Background: The mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that integrates inputs on nutrient status, energy levels, and growth factor stimulation to accordingly regulate cell growth and metabolism. It does this by activating or repressing target proteins covering a broad array of cellular processes. mTOR nucleates two structurally and functionally distinct protein complexes, mTORC1 and mTORC2. Because of their wide-ranging effects in the cell, both mTOR complexes are presumed to have a large number of targets. However, only a relatively small number have been conclusively identified. Methods: With emphasis on mammalian mTOR, we previously reviewed the extensive mTOR literature (1991-2021) and compiled a list of all reported substrates of mTORC1 and mTORC2. We have updated this list for the period 2022-2025. Results/Conclusions: Many of the targets are involved in autophagy, underscoring the major role of mTOR in the regulation of this process. From the perspective of this Special Issue, targets linked to cancer may be responsible for executing an mTOR-driven pro-oncogenic program and merit future study.

Abstract Background Multifocal hepatocellular carcinoma (mfHCC) arises via intrahepatic metastasis (IM) or multicentric occurrence (MO), each with distinct biological behavior and clinical implications, though mfHCC origin is rarely assessed in clinical practice. We aimed to characterize the clinicopathological and molecular features of IM-HCC, MO-HCC, and their surrounding non-tumor liver (NTL) tissues using multi-omics analyses. Methods We analyzed 76 tumor and 44 NTL biopsies from 22 patients, using whole-exome sequencing, RNA-sequencing, and proteomic/phosphoproteomic profiling. Patients were classified as IM, MO or mixed (IM + MO) according to their somatic mutations. A comparator cohort of 48 unifocal HCC and 15 normal livers was used. Clinicopathological parameters, pathway and transcription factor activities, immune infiltration, and targetable alterations were assessed. Results Clonality analysis identified 10 IM, 9 MO, and 3 mixed patients. IM-HCCs showed more frequent macrovascular invasion and extrahepatic metastases, with upregulation of E2F/MYC-related cell cycle pathways, higher expression of metastasis-associated genes (e.g., TTK, BUB1, NUF2), higher CD8 + T-cell exhaustion, and shared actionable mutations (e.g. PTEN). MO-HCCs within patients displayed molecular dissimilarity comparable to tumors from different patients, though they also showed convergent kinase and pathway dysregulation. NTLs of IM-HCC patients had lower fibrosis, extracellular matrix signaling and pro-regenerative pathways (e.g., SOX2, TGFA) than those of MO-HCC. Conclusions The aggressive molecular features and immune exhaustion of IM-HCC support the need for combined therapies, while the convergence of kinase and pathway dysregulation of MO-HCC provides unified therapeutic opportunities. Key differences in fibrogenic and regenerative pathways may influence metastatic potential. Our findings provide insights into the biological behavior and therapeutic opportunities in mfHCC.

Abstract There is growing recognition of how metabolic dependencies influence sensitivity or resistance to neuroblastoma (NB) therapies. In this regard, the impact of Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase which is mutated in approximately 10% of primary NB cases, and a clinical target in NB on cellular metabolism is unclear. In this study, we combined phosphoproteomics and BioID-mediated proximity interaction screening in neuroblastoma cell lines, identifying the sodium bicarbonate transporter SLC4A7, a member of the Solute Carrier (SLC) family, and the multifunctional protein Carbamoyl Phosphate Synthetase 2-Aspartate Transcarbamylase-Dihydroorotase (CAD) downstream of ALK signaling in NB cells. SLC4A7 and CAD are both important molecular components in the metabolism of pyrimidine nucleotides. ALK activation leads to phosphorylation of both SLC4A7 and CAD, resulting in increased sodium bicarbonate transport and pyrimidine biosynthesis that is blocked by the addition of ALK tyrosine kinase inhibitors (TKIs). Combined targeting of both ALK signaling and nucleotide synthesis leads to a more effective inhibition of NB tumor growth in both cell and genetically modified mouse NB models. Finally, we show that SLC4A7 and CAD are also targets in ALK-driven non-small cell lung cancer (NSCLC), revealing a novel therapeutic strategy that leverages metabolomic dependencies to target ALK-driven malignancies such as NB and NSCLC. Citation Format: Ruth Palmer, Wei-Yun Lai, Tzu-Po Chuang, Joel Johansson, Emre Can Tuysuz, Dan E. Lind, Alexander Schmidt, Michael N. Hall, Bengt Hallberg. Anaplastic lymphoma kinase harnesses the SLC4A7 bicarbonate transporter to intensify de novo nucleotide synthesis in neuroblastoma and lung cancer [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 4732.

Identification of a deep intronic PTEN pathogenic variant, which was not detected by standard DNA-targeted panel sequencing but was uncovered by targeted PTEN RNA sequencing using CaptureSeq technology, illustrates the added value of concurrent DNA and RNA analyses in risk assessment for PTEN Hamartoma Tumor Syndrome (PHTS) and in patients given the diagnosis of clinical Cowden Syndrome (CS). These findings have significant clinical implications, including providing the rationale for testing patients meeting clinical criteria for PHTS/CS with concurrent DNA and RNA testing. It also supports reevaluation of patients who test negative by DNA testing alone but with a clinical diagnosis of PHTS/CS with subsequent RNA testing to identify and clinically interpret previously undetected deep intronic PTEN variants. Where cancer treatment and prevention decisions hinge on correct diagnoses, concurrent DNA and RNA testing rather than stepwise testing can permit faster, more accurate results and earlier clinical actionability.

PURPOSE:Growing use of multigene panels (MGPs) is increasing the number of patients identified with multiple pathogenic germline variants (PGVs) in cancer predisposition genes. This study characterizes the landscape of patients with multiple PGVs and identifies clinical settings where multiple PGVs affect management. MATERIALS AND METHODS:This is a single-institution retrospective cohort analysis comprising patients seen in the Department of Clinical Genetics and consented to the Risk Assessment Program (RAP) Registry who were evaluated with a MGP and found to have multiple PGVs. RESULTS:All patients tested between January 1, 2014, and January 1, 2024, and found to have multiple PGV are included. Sixty-four patients (64/7,961, 0.8%) from 58 families carried multiple PGVs, 22/64 (34%) patients carried at least two PGVs in high- or moderate-risk genes, and 33/64 (52%) carried at least two PGVs that result in potential management changes. Five percent (30/557) of all patients with a PGV in BRCA1 or BRCA2 also carried an additional PGV, while 7% (19/284) of patients with a PGV in a mismatch repair (MMR) gene also carried an additional PGV. Ten patients from nine unrelated families had both a PGV in BRCA1 or BRCA2 as well as a PGV in an MMR gene. CONCLUSION:Although the overall percentage of patients undergoing clinical genetic testing with multiple PGVs is small, a significant fraction of these patients could benefit from medical management changes because of the identification of multiple PGVs.

Hepatocellular carcinoma features extensive metabolic reprogramming. This includes alterations in major biochemical pathways such as glycolysis, the pentose phosphate pathway, amino acid metabolism and fatty acid metabolism. Moreover, there is a complex interplay among these altered pathways, particularly involving acetyl-CoA (coenzyme-A) metabolism and redox homeostasis, which in turn influences reprogramming of other metabolic pathways. Understanding these metabolic changes and their interactions with cellular signaling pathways offers potential strategies for the targeted treatment of hepatocellular carcinoma and improved patient outcomes. This review explores the specific metabolic alterations observed in hepatocellular carcinoma and highlights their roles in the progression of the disease. Cancer cells often change their metabolism to support rapid growth. This study examines how liver cancer, specifically hepatocellular carcinoma (HCC), alters its metabolism and explores potential treatments. The authors focus on how HCC cells use glucose, amino acids and fatty acids differently from normal cells. HCC cells rely heavily on glycolysis even when oxygen is present, a phenomenon known as the Warburg effect. They also activate the pentose phosphate pathway to produce molecules needed for growth and survival. Additionally, HCC cells depend on certain amino acids such as glutamine and serine for building blocks and energy. This study highlights that targeting these altered metabolic pathways could be a promising strategy for treating HCC. In conclusion, understanding these metabolic changes in HCC can lead to new treatment approaches. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Post-translational modification of proteins by polyamines (putrescine, spermine and spermidine) is poorly studied due to technical challenges in identification. Polyamination is mainly performed by transglutaminases via transamidation of a polyamine to an acceptor glutamine-residue on the target protein. We performed polyamination reactions on whole cell lysates using 2 different polyamine probes (Biotin-pentylamine and DNP-pentylamine) and identified modified peptides by mass spectrometry. 51 protein targets modified on 66 different sites were identified, with an overlap between hits tagged with the different probes. Many of the targets are involved in translation, cytoskeletal organization or have roles in cancer. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, 310030_192493

IntroductionTumor genomic profiling (TGP) is used to optimize cancer treatment but is underutilized by Black patients, despite having disproportionately higher cancer morbidity and mortality. No interventions using electronic health decision support tools (eHealth DSTs) have been developed to assist patients with understanding this test or address barriers to uptake and communicating preferences with a doctor.MethodsUsing the Ottawa Decision Support Framework, we systematically developed the Gene Pilot eHealth DST with Black cancer patients. We conducted qualitative focus groups (five groups, N = 33) and surveys (N = 121), elicited community advisory board feedback (N = 10) to devise DST content and communication strategies, and then conducted user testing (N = 10). Content was informed by commercial marketing techniques - segmentation, perceptual mapping, vector message modeling – to elucidate how medical mistrust was an important construct to address in Gene Pilot.ResultsUser testing (1–7 scale) indicated Gene Pilot was highly accepted, including ease of use (M = 6.67, SD = 0.50), that it addressed important barriers such as medical mistrust and genetic literacy (6.63, SD = 0.52), and allowed patients to prepare for the decision (M = 6.44, SD = 0.73) and to communicate with their doctor (M = 6.33, SD = 0.73). Suggestions for improved navigability were addressed.ConclusionOverall, Gene Pilot was found to be acceptable, suggesting its readiness for efficacy testing.

Background Identifying and eliminating health disparities is a public health priority. The goal of this analysis is to determine whether cardiac testing or outcome disparities exist by race or sex in patients with detectable to mildly elevated serum troponin. Methods We conducted a secondary analysis of the CMR-IMPACT trial that randomized patients with symptoms suggestive of acute coronary syndrome and a detectable or mildly elevated troponin measure from 4 US hospitals to an early invasive angiography or cardiac MRI strategy. The primary endpoint was the composite of all-cause mortality, myocardial infarction, cardiac hospital readmission, and repeat cardiac ED. Secondary outcomes were components of the composite and revascularization. Results Participants (n = 312, mean age 61 ± 11 years) were 36.2 % non-white and 40.1 % female. The composite outcome occurred in 63.7 % of non-white vs. 49.8 % of white patients (aHR 1.50, 95 % CI 1.08–2.09) and 53.6 % of female vs. 55.6 % of male patients (aHR 0.93, 95 % CI 0.68–1.28). Non-white (aHR 0.57, 95 % CI 0.35–0.92) patients had lower rates of revascularization also less median stenosis (p < 0.001) and stenosis >70 % (p < 0.001) during index cardiac testing. Despite these findings, ACS after discharge was higher among non-white patients (aHR 1.84, 95 % CI 1.11–3.05). Females had lower rates of revascularization (aHR 0.52, 95 % CI 0.33–0.82), but no increase in ACS after discharge (aHR 0.90, 95 % CI 0.55–1.49). Conclusion Non-white patients had higher rates of ACS following discharge despite lower rates of obstructive CAD following standardization of index cardiac testing. Future disparity works should explore care following the index encounter.

