TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Smooth versus Textured Tissue Expanders: Comparison of Outcomes and Complications in 536 Implants.

Background Increasing concerns regarding the safety of textured surface implants have resulted in surgeons transitioning from textured tissue expanders (TEs) to smooth TEs. Given this change has only recently occurred, this study evaluated outcomes between smooth and textured TEs. Methods Women who underwent two-stage breast reconstruction using TEs from 2013 to 2022 were included. TE-specific variables, perioperative information, pain scores, and complications were collected. Chi-squared, t -test, and linear regression analyses were performed. Results A total of 320 patients received a total of 384 textured and 152 smooth TEs. Note that 216 patients received bilateral reconstruction. TEs were removed in 9 cases. No significant differences existed between groups regarding comorbidities. Smooth TEs had a higher proportion of prepectoral placement ( p < 0.001). Smooth TEs had less fills (3 ± 1 vs. 4 ± 2, p < 0.001), shorter expansion periods (60 ± 44 vs. 90 ± 77 days, p < 0.001), smaller expander fill volumes (390 ± 168 vs. 478 ± 177 mL, p < 0.001), and shorter time to exchange (80 ± 43 vs. 104 ± 39 days, p < 0.001). Complication rates between textured and smooth TEs were comparable. Smooth TE had a greater proportion of TE replacements ( p = 0.030). On regression analysis, pain scores were more closely associated with age ( p = 0.018) and TE texture ( p = 0.046). Additional procedures at time of TE exchange ( p < 0.001) and textured TE ( p = 0.017) led to longer operative times. Conclusion As many surgeons have transitioned away from textured implants, our study shows that smooth TEs have similar outcomes to the textured alternatives.

Spatially Resolved Tumor Microenvironment Predicts Treatment Outcomes in Relapsed/Refractory Hodgkin Lymphoma.

PURPOSE: About a third of patients with relapsed or refractory classic Hodgkin lymphoma (r/r CHL) succumb to their disease after high-dose chemotherapy followed by autologous stem-cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL. PATIENTS AND METHODS: We performed imaging mass cytometry (IMC) on 71 paired primary diagnostic and relapse biopsies using a marker panel specific to CHL biology. For each cell type in the TME, we calculated a spatial score measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within the close interaction range. Spatial scores were used as features in prognostic model development for post-ASCT outcomes. RESULTS: Highly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early r/r CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single-cell RNA sequencing data identified unique architecture defined by CXCR5+ Hodgkin and Reed Sternberg (HRS) cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay (RHL4S) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, CD68+ tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk versus low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay. CONCLUSION: We identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

Live Virtual Surgery and Virtual Reality in Surgery: Potential Applications in Hand Surgery Education.

Accelerated in part by the coronavirus disease 2019 pandemic, medical education has increasingly moved into the virtual sphere in recent years. Virtual surgical education encompasses several domains, including live virtual surgery and virtual and augmented reality. These technologies range in complexity from streaming audio and video of surgeries in real-time to fully immersive virtual simulations of surgery. This article reviews the current use of virtual surgical education and its possible applications in hand surgery. Applications of virtual technologies for preoperative planning and intraoperative guidance, as well as care in underresourced settings, are discussed. The authors describe their experience creating a virtual surgery subinternship with live virtual surgeries. There are many roles virtual technology can have in surgery, and this review explores potential value these technologies may have in hand surgery.

Utilizing Computer-Assisted Design and Virtual Surgical Planning for Correction of Unilateral Lambdoid Synostosis.

Unilateral lambdoid synostosis is the rarest form of single-suture craniosynostosis. Although various surgical approaches have been described, cranial vault remodeling remains the predominant approach. To aid in surgical planning, preoperative virtual surgical modeling using a patient's presenting computed tomography scan can be used to increase reconstructive precision and to reduce operative time. Presented is a 7-month-old male with unilateral lambdoid synostosis who underwent medically modeled cranial vault reconstruction.

Molecular determinants of clinical outcomes in a real-world diffuse large B-cell lymphoma population.

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

Distinct Gene Expression Patterns of Calcium Channels and Related Signaling Pathways Discovered in Lymphomas.

Cell surface calcium (Ca2+) channels permit Ca2+ ion influx, with Ca2+ taking part in cellular functions such as proliferation, survival, and activation. The expression of voltage-dependent Ca2+ (CaV) channels may modulate the growth of hematologic cancers. Profile analysis of Ca2+ channels, with a focus on the Ca2+ release-activated Ca2+ (CRAC) and L-type CaV channels, was performed on RNA sequencing data from lymphoma cell lines and samples derived from patients with diffuse large B cell lymphoma (DLBCL). CaV1.2 expression was found to be elevated in classical Hodgkin lymphoma (CHL) cell lines when compared to other B cell lymphoma cell lines. In contrast, CHL exhibited reduced expression of ORAI2 and STIM2. In our differential expression analysis comparing activated B cell-like DLBCL (ABC-DLBCL) and germinal centre B cell-like DLBCL (GCB-DLBCL) patient samples, ABC-DLBCL revealed stronger expression of CaV1.3, whereas CaV1.1, CaV1.2, and CaV1.4 showed greater expression levels in GCB-DLBCL. Interestingly, no differences in ORAI/STIM expression were noted in the patient samples. As Ca2+ is known to bind to calmodulin, leading to calcineurin activation and the passage of nuclear factor of activated T cells (NFAT) to the cell nucleus, pathways for calcineurin, calmodulin, NFAT, and Ca2+ signaling were also analyzed by gene set enrichment analysis. The NFAT and Ca2+ signaling pathways were found to be upregulated in the CHL cell lines relative to other B cell lymphoma cell lines. Furthermore, the calmodulin and Ca2+ signaling pathways were shown to be downregulated in the ABC-DLBCL patient samples. The findings of this study suggest that L-type CaV channels and Ca2+-related pathways could serve as differentiating components for biologic therapies in targeted lymphoma treatments.

Single-cell spatial analysis of tumor immune architecture in diffuse large B-cell lymphoma.

Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2: Characterizing CD4+ T cells in Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Targeting Refractory Mantle Cell Lymphoma for Imaging and Therapy Using C-X-C Chemokine Receptor Type 4 Radioligands.

PURPOSE: Mantle cell lymphoma (MCL) is associated with poor survival. The purpose of this study was to assess whether the C-X-C chemokine receptor type 4 (CXCR4) is a useful target for imaging and radioligand therapy of MCL, using a novel pair of radioligands, [68Ga]Ga and [177Lu]Lu-BL02. EXPERIMENTAL DESIGN: We performed a retrospective analysis of 146 patients with MCL to evaluate CXCR4 expression and its correlation with outcomes. Guided by in silico methods, we designed BL02, a new radioligand labelled with 68Ga or 177Lu for PET imaging and therapy, respectively. We performed imaging and biodistribution studies in xenograft models with varying CXCR4 expression. We evaluated [177Lu]Lu-BL02 in MCL models, and evaluated its potential for therapy in Z138 MCL xenografts. RESULTS: Phosphorylated and nonphosphorylated CXCR4 expression were correlated with poor survival in patients with MCL and characterized by unique underlying molecular signatures. [68Ga]Ga-BL02 uptake correlated with CXCR4 expression, and localized lesions in a metastatic xenograft model. [177Lu]Lu-BL02 showed high uptake in MCL xenografts. Therapy studies with a single dose in the Z138 model showed tumor regression and improved survival compared with a control group. Upon regrowth, the treated mice experienced concurrent metastasis alongside localized xenograft regrowth, and recurrent lesions showed enhanced CXCR4 signaling. CONCLUSIONS: CXCR4 is an independent factor of poor prognosis for MCL and a promising target for imaging and radioligand therapy. [68Ga]Ga-BL02 showed high contrast to visualize CXCR4-expressing xenografts for PET imaging and [177Lu]Lu-BL02 induced rapid tumor regression in a preclinical model of MCL.

Single-cell profiling reveals the importance of CXCL13/CXCR5 axis biology in lymphocyte-rich classic Hodgkin lymphoma.

Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4+ helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1+CXCL13+ T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5+ normal B cells in close proximity to CXCL13+ T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1+CXCL13+ T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1+CXCL13+ T cells as a treatment target in LR-CHL.

Experiences and Perspectives of Transgender Youths in Accessing Health Care: A Systematic Review.

Importance: Transgender and nonbinary youths have a higher incidence of a range of health conditions and may paradoxically face limited access to health care. Objective: To describe the perspectives and needs of transgender youths in accessing health care. Evidence Review: MEDLINE, Embase, PsycInfo, and the Cumulative Index to Nursing and Allied Health Literature were searched from inception to January 2021. Qualitative studies of transgender youths' perspectives on accessing health care were selected. Results from primary studies were extracted. Data were analyzed using thematic synthesis. Findings: Ninety-one studies involving 884 participants aged 9 to 24 years across 17 countries were included. We identified 6 themes: experiencing pervasive stigma and discrimination in health care, feeling vulnerable and uncertain in decision-making, traversing risks to overcome systemic barriers to transitioning, internalizing intense fear of consequences, experiencing prejudice undermining help-seeking efforts, and experiencing strengthened gender identity and finding allies. Each theme encapsulated multiple subthemes. Conclusions and Relevance: This review found that transgender youths contend with feelings of gender incongruence, fear, and vulnerability in accessing health care, which are compounded by legal, economic, and social barriers. This can lead to disengagement from care and resorting to high-risk and unsafe interventions. Improving access to gender-affirming care services with a cultural humility lens and addressing sociolegal stressors may improve outcomes in transgender and nonbinary youths.

Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.

PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.

Single-cell Profiles and Prognostic Impact of Tumor-Infiltrating Lymphocytes Coexpressing CD39, CD103, and PD-1 in Ovarian Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. EXPERIMENTAL DESIGN: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. RESULTS: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. CONCLUSIONS: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.

Mutational landscape of gray zone lymphoma.

The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas.