Mammalian genomes undergo pervasive transcription in both genic and intergenic regions. Trimethylation of histone H3 lysine 4 (H3K4me3) is a deeply conserved and functionally important histone modification enriched at transcriptionally active promoters, where it facilitates RNA polymerase activity. H3K4me3 is also commonly found in intergenic regions, where its role is poorly understood. We interrogated the role of H3K4me3 at intergenic regulatory elements by using epigenetic editing to efficiently deposit H3K4me3 at intergenic loci. We found that H3K4me3 amplifies RNA polymerase activity and is actively remodeled at intergenic regions, shedding light on these important but poorly understood regions of the genome.

Single-cell RNA sequencing (scRNA-Seq) studies identified a novel subpopulation of epithelial cells along the rostrocaudal axis of human intestine, specifically marked by bestrophin 4 (BEST4), that are enriched for genes regulating pH, GPCR acid-sensing receptors, satiety, cGMP signaling, [Formula: see text] secretion, ion transport, neuropeptides, and paracrine hormones. Interestingly, BEST4+ cells in the proximal small intestine express CFTR but have not been widely linked to the previously described CFTR high-expresser cell (CHE) subpopulation in rat and human intestine. ScRNA-Seq studies in rat jejunum identified CHEs and a gene expression profile consistent with human small intestinal BEST4+ and neuropod cells. Protein immunolocalization confirmed that CHEs express CFTR, BEST4, neuropod proteins, high levels of intracellular uroguanylin (UGN), guanylyl cyclase-C (GC-C), and the proton channel otopetrin 2 (OTOP2), and display long basal processes connecting to neurons, confirming that Best4+ cells in the proximal small intestine are CHEs. OTOP2, GC-C, and CFTR traffic robustly into the apical domain of CHEs in response to acidic luminal conditions, indicating their roles in luminal pH regulation. In the ΔF508 cystic fibrosis (CF) rat jejunum, the loss of apical CFTR did not affect BEST4 protein expression in CHEs. However, there was an increased abundance of CHE cells in the ΔF508 rat jejunum compared with wild-type animals. Furthermore, ΔF508 rat CHEs expressed higher levels of GC-C at the apical domain compared with wild-type. These data implicate CHEs in intestinal CF disease pathogenesis.NEW & NOTEWORTHY This is the first study to identify CFTR high-expresser cells in the rat small intestine as neuropod cells capable of sensing and responding to luminal pH, and confirms that Best4+ cells are CHEs in the proximal small intestine. This study also provides the first characterization of CFTR and relevant mRNA and proteins in CHEs in CF rat models that provide insights into the significance of CHEs to CF intestinal disease.

Macrophage migration inhibitory factor (MIF) is an upstream regulatory cytokine that is associated with advanced disease and poor outcomes in multiple cancer types, including melanoma. We investigated whether anti-MIF therapy could enhance the antitumor effects of the immune checkpoint inhibitor anti–programmed cell death 1 (anti–PD-1) in 2 murine tumor models. The therapeutic efficacy of anti-MIF, alone or combined with anti–PD-1, was tested in the YUMMER1.7 melanoma and MC38 colorectal cancer models. Tumor growth and survival were assessed in untreated Mif-knockout (KO) and low-expression human MIF allele (CATT5) mice and compared with wild-type (WT) or high-expression MIF allele (CATT7) mice. Tumor-bearing animals underwent cytokine profiling, tumor immunohistochemistry, flow cytometry, and scRNA-Seq. We also correlated functional variant MIF alleles with melanoma incidence and progression in patients. Our results showed that combined anti-MIF and anti–PD-1 significantly reduced tumor growth, improved survival, and promoted tumor regression, accompanied by enhanced TH1 cytokine levels, increased macrophage activation–related cytokines, and increased type 1 conventional dendritic cells. scRNA-Seq analysis revealed an expansion of intratumor Cd74/C1q/Aif1-expressing macrophages, which exhibited an antitumor phenotype, in response to anti-MIF therapy. MIF-KO and CATT5 mice exhibited reduced tumor burdens compared with WT or CATT7 mice alone and in the presence of anti–PD-1. In patients with melanoma, the high-MIF expression genotype (-173C/C) occurred at higher frequencies compared with healthy controls. These findings highlight that the addition of anti-MIF to anti–PD-1 reduces tumor growth, enhances antitumor responses, prolongs survival, and augments key intratumor immune cell populations involved in immune activation against tumors. This approach merits further consideration for clinical trial development.

