Multiple myeloma (MM) is marked by recurrent cytogenetic abnormalities and mutations that accumulate as the disease progresses. Here, we sought to elucidate the transitions driving tumorigenesis and therapy resistance in MM using a unique cohort of nearly 900 patients spanning premalignant to late-stage refractory MM, comprehensively characterized at molecular and clinical levels. Waves of epigenetic dysregulation drove these critical transitions. In this paradigm, genomic and cytogenetic events unlocked epigenetic plasticity, reshaping MM cell biology to evade tumor microenvironment constraints and therapeutic pressures. Functional perturbation studies in an isogenic proteasome inhibitor-resistant cell line model demonstrated enhanced reliance on transcriptional cofactors, supporting a mechanistic link between chromatin plasticity and therapy adaptation. Collectively, these findings support a unifying framework in which genomic heterogeneity unlocks gene regulatory plasticity, enabling plasma cells to evade microenvironmental constraints and therapeutic pressure. These results provide a mechanistic explanation for sequential relapse without new genomic alterations and nominate epigenetic plasticity-mediated plasma cell adaptation as a therapeutic vulnerability in the heterogeneous genetic background of MM.

Minor histocompatibility antigen (mHAg)-specific alloreactive donor T cells cause graft vs. host disease (GVHD) in matched related donor allogeneic hematopoietic cell transplantation (HCT). In a phase I trial, we expanded and infused (on day -2) mHAg-specific donor regulatory T cells (Treg) together with sirolimus-based pharmacologic prophylaxis to examine safety and preliminary efficacy of this GVHD prevention approach. We employed a 3+3 phase I design escalating Treg dose in 4 levels: 0.5 x 105/kg, 1 x 105/kg, 2 x 105/kg, and 4 x 105/kg. Dose-limiting toxicity (DLT) included grade 4-5 related infusion reaction, grade 4-5 unexpected organ toxicity, grade III-IV acute GVHD, or treatment-related death. Secondary and exploratory measures examined acute and chronic GVHD, survival outcomes, and Treg clone (TCR-Seq) expansion in culture, and in-vivo longevity and expansion post-HCT. 15 subjects were included (N=3 each per dose levels 1-3, and N=6 in dose level 4). No DLT were observed, and 4 x 105/kg Treg was identified as MTD. Median follow up for survivors was 41.7 months (range 14.5-72.8). The day 100 cumulative incidence of grade II-IV acute GVHD was 13% (95% CI 2-35%). NIH moderate/severe chronic GVHD by 1 year was 6.7% (95% CI 0.36-27%) and by 3 years was 20% (95%CI 4.4-44%). Overall survival was 73% (95% CI 54-100%). Treg clones expanded in culture, and demonstrated post-HCT lineage fidelity, persistence, and in-vivo expansion. This translational trial supports mHAg-specific expanded donor Treg as a novel GVHD prevention strategy, and demonstrates expanded donor Treg clones can persist and expand through one-year post-HCT. NCT01795573.

Outcomes are poor for TP53 mutant myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients who undergo allo-HSCT. Notably, minimal data exist on the impact of TP53 MRD monitoring post allo-HSCT. Thus, we completed duplex TP53 MRD sequencing for all patients on our prospective eprenetapopt (APR-246) + azacitidine maintenance study (n=14). Bone marrow aspirates were obtained prior to allo-HSCT, prior to the start of investigational therapy (day +30 to day +100) and after cycle 3 and cycle 12 of therapy. To assess low allele frequency mutations in TP53, a custom-targeted sequencing panel was used with duplex sequencing, targeting 30,000-70,000X duplex coverage to detect variants at a frequency as low as .005%. For all analyses, TP53 MRD negativity cutoff was 0.01%. All study patients had significant TP53 positivity prior to allo-HSCT and 57% post-HSCT. MRD evaluation after end of maintenance (12 cycles) was the strongest predictor of outcomes. Specifically, MRD negativity after cycle 12 strongly predicted OS (33.9 vs 20.4 months; P=.005) and EFS (33.9 vs 10.1 months; P=.004) with a trend for RFS (32.6 vs 13.5 months; P=.06). TP53 MRD was strongly predictive of outcomes, supporting incorporation of this assay in future novel strategies.

This file contains four supplementary tables S1 - S4 and eight supplementary figures S1 - S8.

Background Inherited genetic variation may impact patient response and risk of toxicity following immune checkpoint inhibitor (ICI) therapy. Methods We conducted an agnostic genome-wide analysis study for inherited genetic variants that may predict thyroiditis in patients with melanoma treated with ipilimumab and investigated Polygenic Risk Scores (PRSs) previously reported to be associated with thyroid disease. Germline DNA from 744 participants in a phase 3 adjuvant trial was analyzed following genome-wide genotyping. Results An agnostic genome-wide cohort-level analysis identified top associated single nucleotide polymorphisms (SNPs), and a custom 10-SNP PRS was significantly associated with thyroiditis risk and severity. Among the SNPs identified, five mapped to intronic, four intergenic and one exonic regions. The latter was the lead SNP and mapped to CNOT6L , located near CXCL13 . Among the other variants, most were located within regions with potential relevance to immune regulation and autoimmunity. Separately, multiple thyroid disease-related PRSs derived from Polygenic Score Catalog weights were tested, and several were significantly associated with thyroiditis, but the custom PRS had stronger discrimination for thyroiditis risk (area under the curve 0.82). Conclusions These findings indicate that inherited genetic background contributes to thyroid immune toxicity risk following ICI and support PRS-based approaches for risk-adapted monitoring during immunotherapy.

Abstract Cutaneous T cell lymphoma (CTCL) is a rare malignancy of skin-homing T cells, with Mycosis Fungoides (MF) representing 50-70% of all cases. Although MF typically follows an indolent clinical course, a subset of MF patients progresses to develop large cell transformation (LCT) that is associated with a worse outcome. Disease progression from patch/plaque (PP) lesions to transformed tumor (TT) is accompanied by profound genetic and transcriptional alterations across multiple signaling pathways, including oxidative phosphorylation and MYC, posing a significant challenge for single-agent therapeutic strategies. Epigenetic therapies such as HDAC inhibitors have shown limited efficacy, underscoring the need to identify key transcription factors (TFs) and novel gene regulatory networks (GRNs) that drive CTCL disease progression and may serve as targeted vulnerabilities. In this study, we first performed joint profiling of gene expression and chromatin accessibility using scMultiome on six MF samples (2 PP and 4 TT). By integrating TF expression, chromatin accessibility at targeting regions, and expression of nearby genes, we inferred cell-type specific GRNs across the tumor microenvironment cell types, including the malignant T cells in PP and TT lesions, and other benign immune and non-immune cells. Importantly, this analysis identified JUND-associated GRNs enriched in B cells and ATF6 networks in myeloid cells, demonstrating robust regulatory signal recovery. Applying the same framework, we identified RUNX2 and ATF2 as putative regulators of PP malignant T cells, and LEF1, IKZF2, and TCF7 as key drivers of transformed CTCL cells. We further validated the critical roles of these transcription factors in in-house and publicly available scRNAseq datasets. To investigate the spatially resolved chromatin accessibility landscape in CTCL, we generated the first spatial ATACseq dataset for this rare cancer and profiled paired PP and TT lesions from the same patient. Spatial clustering and inferred copy number analysis revealed distinct epigenetic clone structures, including PP-dominant clones, TT-dominant clones, and shared clones between PP and TT. Notably, GRNs enriched in PP or TT by scMultiome were recapitulated within PP-dominant and TT-dominant spatial ATAC clones, respectively, highlighting consistent regulatory programs across modalities. Together, these results uncover previously unrecognized GRNs associated with MF progression and provide the first spatially resolved epigenetic map of CTCL. Our findings demonstrated potential regulators and clonal heterogeneity that may inform the development of novel therapeutic strategies, including precision epigenetic or combination therapies. Citation Format: Xiaofei Song, Jennifer M. Garbarino, COLIN NG, Lucia Seminario-Vidal, Carly M. Harro, Jodi A. Balasi, Chaomei Zhang, Nan Sun, Douglas C. Marchion, Sean J. Yoder, Jose R. Conejo-Garcia, Pei-Ling Chen. Deciphering gene regulatory network and spatial heterogeneity underlying cutaneous T cell lymphoma large cell transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3203.

The groundbreaking SWOG S1801 and NADINA trials herald the use of neoadjuvant immune checkpoint blockade (NICB) as the standard of care for stage III-IV melanomas. The shift from radiographic to pathologic response scoring offers an unprecedented window of opportunity to interrogate ‘on-treatment’ biospecimens for the immune mechanisms of response and resistance at curative-intent surgeries. To date, single-cell/bulk RNA studies have demonstrated that TCF7+ stem-like CD8+ T cells and tertiary lymphoid structures (TLS) are positive predictors of ICB response. However, how these immune cells organize and communicate within the spatial context of the neoadjuvant tumor microenvironment remains poorly understood. We assembled a retrospective cohort of 68 patients with stage 3 melanoma: 35 NICB (Ipilimumab-nivolumab, nivolumab-relatlimab, nivolumab) (14 complete response, 5 partial response, 14 non-response) and 33 treatment-naive (TxN). We dissected the spatial molecular architecture of all tumor beds (n= 68) and 10 uninvolved LNs (5 post-PD-1/5 TxN) using integrative multiplexed error-robust fluorescence in situ hybridization (MERFISH, 305 genes), Vectra multiplex IF and RNAscope multiomics in 38 whole slides and 3 TMAs (77 cores). We also performed single-cell-FFPE-seq using matched blocks (n=12) as an orthogonal benchmark for the MERFISH data. To analyze these high-dimensional datasets, we built a computational framework that includes 1) cell-typing with scFFPEseq validation, 2) novel spatial quantification methods to compute receptor-ligand (R-L) interactions that account for cell-cell distance and chemical signaling, 3) a graph-based clustering that quantifies germinal centers (GCs)/TLS, B-cell follicles and other spatial topography, and 4) MERFISH/ scRNA imputation via generative AI. Here, we showed that the quantity and size of spatially resolved GC/TLS and B-cell follicles surrounded by TCF7+ stem-like CD4+ and CD8 + T-cells, plasma cells, Tregs and myeloids are associated with positive response to neoadjuvant ICB. Our R-L analysis further revealed the critical importance of CXCR4-CXCL12 between TCF7+ T-cells and M2-myeloids and CCR7-CCL19/21 between TCF7+ T-cells in organizing the immune hubs in NICB response. Lastly, we incorporated scFFPEseq and MERFISH data into a unified latent embedding by environmental variational inference (ENVI) allowing us to impute melanoma phylogenetics, geographically accentuated hypoxic foci, and immune hubs within the spatial MERFISH images. We leveraged cutting-edge spatial -omics technologies and novel computational methods to resolve the immunologic hallmarks of NICB response in metastatic melanoma. We believe our approach will usher in a new paradigm for the investigation of other cancer biospecimens in the new era of standard of care neoadjuvant immunotherapy. Zichao Liu, Xiaofei Song, Jodi Balasi, Wei-Shen Chen, Jiang He, Justin He, Jonathan Nguyen, Carlos Moran-Segura, Joseph Johnson, Chaomei Zhang, Jane Messina, Zena Sayegh, Nan Sun, Douglas Marchion, Sean Yoder, Vernon K. Sondak, Jeffrey H. Chuang, Pei-Ling Chen. Spatially resolved immunologic hallmarks of response to neoadjuvant immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5809.

Abstract Background: High-grade serous ovarian cancer (HGSC) is a deadly gynecologic cancer and is characterized by widespread copy number alterations (CNA). However, knowledge of racial differences in HGSC CNA is limited. Using methylation-inferred CNA, we defined and characterized CNA-based tumor clusters to assess patterns by self-reported race. Methods: Tumor CNA were calculated using Illumina EPIC v1/v2 methylation data for 678 cohort participants (African American Cancer Epidemiology Study, North Carolina Ovarian Cancer Study, Nurses’ Health Study) compared to 296 blood samples. Probes were binned (≥10; ≥100 kb), segmented using circular binary methods, autocorrected, and filtered (|mean log R ratio|≥0.3; >4 markers), to define cytoband-level copy number states (-1-loss /0-neutral /+1-gain). CNA were clustered with Ward’s method. For each cluster, we computed length-weighted cytoband burden (% genome in gain, loss, or neutral states) and region-level ORs to identify CNA-enriched regions. Models clustered by race were then fit to assess differences. Results: Combined analysis of HGSC samples from 403 White (59%) and 275 Black women (41%), identified three CNA clusters: C1 (27%), C2 (57%), and C3 (15%). Clusters showed distinct genomic burdens (Kruskal-Wallis p<10 −55). C1 showed widespread losses, notably in 4q (∼75% vs 14% in C2/C3, q<10-45), which has been reported in HGSOC, and 17p (71% vs 13% in C2/C3, q<10-46), which includes TP53. C3 exhibited extensive chromosomal gains, including 20p/q (∼82%/72%, q <10-38), 1q/1p (∼64%/39%, q<10-32), and 2p/2q (∼56%/42%, q <10-31). Gains on 2p/q have been linked to advanced stage and poor prognosis, potentially via overexpression of AURKA, GNAS, and TPD52L2. Amplified 1q may enhance DHX9 expression, a regulator of genomic stability, while BARD1 (2q) interacts with BRCA1 in DNA repair; aberrant BARD1 expression correlates with poor outcomes. Cluster membership was similar between Black and White women (C1: 30% vs 26%, C2: 53% vs 59%, C3: 17% vs 15%). However, arm-level copy number racial differences were observed. Overall, 21q loss was more frequent in tumors from Black women (28%) than White women (16%; OR=0.5, 95% CI=0.34-0.73, q=0.02). This difference was most pronounced in C2, where 21q loss occurred in 27% of Black women compared to 10% in White women (OR = 0.31, 95% CI=0.17-0.56, qcluster=0.002). Conclusion: Broad arm-level CNAs define molecularly distinct HGSC subgroups with characteristic gain/loss patterns. Race-associated genomic alterations, including 21q loss among Black women, appear to be cluster-dependent genomic events suggesting underlying biological heterogeneity related to focal genomic instability or selective pressures rather than the result of wide-spread changes in overall copy number burden. Citation Format: Irma M. Vlasac, Brett M. Reid, Courtney Johnson, Alicia R. Richards, Christelle M. Colin Leitzinger, Sean J. Yoder, Dana Roeber, Tania Mesa, Andrew Berchuck, Brooke Fridley, Jing-Yi Chern, Jennifer A. Doherty, Kristin Haller, Shelley Tworoger, Jeffrey R. Marks, Joellen M. Schildkrau, Brock C. Christensen, Lauren Cole Peres, Lucas A. Salas Diaz, on behalf of the African American Cancer Epidemiology Study. Characterization of high-grade serous ovarian cancer copy number alterations in Black and White Women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6813.

Immune checkpoint inhibitor (ICI)-mediated dermatitis is a common treatment-related adverse event. We conducted single-nucleotide polymorphisms (SNPs)-array genotyping on germline DNA from 744 participants in a phase three adjuvant trial of ipilimumab in melanoma. Logistic regression was used to estimate associations between inherited genetic markers and the occurrence of dermatitis. We also tested previously reported dermatitis-related polygenic risk scores (PRS) adjusting for genetic ancestry. Dermatitis of any grade occurred in 52% (232 Gr1, 97 Gr2, and 52 Gr3). We identified two SNPs associated with dermatitis and subsequently developed a two-SNP PRS that was associated with dermatitis risk and severity. Both variants mapped to intronic regions of the insulin-like growth factor 1 receptor and A-kinase anchoring protein 12 genes, which have important roles in immune regulation and inflammatory response. Separately, 11 previously reported dermatitis-related PRSs derived from general population cohorts not exposed to immunotherapy were tested and none was associated with ICI-mediated dermatitis in our cohort. The cohort-derived PRS showed modest discrimination for dermatitis risk (AUC 0.63). The incidence of dermatitis was associated with improved outcomes; patients with grade 1-2 or any-grade dermatitis demonstrated improved relapse-free survival (RFS) (hazard ratio: 0.74; P = 0.007 and 0.77, P = 0.01, respectively). The cohort-derived PRS showed a trend toward improved RFS and overall survival. In conclusion, we generated a novel PRS associated with ICI-mediated dermatitis risk and severity that demonstrated a trend towards improved survival outcomes. Our findings support a role for inherited genetic variation in relation to ICI dermatitis and warrant further investigation.

Abstract Background: The recent groundbreaking neoadjuvant immune checkpoint blockade (NICB) clinical trials in melanoma have demonstrated decisively that the administration of ICB prior to intent-to-cure surgeries will become the new standard-of-care for metastatic melanoma and beyond. The paradigm shifts from radiographic to pathologic response assessment also presents an unparalleled “window of opportunity” for the acquisition of abundant “on-treatment” tissues to elucidate the mechanisms and biomarkers of response and for guiding post-surgery personalized therapy decisions. To date, single-cell/bulk RNA studies have demonstrated that TCF7+ stem-like CD8+ T cells and tertiary lymphoid structures (TLS) are positive predictors of ICB response. However, how these immune cells organize and communicate within the spatial context of the neoadjuvant tumor microenvironment remains poorly understood. Methods: We investigated a unique cohort of 91 FFPE biospecimens from 87 patients with stage III metastatic melanoma, including 60 treated with NICB (31 complete response, 7 partial response, 22 non-response) and 27 treatment-naïve. We deployed state-of-the-art technologies, including multiplexed error-robust fluorescent in situ hybridization (MERFISH), single cell sequencing of FFPE blocks, and multiplexed IF for this rich digital resource. Importantly, to analyze these challenging high dimensional spatial datasets, we developed 3 novel computational algorithms, including SCIRA, a scalable quantification method for spatial receptor-ligand (R-L) interactions; GC-SCAN, a graph-based clustering method to detect Germinal Center (GC)/TLS structures in single cell spatial data; and PathNet, an end-to-end AI algorithm for automated GC/TLS detection on H&E slides. Results: Here, we interrogated ∼5.6 million cells and showed that increased GC/mature TLSs, TCF7+ stem-like CD8 and CD4 T-cells, exhausted CD8 T-cells, plasma cells and myeloids in spatially distinct cellular neighborhoods are significantly associated with positive response. Our spatial (R-L) analyses further identified preferential chemokine R-L interactions between GC-B cells and follicular helper T-cell, and TCF7+ stem-like T-cells with CCL19+/CCL21+ fibroblasts in organizing these immune hubs. Lastly, Our PathNet GC/TLS detection algorithm outperforms recent state-of-the-art methods in both specificity and sensitivity, proven potential utility in facilitating clinical response assessment. Conclusions: We leveraged cutting-edge spatial-omics technologies and novel computational methods to resolve the immunologic hallmarks of NICB response in metastatic melanoma. We believe our approach provides a model for how to precisely identify spatially intricate immune interactions that underline treatment response in the rapidly advancing era of standard of care neoadjuvant immunotherapy. Citation Format: Zichao Liu, Xiaofei (Sophia) Song, Jodi A. Balasi, Wei-Shen Chen, Jiang He, Justin He, Jonathan Nguyen, Carlos M. Morán-Segura, Joseph O. Johnson, Chaomei Zhang, Jane L. Messina, Zena Sayegh, Douglas C. Marchion, Sean J. Yoder, Vernon K. Sondak, Jeffrey H. Chuang, Pei-Ling Chen. Spatially resolved immunologic hallmarks of response to neoadjuvant immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6564.

ABSTRACT:In this response-adapted clinic trial with daratumumab monotherapy for older patients with newly diagnosed multiple myeloma (MM), we identified target antigen expression, a plasma cell phenotype, and an activated immune tumor microenvironment (iTME) as critical features associated with response to CD38 monoclonal antibody therapy. Here, patients achieving a partial response after 2 cycles continued daratumumab, otherwise lenalidomide or bortezomib was added. This strategy resulted in an overall response rate of 97% and low rates of adverse events, with 37% of patients able to continue daratumumab monotherapy. Importantly, we found that higher CD38 expression, plasma cell gene expression programming, and an activated iTME were associated with patients who were able to continue daratumumab therapy alone. In contrast, patients requiring the addition of lenalidomide or bortezomib had increased expression of adhesion, tumor necrosis factor signaling, KRAS signaling, and B-cell programs, as well as an immunosuppressed iTME. Tracking of clonal dynamics illustrated the selection of subclones enriched for de novo resistance gene expression programs after only 2 cycles of daratumumab monotherapy. Upon relapse, daratumumab refractory MM cells were characterized by the expansion of preexisting minor subclones with mixed transcriptomic programs containing the plasma cell phenotype with decreased CD38 expression and maintenance of resistance programs, suggesting development of acquired resistance involves an uncoupling of transcriptional programs present in therapy-naïve tumors. To our knowledge, this is the first study to demonstrate the effectiveness of response-adapted daratumumab treatment and describe critical biomarkers of single-agent daratumumab sensitivity in vulnerable patients with therapy-naïve MM. This trial was registered at www.ClinicalTrials.gov as #NCT04151667.

We demonstrate that carcinogenesis and multi-therapy resistance in multiple myeloma (MM)-a treatable yet incurable plasma cell malignancy-are driven by epigenetic dysregulation. In this new paradigm, genomic and cytogenetic events unlock epigenetic plasticity, reshaping MM cell biology to evade tumor microenvironment constraints and therapeutic pressure. These conclusions are derived from a newly assembled cohort of nearly 1,000 patients, spanning premalignant to late-stage refractory MM, comprehensively characterized at molecular and clinical levels. Our findings provide a unifying framework to explain inter-patient genomic heterogeneity and the emergence of therapy resistance in sequential samples without new genomic alterations. In conclusion, we propose targeting epigenetic plasticity-mediated plasma cell evasion as a promising therapeutic strategy in MM.

Publications on the use of RSI/12CK GES in retrospective cohorts

ABSTRACT:The radiation sensitivity index (RSI) and 12-chemokine gene expression signature (12CK GES) are two gene expression signatures (GES) that were previously developed to predict tumor radiation sensitivity or identify the presence of tertiary lymphoid structures in tumors, respectively. To advance the use of these GESs into clinical trial evaluation, their assays must be assessed within the context of the Clinical Laboratory Improvement Amendments (CLIA) process. Using HG-U133Plus2.0 arrays, we first established CLIA laboratory proficiency. Then the accuracy (limit of detection and macrodissection impact), precision (variability by time and operator), sample type (surgery vs. biopsy), and concordance with a reference laboratory were evaluated. RSI and 12CK GES were reproducible (RSI: 0.01 mean difference, 12CK GES: 0.17 mean difference) and precise with respect to time and operator. Taken together, the reproducibility analysis of the scores indicated a median RSI difference of 0.06 (6.47% of range) across samples and a median 12CK GES difference of 0.92 (12.29% of range). Experiments indicated that the lower limit of input RNA is 5 ng. Reproducibility with a second CLIA laboratory demonstrated reliability with the median RSI score difference of 0.065 (6% of full range) and 12CK GES difference of 0.93 (12% of observed range). Overall, under CLIA, RSI and 12CK GES were demonstrated by the Moffitt Cancer Center Advanced Diagnostic Laboratory to be reproducible GESs for clinical usage. SIGNIFICANCE:The RSI and 12CK GES are two GESs that predict tumor radiation sensitivity or the presence of tertiary lymphoid structures in tumors, respectively. These GESs were assessed within the CLIA process for future clinical use. We established proficiency, reproducibility, and reliability characteristics for both signatures in a controlled setting, indicating these GESs are suitable for validation within future clinical trials.

Supplemental Table 2 includes tissue/RNA assessment values.

Penile squamous cell carcinoma (PSCC) is a rare malignancy with limited understanding of the tumor immune microenvironment (TIME). The interplay between PSCC and the immune system across disease progression and HPV infection status remains poorly characterized. This study aims to assess the TIME changes from localized to advanced disease and between HPV-positive versus negative tumors to identify potential immune evasion mechanisms in advanced PSCC. scRNA-seq was performed on ten PSCC tissue samples from penile, lymph node and distant metastatic sites with four matched penile and lymph node samples to understand the cellular heterogeneity within PSCC tumors. Analysis of immune cell populations and transcriptional hallmarks were performed stratified by localized (pT1-3, N0) versus advanced (N1-3, M0 or any N, M1) disease states and HPV infection status. We observed significant differences in immune cell infiltration between localized and advanced PSCC disease states and by HPV status. Advanced disease states demonstrated an exhausted immune phenotype, characterized by terminally exhausted CD8+ T cells, M2-like macrophages and hypoxic signature, while localized disease states demonstrated an active innate immune system characterized by increased DCs. HPV-negative tumors displayed low immune cell infiltration while HPV-positive tumors demonstrated an immune exhausted phenotype. These findings offer valuable insights into the evolving PSCC immune landscape, paving the way for the development of potential therapeutic approaches for advanced PSCC.

9562 Background: The groundbreaking neoadjuvant ICB clinical trials have established beyond doubt that ICB before surgery will become the new standard of care for metastatic melanoma. Importantly, the shift from radiographic to pathologic response scoring offers an unprecedented window of opportunity to interrogate a goldmine of ‘on-treatment’ biospecimens. To date, single-cell profiling and region-based spatial transcriptomics (ST) have highlighted the importance of tertiary lymphoid structures (TLS) and stem-like T-cells as positive predictors of ICB response. However, the molecular mechanisms by which tumor infiltrating lymphocytes communicate and organize multicellular immune hubs within the spatial context of the tumor ecosystem remains poorly understood. Methods: To identify robust biomarkers of response to neoadjuvant ICB, we assembled a cohort of 58 stage III melanoma patients treated with neoadjuvant ICB [24 ipilimumab-nivolumab (IPI-NIVO), 21 NIVO-relatlimab (RELA), 13 PD1 mono] and deployed transformative technologies and computational methods, including single-cell FFPE sequencing, multiplexed FISH single-cell ST (MERFISH 305 genes), digital pathology, 3D open-top light-sheet imaging and AI-based computational pathology, to decipher the complex neoadjuvant ICB tumor ecosystem in response to therapy. We also developed 2 critical computational tools, SCIRA (Spatial Cellular Interaction and Receptor Activation), to compute receptor-ligand (R-L) interactions in whole slide images, and GC-SCAN (graph-based spatial clustering against noise), a graph-based algorithm that quantifies locally clustered structures from spatial -omics data. Results: Our results showed that the quantity and size of hyper-expanded germinal center/TLS, with increased GC: non-GC B-cell ratio, plasma cells and spatially resolved stem-like T-cells are strongly associated with response. IPI-NIVO elicited significantly stronger GC proliferation compared to NIVO-RELA and PD1 monotherapy, suggesting anti-CTLA4 can robustly induce germinal center/TLS proliferation. SCIRA spatial R-L analyses revealed the critical chemokine R-L interactions that organize the GC- and T-cell zones in response to therapy. Lastly, 3D light-sheet imaging revealed remarkable morphologic heterogeneity in 3D, with interconnected GC-TLS networks that are indicative of long-range molecular gradients. Conclusions: Our investigations herein have provided a comprehensive characterization of the immune architectures, cellular communications and 3D large-scale morphologic organizations of the TME that drive response to neoadjuvant ICB therapy. We believe the results of this study will enable the development of robust predictive biomarkers to guide the design of next generation combination ICB therapies in the clinical trial setting for melanoma and other cancer types.

Experimental design of CLIA validation experiments. The experiments were organized into three categories: accuracy, precision, and sample content. Each of these categories assessed different sources of variability expected to impact signature scores

Several therapeutic agents have been approved for treating multiple myeloma, a cancer of bone marrow-resident plasma cells. Predictive biomarkers for drug response could help guide clinical strategies to optimize outcomes. In this study, we present an integrated functional genomic analysis of tumor samples from patients multiple myeloma that were assessed for their ex vivo drug sensitivity to 37 drugs, clinical variables, cytogenetics, mutational profiles, and transcriptomes. This analysis revealed a multiple myeloma transcriptomic topology that generates "footprints" in association with ex vivo drug sensitivity that have both predictive and mechanistic applications. Validation of the transcriptomic footprints for the anti-CD38 mAb daratumumab (DARA) and the nuclear export inhibitor selinexor (SELI) demonstrated that these footprints can accurately classify clinical responses. The analysis further revealed that DARA and SELI have anticorrelated mechanisms of resistance, and treatment with a SELI-based regimen immediately after a DARA-containing regimen was associated with improved survival in three independent clinical trials, supporting an evolutionary-based strategy involving sequential therapy. These findings suggest that this unique repository and computational framework can be leveraged to inform underlying biology and to identify therapeutic strategies to improve treatment of multiple myeloma. Significance: Functional genomic analysis of primary multiple myeloma samples elucidated predictive biomarkers for drugs and molecular pathways mediating therapeutic response, which revealed a rationale for sequential therapy to maximize patient outcomes.