection between HMGCS2, COASY, and PI3K-pathway seems promising. Future scientific research should focus on further validating these genetic resistance markers., Competing Interests: Conflict of Interest We declare that there is no financial or personal relationships that could inappropriately influence our work., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: Our goal was to describe a precision medicine program in a regional academic hospital, characterize features of included patients and present early data on clinical impact., Materials and Methods: We prospectively included 163 eligible patients with late-stage cancer of any diagnosis from June 2020 to May 2022 in the Proseq Cancer trial. Molecular profiling of new or fresh frozen tumor biopsies was done by WES and RNAseq with parallel sequencing of non-tumoral DNA as individual reference. Cases were presented at a National Molecular Tumor Board (NMTB) for discussion of targeted treatment. Subsequently, patients were followed for at least 7 months., Results: 80% ( N  = 131) of patients had a successful analysis done, disclosing at least one pathogenic or likely pathogenic variant in 96%. A strongly or potentially druggable variant was found in 19% and 73% of patients, respectively. A germline variant was identified in 2.5%. Median time from trial inclusion to NMTB decision was one month. One third ( N  = 44) of patients who underwent molecularly profiling were matched with a targeted treatment, however, only 16% were either treated ( N  = 16) or are waiting for treatment ( N  = 5), deteriorating performance status being the primary cause of failure. A history of cancer among 1st degree relatives, and a diagnosis of lung or prostate cancer correlated with greater chance of targeted treatment being available. The response rate of targeted treatments was 40%, the clinical benefit rate 53%, and the median time on treatment was 3.8 months. 23% of patients presented at NMTB were recommended clinical trial participation, not dependent on biomarkers., Conclusions: Precision medicine in end-stage cancer patients is feasible in a regional academic hospital but should continue within the frame of clinical protocols as few patients benefit. Close collaboration with comprehensive cancer centers ensures expert evaluations and equality in access to early clinical trials and modern treatment.

Patients diagnosed with epithelial ovarian cancer (OC) have a 5-year survival rate of 49%. For early-stage disease, the 5-year survival rate is above 90%. However, advanced-stage disease accounts for most cases as patients with early stages often are asymptomatic or present with unspecific symptoms, highlighting the need for diagnostic tools for early diagnosis. Liquid biopsy is a minimal invasive blood-based approach that utilizes circulating tumor DNA (ctDNA) shed from tumor cells for real-time detection of tumor genetics and epigenetics. Increased DNA methylation of promoter regions is an early event during tumorigenesis, and the methylation can be detected in ctDNA, accentuating the promise of methylated ctDNA as a biomarker for OC diagnosis. Many studies have investigated multiple methylation biomarkers in ctDNA from plasma or serum for discriminating OC patients from patients with benign diseases of the ovaries and/or healthy females. This systematic review summarizes and evaluates the performance of the currently investigated DNA methylation biomarkers in blood-derived ctDNA for early diagnosis of OC. PubMed's MEDLINE and Elsevier's Embase were systematically searched, and essential results such as methylation frequency of OC cases and controls, performance measures, as well as preanalytical factors were extracted. Overall, 29 studies met the inclusion criteria for this systematic review. The most common method used for methylation analysis was methylation-specific PCR, with half of the studies using plasma and the other half using serum. RASSF1A, BRCA1, and OPCML were the most investigated gene-specific methylation biomarkers, with OPCML having the best performance measures. Generally, methylation panels performed better than single gene-specific methylation biomarkers, with one methylation panel of 103,456 distinct regions and 1,116,720 CpGs having better performance in both training and validation cohorts. However, the evidence is still limited, and the promising methylation panels, as well as gene-specific methylation biomarkers highlighted in this review, need validation in large, prospective cohorts with early-stage asymptomatic OC patients to assess the true diagnostic value in a clinical setting., (© 2023. The Author(s).)

Purpose: Platinum-containing therapy is standard treatment for relapsed Diffuse Large B-Cell Lymphoma (DLBCL). However, the efficacy of treatment is limited by drug resistance leading to relapse. Cisplatin resistance has been linked to impairments of the DNA damage response, and several DNA repair proteins have been identified as clients of the molecular chaperone Hsp90. Here, we investigated the combinatory treatment of cisplatin and the Hsp90 inhibitor, 17AAG, in DLBCL cells to evaluate if inhibition of Hsp90 could sensitize DLBCL cells to cisplatin treatment., Methods: Cell viability was assessed for cisplatin and 17AAG as monotherapies and for 25 different combinations in 7 DLBCL cell lines, where the Bliss Independence Model and the Combination Index were applied to assess their interaction. Induction of apoptosis and DNA damage response were evaluated by measuring Annexin V and γH2AX levels after 48 h of exposure., Results: 17AAG synergized with cisplatin in DLBCL cells as detected in both interaction assessment models, resulting in a lower viability after 48 h for the combination-treated cells compared to both vehicle and single drug-treated cells. The combination also induced a stronger apoptotic response and an increase in DNA damage in 17AAG, cisplatin- and combination-treated cells compared to vehicle-treated cells, with the effect of the combination generally being higher than compared to both single drugs., Conclusion: This study demonstrates that 17AAG sensitizes DLBCL cells to cisplatin treatment. This effect is correlated with increased apoptotic and DNA damage response, potentially mediated by downregulation of Hsp90 clients in DNA repair pathways. Thus, cisplatin resistance could plausibly be overcome by combining the treatment with an Hsp90 inhibiting drug., (© 2022. The Author(s).)

Purpose: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers., Experimental Design: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of DLBCL. We performed correlative and prognostic analyses within and across DLBCL genetic subtypes and MYC/BCL2 double expressors., Results: We found that MYC/BCL2 double-high-expression (DhE) had significant adverse prognostic impact within the EZB genetic subtype and LymphGen-unclassified DLBCL cases but not within MCD and ST2 genetic subtypes. Conversely, KMT2D mutations significantly stratified DhE but not non-DhE DLBCL. T-cell infiltration showed favorable prognostic effects within BN2, MCD, and DhE but unfavorable effects within ST2 and LymphGen-unclassified cases. FN1 and PD-1-high expression had significant adverse prognostic effects within multiple DLBCL genetic/phenotypic subgroups. The prognostic effects of DhE and immune biomarkers within DLBCL genetic subtypes were independent although DhE and high Ki-67 were significantly associated with lower T-cell infiltration in LymphGen-unclassified cases., Conclusions: Together, these results demonstrated independent and additive prognostic effects of phenotypic MYC/BCL2 and microenvironment biomarkers and genetic subtyping in DLBCL prognostication, important for improving DLBCL classification and identifying prognostic determinants and therapeutic targets., (©2022 American Association for Cancer Research.)

Background: Multiple myeloma remains an incurable disease with multiple relapses due to residual myeloma cells in the bone marrow of patients after therapy. Presence of small number of cancer cells in the body after cancer treatment, called minimal residual disease, has been shown to be prognostic for progression-free and overall survival. However, for multiple myeloma, it is unclear whether patients attaining minimal residual disease negativity may be candidates for treatment discontinuation. We investigated, if longitudinal flow cytometry-based monitoring of minimal residual disease (flow-MRD) may predict disease progression earlier and with higher sensitivity compared to biochemical assessments., Methods: Patients from the Nordic countries with newly diagnosed multiple myeloma enrolled in the European-Myeloma-Network-02/Hovon-95 (EMN02/HO95) trial and undergoing bone marrow aspiration confirmation of complete response, were eligible for this Nordic Myeloma Study Group (NMSG) substudy. Longitdudinal flow-MRD assessment of bone marrow samples was performed to identify and enumerate residual malignant plasma cells until observed clinical progression., Results: Minimal residual disease dynamics were compared to biochemically assessed changes in serum free light chain and M-component. Among 20 patients, reaching complete response or stringent complete response during the observation period, and with ≥3 sequential flow-MRD assessments analysed over time, increasing levels of minimal residual disease in the bone marrow were observed in six cases, preceding biochemically assessed disease and clinical progression by 5.5 months and 12.6 months (mean values), respectively. Mean malignant plasma cells doubling time for the six patients was 1.8 months (95% CI, 1.4-2.3 months). Minimal malignant plasma cells detection limit was 4 × 10-5., Conclusions: Flow-MRD is a sensitive method for longitudinal monitoring of minimal residual disease dynamics in multiple myeloma patients in complete response. Increasing minimal residual disease levels precedes biochemically assessed changes and is an early indicator of subsequent clinical progression., Trial Registration: NCT01208766., (© 2022. The Author(s).)

