Citations

Abstract Background Meningiomas exhibit clinical heterogeneity. Radiotherapy (RT) remains the only adjuvant therapy, but tumor-control is variable, and biomarkers are limited. NRG/RTOG-0539 is the first prospective phase 2 trial to stratify meningioma patients for adjuvant RT based on clinical risk. Here, we apply modern molecular tools to this cohort and identify correlates of RT response. Methods Tumor tissue from 100 RTOG-0539 patients was profiled using DNA methylation arrays, RNA sequencing, and whole-exome sequencing. Recurrence scores, Molecular Groups, gene expression, and copy number alterations were compared across clinical groups and between RT responders and non-responders; non-response was defined as progression or death within 3 years. Results Modern grading criteria, including brain invasion, TERT mutation, CDKN2A/B deletion, and 1p/1q status, would reclassify 10% of tumors and alter treatment group assignment in 7%. Non-responders to RT exhibited more frequent 1p and 14q loss, and more copy number alterations. Transcriptomic and epigenetic profiling revealed immune-related signatures in responders and cell cycle–related pathways in non-responders, several of which overlapped with targets of vorinostat, a histone deacetylase inhibitor previously validated in aggressive meningioma models. The Proliferative Molecular Group was an independent predictor of post-RT recurrence in multivariable analysis, outperforming WHO grade. Conclusion Multi-omic analysis of the NRG/RTOG-0539 cohort shows that updated WHO grading criteria, incorporating molecular and cytogenetic features, improve risk stratification. However, molecular classification, particularly the Proliferative group, remains an independent and stronger predictor of RT response. These findings support integrating molecular biomarkers alongside modern grading frameworks to guide treatment and trial design in meningioma. Clinical Trial Information NCT00895622.