Tumor response rates to targeted therapy are generally low. This poses an urgent medical need to identify molecular mechanisms determining responsiveness to inform therapy. We describe the first longitudinal deepscale multi-omic analysis (genomics, transcriptomics, proteomics and phosphoproteomics) of tumor biopsies from eight hepatocellular carcinoma (HCC) patients treated with the targeted kinase inhibitor sorafenib. Three patients were sorafenib responders and five were nonresponders. Resistance did not correlate with a single mutated gene but with genomic instability, especially gain of Chr1q. The patients clustered by therapy response, based on proteomics and phosphoproteomics. Nonresponder tumors showed increased EMT, glycogen storage, splicing and ribosome biogenesis, and decreased carbohydrate and drug metabolism. Resistance also correlated with deregulated phosphorylation of AMPK and NOTCH pathway components. Our data suggest a correlation between dedifferentiation and resistance not evident based on classical clinical staging. We propose potential novel biomarkers to identify and drug combinations to treat sorafenib resistant patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by European Research Council Synergia grant 609883 MERiC (M.H., N.B., M.N.H). Other funding: Swiss National Science Foundation (M.N.H), Goldschmidt-Jacobson-Stiftung (E.D.) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethics committee of the Canton Basel, Switzerland gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes 1) The genomics (CNA and SNV) and transcriptomics data of the longitudinal study cohort have been deposited in the European Genome-phenome archive (EGA) with the accession IDs EGAS00001005661 (WES) and EGAS00001005662 (RNA). 2) The proteomics and phosphoproteomics data of the longitudinal study cohort have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al, 2019) partner repository with the dataset identifier PXD028593. 3) The proteomics and phosphoproteomics data of the independent cohort (data partially published in Ng et al., 2021) have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al, 2019) partner repository with the dataset identifier PXD034593 and PXD035238.

e15655 Background: LS is among the most common hereditary CA syndromes, characterized by autosomal dominant inheritance of mismatch repair deficiency with increased lifetime risk of multiple CA types. While intensive screening can lower risk of LS-associated CAs, additional preventive measures could be broadly beneficial for motivated patients (pts). Our recent study (Singhal S et al, JCO Precision Oncology 2024), however, found that pts with LS reported only modest uptake of daily ASA/NSAIDs, a proven prevention option. In this study, we examine perceptions of dietary interventions as an alternative CA prevention option in a multi-national cohort of pts w/LS. Methods: PREVENTLynch study recruited pts w/LS to complete an online survey about screening behaviors and perceptions of novel prevention options. Pts were recruited through the Fox Chase Cancer Center (FCCC) Risk Assessment Program Registry and invitations posted via LS advocacy websites. In addition to reporting demographic factors, personal history, and screening behaviors, pts rated five dietary interventions: resistant starch supplements (RS), vitamin supplements (VS), online nutritional feedback (ONF), intermittent fasting (IF), and low inflammatory index diet (LID) (scale 0-10, low-to-high). Pts evaluated each intervention for perceived inconvenience, concern for side effects, reassurance to reduce CA risk, and study participation interest. Perception scores were colonoscopy (CO) benchmarked. Differences were tested by rank sum testing (Wilcoxon). The study was IRB approved (FCCC #20-8014). Results: A total of 296 responses were received. Participants were diverse by age [8% < 35 yr, 74% 35-65 yr, 18% > 65 yr] and location (39 states; 15% non-US based) but predominantly female (84%) and non-Hispanic white (92%). VS (1.77), ONF (1.94) and RS (2.00) were significantly more convenient than CO (3.40) (p < 0.001). Perceived side effects of ONF (1.81) (p < 0.001) were lowest compared to CO (2.46), especially in US participants (1.73) vs non-US (2.31) (p < 0.05). In contradistinction, CO reassurance (8.25) was significantly higher than all interventions (VS 5.04, RS 4.85, NF 5.72, OF 4.95, LI 5.04, all p < 0.001). Notably, pts with neoplasia history had the lowest reassurance from IF (4.73, p < 0.001), while non-US participants rated VS (3.49 vs 5.28), IF (4.08 vs 5.10) and LI (4.4 vs 5.24) (all p < 0.001) significantly less favorably. Study participation interest was highest for RS (7.31) and NF (7.06) and lowest for IF (6.31). Conclusions: Dietary interventions show promise as low-risk adjuncts to CO surveillance with favorable perceived acceptability and side effects among LS pts. Our findings highlight the importance of considering pt preferences when developing and testing risk-reducing interventions to maximize real-world uptake and downstream effectiveness.