Multiple studies have demonstrated that diffuse large B-cell lymphoma (DLBCL) can be divided into subgroups based on their biology; however, these biological subgroups overlap clinically. Using machine learning, we developed an approach to stratify patients with DLBCL into four subgroups based on survival characteristics. This approach uses data from the targeted transcriptome to predict these survival subgroups. Using the expression levels of 180 genes, our model reliably predicted the four survival subgroups and was validated using independent groups of patients. Multivariate analysis showed that this patient stratification strategy encompasses various biological characteristics of DLBCL, and only TP53 mutations remained an independent prognostic biomarker. This novel approach for stratifying patients with DLBCL, based on the clinical outcome of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone therapy, can be used to identify patients who may not respond well to these types of therapy, but would otherwise benefit from alternative therapy and clinical trials., (© 2022. The Author(s).)

Aim: To estimate current real-world costs of drugs and supportive care for the treatment of multiple myeloma in a tax-based health system. Methods: Forty-one patients were included from a personalized medicine study (2016-2019). Detailed information was collected from patient journals and hospital registries to estimate the total and mean costs using inverse probability weighting of censored data. Results: Total observed (censored) costs for the 41 patients was €8.84 million during 125 treatment years, with antineoplastic drugs as the main cost driver (€5.6 million). Individual costs showed large variations. Mean 3-year cost per patient from first progression was €182,103 (€131,800-232,405). Conclusion: Prediction of real-world costs is hindered by the availability of detailed costing data. Micro-costing analyses are needed for budgeting and real-world evaluation of cost-effectiveness.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma with high mutation burdens but a low response rate to immune checkpoint inhibitors. In this study, we performed targeted next-generation sequencing and fluorescent multiplex immunohistochemistry, and investigated the clinical significance and immunological effect of mutation numbers in 424 DLBCL patients treated with standard immunochemotherapy. We found that KMT2D and TP53 nonsynonymous mutations (MUT) were significantly associated with increased nonsynonymous mutation numbers, and that high mutation numbers (MUT high ) were associated with significantly poorer clinical outcome in germinal center B-cell-like DLBCL with wild-type TP53 . To understand the underlying mechanisms, we identified a gene-expression profiling signature and the association of MUT high with decreased T cells in DLBCL patients with wild-type TP53 . On the other hand, in overall cohort, MUT high was associated with lower PD-1 expression in T cells and PD-L1 expression in macrophages, suggesting a positive role of MUT high in immune responses. Analysis in a whole-exome sequencing dataset of 304 patients deposited by Chapuy et al. validated the correlation of MUT- KMT2D with genomic complexity and the significantly poorer survival associated with higher numbers of genomic single nucleotide variants in activated B-cell-like DLBCL with wild-type TP53 . Together, these results suggest that KMT2D inactivation or epigenetic dysregulation has a role in driving DLBCL genomic instability, and that genomic complexity has adverse impact on clinical outcome in DLBCL patients with wild-type TP53 treated with standard immunochemotherapy. The oncoimmune data in this study have important implications for biomarker and therapeutic studies in DLBCL., Competing Interests: HN, MLN, and QA are employees of NeoGenomics Laboratories, Inc.. MA is an employee of Genomic Testing Corporative, LCA. Other authors declare no conflicts of interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid neoplasm among adults,and approximately 30-40% of patients will experience relapse while 5-10% will suffer from primary refractory disease caused by different mechanisms, including treatment-induced resistance. For refractory and relapsed DLBCL (rrDLBCL) patients, early detection and understanding of the mechanisms controlling treatment resistance are of great importance to guide therapy decisions. Here, we have focused on genetic variations in immune surveillance genes in diagnostic DLBCL (dDLBCL) and rrDLBCL patients to elaborate on the suitability of new promising immunotherapies., Methods: Biopsies from 30 dDLBCL patients who did not progress or relapse during follow up and 17 rrDLBCL patients with refractory disease or who relapsed during follow up were analyzed by whole-exome sequencing, including matched individual germline samples to include only somatic genetic variants in downstream analysis of a curated list of 58 genes involved in major immune surveillance pathways., Results: More than 70% of both dDLBCLs and rrDLBCLs harbored alterations in immune surveillance genes, but rrDLBCL tumor samples have a lower number of genes affected compared to dDLBCL tumor samples. Increased gene mutation frequencies in rrDLBCLs were observed in more than half of the affected immune surveillance genes than dDLBCLs., Conclusion: Genetic variants in the antigen-presenting genes affect a higher number of rrDLBCL patients supporting an important role for these genes in tumor progression and development of refractory disease and relapse., (© 2021. The Author(s).)

DNA released from cells into the peripheral blood is known as cell-free DNA (cfDNA), representing a promising noninvasive source of biomarkers that could be utilized to manage Diffuse Large B-Cell Lymphoma (DLBCL), among other diseases. The procedure for purification and handling of cfDNA is not yet standardized, and various preanalytical variables may affect the yield and analysis of cfDNA, including the purification kits, blood collection tubes, and centrifugation regime. Therefore, we aimed to investigate the impact of these preanalytical variables on the yield of cfDNA by comparing three different purification kits DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). Two blood collection tubes (BCTs), EDTA-K2 and Cell-Free DNA (Streck), stored at four different time points before plasma was separated and cfDNA purified, were compared, and for EDTA tubes, two centrifugation regimes at 2000 × g and 3000 × g were tested. Additionally, we have tested the utility of long-term archival blood samples from DLBCL patients to detect circulating tumor DNA (ctDNA). We observed a higher cfDNA yield using the QIAamp Circulating Nucleic Acid Kit (Qiagen) purification kit, as well as a higher cfDNA yield when blood samples were collected in EDTA BCTs, with a centrifuge regime at 2000 × g . Moreover, ctDNA detection was feasible from archival plasma samples with a median storage time of nine years., Competing Interests: The authors have no conflicts to disclose., (Copyright © 2021 Marijana Nesic et al.)

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Díez, Pérez-Andrés, Bøgsted, Azkargorta, García-Valiente, Dégano, Blanco, Mateos-Gomez, Bárcena, Santa Cruz, Góngora, Elortza, Landeira-Viñuela, Juanes-Velasco, Segura, Manzano-Román, Almeida, Dybkaer, Orfao and Fuentes.)

Aim : Bendamustine is primarily used for treatment of indolent lymphomas but has shown efficacy in some patients with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). Molecular-based patient stratification for identification of resistant patients, who will benefit from alternative treatments, is important. The aim of this study was to develop a resistance gene signature (REGS) from bendamustine dose-response assays in cultures of DLBCL and MM cell lines, enabling prediction of bendamustine response in DLBCL and MM patients. Methods : Bendamustine response was determined in 14 DLBCL and 11 MM cell lines. Using baseline gene expression profiles and degree of growth inhibition after bendamustine exposure, a bendamustine REGS was developed and examined for the risk stratification potential in DLBCL ( n = 971) and MM ( n = 1,126) patients divided into prognostic subtypes. Results : Bendamustine resistance significantly correlated with resistance to cyclophosphamide in DLBCL and melphalan in MM cell lines. The bendamustine REGS showed significantly lower bendamustine resistance probabilities in DLBCL patients with GCB subtype tumors and in tumors of the differentiation dependent centrocyte and plasmablast subtypes. In MM patients, pre-BII classified tumors displayed high bendamustine resistance probabilities and the plasma cell subtype had lower bendamustine resistance probability than memory cells. Furthermore, tumors belonging to the 4p14, MAF, and D2 TC subclasses consistently displayed high bendamustine resistance probabilities. Conclusion : Significant differences in predicted response to bendamustine were found in molecular subtypes of DLBCL and MM, encouraging validation in prospective bendamustine-treated cohorts with available gene expression profiles and follow-up data., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2021.)