Abstract Despite a characteristic indolent course, a substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. Thus far, efforts to identify factors that predict survival have been unsuccessful. However, we and others have demonstrated the prognostic relevance of CD4+ T cells in the tumor microenvironment (TME) of FL and developed a bio-clinical risk model (called BioFLIPI) that offers improved risk stratification (Mondello et al, BCJ 2021). However, the mechanisms defining different BioFLIPI scores remain to be fully understood. To identify the programs associated with the BioFLIPI scores, we first interrogated the RNA-seq of purified B cells from 67 newly diagnosed FL1-3A (BioFLIPI 1 n=21; BioFLIPI 2-3 n=36; BioFLIPI 4 n=10). We found 1,156 upregulated genes and downregulation of 930 genes in BioFLIPI 4 vs 1, including well-known immune molecules such as CD70, IL10RA, CD69, and CCR6 (FDR<0.05). Gene set enrichment analysis (GSEA) using the MSigDB confirmed a negative enrichment in multiple immune response and interferon gamma signatures and revealed striking positive enrichment for cell proliferation and antigen presentation pathways in BioFLIPI 4 compared to BioFLIPI 1 tumors. The same pathways were dysregulated in BioFLIPI 2-3 but a lower magnitude. To investigate the somatic mutations associated with different BioFLIPI scores, we performed WES of the same 67 FLs and further expanded this cohort for a total of 90 patients (BioFLIPI 1 n=26; BioFLIPI 2-3 n=51; BioFLIPI 4 n=13). We found enrichment for mutations in several regulators of the germinal center in BioFLIPI 4 and 2-3, such as MEF2B (p=0.029), FOXO1 (p=0.038) and IRF8 (p=0.035), suggesting the cooperation of multiple oncogenic events promoting lymphomagenesis. In contrast, BioFLIPI 1 tumors harbored a higher rate of mutations in TNFRSF14 gene, known to enhance anti-lymphoma immune response. To investigate whether BioFLIPI modulates immune signatures of the microenvironment, we performed TME deconvolution by applying CIBERSORTx to bulk RNA-seq of these 67 FLs. We found that FLs with BioFLIPI 4 and 2-3 were enriched for CD4+ Tcm exh (p=0.02), Tem exh (p=0.04), Tfhexh (p=0.07) and NK cells (p=0.01) compared to BioFLIPI 1. In line with this data, CyTOF performed on a subset of these patients (BioFLIPI 1 n=12; BioFLIPI 2-3 n=25; BioFLIPI 4 n=6) confirmed progressive increase of CD4+ Tcm exh (p=0.006), Tem exh (p=0.009) and Tfh exh (p=0.025) cells from BioFLIPI 1 to BioFLIPI 4. Single cell RNA/BCR-seq of 21 FLs (BioFLIPI 1 n=6; BioFLIPI 2-3 n=12; BioFLIPI 4 n=3) corroborated a higher abundance of Tem exh, cytotoxic (CTL) Tem exh, Tfhexh and NK cells in BioFLIPI 4 and 2-3. Conversely, CD8+ naïve T cells were prevalent in BioFLIPI 1 tumors. Differential gene expression in T cell clusters from BioFLIPI 4 vs BioFLIPI 1 FL revealed a marked upregulation of multiple immune genes involved in antigen presentation (e.g., HLA-DRB, HLA-DQA, HLA-DPA/B) and immune response (e.g., ETS1, TNFSF8, IL7R). GSEA showed a positive enrichment for antigen presentation signatures and negative enrichment for TNFα signaling and Oxphos metabolism. Similar findings were observed in BioFLIPI 2-3 FLs. Using CellChat analysis, we found a stronger interaction between malignant B cells and Tem exh and CTL Tem exh cells in BioFLIPI 4 and 2-3 vs BioFLIPI 1 tumors. This was linked to an increased strenght of MHCI-CD8 interaction and concordant decrease of MHCII-CD4. Notably, the suppressive BTLA-TNFRSF14 signaling axis was present in BioFLIPI 4 and 2-3 but not in BioFLIPI 1 FLs. This was accompanied by a decreased signaling through TNF/LTA-TNFRSF1B, CD80/CD86-CD28/CTLA4. Linking the BioFLIPI-specific immunogenicity to lymphomagenesis, we found that FL patients with BioFLIPI 4 had two-three folds higher risk of transformation to an aggressive lymphoma (12% vs 7%) or death (21% vs 3%) than those with BioFLIPI 1, suggesting that this immune dysfunction favors the expansion of a dominant tumor clone and promotes a more aggressive form of FL. Collectively, our findings demonstrate that BioFLIPI scores in FL reflect distinct patterns of tumor immunogenicity that shape the TME. Notably, high BioFLIPI scores are paradoxically associated with enhanced antigen presentation alongside a suppressed immune response. This suggests a sustained antigen-specific T cell response that may drive immune exhaustion, promote tumor progression, and ultimately lead to histologic transformation

Abstract Description Follicular Lymphoma (FL) is a germinal center-derived malignancy representing approximately 20% of all non-Hodgkin’s lymphomas. Despite a relatively indolent disease course, FL is a biologically and clinically heterogeneous disease which poses significant clinical challenges due to its propensity for relapse and transformation into aggressive and untreatable forms of lymphoma. Here, we have leveraged computational techniques with WES, RNA-sequencing, and immune profiling data from FL tumors to gain further insight into the biologic basis for FL heterogeneity. We performed computational reconstruction of the B cell receptor repertoire on B cell sorted RNA-sequencing data from normal lymph nodes (n = 5) and FL tumors (n = 89). To gain a better understanding of FL B cell biology at the single cell level, we further investigated the clonal architecture of dominant malignant clonotypes in FL tumors using computational analysis of paired single cell V(D)J and 5’ GEX sequencing data from FL tumors (n = 21) and non-malignant lymph nodes (n = 3), allowing us to characterize the germinal center programs in malignant versus non-malignant B cells. Our data suggest that a subset of FL tumors consist of complex polyclonal B cell populations, some of which represent a pre-malignant FL precursor. Our data provide a deepened understanding of FL biology, including precursor cells, and contributes a refined transcriptomic characterization of malignant and non-malignant B cells in Follicular Lymphoma. Funding Sources This work was supported by the National Institutes of Health (R01 CA212162-01A1 to A.J.N and J.R.C., and P50 CA097274 to J.R.C., and A.J.N) Topic Categories Computational and Systems Immunology (COMP)

The NCCN Guidelines for Biliary Tract Cancers (BTCs) provide recommendations for the evaluation and comprehensive care of patients with gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma. The multidisciplinary panel of experts is convened at least once annually to review requests from internal and external entities as well as to evaluate new data on current and emerging therapies. This manuscript focuses on the adjuvant chemotherapy and chemoradiation treatment options for BTCs as well as the systemic treatment recommendations for patients with advanced BTCs.

Abstract Purpose: NFE2L2/KEAP1/CUL3 mutations have been validated for radioresistance in cell-based assays and animal models. However, clinical validation of these biomarkers has been challenging because of multimodality treatment regimens. This study aims to investigate the association between NFE2L2/KEAP1/CUL3 mutations and patient outcomes, including local failure, locoregional failure, disease-free survival (DFS), and overall survival, using samples from a phase III trial in which patients were treated with radiation monotherapy at two controlled doses. Patients and Methods: We investigated NFE2L2/KEAP1/CUL3 mutations in 250 randomized patients with T2N0 glottic squamous cell carcinoma receiving definitive radiotherapy in the NRG/RTOG 9512 trial. A total of 119 patients had available biospecimens that were subjected to amplicon-based next-generation sequencing to assess for the presence of NFE2L2/KEAP1/CUL3 mutations without regard to outcomes. Mutations in NFE2L2/KEAP1/CUL3 were assessed blinded to clinical outcomes. Cox models (two-sided α = 0.05) were used to evaluate the association with clinical outcomes, performed by an independent statistical team. Results: Nineteen of 119 patients (16.0%) had NFE2L2/KEAP1/CUL3 mutations. Patient, treatment, and tumor characteristics were similar between those with and without mutations. Patients with mutation compared with those without had significantly more local failure [HR = 3.50; 95% confidence interval (CI), 1.56–7.89; P = 0.0025] and locoregional failure (HR = 3.80; 95% CI, 1.80–8.03; P = 0.0005). DFS was significantly worse for the mutated compared with the nonmutated group in the first 2 years (HR = 2.88; 95% CI, 1.46–5.66; P = 0.0022). The median DFS was shorter in the mutation group (10.3 months) versus those with intact NFE2L2/KEAP1/CUL3 (4.2 years). Conclusions: NFE2L2/KEAP1/CUL3 mutations may predict radiation treatment failure in T2N0 glottic cancer. See related commentary by Rao, p. 1563

Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.