e22598 Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PTHS) is a family of related genetic disorders including Cowden Syndrome (CS), Bannayan-Riley-Ruvalcaba Syndrome (BRRS), and Proteus Syndrome (PS) associated with distinct phenotypes and increased CA risk. Germline PTEN PVs remain unidentified in many patients (pts), including 40% w/BRRS, 80% w/PS, and 15% w/CS. Standard DNA testing may fail to identify clinically relevant deep intronic PTEN PVs detectable by comprehensive testing that includes RNA analysis. Using a commercial laboratory database, we identify pts tested by DNA/RNA and found to harbor deep intronic PVs associated with alternative RNA splicing of PTEN and predicted to cause PHTS. Methods: Pts tested by paired DNA/RNA testing (2019-2024, Ambry Genetics) and found to have a deep intronic PV leading to a splice site variant were identified. DNA/RNA results were linked to clinical data from a test requisition form. RNA was isolated using standard methodology and converted to cDNA by RT-PCR. Sequence enrichment of the targeted coding exons and adjacent intronic nucleotides is carried out by a bait-capture methodology using long biotinylated oligonucleotide probes followed by PCR and NGS. Absence or presence of RNA transcripts meeting quality thresholds is incorporated as evidence towards classification of DNA variants. Study was IRB reviewed (FCCC IRB #24-9030/Ambry WCG IRB exempt). Results: Index case: 64 yo F w/hx of breast CA (42 & 53) and thyroidectomy for nodules and papillary thyroid CA was referred for genetic counseling following colonoscopy showing extensive ganglioneuromatosis. Exam found macrocephaly (59.5 cm), tongue papules, likely facial trichilemmomas, and axillary acrochordons. RNA testing identified a deep intronic PV at c.209+2047A>G resulting in alternative PTEN splicing. In the combined DNA/RNA testing database (n=489,960), 17 additional individuals/families undergoing PTEN testing were identified with a deep intronic PV/LPV (n=14) or suspicious VUS (n=3). In total, 12 probands reported CA (9 <50 yrs) including cases of early breast (n=4, 38-63 yrs), thyroid (n=1, 25 yrs), salivary (n=1, 31 yrs), uterine (n=1, 46 yrs), bladder (n=1, 29 yrs), prostate (n=1, 66 yrs) and ovary CA (n=3, 28-32 yrs). Other PHTS features reported in probands/families: macrocephaly (n=7), colon ganglioneuromas and/or polyposis (n=7), goiter/thyroid nodules (n=6), penile freckling (n=2), skin findings (n=3), and intellectual disability (n=2). Potentially clinically relevant co-occurring variants were detected in NF1 (x3), GALNT12 (x2), BRCA2 and SDHC . Conclusions: Deep intronic PTEN PVs missed by DNA sequencing but detectable by RNA testing and associated with early-onset CA explain a portion of negative results in pts meeting clinical criteria for CS and/or manifesting phenotypic features of PHTS.

10574 Background: Approximately 10% of cancers stem from inherited germline pathogenic variants (gPV), predominantly in DNA repair genes including ATM and BRCA1 or BRCA2 . Identification of a gPV in high-risk families can guide management, however the clinical implications of variants of uncertain significance (VUS) identified by genetic testing remain unclear. Our aim is to develop a DNA damage response (DDR) activity profile of high-risk populations with/without cancer with ATM or BRCA2 gPV to assist in determining the relevance of ATM or BRCA2 gPV/VUS. Methods: We profiled several key proteins that play a role in DDR using a Luminex-based Multianalyte immunoassay (hereafter referred to as DDR xMAP) in peripheral blood monocytes (PBMCs) derived from whole blood. The standardized DDR xMAP assay was first applied to PBMC specimens from sporadic colorectal cancer (CRC, n = 95) patients and cancer-free age-matched controls (n = 47) at baseline. The DDR xMAP was used to profile seven DDR proteins, phosphorylated Chk1 S345 , Chk2 T68 , γH2AX S139 , p53 S15 and total ATR, MDM2, p21. Univariate classification and regression tree analysis was used to identify statistically significant cut points in DDR analyte levels. We then measured the DDR analyte levels in individuals with BRCA2 gPVs with (n = 11) and without a diagnosis of cancer (n = 11) as well as in cancer-free non-carrier controls (n = 15) at baseline. We compared these values using the two-sided Mann-Whitney test and the Benjamini-Hochberg false discovery rate method to account for multiple markers. Results: Using the initial set of CRC cases and healthy controls, we identified statistically significant cut points in multiple DDR analyte levels including total ATR ( > 81.8), Chk1 S345 ( > 28.0) and γH2AX S139 ( > 51.3) that can individually distinguish between CRC cases and cancer-free controls (P < 0.001). Next, in preliminary analysis of patients with BRCA2 gPV with and without cancer had increased levels of all DDR analytes (P < 0.05) compared to non-carrier cancer-free controls, except for γH2AX S139 . Levels of proteins involved in replication stress response, ATR and downstream Chk1 S345 , were elevated. Total MDM2 levels, negative regulator of p53, were highly elevated in BRCA2 gPV carriers. There was not a significant difference between DDR analytes for those with BRCA2 gPV with and without cancer. Conclusions: Profiling key DDR markers can significantly distinguish between CRC patients and cancer-free controls. DDR analytes are significantly increased in PBMC specimens from individuals with BRCA2 gPV (with or without a diagnosis of cancer) compared to non-carrier cancer-free controls. Ongoing research in high-risk groups will establish cut points for this assay, improving our understanding of DDR activity in individuals with ATM/BRCA2 gPV and clarifying the clinical significance of gPV/VUS.

Cancer interception represents a new approach, aimed at targeting precancerous lesions and therefore preventing cancer occurrence. Lynch syndrome (LS) is one of the most prevalent hereditary cancer syndromes, with an estimated prevalence of one in 300, and high-risk predisposition to several types of cancer, with up to 80% lifetime risk to develop CRC. LS is caused by germline mutations in one of the DNA mismatch repair (MMR) genes. Loss of MMR proteins causes an accumulation of mutations in the microsatellite sequences, known as Microsatellite instability (MSI), that can lead to shared frameshift mutations (FSP). Nous-209 is a neoantigen directed immunotherapy encoding 209 FSP shared across sporadic and hereditary MSI tumors and precancer lesions in clinical development for interception and treatment of MSI tumors (Leoni et al., 2020; NCT04041310). Here, we report the full set of safety and immunogenicity results of a Phase Ib/II study of Nous-209 monotherapy in LS carriers. NCT05078866 is a Phase Ib/II single-arm, open-label, trial of Nous-209 for cancer interception in LS carriers assessing as co-primary endpoints the safety and immunogenicity against the FSP neoantigens encoded by Nous-209. Nous-209 is administered intramuscularly: one prime with a Great Ape Adenovirus (GAd20-209-FSPs) on day 1, followed by a boost with a Modified Vaccinia Ankara (MVA-209-FSPs) at week 8. Immunogenicity was measured by ex-vivo IFN-γ ELISpot assay utilizing pre- and post-vaccination peripheral blood mononuclear cells (PMBCs) samples collected at different timepoints. The trial fully enrolled 45 LS carriers, all receiving GAd20-209-FSPs and MVA-209-FSPs. Most pts (21 of 45, 46.7%) harbored MSH2 pathogenic germline variants. 19 of 45 (42.2%) pts had a prior history of cancer. Treatment was safe and well tolerated with no treatment-related serious adverse events (AEs) and no unexpected reactogenicity. The most common AEs were injection site reactions (any grade: 84% post-prime, 73% post-boost; grade 3: 0%) and fatigue (any grade: 78% post-prime, 51% post-boost; grade 3: 4%). Neoantigen specific immune responses post Nous-209 were observed in 100% of evaluable participants (37/37), with induction of robust T cell immunity reaching a mean of T cell response at peak of ∼ 1100 IFN-γ spot forming cells (SFC) per million of PBMC, polyfunctional and long lasting CD8 and CD4 T cell responses broadly recognizing multiple FSPs. The use of peptide binding HLA predictions allowed the precise identification of several immunogenic FSPs, shown to be present in independent datasets of MSI premalignant lesions and cancers in LS carriers. The complete safety and immunogenicity results from this Phase Ib/II trial support the further development of Nous-209 monotherapy, highlighting its potential to efficiently stimulate the immune system and intercept cancer in LS carriers. Jason Willis, Anna Morena D'Alise, Michael J. Hall, Marcia Cruz-Correa, Gregory E. Idos, Selvi Thirumurthi, Veroushka Ballester, Guido Leoni, Irene Garzia, Laura Antonucci, Lorenzo De Marco, Elisa Micarelli, Sven Gogov, Wenli Dong, J. Jack Lee, Lana A. Vornik, Araceli Garcia-Gonzalez, Laura Reyes-Uribe, Ellen Richmond, Asad Umar, Powel H. Brown, Luz Maria Rodriguez, Elisa Scarselli, Eduardo Vilar. Nous-209 off-the-shelf neoantigen immunotherapy induces robust neoantigen T cell response with the potential to intercept cancer in Lynch syndrome carriers [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 6427.

Background/Objectives: Medical mistrust (MM) is associated with adverse health outcomes, but few studies have assessed MM in cancer patients. MM is frequently measured using the Medical Mistrust Inventory (MMI), measuring institutional MM (e.g., government), or the Group-Based Medical Mistrust Scale (GBMMS), measuring race-based MM. We sought to assess the prevalence of MM among cancer patients diverse by age, sex, race/ethnicity, and socioeconomic status (SES), recruited from an urban safety net hospital and a suburban comprehensive cancer center. Methods: Patients completed a one-time survey. The primary outcome was MM as measured by the GBMMS and MMI tools. Covariates included demographics, treatment campus (urban vs. suburban), and psychosocial measures relevant to MM. Results: Purposeful sampling recruitment resulted in 200 participants (survey completion: 74.6%). The median age was 60 years, with 62% female, 45% African-American, 15% Hispanic, 47.5% education ≤ HS diploma, and 51.5% income ≤ USD 50,000/yr. Elevated MMI and GBMMS scores (moderate-to-high) were seen, respectively, in Hispanic (20.7% and 33.4%) and African-American (AA) patients (31.8% and 48.9%), compared with White patients (14.3% and 9.9%). The MMI and GBMMS tools captured complimentary aspects of MM in cancer patients (Spearman’s 0.531, p < 0.0001). MMI was associated with lower education (0.034) and race (p = 0.04), while GBMMS was strongly associated with race (p < 0.001), urban campus (p = 0.035), and mistrust of government/health organization information (both p < 0.05). Higher MMI/GBMMS scores were both associated with research mistrust and mistrust of information from physicians. Conclusions: Institutional and race-based MM are prevalent among cancer patients diverse by age, sex, race/ethnicity, and SES. Lower education was associated with institutional MM but not race-based MM.

Polyamines are abundant metabolites that are involved in many cellular processes. Despite playing wide-ranging and essential roles in the cell, only a few examples of a specific polyamine function are known. Polyamination is the post-translational modification of a protein by polyamines (putrescine, spermidine or spermine). This reaction is catalyzed by transglutaminases (primarily TGM2) via a transamidation reaction that conjugates a polyamine to an acceptor glutamine in a target protein. Protein polyamination is poorly characterized due to technical challenges in detecting the polyaminated adduct, and is neglected in most proteomic surveys. We performed polyamination reactions using whole cell lysates from a mouse liver cancer cell line with elevated TGM2 expression. Two differently tagged polyamine analogs (Biotin-pentylamine and DNP-pentylamine) with distinct molecular masses were used, and the respective modified peptides were identified by mass spectrometry. 51 protein targets modified on 66 different sites were identified, in some cases with both donor polyamines. Many of the targets are involved in translation or cytoskeletal organization, and implicated in cancer.

All patient clinical, demographic, and molecular data in this manuscript (de-identified).

