Moffitt Cancer Center

Immunotherapy with checkpoint inhibitors targeting the PD1/PD-L1 and CTLA4 pathways has limited activity in patients with microsatellite stable (MSS) colorectal adenocarcinoma (CRC). In a prior study, the combination of cetuximab and pembrolizumab failed to improve outcomes for patients with advanced RAS wild-type (RASwt) CRC. In this post hoc secondary analysis, we show that the cetuximab and pembrolizumab-treated patients with TP53 mutant (p53mt) tumors had significantly higher progression-free survival (PFS) and a decrease in tumor burden compared to patients with TP53 wild-type (p53wt) tumors but no difference in overall survival compared to patients with p53wt tumors. The gene set enrichment analysis showed a uniform upregulation of multiple metabolic and immune gene sets, including NK-mediated immunity and IL-12 pathway, while the IL6 pathway was downregulated. There were no overlapping transcriptional alterations between the p53mt and p53wt groups with treatment that remain constant despite the therapeutic intervention. Functional overlap with treatment in both groups in the proliferative, immune, and metabolic pathways were identified. In the baseline tumor samples, the number of PD-L1+ tumor cells was significantly higher in p53mt tumors while the number of OX40-/AE1_AE3-/PD-L1- non-tumor cells, positive for either LAG3, CTLA4 or TIM3, was significantly higher in p53wt tumors. In conclusion, TP53 status was prognostic of improved PFS with cetuximab plus pembrolizumab in RASwt CRC. Future studies evaluating immune-oncology agents in patients with MSS, RASwt CRC should include TP53 as an integrated biomarker and evaluate its performance as a positive predictive biomarker (ClinicalTrials.gov NCT02713373).

Background: Identifying patients most likely to benefit from immune checkpoint inhibitors (ICIs) remains a significant challenge in advanced melanoma. We evaluated the association between tumor somatic mutations and clinical outcomes, focusing on relapse-free survival (RFS) and overall survival (OS) in locoregionally advanced melanoma patients treated with neoadjuvant ipilimumab. Methods: Tumor specimens and matched peripheral blood samples from 22 patients underwent whole-exome sequencing (WES) to identify non-synonymous somatic mutations. Tumor mutational burden (TMB) was quantified, and specific mutations were analyzed for associations with survival outcomes. Results: The analysis revealed a mutational landscape dominated by single-nucleotide missense mutations with a median TMB of 11.4 mutations/MB. BRAF and NRAS mutations were detected in 73% of patients and exhibited mutual exclusivity and concurrence patterns (p < 0.05). Positional clustering identified NRAS and SLC35B4 as key contributors to melanoma (FDR p-value < 0.05). Log-rank analysis indicated that mutations in ODZ1, USP34, CEP192, EML5, KIAA1797, ATAD5, and ANKHD1-EIF4EBP were associated with shorter survival outcomes (RFS or OS). The associations remained significant in both univariate and multivariable Cox regression models adjusted for TMB. These genes can be broadly grouped into functional categories relevant to tumor progression and immune modulation. In applying multiple testing correction, none maintained statistical significance, indicating that these findings should be interpreted as exploratory and require validation in independent cohorts. Conclusions: This study identified tumor genomic alterations associated with clinical outcomes in melanoma patients treated with neoadjuvant ipilimumab, suggesting their potential role in anti-tumor immunity. These findings warrant further investigation in larger cohorts and across other ICIs in melanoma and other malignancies.

Menstrual hygiene products (MHPs) may offer a high-reach, frequent, long-lasting yet underutilized channel for health education, particularly in low-resource settings. This study aimed to explore women's breast health awareness (BHA) and their attitudes towards and preferences for BHA messages on MHPs. Women aged 18-49 years in Ghana and Tanzania (n = 438; 216 Ghana, 222 Tanzania), stratified by age and urban/rural setting, completed a questionnaire assessing socio-demographics, BHA, and preferences for BHA messages with MHP. BHA was assessed with an adapted 31-item Breast Cancer Awareness Measure Tool covering 4 domains: breast cancer signs and symptoms, confidence, skills, and behaviour in noticing breast changes, anticipated help-seeking delays, and risk factors. Items were scored on a 10-point scale. Here we show that overall BHA is slightly higher in Ghana than Tanzania (urban/rural: 6.2/6.4 vs. 5.6/5.3). Awareness is highest for signs and symptoms (median score range hereafter: 5.5-7.8) and lowest for risk factors (2.0-3.8). While women show confidence in noticing breast changes (8.0-10.0) and seeking help (10-10), their skills are insufficient (1.0-5.0) and previous help-seeking behaviours were low (0-0). Women would overwhelmingly (91%) choose MHPs with BHA messages over MHPs without, preferring varied presenting formats, mostly between outside of the package, on individual pad covers, and a leaflet. Findings highlight generally low BHA level in Ghana and Tanzania. The strong acceptability of having BHA messages on MHP presents a scalable and sustainable opportunity to improve BHA with the goal of reducing BC incidence and mortality.

BackgroundT-cell immunoglobulin and mucin-domain containing 3 (TIM-3) is an inhibitory receptor linked to decreased antitumor activity of immune cells. S095018 is a human anti-TIM-3 IgG2 antibody that blocks the binding of phosphatidyl serine to TIM-3. Sym021 is a humanized IgG1 antibody that inhibits the binding of programmed cell death protein-1 (PD-1) to its ligands programmed death-ligand 1 (PD-L1) and PD-L2.MethodsS095018 in combination with Sym021 was tested in patients with advanced/metastatic biliary tract cancers (BTC) whose disease progressed under treatment with at least 1 line of systemic therapy and who had not received prior treatment with PD-(L)1 inhibitors (NCT04641871). Patients received 3 mg/kg of Sym021 and 10 mg/kg of S095018 once every 2 weeks. Primary endpoints included overall response rate and incidence/severity of adverse events. Key secondary endpoints included pharmacokinetics, immunogenicity assessment, progression-free survival (PFS) and overall survival (OS).Results35 patients with stage IV BTC received S095018 in combination with Sym021. A partial response was achieved in 2 patients (5.7%) and stable disease in 11 patients (31.4%) for a disease control rate of 37%; 4 patients were not evaluable for response. Median PFS and OS were 1.9 months (90% CI 1.8 to 3.7) and 13.4 months (90% CI 8.2 to 27.1), respectively. The most common treatment-emergent adverse events of any grade included fatigue, pruritus, infusion-related reaction, and increases in amylase (8.6% each). Exploratory biomarker analyses in paired tumor biopsies showed an increase in intratumoral CD8 T-cell density and an upregulation of gene signatures related to interferon-γ signaling, antigen presentation, and T-cell activation with treatment without, however, clear association with efficacy endpoints.ConclusionsDual PD-1/TIM-3 inhibition was tolerable but exhibited modest antitumor activity in patients with advanced/metastatic recurrent BTC who had not received prior anti-PD-(L)1 treatment.

Chromosomal instability, characterized by pervasive copy number alterations (CNAs), significantly contributes to cancer progression and therapeutic resistance. CNAs drive intratumoral genetic heterogeneity, creating distinct subpopulations whose interactions shape tumor evolution through frequency-dependent selection. Here, we introduce ECO-K (Ecological-Karyotypes), an inverse game theory framework that quantifies frequency-dependent interaction coefficients among karyotypically defined subpopulations under the assumption that their fitness is frequency-dependent. Applying this approach to serially-passaged, triple-negative breast cancer cell lines and patient-derived xenografts (PDXs), we estimated interaction matrices consistent with the observed time-series dynamics. In one PDX lineage, the inferred matrices consistently assigned large interaction coefficients to a subpopulation characterized by chromosome 1 loss and chromosome 14p gain, suggesting it may act as an ecological hub within the frequency-dependent model. Our framework provides testable predictions of intratumoral ecological dynamics, highlighting opportunities to strategically target key subpopulations to disrupt tumor evolution.

BackgroundUrothelial carcinoma (UC) is a biologically heterogeneous disease, and current molecular classifications have limited integration into clinical decision‐making. To further pursue precision oncology efforts in UC, we developed a molecular classification framework applicable to transcriptomic and proteomic data from non–muscle‐invasive bladder cancer (NMIBC), muscle‐invasive bladder cancer (MIBC) and urothelial cancer cell lines.MethodsUsing a whole‐transcriptome self‐organised map and regularised semi‐supervised clustering of 4439 bulk NMIBC and MIBC transcriptomes and proteomes, and 33 UC cell lines, we identified three molecular UC clusters. Making use of both in silico and in vitro approaches, we selected promising treatment approaches for each cluster.ResultsThe three developed clusters displayed distinct signatures of mRNA, proteins, biological processes, metabolism and essential driver genes. They also differed in prognosis and machine learning‐predicted treatment vulnerabilities and resistance. High‐risk, stroma‐rich Cluster #1 cancers were predicted to respond to selected cytotoxic drugs, ferroptosis inducers and PARP inhibitors. For the aggressive, fast‐proliferating, immune‐infiltrated Cluster #2 tumours with basal/squamous differentiation, cytotoxic agents and EGFR/ERBB‐ and MEK/ERK‐targeting therapies were proposed. Cluster #3 cancers of predominantly luminal papillary phenotype with scarce stroma and immune infiltration were enriched with NMIBC and low‐risk malignancies. For patients with Cluster #3 tumours, selected epigenetic drugs or EGFR/FGFR inhibitors may represent attractive treatment options.ConclusionsOur novel molecular taxonomy holds promise as a practical framework for patient risk stratification and clinical trials in UC. Our molecular classification scheme may facilitate personalised transcriptome‐ and proteome‐based risk assessment and clinical trial design for the development of various therapeutics.key pointsWe developed three UC clusters, applicable for MIBC and NMIBC, which were validated using transcriptomic- and proteomic datasets.Publically available UC cell lines were assigned to the clusters, to have in vitro models representing each cluster.The clusters differ in molecular and biological signatures, with distinct prognostic and therapeutic characteristics.

Abstract Emerging data from clinical trials suggests that mechanisms that reactivate the MAPK pathway via gene amplification events can drive resistance to RAS inhibitors. This includes amplification of KRAS itself, as well as RAF1, RICTOR , and MYC. How amplification events affect signaling networks and the implications for therapeutic opportunities to overcome resistance remain unclear. To model this, we employed RMC-7977, a RAS(ON) multi-selective inhibitor structurally related to the clinical candidate daraxonrasib (RMC-6236), and established a RMC-7977–resistant (RMC-7977-R) H358 isogenic model that exhibit RAF1 amplification, as confirmed by amplicon-level analysis of whole-exome sequencing (WES), western blotting, and RNA sequencing. We evaluated this resistant cell line responses to a panel of RAS pathway–targeting agents, including RAF inhibitors (LXH-254 and exarafenib), MEK1/2 PROTAC degrader (MS934), pan-RAF-MEK non-degrading molecular glue (NST-628), RAF/MEK clamp (avutometinib) and MEK1/2 inhibitor (trametinib). Cell viability analysis demonstrated increased sensitivity of RMC-7977 resistant cells to RAF inhibitors compared to the parent H358. Combinations of MEK-targeting agents with either RAF inhibitors or RMC-7977 more effectively suppressed ERK phosphorylation in resistant cells compared to RAF inhibitors combinations with RMC-7977. However, combining RAF inhibitors with RMC-7977 or MEK-targeting agents (MS934, NST-628, avutometinib, or trametinib) more potently inhibited cell proliferation in resistant cells compared with combinations of RMC-7977 with MEK-targeting agents. In the context of siRNA-mediated RAF1 knockdown in RMC-7977 resistant cell line, trametinib treatment reduced ERK phosphorylation and inhibited cell proliferation more deeply than RMC-7977. This suggested that RAF1 may have additional signaling mechanisms beyond activation of MEK/ERK. To elucidate the mechanistic basis of LXH-254 activity in RAF1-amplified cells, we performed RNA sequencing and transcriptional analyses in cells treated with LXH-254, which revealed enrichment of G2/M checkpoint, MYC target, mitotic spindle, E2F target, and DNA repair signatures. Consistent with these observations, RAF inhibitors induced G2/M cell cycle arrest and apoptosis in RAF1-amplified cells, and the apoptotic response was enhanced when combined with RMC-7977 or MEK-targeting agents. Resistant cells exhibited increased nuclear RAF1 expression compared with parental H358. Co-immunoprecipitation experiments reveal enhanced interactions between RAF1 and the mitotic kinases PLK1 and AURKA in resistant cells. Our data indicates that RAF1 amplification rewires signaling states by affecting nuclear G2/M signaling networks in addition to the MEK/ERK pathway. More broadly, our results suggest that RAF1 gene amplification associated with RAS inhibitor resistance may produce neomorphic signaling states that affect therapeutic sensitivity. Citation Format: Hitendra S. Solanki, Denis Imbody, Bina Desai, Andriy Marusyk, Eric B. Haura. RAF1 gene amplification drives RAS inhibitor resistance via nuclear translocation and interactions with PLK1 and Aurora A kinases [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B026.