A growing number of older adults live in unaffordable and unstable housing; however, whether programs designed to counter housing insecurity contribute to earlier-stage cancer diagnosis remains largely unknown. To examine the association between the receipt of federal housing assistance, which limits household spending on rent and utilities, and cancer stage at diagnosis among older adults in the US. This comparative cohort study used the Surveillance, Epidemiology, and End Results cancer registry program and Medicare database linked with data from the US Department of Housing and Urban Development (HUD). Participants were individuals aged 66 to 95 years who received new diagnoses of breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), or prostate cancer between 2007 and 2019. Data were acquired in 2023, and the data analysis was performed from June 2023 through March 2025. Receipt of federal housing assistance. The primary outcome was stage at cancer diagnosis, defined as in situ (for breast cancer only), localized, regional, or distant. Odds ratios (ORs) for the association between having HUD assistance at diagnosis and cancer stage at diagnosis were estimated using multinomial (nonproportional odds) regression. Individuals with HUD assistance were propensity score matched to individuals without housing assistance. A total of 52 532 individuals (mean [SD] age at diagnosis, 76.3 [6.8] years; 33 608 women [64.0%]) with housing assistance at diagnosis were included: 16 064 had breast cancer, 10 807 had colorectal cancer, 17 156 had NCSLC, and 8505 had prostate cancer; 38 183 (72.7%) were enrolled in Medicaid, and 38 539 (73.4%) had Part D low-income cost sharing. Compared with matched controls, fewer individuals with housing assistance received a diagnosis of distant breast cancer (1071 patients [6.7%] vs 3485 patients [7.2%]; adjusted OR [aOR], 0.85; 95% CI, 0.82- 0.90), distant colorectal cancer (2398 patients [22.2%] vs 7562 patients [23.3%]; aOR, 0.90; 95% CI, 0.83-0.98), and distant NSCLC (8810 patients [51.4%] vs 27 901 patients [54.2%]; aOR, 0.83; 95% CI, 0.79-0.86) compared with localized cancers. Housing assistance was not significantly associated with stage at diagnosis for individuals with prostate cancer. The association between housing assistance and stage at cancer diagnosis varied across the different types of housing assistance, including the Housing Choice voucher program, multifamily housing, and public housing. The findings of this cohort study of older adults with cancer suggest that federal housing assistance was associated with earlier-stage diagnosis of breast cancer, colorectal cancer, and NSCLC, highlighting its potential role in mitigating the adverse associations of housing insecurity with cancer outcomes.

In the randomized, phase 3 KEYNOTE-119 study, overall survival (OS) was not significantly improved with pembrolizumab 200 mg Q3W versus investigator’s choice of chemotherapy in participants with previously treated metastatic TNBC. In this exploratory analysis, we evaluated associations of tumor-infiltrating lymphocytes (TILs), T-cell‒inflamed gene expression profile (TcellinfGEP), BRCA1/BRCA2 mutation (BRCAm) status, homologous recombination deficiency (HRD) status, and tumor mutational burden (TMB) with clinical outcomes. TIL level was associated with improved objective response rate (ORR), progression-free survival (PFS), and OS with pembrolizumab but not with chemotherapy or after adjusting for TcellinfGEP. Associations were also identified between TcellinfGEP and improved ORR, PFS, and OS with pembrolizumab. Participants with TMB ≥ 10 mut/Mb showed a trend toward increased benefit with pembrolizumab versus chemotherapy. No association was seen between BRCAm/HRD status and treatment response. These findings suggest a positive association between TILs, TcellinfGEP, and TMB with clinical outcomes in patients with metastatic TNBC receiving pembrolizumab. ClinicalTrials.gov Identifier: NCT02555657 (date of registration: September 18, 2015).

274 Background: Breast cancer accounts for 30% of female cancers in the United States, resulting in many women undergoing treatment in their communities. Regionalization in cancer care organizes complex oncology treatment towards high-volume centers of excellence, yet barriers to equitable access remain. We sought to determine patient factors and disease characteristics associated with greater survival benefit from high-volume care, and those for whom this may not be necessary. Methods: We queried the National Cancer Database (2007-2017) for individuals who underwent surgery for stage 0-III breast cancer. Using restricted cubic spline methodology, we modeled the relationship between rolling annual facility-level breast surgical volume and adjusted hazard of overall survival to identify volume thresholds associated with improved overall survival. We compared patient-level characteristics across the high- vs low-volume threshold and performed an adjusted survival analysis with interaction terms for factors including demographics, socioecological factors, and disease characteristics to assess for effect modification. Results: Overall, 308,271 individuals with stage 0-III breast cancer were identified. 99.2% of whom were female and 74.4% of whom were non-Hispanic White. A survival benefit in the volume-outcome relationship was noted at the inflection point of 201 cases per year, differentiating “high-volume” from “low volume” centers. The adjusted hazard ratio [HR] of overall mortality for utilizing a high-volume center was 0.86 (95% confidence interval [CI] 0.85, 0.88). Characteristics that demonstrated an interaction with surgical volume and survival included tumor grade [well-differentiated HR 0.80 (95% CI 0.75, 0.84); moderately-differentiated HR 0.73 (95% CI 0.70, 0.75); poorly-differentiated HR 0.89 (95% CI 0.86, 0.92)], hormone receptor status [ER+/PR+/HER2- HR 0.84 (95% CI 0.81, 0.87); HER2+ HR 0.69 (95% CI 0.64, 0.74); ER-/PR-/HER2- HR 0.92 (95% CI 0.86, 0.98)], and patient age [18-54 years HR 0.91 (95% CI 0.86, 0.95); 55-69 years HR 0.84 (95% CI 0.81, 0.87); 70-89 years HR 0.88 (95% CI 0.84, 0.90)]. Notably, neither patient comorbidities nor distance traveled to the treating facility were associated with a different degree of improvement in survival at high-volume centers. Conclusions: Breast cancer treatment at high volume centers remains associated with an improved overall survival for all women, regardless of patient demographics and disease. Further research is needed to identify individuals from across common cancer types, as well as aspects of respective multidisciplinary treatment, where the benefits of regionalized care outweigh the burden to patients and families.

577 Background: Prostate specific membrane antigen positron emission tomography (PSMA-PET) increases the detection of metastatic prostate cancer relative to conventional imaging. To understand whether PSMA-PET is associated with greater use of therapies for metastatic disease we evaluated initial treatment among contemporary commercial insurance beneficiaries undergoing staging evaluation for prostate cancer. Methods: We conducted a retrospective cohort study among Blue Cross Blue Shield insurance beneficiaries with an incident diagnosis of prostate cancer from July 1st, 2021, through March 31, 2023, who received either PSMA-PET or bone scintigraphy imaging in the six-month period before and after diagnosis based on billing codes. The primary study outcome was the receipt of androgen pathway receptor inhibitor (ARPI) therapy in the one-year period following diagnosis using national drug codes. Secondary outcomes included local therapy and androgen deprivation therapy (ADT) use. Chi-square (χ²) tests were used to compare distributions of treatment by imaging modality. We used mixed effects logistic regression to examine the association between imaging modality (PSMA-PET versus bone scan) and initial treatment, accounting for beneficiary age, diagnosis period, comorbidity, frailty, local therapy, census region, and social deprivation index for clustering of beneficiaries within hospital referral regions. Results: Among 4,952 beneficiaries newly diagnosed with prostate cancer, 3,690 received bone scans and 1,262 received PSMA-PET imaging. The median age in both groups was 62 years. Patients receiving PSMA-PET were less likely to undergo radical prostatectomy. (40.8% versus 48.1%, χ² p<0.001) than beneficiaries imaged with bone scan. Receipt of ARPI was more common among beneficiaries who received a PSMA-PET scan (25.0%) versus bone scan (9.7%, χ² p<0.001). ADT use was also more common in PSMA-PET imaged versus bone scan imaged individuals (58.9% versus 39.4%, χ² p<0.001). PSMA-PET was independently associated with use of initial ARPI therapy, (odds ratio vs bone scan: 3.83, 95% confidence interval 3.11-4.71, p<0.001). Conclusions: In this claims-based study of a single large U.S. commercial health insurer, receipt of PSMA-PET was associated with increased use of therapies for metastatic prostate cancer. These findings highlight the growing role of molecular imaging in the diagnostic landscape of prostate cancer, and support further efforts to estimate effects of imaging on clinical outcomes and healthcare costs.

490 Background: In response to efficiency concerns, the NCI cooperative group trial system was redesigned in 2014 to streamline trial design and conduct. As timely evidence is vital to high-quality cancer care, we examined trial accrual and timeliness in recent NCI cooperative group trials, focusing on meeting accrual targets and time to completion. Methods: We used the Aggregate Analysis of ClinicalTrials.gov (AACT) database to identify phase II or III interventional trials that were initiated between 2011 and 2019 and had an NCI cooperative group listed as the sponsor, responsible party, or principal investigator. To assess a study’s time to completion, we quantified the time between its “start date” and “primary completion date” (date when the final data to fulfill the primary outcome were ascertained) listed on ClinicalTrials.gov. The primary outcome was trial primary completion within 5 years of initiation (for trials initiated before 2019); we also assessed 8-year trial completion (for trials initiated prior to 2016). To examine the timeliness of recent NCI cooperative group trials, we used chi-square tests and adjusted logistic regressions to compare the five-year completion status of trials initiated in 2011–2013 (T1: prior to the 2014 redesign of the National Clinical Trials Network (NCTN)) versus 2016–2018 (T2: after NCTN redesign), adjusting for trial phase and cancer type. A subgroup analysis was conducted among phase III trials. Results: We included 323 studies, and 196 were classified as phase II, 17 were phase II/III, and 110 were phase III. Hematologic (n=49), breast (n=36), and non-small cell lung (n=39) were the most common cancer types researched. NRG Oncology (n=75), Alliance for Clinical Trials in Oncology (n=64), and Eastern Cooperative Oncology Group (n=61) were the most represented cooperative groups. Overall, 40 studies had a final study status of either terminated (0.8%), withdrawn (0.2%), or unknown (0.2%). 50.2% of studies reached their primary outcome within five years (24.5% of phase III and 62.2% of phase II trials). Among the 194 studies initiated prior to 2016, 147 (75.8%) were completed at 8 years (60% of the 70 phase III trials; 84.8% of the 112 phase II trials). Of the 2011–2013 (T1) trials (n=119), 5-year completion status was 46.2%, compared to 58.9% of the 95 studies in T2 (p=0.09). Among phase III trials, the 5-year completion status was 25.6% (T1) and 28.5% (T2) (p=1.00). Adjusting for trial phase and cancer type, there was no significant association between time period and five-year completion status (Odds ratio for T2 vs T1: 1.27; 95% CI: 0.66 to 2.48). Conclusions: Half of NCI cooperative group trials reached primary completion within 5 years, with only one quarter of phase III trials completed in this same time frame. There was no significant improvement in time to completion of phase III trials after the 2014 NCTN redesign, although the COVID epidemic may have hampered trial conduct during the latter time period.

273 Background: Racial and ethnic groups underrepresented in medicine (URiM) comprise 31% of the US population but 8% of oncologists. Although there have been initiatives to increase diversity of the oncology workforce, recent challenges have raised concerns over the degree to which the oncology workforce will be reflective of the broader population in the coming years. This has implications for patient access to concordant physicians and quality of cancer care. This modeling study projects the racial and ethnic diversity of the cancer physician workforce through 2060 under three distinct scenarios. Methods: We used data from the American Medical Association (physician specialty and age) linked to data from the Association of American Medical Colleges (physician self-reported race and ethnicity). We classified cancer oncology specialties as Medical, Radiation, and Surgical Oncology, General Surgery, and Palliative Care. URiM was defined as American Indian/Alaska Native (AIAN), Black, Hispanic, or Native Hawaiian/Pacific Islander (NHPI). We determined the URiM distribution of the oncology workforce in 2020 and created stock and flow models to project changes by decade from 2030 to 2060, factoring in inflows from graduating trainees and expanded training slots, and outflows due to retirement. We modeled three scenarios with varying trends in the growth rate of URiM trainees: Baseline (URiM distribution remains the same as 2020 levels); Trajectory (distribution changes each decade at the same rate of change observed from 2010-2020); and Doubling (URiM growth occurs at twice the rate of non-URiM). Results: In 2020, there were 66,450 practicing cancer care physicians. Of these, 11.3% identified as URiM (9.7% of medical oncologists, 8.8% of radiation oncologists, 8.3% of surgical oncologists, 12.3% of general surgeons, 11.5% of palliative care physicians). The trend sample from 2010-2020 of physicians within 5 years of training completion showed a +1.2% increase in URiM representation (11.7%–12.9%), +1.2% in Hispanic (6.2%–7.4%), and +0.05% in Black (4.95%–5.00%). In the baseline scenario (no change in the % URiM trainees), in 2060, an estimated 12.5% of the workforce would be URiM, compared to 43.2% of the US population (Table). Continuing 2010-2020 trends would raise URiM representation to 16.5% in 2060. Doubling URiM growth would reach 19.1%. Achieving census parity would require increasing URiM trainee representation by 8% per decade. Conclusions: Achieving oncology physician workforce alignment with U.S. demographics by 2060 will require deliberate, systemic action—not just incremental change. 2060 Oncology workforce composition by scenario. Baseline (%) Trajectory (%) Doubling (%) US 2060 Population (%) AIAN 0.5 0.2 0.8 0.7 Asian 27.5 31.4 25.4 9.6 Black 4.9 5.1 7.5 14.1 Hispanic 7.0 11.2 10.7 28.2 NHPI 0.12 0.00 0.19 0.27 White 60.0 52.1 55.5 47.2 URiM 12.5 16.5 19.1 43.2

363 Background: In families of children with cancer, household material hardship (HMH) and health literacy challenges are associated with increased parent distress and lower care satisfaction, respectively. We aimed to describe how HMH and health literacy challenges may influence the advanced childhood cancer care experience. Methods: We conducted a secondary analysis of data collected at 8 U.S. hospitals, using the “Pediatric Cancer Care Experience” (PACE) questionnaire, which was completed by parents/caregivers of children with advanced, or incurable, cancer. We defined HMH as self-reported difficulty with at least one of four domains (housing, food, utilities, and/or transportation) and further characterized the number of HMH domains (0, 1, or >2). We defined health literacy challenges as an affirmative response to at least one of two validated screening questions. To capture key elements of the care experience, we employed three subscales as our primary outcomes of interest: (1) therapeutic alliance, (2) collaborative care, and (3) prognostic communication and shared decision making. Each subscale was comprised of items from PACE with 5-point Likert responses. We computed mean response scores for each subscale across the entire cohort. We then conducted bivariate analyses to explore associations between HMH, parent/caregiver health literacy, and subscale scores. Results: Among 158 respondents, a majority were female (70%), non-Hispanic White (56%), and spoke English as their first language (89%). Over one-third (37%) experienced HMH, with hardship most often reported in >2 domains (n = 40/59, 68%). The most common HMH domain was housing (32%) followed by food (24%), utilities (18%), and transportation (10%). Thirty-eight percent of the cohort reported health literacy challenges, with a higher frequency among respondents who speak a first language other than English (p = 0.010). Care experience subscale scores did not vary significantly by HMH. However, respondents reporting health literacy challenges had lower mean scores for all three subscales, compared to respondents who did not report health literacy challenges: therapeutic alliance (mean subscale score 4.51 [95% CI 4.37, 4.66] vs. 4.78 [4.72, 4.85], p < 0.01), collaborative care (4.55 [4.42, 4.68] vs. 4.76 [4.68, 4.83], p = 0.01), and prognostic communication and shared decision making (4.13 [3.96, 4.31] vs. 4.48 [4.39, 4.58], p < 0.01). Conclusions: Care experience, as measured by PACE subscales characterizing therapeutic alliance, collaborative care, and prognostic communication and shared decision-making, did not vary significantly with HMH but was reduced in the setting of family-reported health literacy challenges. Findings suggest that interventions to support families facing health literacy challenges may improve the advanced cancer care experience.