Characterization of POLE mutations in the CLS and TCGA dataset. A, Flowchart and analysis tree for colorectal cancer (CRC), endometrial cancer (EC), and ovarian cancer (OC) tumors by POLE mutations, TMB, and MSI/MSS status. Among 1,870 colorectal cancer, 4,481 endometrial cancers, and 8,910 ovarian cancer tumor genomic profiles, a total of 447 carried POLE mutations. Clinically relevant TMB cut-off points were used to define the TMB-H (≥10 mut/Mb) and TMB-L (<10 mut/Mb) cohorts. POLE mutation cohorts along with TMB and MSI/MSS status were defined. TMB-L tumors with POLE variants but no established POLE ExoD driver are referred to as “POLE Variants TMB-L” (Group 1, MSS or MSI). TMB-H tumors with known POLE ExoD driver only were referred to as “POLE ExoD Driver” (Group 2, MSS or MSI). TMB-H tumors with co-occurring POLE ExoD driver and POLE variant(s) were referred to as “POLE ExoD Driver + POLE ExoD Variant” (Group 3, MSS or MSI). TMB-H tumors with only POLE variant(s) and no POLE ExoD driver were referred to as “POLE Variant TMB-H” (Group 4, MSS or MSI). B, Age distribution of patients in the CLS cohort with POLE-mutated tumors (n = 447) designated as Group 1 (green), Group 2 (red), Group 3 (purple), and Group 4 (blue). mTMB comparisons between Group 2 and 3 colorectal cancers (C), endometrial cancers (D), and ovarian cancers (E). mTMB comparisons between Group 2 and 3 genomic profiles of colorectal cancer (F), endometrial cancer (G), and ovarian cancer (H). MSI-H tumor profiles were removed from this analysis. TCGA cohort mTMB comparisons between Group 2 and 3 tumors, in I MSI-H or MSS tumor profiles were included and in J only MSS tumor profiles were included. Because of smaller sample size per tumor type, analyses were pooled. A Mann–Whitney test was performed and ***, P < 0.001; *, P < 0.05; NS, nonsignificant.

77 Background: LS is among the most common cancer (CA) syndromes affecting 1:280 individuals and portends high lifetime CA risk. Clinical trials are currently evaluating whether frameshift peptide vaccination (FSPVAX) could reduce LS CA risk. FSPVAX enhances immune response to diverse FSP neoantigens in MSI-H tumors and has shown promising results in an MSH2-mutant mouse model and metastatic MSI-H CRC. However, negative public attitudes towards vaccination and evidence suggesting low uptake of ASA chemoprevention (CP) among patients with LS could undermine success of this novel therapy. Here, we benchmark attitudes towards FSPVAX for CA prevention against colonoscopy (COLO) and aspirin or NSAID chemoprevention (CP). Methods: The PREVENTLynchII survey (IRB 20-8014) was distributed electronically to 1) participants in the Fox Chase Cancer Center (FCCC) Risk Registry and 2) members of two online LS support groups. Patients were invited to complete the one-time survey after providing informed consent. Demographic and personal CA history were collected. Attitudes were measured on 9-point scales indicating low vs high agreement with belief statements: e.g. “Colonoscopy is a convenient way to lower my risk of LS-related CA.” Results: Overall 296 participants at FCCC, nationally, and internationally completed the survey. Median age was 52.6 years [23-78], 84% female, and 95% White. Affected genes included MLH1 (20.3%), MSH2/EPCAM (35.5%), MSH6 (28.7%), and PMS2 (15.5%). Use of CP was uncommon (32.1% and 5.1% respectively). Stratified analyses of COLO perceptions by sex, age, and personal history of CA showed no differences. Interestingly, US pts viewed ASA as more convenient than non-US, and with lower risk of side effects (p=0.007), but were no more likely to use or recommend CP. Compared to COLO, FSPVAX was overall viewed as more convenient, but side effect concerns were higher, and perceptions of efficacy were lower (all p<0.001), while compared to CP, FSPVAX was less convenient and fostered greater concerns about side effects, but was viewed as having higher potential preventive efficacy (all p<0.001). FSPVAX perceptions showed high variability: pts >50 (p=0.003) and CA pts (p=0.01) saw FSP as more convenient (p=0.003) than younger and unaffected pts. CA pts reported higher reassurance than unaffected (p=0.04), while those >50 were more likely to participate in an FSPVAX study (p=0.04). US pts perceived more side effect risk of FSPVAX than non-US (p=0.003). Conclusions: Concerns about ASA CP and FSPVAX convenience, side effects, and efficacy are common among US and international patients with LS. [Table: see text]

Mutations in Group 3 tumors with P286R or V411L plus one variant and mTMB comparisons.