Importance Neoadjuvant therapy (NT) is an increasingly used treatment strategy for patients with localized pancreatic ductal adenocarcinoma (PDAC). Little research has been conducted on cancer care delivery during NT, and the standards for optimal delivery of NT have not been defined. Objective To develop consensus best practices for delivering NT to patients with localized PDAC. Design, Setting, and Participants This study used a modified Delphi approach consisting of 2 rounds of voting, and a series of virtual conferences (from October to December 2023) to reach expert consensus on candidate best practice statements generated from a systematic review of the literature and expert opinion. An interdisciplinary panel was formed including 47 North American experts from surgical, medical, and radiation oncology, radiology, pathology, gastroenterology, integrative oncology, anesthesia, pharmacy, nursing, cancer care delivery research, and nutrition as well as patient and caregiver stakeholders. Main Outcome and Measures Statements that reached 75% agreement or greater were included in final consensus statements. Results Of the 47 participating panel members, 27 (57.64%) were male, and the mean (SD) age was 47.6 (8.2) years. Physicians reported completing training a mean (SD) 14.6 (8.6) years prior and seeing a mean (SD) 110.6 (38.4) patients with PDAC annually; 35 (77.7%) were in academic practice. Final consensus was reached on 82 best practices for delivering NT. Of these, 38 statements focused on pre-NT practices, including diagnosis and staging (n = 15), evaluation and optimization (n = 20), and decision-making (n = 3); 29 statements defined best practices during NT, including initiation (n = 3), delivery of therapy (n = 8), restaging practices (n = 12), and management of complications during NT (n = 6); and 15 best practices were identified to guide treatment post-NT, focusing on surgery (n = 7), pathology (n = 4), and follow-up (n = 3). Conclusions Using a modified Delphi consensus technique, best practice guidelines were developed focusing on the optimal standards for delivering NT to patients with localized PDAC. Given the prognostic importance of completing multimodality therapy, efforts to standardize and optimize the delivery of NT represent an immediate opportunity to decrease care variation and improve outcomes for patients with PDAC. Future research should focus on validating and implementing best practice standards into clinical practice.

ABSTRACTDiffuse large B‐cell lymphoma (DLBCL) patients that fail to achieve a complete metabolic response with frontline immunochemotherapy have a poor prognosis. Genomic profiling has led to a broader understanding of the molecular drivers in DLBCL, but it is unknown how well current classifiers identify patients that will experience primary treatment resistance (PTR). Using whole exome and RNA sequencing data from newly diagnosed DLBCL patients, we evaluated the genomic landscape of PTR and compared it to that of non‐PTR DLBCL. We found a significant increase in the frequency of TP53 (34% vs. 15%, p = 0.005) and ARID1A mutations (21% vs. 7%, p = 0.007) in PTR cases, with pathway analysis further demonstrating a downregulation of TP53 and an increase in chromatin modifying pathways. These results suggest that TP53 and ARID1A may be key mediators of PTR and important pathways contributing to the poor outcomes. We found that the current molecular classifiers were unable to identify PTR cases at diagnosis. However, our newly identified high‐risk signature identified 46% of PTR cases at diagnosis. Overall, these results contribute to our understanding of the genomic landscape of patients with primary treatment resistance.

Abstract Currently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

Background The mortality risk attributable to moderate aortic stenosis (AS) remains incompletely characterized and has historically been underestimated. We aim to evaluate the association between moderate AS and all‐cause death, comparing it with no/mild AS (in a general referral population and in patients with heart failure with reduced ejection fraction). Methods and Results A systematic review and pooled meta‐analysis of Kaplan–Meier‐derived reconstructed time‐to‐event data of studies published by June 2023 was conducted to evaluate survival outcomes among patients with moderate AS in comparison with individuals with no/mild AS. Ten studies were included, encompassing a total of 409 680 patients (11 527 with moderate AS and 398 153 with no/mild AS). In the overall population, the 15‐year overall survival rate was 23.3% (95% CI, 19.1%–28.3%) in patients with moderate AS and 58.9% (95% CI, 58.1%–59.7%) in patients with no/mild aortic stenosis (hazard ratio [HR], 2.55 [95% CI, 2.46–2.64]; P <0.001). In patients with heart failure with reduced ejection fraction, the 10‐year overall survival rate was 15.5% (95% CI, 10.0%–24.0%) in patients with moderate AS and 37.3% (95% CI, 36.2%–38.5%) in patients with no/mild AS (HR, 1.83 [95% CI, 1.69–2.0]; P <0.001). In both populations (overall and heart failure with reduced ejection fraction), these differences correspond to significant lifetime loss associated with moderate AS during follow‐up (4.4 years, P <0.001; and 1.9 years, P <0.001, respectively). A consistent pattern of elevated mortality rate associated with moderate AS in sensitivity analyses of matched studies was observed. Conclusions Moderate AS was associated with higher risk of death and lifetime loss compared with patients with no/mild AS.