Tobacco use remains the leading preventable cause of cancer mortality worldwide, with marked disparities in cancer incidence and outcomes across racial and ethnic groups. This study investigates the genetic determinants of tobacco carcinogen metabolism among smokers, focusing on ancestry-informative markers (AIMs) near genes involved in metabolic activation (Phase I), detoxification (Phase II), and transport (Phase III) of tobacco-specific carcinogens. Using data from 274 smokers recruited in the United States and the Caribbean, we measured urinary NNAL (a metabolite of NNK) to classify metabolic phenotypes and performed genotyping with a customized array enriched for AIMs. Most participants (94%) were classified as poor metabolizers of NNAL, with a higher prevalence among Black/African American smokers. Multivariable analyses, after adjusting for age, sex, assay batch, and number of cigarettes smoked per day, identified 14 AIMs near Phase I, II, and III pathway genes significantly associated with NNAL metabolizer phenotype, with African ancestry alleles conferring increased odds of poor metabolism. Functional annotation revealed that most significant AIMs overlapped regulatory regions, and the CYP3A5 rs776746 variant demonstrated structural disruption likely to impair enzymatic activity. Majority of participants had high African ancestry and were poor NNAL metabolizers, consistent with the observed association between African ancestry and poor NNAL metabolism. These findings highlight the biological basis for tobacco-related cancer disparities and underscore the importance of integrating genetic ancestry and metabolic phenotyping in risk assessment and personalized prevention strategies for tobacco-associated cancers.

98 Background: Pts w/LS have high lifetime risk of colorectal cancer (CRC). Intensive colonoscopy surveillance is standard in LS, but recent guideline updates have de-escalated screening intervals and favor segmental over total colectomy for LS pts w/CRC, increasing risk of interval cancers (CAs) and more advanced CAs at diagnosis. Retrospective studies have linked statin use to reduced risk of CRC. Preclinical data from our team identified lower incidence of intestinal polyps in mice exposed to Atorva+/-ASA especially when exposure preceded any neoplasia. Based on these findings, we initiated a two-arm trial to examine metabolic, epithelial and molecular impact of Atorva+/-ASA in pts w/LS. Effective recruitment, retention, adherence, and acceptability are critical to research. Methods: Pts w/LS were identified via the Fox Chase Cancer Center (FCCC) Risk Assessment Program (RAP) registry. Pts had a germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM and were undergoing screening colonoscopy at FCCC. This was a non-randomized 2-arm trial: after biosample (blood) and baseline colonoscopy w/biopsy, pts w/hx of adenoma or CRC were enrolled on Arm B (Atorva 20 mg+ASA 325 mg x 6 wks), while those with no neoplasia hx were enrolled on Arm A (Atorva 20 mg monotherapy x 6 wks). A follow-up sigmoidoscopy was conducted at 6 wks. Adherence was determined by self-report and medication count. Acceptability was measured on a 7-point Likert scale. The study was IRB approved 18-1039. Results: From N=420 total subjects, 168 were ineligible for the study, 111 actively refused, and 76 passively refused. 65 pts consented to the study, and 66% (43/65) completed all components. 13/65 pts were removed from study before starting the intervention [5/13 w/new CA diagnosis; 7/13 w/study non-compliance;1/13 w/ineligibility], while 9/65 additional were removed after starting drug [6/9 consent withdrawal w/side effects;1/9 w/medical reason; 2/9 removed w/non-compliance]. 20 pts completed Arm A and 23 pts completed Arm B. Adherence to ASA (Arm B) and Atrova doses (Arms A&B) and acceptability ratings were both high [>90% pts met ≥80% dose adherence; mean acceptability was 1.46, range 0.31-2.39]. Conclusions: Drug adherence and study acceptability were favorable in pts who completed the study. Future analyses will assess biomarkers at the tissue and molecular level. Clinical trial information: NCT04379999 . Arm, medication, % doses adherent % pts Arm A Atorva 100% 88% 80-99% 12% Arm B Atorva 100% 65% 80-99% 31% <80% 4% ASA 100% 73% 80-99% 18% <80% 9% Acceptability (def yes=1, def no=7) Mean (SD) Do you feel participating in the study had a positive impact on your CA care? 2.39 (1.82) Do you regret that you chose to participate? (reverse scored) 0.31 (0.95) Would you recommend this study to other pts? 1.36 (0.63) Please rate your interest in participating in a future study if it involved taking ASA or Atorva for 1 year? 2.15 (1.23)

