Adrenal and extra-adrenal paragangliomas (PPGLs) are highly heritable non-epithelial neuroendocrine neoplasms. Through a retrospective chart review of 110 individuals diagnosed with PPGLs at the University Health Network in Toronto, Canada (2011–2023), we characterized germline findings, tumor features, self-reported ethnicity, and variant reclassification across a multi-ethnic cohort using targeted germline panel sequencing, whole genome sequencing (WGS), and optical genome mapping (OGM). Panel sequencing identified pathogenic or likely pathogenic (P/LP) germline variants in 28.18

ABSTRACT:We previously reported a chemogenomics screen that unexpectedly identified phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) as a vulnerable target in multiple myeloma (MM). PIKfyve is an essential regulator of lysosomal function and autophagy. Given the high basal requirement for autophagy in MM for sustainable immunoglobulin synthesis, targeting autophagy holds clinical potential as a novel therapeutic avenue. Here, we report the development and characterization of PIK001 and analogs, potent and selective novel small-molecule inhibitors of PIKfyve. PIK001 demonstrated potent anti-MM activity in vitro, as well as synergistic activity with established anti-MM agents (including venetoclax and selinexor), while retaining efficacy in lenalidomide-resistant models. Multiomic characterization of isogenic cell lines sensitive and resistant to PIK001 identified a catalytic domain mutation (PIKFYVE N1939K) and heterogenous alterations in autophagy capabilities. Importantly, we noted that PIK001 exposure also resulted in significantly increased cholesterol metabolism and upregulation of major histocompatibility complex (MHC) class I expression, with potential implications in tumor immunity. Beyond MM, PIKfyve inhibition also shows selective cytotoxicity in acute myeloid leukemia, melanoma, and renal cancer, highlighting broader therapeutic potential. These findings establish PIKfyve inhibition as a valid target for MM and other hematologic malignancies, provide insights into mechanisms of sensitivity and resistance, and lay the foundation for further preclinical (particularly the role of cholesterol metabolism and tumor immunity) and clinical development.

Five rare variants in BRIP1/FANCJ, initially identified in ovarian cancer (OC) or breast cancer (BC) cases by the adult hereditary cancer clinics, were investigated for their candidacy as clinically relevant variants. These variants were investigated genetically in a population exhibiting genetic drift and molecularly assayed for biological impact. Using in silico tools, population-based genetic databases and other resources, three of the five reported BRIP1 variants were likely to be damaging: c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61. The carrier frequencies ranged from 0 to 0.7% in ancestry-defined cancer groups comprising 47 OC families, 49 hereditary breast and ovarian cancer syndrome families, 142 hereditary breast cancer syndrome families, 435 sporadic OC cases and 563 sporadic BC cases and 0-0.2% in 1025 population-matched controls. Multiple carriers of the these variants were identified in additional population-matched cancer cases. Of the five reported BRIP1 variants, p.Thr266Met, p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61, which were predicted to be damaging, conferred cellular sensitivity to mitomycin C and cisplatin unlike p.Ser139Ala and p.Ala406Ser. Collectively, our investigation implicates BRIP1 c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61 as deleterious variants in OC and BC.

Abstract Background and Aims: Glioblastoma driven by DNA Replication Repair Deficiency (RRD) account for 10% of all high-grade glioma in children and young adults, harbour high tumor mutation burden (TMB) and can respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority ultimately progress, highlighting the need for combinatorial therapies for sustained immune-surveillance. Methods: We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combined ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. Results: Human RRD-glioblastoma (n=80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2LoxP/LoxP-POLES459F/+) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1-/-/NestinCre+/Trp53LoxP/LoxP and therapy-induced hypermutant ENU/Trp53-/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming, in-vivo, that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Seven children with refractory RRD-glioblastoma who had progressed after anti-PD1 treatment were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition for similar patients. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. Conclusions: LAG3 is an effective target in refractory RRD-glioblastoma. Combined inhibition with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses. The combination will now be tested in biomarker-driven clinical trials in RRD-glioblastoma and other immune-inflamed solid tumors. Citation Format: Anirban Das, Vienna Mazzoli, Owen Crump, Olha Kos, Nuno M. Nunes, Lucie Stengs, Amanda Li, Adrian Levine, Yoshiko Nakano, Hope Friedman, Katharine O'Flaherty, Alexander Stein, Gadi Abebe-Campino, Annika Bronsema, Vanessa Bianchi, Melissa Edwards, Stergios Zacharoulis, Birgit Ertl-Wagner, Daniel A. Morgenstern, Trevor J. Pugh, Pamela Ohashi, Eric Bouffet, Cynthia E. Hawkins, Peter B. Dirks, Uri Y. Tabori. Translating combined PD1 and LAG3 inhibition from preclinical models to patients with refractory, DNA replication repair deficient (RRD) glioblastoma: An IRRDC study [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 2796.

Abstract Background RRD-glioblastoma harbour high tumor mutation burden (TMB) and respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority progress, highlighting the need for novel therapies for sustained immune-surveillance. Methods We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combination ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. Results Human RRD-glioblastoma (n = 80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2LoxP/LoxP-POLES459F/+) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1-/-/NestinCre+/Trp53LoxP/LoxP and therapy-induced hypermutant ENU/Trp53-/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming in-vivo that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Thirteen children with refractory RRD-glioblastoma who had progressed on anti-PD1 were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. Conclusions LAG3 is an effective target in refractory RRD-glioblastoma. Combination with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses, paving way for the next clinical trial using this combination.

cBioPortal for Cancer Genomics is a widely used platform for exploratory, interactive visualization and analysis of large-scale clinico-genomic datasets. cBioPortal provides a range of visualizations and analyses including interactive cohort exploration, OncoPrints, mutation “lollipop” plots, survival analysis, alteration enrichment analysis, and detailed patient-level visualizations. cBioPortal also integrates variant annotations from a variety of sources to facilitate interpretation. The public cBioPortal (https://www.cbioportal.org) is accessed by >40,000 unique visitors each month and hosts data from >460 studies. All data is also available in the cBioPortal Datahub: https://github.com/cBioPortal/datahub. In 2024 we added 76 new studies (∼30,000 samples), including data from the NCI Genomic Data Commons. In addition, >94 instances of cBioPortal are installed at academic institutions and companies worldwide. cBioPortal partners with AACR Project GENIE to provide access to the GENIE cohort in a dedicated instance (https://genie.cbioportal.org). Users can explore the full GENIE cohort of >229,000 clinically sequenced samples from 19 institutions, as well as cohorts with comprehensive clinical annotations including response, outcome, and treatment history, from the GENIE Biopharma Collaborative (BPC). BPC cohorts for NSCLC (∼2,000 samples) and colorectal cancer (∼1,500 samples) are available, with more to come. The past year has brought a variety of enhancements to cBioPortal. A new data type selector on the home page enables users to find studies with specific types of data. The interactive cohort exploration has new ways to explore data with the addition of gene-specific charts to summarize the types of mutations in a gene and the integration of the Plots tab for customizable graphs of any two data attributes. The OncoPrint can now display per group alteration frequency based on any categorical attribute. Variant interpretation is enhanced with the integration of AlphaMissense as a novel annotation source and an update to the latest MutationAssessor data. The patient page also has new visualizations, including mutational signatures and the integration of Chromoscope to visualize structural variations. We also made significant changes to the backend code to improve both the developer and user experience. The backend code was repackaged and upgraded to simplify and improve the development process. In addition, we are working on switching to an Online Analytical Processing (OLAP) database which will bring significant performance improvements. cBioPortal is open source: https://github.com/cBioPortal. Development is a collaborative effort among groups at Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Children’s Hospital of Philadelphia, Princess Margaret Cancer Centre, Caris Life Sciences, Bilkent University, SE4BIO and The Hyve. We welcome open source contributions from others in the cancer research community. Ino de Bruijn,Tali Mazor,Rima AlHamad,Calla Chennault,Corey Dubin,Jeremy Easton-Marks,Zhaoyuan Fu,Benjamin Gross,Charles Haynes,David M. Higgins,Jason Hwee,Prasanna K. Jagannathan,Mirella Kalafati,Karthik Kalletla,Zeynep Karagöz,James Ko,Tim Kuijpers,Sowmiyaa Kumar,Priti Kumari,Ritika Kundra,Bryan Lai,Xiang Li,James Lindsay,Aaron Lisman,Qi-Xuan Lu,Ramyasree Madupuri,Zain-ul-Abideen Nasir,Angelica Ochoa,Yusuf Ziya Özgül,Oleguer Plantalech,Matthijs N. Pon,Baby A. Satravada,Jessica Singh,Selcuk Onur Sumer,Pim van Nierop,Floris Vleugels,Avery Wang,Manda Wilson,Hongxin Zhang,Gaofei Zhao,Ugur Dogrusoz,Allison Heath,Adam Resnick,Trevor J. Pugh,Chris Sander,Ethan Cerami,JianJiong Gao,Nikolaus Schultz. cBioPortal for cancer genomics [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 1117.

Glioblastoma (GBM) is an aggressive brain cancer with a poor survival rate. Despite hundreds of clinical trials, there is no effective targeted therapy. Glioblastoma stem cells (GSCs) are an important GBM model system. In culture, these cells form spatial structures that share morphological aspects with their source tumors. We collected 17,000 phase-contrast images of 15 patient-derived GSC lines growing to confluence. We find that GSCs grow in characteristic multicellular patterns depending on their transcriptional state. Interpretable computer vision algorithms identified specific image features that predict transcriptional state across multiple cell confluency levels. This relationship will be useful in developing GSC screens where image features can be used to identify how GSC biology changes in response to perturbations simply by imaging cultured cells on plates.

We previously reported a chemogenomics screen that unexpectedly identified phos-phatidylinositol-3-phosphate 5-kinase (PIKfyve) as a vulnerable target in multiple myeloma (MM). PIKfyve is an essential regulator of lysosomal function and autophagy. Given the high basal requirement for autophagy in MM for sustainable immunoglobulin synthesis, targeting autophagy holds clinical potential as a novel therapeutic avenue. Here, we report the development and characterization of PIK001 and analogs, potent and selective novel small-molecule inhibitors of PIKfyve. PIK001 demonstrated potent anti-MM activity in vitro, as well as synergistic activity with established anti-MM agents (including venetoclax and selinexor), while retaining efficacy in lenalidomide-resistant models. Multiomic characterization of isogenic cell lines sensitive and resistant to PIK001 identified a catalytic domain mutation (PIKFYVE N1939K) and heterogenous alterations in autophagy capabilities. Importantly, we noted that PIK001 exposure also resulted in significantly increased cholesterol metabolism and upregulation of major histocompatibility complex (MHC) class I expression, with potential implications in tumor immunity. Beyond MM, PIKfyve inhibition also shows selective cytotoxicity in acute myeloid leukemia, melanoma, and renal cancer, highlighting broader therapeutic potential. These findings establish PIKfyve inhibition as a valid target for MM and other hematologic malignancies, provide insights into mechanisms of sensitivity and resistance, and lay the foundation for further preclinical (particularly the role of cholesterol metabolism and tumor immunity) and clinical development.

Supplementary Table S2: Sample Summary. List of patients and samples (timepoints) included in the current study.

Fluorescence in situ hybridization (FISH) remains the gold-standard clinical assay to detect genetic abnormalities in multiple myeloma (MM). However, FISH panel design, use of conventional chromosome banding analysis and reporting practices have been reported to vary among laboratories. Therefore, standardization in FISH testing and reporting practices is needed to improve report clarity and avoid misinterpretation. The recommendations in this paper represent a consensus of our Cancer Genomics Consortium Plasma Cell Neoplasm Working Group, comprising a joint panel of cytogenetic laboratory directors and clinical investigators with expertise in the diagnosis, risk stratification, and treatment of multiple myeloma. Prior to developing these consensus recommendations, we performed a full literature review and conducted a survey of 102 oncologists to assess current variations and challenges in MM cytogenetic/FISH testing and reporting. Our guidelines establish best practices for the optimization of FISH panel selection, and recommendations for standardized reporting of cytogenetic results to align with the 2025 International Myeloma Society (IMS)/International Myeloma Working Group (IMWG) Updated Risk Stratification.

Glioblastoma or GBM is an aggressive brain cancer with a 5 year survival rate below 10 percent. Glioma stem cells or GSCs drive GBM formation, growth, and resistance. Previously, analysis of 17,601 phase contrast images from 15 GBM patients revealed that neurodevelopmental GSCs form smaller, uniform clusters, while mesenchymal and injury-response GSCs exhibit complex, irregular growth patterns. This new study examines the spatial distribution and local cellular niche of GSCs along a neurodevelopmental gradient in primary GBM. We develop a geospatial map of GSCs in relation to other cell-types, identifying community patterns of organization. We profiled 14 primary, treatment-naive GBM tissue sections (5 micrometer thick) on a spatial transcriptomics platform (Xenium), utilizing a custom 414 gene panel to score the GSC, GBM and other cell signatures. We segmented cell boundaries using both Baysor and Proseg algorithms and performed hematoxylin-eosin (HE) staining of tissue sections post-Xenium run. We performed spatial clustering using SpaGCN and Banksy, followed by spatial distribution and spatial point pattern analysis (Ripley’s, Moran’s I). Across the 14 tissues, we segmented 5, 705, 825 cells and annotated each cell with a cell-type label using pre-defined gene sets. This resulted in 15.2 percent GSC-like cells, 9.0% oligodendrocytic-OPC-like, 8.7% astrocytic-mesenchymal-like, 6.7% neuronal-like, 15.0% immune, 7.1% endothelial, and the rest of the cells showing mixed signatures. In total we identified 110-140 spatial clusters, which we classified into 8 domains by grouping highly correlated spatial clusters using Pearson correlation. All cell-types were differentially distributed both proportionally and spatially. Domains with highly correlated grouped clusters exhibited distinct spatial arrangements of cells as well. For instance, GSC-like cells were confined to unique spatial domains with specific geometric properties across cellular hierarchies. Likewise, immune and endothelial cells displayed enrichment in preferred domains, with some co-localized with GSC-enriched regions while others were devoid of these cell types. Therefore, we find that these geospatial topology maps could identify spatial enrichment patterns and distributional trends. This study deciphers GBM’s geospatial topology, where annotated cell-types are mapped to specific niches. Though the ultimate aim of the study will be to eliminate GSC-like cells in GBM, we posit that by revealing cell community patterns, we aim to shift therapeutic strategies from targeting single cells or molecules to biomarkers that address entire cell communities within which these GSCs are spatially and functionally connected to. This framework lays the groundwork for future biomarker discovery and therapeutic interventions in GBM. Shamini Ayyadhury, Fatemah Al Solaiman, Yuna Lee, Alyona Ivanova, Ana Nikolic, Farzaneh Aboulizadeh, Melanie Peralta, Patty Sachamitr, Michelle M. Kushida, Nicole I. Park, Fiona J. Coutinho, Owen Whitley, Panagiotis Prinos, Cheryl H. Arrowsmith, Sam Weiss, Sheila Mansouri, Gelareh Zadeh, Peter B. Dirks, Troy Ketela, H. Artee Luchman, Gary D. Bader, Trevor J. Pugh. Discovering the geospatial framework in glioblastoma: Uncovering hotspots for glioma stem cell hide-out and their therapeutic implications [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 767.

Clinical sequencing of tumor samples is now a component of routine cancer care. By identifying genomic alterations that contribute to tumor initiation or progression, clinical cancer genomic sequencing may be used to identify predictive biomarkers of drug response, refine patient cancer diagnoses, assess heritable cancer risk, or inform patient prognosis. Most genomic alterations are accurately annotated with tools such as the Variant Effect Predictor (VEP) that infer the effects of these alterations on the mRNA and protein by following basic rules of transcription, mRNA post-transcriptional processing, and translation. However, a select subset of variants’ effects cannot be predicted as easily by these rules. While many of these “variants with unexpected effects (VUE)” are functionally characterized and documented in the literature, these VUEs are often mis-annotated during routine clinical cancer genomic sequencing. Importantly, certain VUEs may have therapeutic implications, which, if mis-annotated may lead to suboptimal treatment decisions for individual patients with cancer. To address this unmet clinical need, we created a centralized database resource, the repository for Variants with Unexpected Effects (reVUE - cancerrevue.org), which curates and programmatically stores VUEs to enable the annotation of these variants during routine clinical cancer genomic sequencing. The reVUE resource consists of (1) an intuitive website listing curated VUEs with their observed effects as demonstrated by functional characterization in peer-reviewed literature and (2) an application programming interface (API) for programmatic annotation of variants. We successfully curated 109 VUEs spanning 22 genes from 31 articles, and continue to expand this database. Several curated VUEs were associated with clinical treatment implications, including KIT, MET, ATM, EGFR, and BRCA1/2. The reVUE database has also been integrated into the publicly available bioinformatic ecosystem of cancer variant annotation and interpretation tools that currently includes Genome Nexus, OncoKB, and cBioPortal. By addressing the critical challenge of the accurate annotation of genomic variants with unanticipated protein effects, reVUE enhances our understanding of complex variant interpretation and contributes directly to improved patient care. Notably, the software and all annotated variants are publicly available, allowing for community contributions, and enabling seamless integration into other genomics tools, clinical workflows, and research pipelines. Xiang Li, Ino de Bruijn, Thomas Y. Cong, Walid Chatila, Hongxin Zhang, Moriah Nissan, Amanda Dhaneshwar, Sara E. DiNapoli, Erika Gedvilaite, Bryan Lai, Selcuk Onur Sumer, Aditi Gopalan, Tonatiuh Gonzalez, Madelaine Rangel, Trevor J. Pugh, Rose Brannon, Michael F. Berger, Jianjiong Gao, Nikolaus Schultz, Debyani Chakravarty. reVUE: repository for variants with unexpected effects [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 5043.

Table S1: Patient Radiotherapy Information and Multivariate Analysis.

PURPOSE:To assess modified folinic acid/leucovorin, fluorouracil, irinotecan, oxaliplatin (FOLFIRINOX [mFFX]) versus gemcitabine/nab-paclitaxel (GnP) in de novo metastatic pancreatic ductal adenocarcinoma (PDAC) and explore predictive biomarkers. PATIENTS AND METHODS:Patients were randomly assigned 1:1 to mFFX or GnP with exclusion of germline pathogenic variants in BRCA1/2 or PALB2. The primary end point was progression-free survival (PFS) between arms with 0.3 significance. The per-protocol (PP) population included patients who received one dose of chemotherapy. Pretreatment biopsies underwent whole-genome/transcriptome sequencing and patient-derived organoid (PDO) development, providing correlate recommendations at a molecular tumor board and outcomes assessed according to RNA signatures (basal-like v classical). RESULTS:Of 160 patients randomly assigned (80 mFFX, 80 GnP), 140 patients were in the PP population (71 mFFX, 69 GnP), with median follow-up of 8.3 months. The median PFS was 4.0 months for mFFX versus 5.3 months for GnP (hazard ratio [HR], 1.37 [95% CI, 0.97 to 1.92]; P = .069) in intention-to-treat. Median overall survival (OS) was 8.5 months with mFFX and 9.7 months with GnP (HR, 1.57 [95% CI, 1.08 to 2.28]; P = .017). Genomic data were generated in 94%, transcriptomes in 74%, and PDOs in 50%. The median PFS for those with basal-like was 3.0 (mFFX) and 5.5 (GnP) months (P = .17), and classical PDAC was 6.3 (mFFX) versus 5.4 (GnP) months (P = .36). The median OS in basal-like was 7.5 (mFFX) and 8.9 (GnP) months (P = .75) versus in classical OS was 9.7 (mFFX) and 13.9 (GnP) months (P = .047). Overall, 75 (54%) of patients received second-line treatment, 33/75 (44%) correlate-guided. The median time on second-line treatment was only 2.1 months with a median OS of 5.4 months for a correlate-guided choice versus 4.4 months on a standard chemotherapy approach (P = .45). CONCLUSION:In the phase II Pancreatic Adenocarcinoma Signature Stratification for Treatment-01 (PASS-01) trial population, PFS was similar between GnP and mFFX; however, OS and safety trends favored GnP. The second-line setting appears inadequate to offer precision choices, given the short survival observed.

Despite advancements in the treatment of multiple myeloma (MM), most patients ultimately relapse due to innate and acquired drug resistance. Our search for novel therapeutic strategies led to the development of PIK001, a potent and selective PIKfyve inhibitor, as a promising therapeutic approach targeting lysosomal function and autophagy. In addition to its robust single agent anti-MM activity, PIK001 presented synergistic activity with relevant anti-MM therapeutics, including selinexor, venetoclax, and pomalidomide (IMiDs), in vitro. Notably, PIK001 retained efficacy in IMiD-resistant isogenic human myeloma cell line (HMCL) models, underscoring its potential in drug-resistant MM. To investigate the determinants of PIKfyve resistance, we generated PIK001-resistant HMCLs by culturing three PIK001-sensitive HMCLs (KMS26, KMS11, and JJN3) in escalating doses of the PIKfyve inhibitor (up to 5uM). These isogenic models of resistance were characterized by whole genome and transcriptome sequencing and mass spectrometry-based proteomics. Ex vivo downstream effects of PIK001 treatment with 500nM for 16h were further assessed in patient-derived CD138+ MM samples using single-cell multiomic sequencing. PIK001 resistance was associated with a marked upregulation of genes and proteins involved in lysosomal function, autophagy regulation, and cholesterol homeostasis, along with downregulation of MYC targets. These findings were also found in primary patient samples following PIK001 treatment. KMS26 PIK001-resistant showed a clonal PIKFYVE kinase domain mutation, previously described in a resistant diffuse large B-cell lymphoma cell line. Importantly, we observed a two-fold increase in canonical and noncanonical Major Histocompatibility Complex (MHC) class I and a four-fold increase in MHC class II gene and protein expression in the PIK001-resistant compared to PIK001-sensitive KMS11. Increased cell surface expression of MHC Class I and II in KMS11 PIK001-resistant was confirmed by flow cytometry. PIK001-resistant KMS26 and JJN3 also presented an upregulation of cell surface expression of MHC Class I and, to a lesser extent, II. Similarly, PIK001 treatment also resulted in increased in MHC Class I gene expression on primary patient samples. Since downregulation or loss of MHC Class I has been shown as a mechanism of immune evasion in cancer, these findings suggest that PIKfyve inhibition may enhance MM immunotherapy responses by upregulating MHC surface expression. This hypothesis aligns with recent studies demonstrating increased tumor-specific MHC Class I expression and improved cancer immunotherapy efficacy following PIKfyve inhibition in solid tumors. Together, these results highlight the potential of PIKfyve inhibitors to synergize with existing anti-MM therapeutics and sensitize MM cells to MHC-dependent immunotherapies via autophagy disruption. Cecilia Bonolo de Campos, Dor D. Abelman, Ruijuan He, Tessa Pelino, Ding Yan Wang, David S. Scott, Zhihua Li, Michael St Paul, Trevor J. Pugh, Olga Issakova, Nikolai Sepetov, Tak W. Mak, Suzanne Trudel, A Keith Stewart. Autophagy disruption via PIKfyve inhibition as a novel strategy to enhance immunotherapy responses in multiple myeloma [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 6908.

Adoptive cell transfer (ACT) of tumour-infiltrating lymphocytes (TIL) is an investigational treatment for solid tumours, with preliminary results showing objective clinical responses in some metastatic melanoma patients. The ability to sequence and track the T cell repertoire throughout ACT of TILs provides a method to identify T cell repertoire features associated with patients’ benefit from ACT. Identification of response biomarkers for patients receiving ACT of TILs has been limited. Conflicting evidence is observed in biomarkers such as the number of TILs in the infusion product, with some studies suggesting a relationship with response and others not. Meanwhile, certain potential biomarkers, such as the diversity of the post-infusion peripheral repertoire, have not yet been studied. In this study, we sought to determine 1) the efficacy of using CapTCR-seq to track TILs in serial blood draws over the course of ACT immunotherapy 2) whether peripheral T cell repertoire statistics are associated with ACT response. In this study, 9 patients with cutaneous (n = 7) or mucosal (n = 2) melanoma received TIL ACT after chemotherapeutic depletion. Hybrid-capture CapTCR-seq was conducted on pre-/post-transfer peripheral blood mononuclear cells (PBMC) and cell-free DNA. Comparison between PBMC DNA, PBMC RNA, and circulating free DNA (cfDNA) repertoires demonstrated an increased presence of shared T cell clonotypes post-infusion when compared with baseline samples. Higher abundance of TIL clonotypes in the PBMC baseline and post-infusion DNA T cell repertoires and the presence of shared DNA T cell clonotypes between timepoints was seen in responders when compared with non-responders according to RECIST criteria. These results demonstrate effective tracking methodologies and suggest a predictive role for baseline repertoire statistics in response to the ACT of TILs.

The randomized phase II MA.38 trial estimated the relative progression-free survival (PFS) associated with second-line endocrine therapy plus palbociclib administered on a 100 mg continuous daily dosing (CDD) schedule compared with the standard dose schedule (SDS) of 125 mg (days 1-21 of a 28-day cycle). A total of 180 patients were allocated 1:1 to protocol therapy. Molecular profiling was performed on the archival tissue and cell-free DNA (cfDNA) at enrollment, 3 months, and 6 months. The primary analysis for PFS demonstrated a similar outcome for the CDD versus SDS treatment strategy: HR = 0.93 (90% confidence interval, 0.66-1.30). Secondary efficacy measures for CDD versus SDS included the following: overall survival, HR = 1.07 (90% confidence interval, 0.67-1.69); response rate, 16.1% versus 18.0% (P = 0.66); median duration of response, 4.2 months (range, 2.8-13.9 months) versus 5.6 months (range, 2.4-13.9 months; P = 0.86); and clinical benefit rate, 53.2% versus 57.3% (P = 0.89). cfDNA profiling of the baseline enrollment sample prior to palbociclib commencement showed low tumor fraction (HR = 2.28; P = 9.9 × 10-6); higher short/long fragment length ratios (HR = 1.19; P = 0.049) and cfDNA variants in FGFR4 (HR = 3.65; P = 0.012) were prognostic and associated with inferior PFS. Variants in TP53 (HR = 2.48; P = 0.006) and ESR1 (HR = 3.42; P = 0.005) detected at 12 weeks on treatment were also associated with poor PFS. CDD palbociclib 100 mg dosing was not associated with improved efficacy compared with the standard intermittent 125 mg dosing schedule. Additionally, we identified prognostic biomarkers in alignment with prior research and demonstrated the value of cfDNA dynamics, including fragment length ratios and tumor fraction as a measure of treatment response. SIGNIFICANCE:A continuous 100 mg dosing schedule of palbociclib was tolerable but not associated with improved efficacy signals versus the standard intermittent 125 mg (days 1-21 of a 28-day cycle) schedule. Mutations detected in liquid biopsies and changes in cfDNA dynamics were linked to poor outcomes and may identify patients with treatment-resistant cancer.

Glioblastoma (GBM) is an aggressive brain tumor with a highly invasive nature. Despite the clinical relevance of this behavior, the molecular underpinnings of infiltrating GBM cells in the peritumoral zone remain underexplored in patients. Here, we show that peritumoral progenitor-like GBM cells activate transcriptional programs associated with increased invasivity, synaptic activity, and NOTCH signaling. These cells spatially colocalize with neurons and exhibit an increased propensity for neuronal crosstalk. The epigenetic encoding of these infiltrative cells mirrors that of uncommitted oligodendrocyte progenitor cells (OPCs) in the developing human brain, a neurodevelopmental state marked by increased synaptic and migratory potential. Functional perturbation of a nominated regulatory factor, ZEB1, confirmed its role in maintaining the invasive and uncommitted developmental potential of infiltrative GBM cells. Our findings provide insights into the neurodevelopmental hijacking that drives GBM infiltration in patients, rationalizing further investigation into targeting differentiation potential as a therapeutic strategy.

Lung cancers and melanomas have many somatically mutated self-proteins that would be expected to trigger an immune rejection response, yet therapeutic responses can only be induced in a subset of patients. Here, we investigated the possibility that inherited differences in immune tolerance checkpoints contribute to variability in outcomes. Whole genome sequencing revealed biallelic germline loss-of-function (LOF) mutations in the immune tolerance checkpoint gene, NOD2, in an exceptional immune responder to targeted radiotherapy for metastatic melanoma. In 40 exceptional immune responders to anti-PD1 monotherapy for non-small cell lung cancer (NSCLC), genome sequencing showed 30% had inherited a NOD2 LOF variant, more than twice the population frequency (P = 0.0021). Conversely, a gain-of-function RIPK2 allele known to increase NOD2 signaling was inherited by 61% of nonresponders from the same cohort, compared to 10% of exceptional responders and much higher than the population frequency (P < 0.0001). Within the overall recruited cohort of 144 NSCLC anti-PD1 patients, individuals with immune-related adverse events (irAE) had better overall survival, further improved in those with NOD2 LOF. In independent anti-PD1 monotherapy cohorts with a range of cancers, inherited NOD2 LOF was associated with complete or partial response (P = 0.0107). Experimental validation in mice showed germline Nod2 LOF enhanced therapeutic immune responses elicited by anti-PD1 monotherapy against a high mutation burden colorectal cancer, increasing tumor infiltration by effector memory CD8 T cells. Collectively these results reveal common inherited human variation in an immune tolerance checkpoint is a determinant of cancer immune responses elicited by pharmacological inhibition of another checkpoint.