Most gastrointestinal stromal tumor (GIST) patients develop clinical resistance to approved tyrosine kinase inhibitors (TKI). The development of effective treatment strategies for refractory GIST requires identification of novel targets for investigational agents and well-characterized preclinical models, like patient-derived xenograft (PDX) models, to evaluate them. While GIST PDX models have been shown to mirror patient tumors in cell morphology and driver mutation status, studies across tumor types indicate that PDX models may undergo limited genomic changes during engraftment and passaging. We performed a comprehensive genomic analysis of six KIT-mutant GIST PDX models derived from TKI-refractory patients. Pairwise comparison of copy number variation (CNV) profiles indicated strong correlation between PDX and matched primary tumors. Four models exhibited changes in the number of CNV events relative to the primary tumors, suggesting clonal selection and tumor heterogeneity during engraftment and passaging. Analysis of cancer-associated gene aberrations showed stability of primary KIT driver mutations, with minor fluctuations in the allelic frequencies of acquired mutations, suggesting initial heterogeneity of GIST tumors and potential enrichment of sub-clonal populations through passaging. To functionally assess model fidelity in vivo, three PDX models were treated with the approved second-line TKI sunitinib. The models exhibited differential responses that could not be fully explained by driver mutation status alone, highlighting the need for further investigation in larger cohorts. These findings highlight the importance of comprehensive genomic characterization of PDX models prior to their use in preclinical studies, supporting more accurate interpretation of experimental results and preserving their value as research tools.

548 Background: Combination tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (IO) are an established standard of care for patients with metastatic renal cell carcinoma (mRCC). We report updated analysis of a multi-center, investigator-initiated (IIT), phase I/II study of axitinib (axi) with nivolumab (nivo) in the previously treated patient cohort. Methods: The study investigated the combination of axi/nivo in an initial dose finding phase I portion and a phase II portion including 2 parallel arms: treatment naïve mRCC patients and mRCC patients previously treated with TKIs or IO/IO combination (NCT03172754). We are presenting final results from the previously treated cohort. Included patients had pathology with any clear cell component, ECOG performance status of 0-1, no known or symptomatic brain metastases, and no history of autoimmune disease. The recommended phase 2 dose of axi was 5 mg BID and patients were treated for up to 2 years then could stop one or both therapies. The primary endpoint of the phase II portion was objective response rate (ORR) per investigator assessment. Results: Twenty-six patients were accrued to the previously treated arm, all evaluable for efficacy and toxicity. The median age was 62 yrs (range: 42-81), 80.8% were male and 88.5% were white. Twenty pts had 1 prior line of therapy (18 TKI alone), 6 pts had 2 or more prior lines of Tx. Two pts had prior nivo with ipilimumab. Median follow-up was 47.2 months. The ORR was 30.8% (all PRs) and 57.7% achieved stable disease. The primary PD rate was 11.5%. Median OS was 48.4 months and median PFS was 13.0 months. Six pts (23.1%) completed two years of Tx on trial and elected to stop one or both drugs (2 elected to stay on axi alone). Five pts remain progression-free and have received no subsequent therapy with a median progression-free interval of 23.2 months. None had received prior IO. Adverse event (AE) data was similar to published data for IO/TKI combinations. There was one Gr4 TRAE of elevated lipase and 12 pts (46%) experienced a Gr3 TRAE. One pt (4%) discontinued the study due to TRAEs. Conclusions: Final results from the previously treated cohort of this IIT of axi/nivo for pts with mRCC demonstrated an ORR of 30.8% and a DCR of 88.5% with no unexpected AEs. Though there were no CRs, disease control rate and median OS were encouraging in this previously treated population. This is the only reported IO/TKI trial that allowed for stopping of all Tx at 2 years, with 6 pts (23.1%) meeting this milestone, 5 of whom (19% of all pts) remain progression-free for a median period close to 2 years. Clinical trial information: NCT03172754 .

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.

e16574 Background: Enfortumab vedotin (EV) monotherapy and its combination with pembrolizumab (EV/P) are established treatment options in metastatic urothelial carcinoma (mUC). However, significant toxicities associated with EV and EV/P often lead to treatment discontinuation. Despite this, some patients maintain disease control beyond discontinuation, suggesting the potential for treatment re-challenge. This study aimed to evaluate pts who discontinued EV for >8 weeks to examine the time to disease progression after discontinuation and the efficacy of EV re-challenge. Methods: This retrospective study analyzed clinical data from 86 pts who received either EV or EV/P for mUC in either the first or subsequent lines of therapy between January 2018 and June 2024.Descriptive statistics were employed for data analysis, and the Kaplan-Meier estimator was used to calculate survival probabilities. The interquartile range (IQR) was calculated to assess the median duration. Results: Among 86 pts, 43 received EV alone and 43 received EV/P, with a median age of 74. Most pts were male (69, 80%), with 59 reporting a history of smoking. Baseline neuropathy was present in 19 pts (22%). Prior cystectomy or nephroureterectomy was reported in 34 pts (39.5%). Overall, 59 pts (68%) discontinued EV for at least 8 weeks, 36 pts (84%) in EV alone, 23 pts (53%) in EV/P group. Most EV discontinuations were due to toxicity (52 pts, 60%). 12 pts (21%) discontinued due to complete response. The median duration of tx before EV discontinuation was 3.7 months (IQR 4.2), with 3.2 months (IQR 3.9) in the EV-alone group and 5.0 months (IQR 4.7) in the EV/P group. Following discontinuation, progression occurred in 34 of 56 (57%) pts, 23 in EV alone, and 11 in EV/P group. 39% pts remained disease-free despite discontinuing EV at the time of last follow-up. The median time to progression after treatment discontinuation was 5.92 months (IQR 5.06–10.85) overall, with 7.82 months (IQR 3.62–NA) in the EV/P group and 5.88 months (IQR 4.41–NA) in the EV-alone group. EV rechallenge was attempted in 23 of 56 (41%) pts discontinuing EV, and the median duration of EV rechallenge was 3.0 months (IQR 1.9).Upon EV re-challenge (N = 23), 5 pts had partial responses, 5 pts had stable disease, and 12 pts had progressive disease. No significant correlation was observed between the duration of EV tx before the tx discontinuation and response to EV upon re-challenge in either group (two-sided Mann-Whitney test p-value = 0.59. Conclusions: This study highlights that tx discontinuation due to toxicities is common in mUC pts receiving EV and EV/P. About 60% pts experienced disease progression after EV discontinuation with a median time to progression of 6 months. EV re-challenge was associated with responses in some pts and should be considered by those who may tolerate it.

Purpose:This study aimed to stratify patients with locally advanced rectal cancer (LARC) based on their response to neoadjuvant chemoradiation therapy (nCRT) using DNA damage response (DDR)-related proteins measured in peripheral blood monocytes (PBMCs). We optimized and validated an innovative assay to quantify these proteins, providing a predictive framework for nCRT response. Experimental Design:We used PBMCs collected from LARC patients either before or after standard course of ∼5.5 weeks of nCRT, with patients categorized by neoadjuvant rectal (NAR) score. DDR was assessed by immunofluorescence (γH2AX S139 foci), and by Luminex multi-analyte platform (xMAP) assay providing semi-quantitative assessment of phosphorylated Chk1 S345 , Chk2 T68 , γH2AX S139 , p53 S15 and total ATR, MDM2, p21. Assay performance was evaluated using reference controls and banked PBMCs from healthy controls (n=50). Results:PBMCs from poor responders (PoR; NAR >14; n=21) had significantly lower γH2AX S139 foci than complete responders (CR; NAR <1; n=21) (p<0.0001), with no significant differences between pre- and post-nCRT samples (p=0.4961). The xMAP assay performance assessment showed linear sample curves, precision with acceptable inter- and intra-assay coefficients of variability, and high reproducibility with ∼1% outliers in replicates. Clinical associations using the xMAP assay found levels of six proteins (ATR, MDM2, Chk1 S345 , Chk2 T68 , γH2AX S139 , p53 S15 ) significantly differentiating CRs from PoRs (p ≤ 1e-5). Univariate CART analysis determined thresholds that segregated PoRs from CRs with high precision (p<0.001). Conclusion:We optimized an assay to assess DDR proteins in PBMCs and identified specific proteins, along with their threshold levels, that can accurately predict response to nCRT in patients with LARC. Translational Relevance:Although neoadjuvant chemoradiation therapy followed by surgery is the standard of care for patients with locally advanced rectal cancer (LARC), many patients do not benefit from this treatment and suffer from its side effects. The motivation for this study was to reliably identify patients with LARC who will or will not respond to treatment, thereby permitting more effective direction of therapy only to likely responders. In this report, we describe identification and optimization of a novel multianalyte assay for patients diagnosed with LARC. This assay uses a Luminex xMAP platform to detect DNA damage response (DDR) signaling proteins in peripheral blood monocytes of pre-treatment patients. This assay, detecting the DDR proteins, effectively segregates responders from non-responders (p ≤ 1e-5), supporting optimization of treatment efficacy and reduction of unnecessary toxicity, thus advancing personalized medicine in oncology.

Abstract Background: Neoadjuvant chemoradiotherapy (nCRT) is standard therapy for locally advanced rectal cancer (LARC). While only a minority of patients are expected to have a complete response, all are at risk of toxicity. Currently no biomarkers reliably predict response to nCRT in LARC. Further, standard therapy in locally advanced head and neck squamous cell carcinoma (HNSCC) is adjuvant CRT. A major cause of poor outcomes in HNSCC is regional persistence or recurrence post-CRT. No biomarkers, apart from HPV, predict CRT response in HNSCC. CRT causes double-strand breaks (DSBs) and methylation of histones on lysine residues is known to affect chromatin, influencing DSB repair. We hypothesized that single nucleotide polymorphisms (SNP) in the histone 3 lysine 9 (H3K9) demethylase-encoding JMJD1C influenced CRT response. Methods: LARC patients (n=84) were divided into poor (PoR) and complete (CR) responders based on Neoadjuvant Rectal score. Lymphocyte DNA was sequenced, and LARC patient-derived cells were treated with DSB-inducing agents to assess viability. Sequencing was confirmed in an independent cohort of CRT-treated HNSCC patients (n=90) divided into: disease-free (n=30), recurrent disease (n=28), and never-disease-free (n=32). HNSCCs were in the larynx or oral cavity. Not all patients received HPV-testing; p16 expression was positive for 19/43 tumors. CRISPR/Cas9 editing of HPV-negative HNSCC lines (SCC9, Cal27) generated JMJD1C WT or SNP isogenic pairs. Bulk RNA-sequencing, colony survival, and immunofluorescence studies were performed. Results: In LARC patients, CRs were enriched for a coding, missense germline SNP in JMJD1C versus PoRs (discovery n=30, p<0.00001; validation n=54, p=0.001, Fisher’s Exact). When subjected to DSB, LARC patient-derived cells with the SNP displayed greater sensitivity than their WT counterparts (n=12, p<0.001, Mann-Whitney). In HNSCC, a correlation was identified between the same JMJD1C SNP and no recurrence (p=0.0003, Fisher’s Exact). HNSCC SNP cells showed changes in mRNA levels of DNA repair and immune response genes versus HNSCC WT cells (q-value<0.05). Upon irradiation, kinetics of foci resolution of the DNA damage response protein MDC1, specific to DSBs, were slower in SNP versus WT HNSCC cells (p<0.05, Mann Whitney). Further, there was reduced colocalization of BRCA1 with RAP80, a crucial factor for homology-directed DSB repair, in SNP versus WT HNSCC cells (p<0.01, Mann-Whitney).Targeted introduction of the SNP into HNSCC cell lines reduced viability and radiation response. Conclusion: Our study identifies a polymorphic coding variant in JMJD1C, as a predictive biomarker for CRT effectiveness in LARC and HNSCC, and defines a DNA repair mechanism by which JMJD1C influences response to irradiation. Citation Format: Adria Hasan, Elena V. Demidova, Philip Czyzewicz, Shreya M. Shah, Karthik Devarajan, Thomas J. Galloway, Margret B. Einarson, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Molecular investigation of a polymorphic variant in JMJD1C that associates with chemoradiotherapy outcomes in locally advanced rectal and head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5143.

BACKGROUND:Differential responses to neoadjuvant therapy (NAT) exist in pancreatic ductal adenocarcinoma (PDAC); however, contributing factors are poorly understood. Tobacco smoke is a common risk factor for PDAC, with nicotine-induced chemoresistance observed in other cancers. This study aimed to explore the potential association between tobacco use and NAT efficacy in PDAC. METHODS:A single-center, retrospective analysis was conducted that included all consecutive patients with PDAC who underwent surgical resection after NAT with a documented smoking history (N = 208). NAT response was measured as percentage fibrosis in the surgical specimen. Multivariable models controlled for covariates and survival were modeled using the Kaplan-Meier method. RESULTS:Postoperatively, major responses to NAT (>95% fibrosis) were less frequently observed in smokers than in nonsmokers (13.7% vs 30.4%, respectively; P = .021). Pathologic complete responses were similarly less frequent in smokers than in nonsmokers (2.1% vs 9.9%, respectively; P = .023). On multivariate analysis controlling for covariates, smoking history remained independently associated with lower odds of major fibrosis (odds ratio [OR], 0.25; 95% CI, 0.10-0.59; P = .002) and pathologic complete response (OR, 0.21; 95% CI, 0.03-0.84; P = .05). The median overall survival was significantly longer in nonsmokers than in smokers (39.1 vs 26.6 months, respectively; P = .05). CONCLUSION:Tobacco use was associated with diminished pathologic responses to NAT. Future research to understand the biology underlying this observation is warranted and may inform differential NAT approaches or counseling among these populations.

Abstract Background: Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is standard of care for locally advanced rectal cancer (LARC). About 20% patients receiving nCRT alone experience a pathologic complete response (pCR), while up to 25% patients exhibit a poor response. Currently, there is no biomarker to predict response to nCRT. We hypothesized that inherited ability to recognize and repair DNA damage, especially lethal double-strand breaks (DSBs), manifest in lymphocytes, would yield a reproducible test predicting therapeutic response to nCRT. Methods: Expression of DNA damage response (DDR) proteins was studied using peripheral blood lymphocytes (PBLs) from LARC patients and healthy controls. PBLs from LARC patients collected either before nCRT or after a standard course of ~5.5 weeks of nCRT were assessed by immunofluorescence (IF) for phospho-γH2AXSer139 and by Luminex multi-analyte platform (xMAP) approaches. The xMAP assay was modified from a qualitative to a semi-quantitative assay that measures the following DDR-associated proteins- total ATR, phospho-γH2AXSer139, total MDM2, phospho-Chk1S345, phospho-Chk2T68, phospho-p53S15, total p21. The analytical performance of the assay was assessed with vendor-provided reference controls and banked PBLs from healthy controls (n=50). LARC patients were segregated by neoadjuvant rectal (NAR) score to determine if tested biomarkers correlated with nCRT response. We used two independent sets of LARC biospecimens for xMAP analysis (set 1, n=48; set 2, n=44). Results: IF-based assessment: Using PBLs from LARC patients, we found that poor responders (PoR; NAR score>14; n=21) had significantly lower γH2AX foci than complete responders (CR; NAR score<1; n=21) (P<0.0001, logistic regression). No significant difference was observed in positive γH2AX foci from PBLs drawn pre-nCRT versus post-nCRT from the same patient (p=0.4961, n=11, Wilcoxon test). xMAP assay performance: The performance assessment showed linear sample curves, precision with acceptable inter- and intra-assay coefficients of variability, and high reproducibility with ~1% outliers identified in replicates. Clinical associations using xMAP assay: levels of six proteins (ATR, p-γH2AXSer139, MDM2, p-Chk1S345, p-Chk2T68, p-p53S15) significantly differentiated CRs from PoRs (p-values and False Discovery Rate ⇐ 1e-5). The strongest associations with nCRT response were observed for: ATR, MDM2 and γH2AX. Conclusion: 1) we standardized an xMAP assay to assess DDRs in human PBLs, and 2) found novel biomarkers that predict response to nCRT in LARC patients using minimally invasive PBL biospecimen. In ongoing work, we are using a larger biospecimen set to establish clinical validity parameters. Citation Format: Adria Hasan, Elena V. Demidova, Philip Czyzewicz, Karthik Devarajan, Margret B. Einarson, Donald Baldwin, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Novel biomarkers that predict response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2506.

Abstract Background: Adjuvant chemoradiation therapy (CRT) is the standard treatment for locally advanced head and neck squamous cell carcinomas (HNSCCs). Over 50% of HNSCC cases are diagnosed at the locally advanced stage, with in-field persistence and recurrence major causes of morbidity and poor survival outcome. Apart from human papillomavirus (HPV) status, there are no predictive biomarkers for CRT outcomes. CRT lethality arises from unrepaired double strand breaks (DSBs) in DNA. Recent work has suggested that dynamic changes in lysine modification of chromatin regulate DSB repair. This study investigated the association of germline single nucleotide polymorphisms (SNPs) in lysine-modifying genes with DSB repair and CRT outcomes in HNSCC. Methods: Blood specimens and associated retrospective clinical data from HNSCC patients (n=90; age range=41-89, median age 64; males=69, females=21) were obtained under IRB# 21-9921. The patients had HPV-negative tumors in the larynx or oral cavity, and had received radiation, with some receiving concurrent chemotherapy. Patients receiving concurrent chemotherapy were: 40% in disease-free group, 21% in recurrence disease group, and 38% in never disease-free group. The patients were segregated into 3 groups: disease-free (n=30), recurrent disease (n=28), and never-disease free (n=32). DNA from blood was genotyped for specific germline SNPs in lysine-modifying genes. Isogenic HNSCC lines (Cal-27, SCC9) carrying either the wild type (WT) or homozygous SNP were generated using CRISPR/Cas9 technology, and bulk RNA sequencing and immunofluorescence (IF)-based studies were performed to assess cellular pathways affected. Results: A homozygous variant SNP in the coding region of JMJD1C was significantly enriched in the disease-free group (n=21/30) versus the recurrent disease (n=3/28) and never disease-free (n=2/32) groups (p=0.0003, OR=0, Fisher’s Exact test). Analysis of SNP frequency in large population datasets found that while it was abundant in European Americans (EAs), it is relatively rare in African Americans (AAs) (EA= 0.3139 vs. AA=0.072; gnomAD population). Cal-27 JMJD1C SNP cells were significantly enriched for altered mRNA levels of genes associated with DNA repair, replication, cell cycle, oxidative phosphorylation, and histone kinase activity versus CAL-27 with WT JMJD1C (q-value<0.05). The SCC9 JMJD1C SNP cells were significantly enriched in extracellular matrix organization, cytokine-cytokine receptor interaction, and humoral immune response versus SCC9 with WT JMJD1C (q-value<0.05). On irradiation, the JMJD1C SNP cells had robust activation of the DSB repair pathway (BRCA1- mediated repair foci) versus the WT cell lines. Conclusion and future work: This work provides novel insight into a germline SNP in a lysine-modifying gene effecting DSB repair and associating with CRT outcomes in HNSCC. It may serve as a predictive biomarker for recurrence. Future research will explore racial disparities in tumor recurrence and confirm results in larger HNSCC datasets. Citation Format: Adria Hasan, Elena V. Demidova, Shreya M. Shah, Philip Czyzewicz, Karthik Devarajan, Thomas Galloway, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-032.

PURPOSE:The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS:Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS:A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS:High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

Perspective on this Article from Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations

This phase I, dose-escalation trial evaluates the safety of combining interferon-gamma (IFN-γ) and nivolumab in patients with metastatic solid tumors. Twenty-six patients are treated in four cohorts assessing increasing doses of IFN-γ with nivolumab to evaluate the primary endpoint of safety and determine the recommended phase two dose (RP2D). Most common adverse events are low grade and associated with IFN-γ. Three dose limiting toxicities are reported at the highest dose cohorts. We report only one patient with any immune related adverse event (irAE). No irAEs ≥ grade 3 are observed and no patients require corticosteroids. The maximum tolerated dose of IFN-γ is 75 mcg/m 2 , however based on a composite of safety, clinical, and correlative factors the RP2D is 50 mcg/m 2 . Exploratory analyses of efficacy in the phase I cohorts demonstrate one patient with a complete response, and five have achieved stable disease. Pre-planned correlative assessments of circulating immune cells demonstrate intermediate monocytes with increased PD-L1 expression correlating with IFN-γ dose and treatment duration. Interestingly, post-hoc analysis shows that IFN-γ induction increases circulating chemokines and is associated with an observed paucity of irAEs, warranting further evaluation. ClinicalTrials.gov Trial Registration: NCT02614456.

Perspective on this Article from Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue