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224 Background: Metabolic pathways are reprogrammed during CRC development, leading to increased glycolysis, glutaminolysis, and fatty acid synthesis. Understanding the impact of metabolic reprogramming on treatment outcomes is paramount. Hence, we investigated whether the tumor gene expression of 4 selected major metabolic genes ( PKM, SLC2A1 , SLC16A1 , and CAV1 ) could affect treatment response in pts enrolled in the CALGB/SWOG 80405 trial. Methods: 433 mCRC pts treated with either bevacizumab (bev, n = 226) or cetuximab (cet, n = 207) in combination with first-line chemotherapy were analyzed. RNA was isolated from FFPE tumor samples and sequenced on the HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared between groups of pts categorized by tertiles of gene expression (high [H], medium [M] and low [L]). Logrank P -values provide a non-parametric unadjusted assessment of differences. Likelihood ratio tests (LRT) were computed from multivariate Cox proportional hazards models, adjusting for age, sex, ECOG, tumor side, number of metastatic sites, KRAS , CMS, and treatment. Results: PKM gene expression was associated with cet treatment outcomes with PKM- low tumors showing significantly longer PFS and OS (median PFS: 14.3 vs 9.8 vs 8.1 months, L vs M vs H, P < 0.0001, LRT P = 0.011; median OS: 45.9 vs 31.2 vs 20.9 months, P < 0.0001, LRT P = 0.12). Low SLC2A1 was also associated with longer OS in cet-treated pts (32.4 vs 35.8 vs 25.2 months, P = 0.02, LRT P < 0.0001). Similar results were observed in bev-treated pts where low SLC2A1 expressing tumors had longer OS (37.4 vs 26.1 vs 29 months, P = 0.037, LRT P = 0.069) and a non-significant trend for longer PFS (13.1 vs 11 vs 9.5 months, P = 0.076). CAV1 low gene expression was associated with longer OS in cet-treated pts (37.4 vs 34 vs 21.5 months, P = 0.0048, LRT P = 0.013), while no significant associations were found in bev-treated (interaction LRT P = 0.0009). No significant results were observed for SLC16A1 . Conclusions: The intratumoral expression of metabolic genes was prognostic and predictive in mCRC pts treated with first-line therapy. GLUT1 (encoded by SLC2A1 ) and PKM2 (encoded by PKM ) promote aerobic glycolysis of cancer cells, known as Warburg effect, supporting rapid cell proliferation and survival. CAV1 has been shown to be involved in mitochondrial bioenergetics and fatty acid metabolism and to play either a tumor suppressor or oncogenic role depending on the cancer type and stage. Lower expression of SLC2A1 and PKM was associated with improved survival and increased benefit from cet treatment in our cohort. Low CAV1 expression was also associated with increase survival suggesting a cancer-promoting role in mCRC. Our results suggest that targeting cancer cell metabolism through novel inhibitors of the aforementioned pathways may be a promising therapeutic strategy.

269 Background: Sirtuins (SIRT1-7) are class III histone deacetylases with different roles in CRC as tumor suppressive and oncogenic, mainly influencing proliferation, migration, invasion, DNA damage and chemoresistance. In the present study, we explored the association between tumor gene expression of sirtuins and first-line treatment outcomes in mCRC pts. Methods: We evaluated the impact of the tumor gene expression of sirtuin genes ( SIRT1 -7) on treatment outcomes (overall survival: OS; progression free survival: PFS) in 433 pts enrolled in the randomized, phase III CALGB/SWOG 80405 (NCT00265850) trial. RNA extracted from FFPE tumor samples was sequenced using the HiSeq 2500 (Illumina). Subgroup analyses were based on treatment (bevacizumab [bev] (N = 226) vs. cetuximab [cet] (N = 207)). For each gene, expression was divided into tertiles of high, medium and low. The associations between tumor gene expression and outcomes (PFS and OS) were assessed using log-rank test in univariate analysis. Multivariable Cox proportional hazards regression model was used to compute hazard ratios (HR) and 95% confidence intervals while adjusting for baseline characteristics. Treatment-gene interactions were analyzed using the likelihood ratio test. Results: Of the seven genes analyzed, SIRT3 high was associated with both longer PFS (HR: 0.59 [95% CI: 0.46-0.74], P < 0.001) and OS (HR:0.64 [95% CI: 0.50-0.83], P = 0.002) overall. Treatment-gene interaction testing found significant interactions for SIRT1 and SIRT5 with PFS ( P values: 0.021 and 0.016, respectively) and OS ( P values: 0.038 and 0.014, respectively) with targeted therapies (bev vs. cet). Multivariate analysis showed SIRT1 high associated with longer PFS (HR: 0.62 [95% CI: 0.42-0.89], P = 0.014) and OS (HR: 0.59 [95% CI: 0.40-0.89], P = 0.036) in cet-treated pts; in contrast with shorter observed PFS (HR: 1.38 [95% CI: 0.96-1.99], P = 0.22) and OS (HR: 1.31 [95% CI: 0.90-1.93], P = 0.23) in bev-treated pts. SIRT5 high showed similar results and associated with longer PFS (HR: 0.64 [95% CI: 0.45-0.91], P = 0.039) and OS (HR: 0.57 [95% CI: 0.39-0.83], P = 0.013) in cet-treated pts, with no similar associations in bev-treated pts. Conclusions: Our results highlight a potential role for SIRT3 as prognostic and, SIRT1 and SIRT5 as predictive biomarkers for first-line treatment in mCRC, with differential effects based on targeted agents. Significant interaction favored cet treatment in SIRT1 high and SIRT5 high tumors. However, previous reports suggest an opposite role of SIRT5 that has been linked to chemotherapy and/or cet resistance in CRC via the inactivation of succinate dehydrogenase complex subunit A. Therefore, further studies are warranted to dissect the mechanism behind the interaction of SIRT5 with cetuximab activity and establish the potential of targeting SIRT1/5 in CRC.

254 Background: SLFN11 was recently identified as a dominant determinant of response to DNA-damaging anticancer agents. Several studies showed SLFN11 expression led to better sensitivity to topoisomerase inhibitors and PARP inhibitors. However, clinical evidence in mCRC has been lacking. Methods: Two independent cohorts (CALGB/SWOG 80405 trial and a real-world patient [Caris CODEai] cohort) were included in this study. Patients were stratified into SLFN11 -high and -low groups based on the median SLFN11 gene expression levels. Survival outcomes were compared between SLFN11 -high and SLFN11 -low in irinotecan-treated and oxaliplatin-treated patients. Median time was estimated using Kaplan-Meier method and Cox models were used for covariate adjustment. Endpoints of interest were progression-free survival (PFS) and overall survival (OS). Results: In the CALGB/SWOG 80405 trial cohort, 433 patients were included (110 treated with FOLFIRI-based chemotherapy and 323 treated with FOLFOX-based chemotherapy). In patients treated with FOLFIRI-based chemotherapy, SLFN11 -low patients exhibited numerically better PFS (median PFS, 13.4 vs 11.2 months, log-rank p = 0.03, adjusted hazard ratio [HR] = 0.86, 95% confidence interval [CI] 0.55-1.39, p = 0.50) and OS (median OS, 39.6 vs 30.3 months, log-rank p = 0.02, adjusted HR = 0.73, 95% CI 0.46-1.16, p = 0.19) compared to SLFN11 -high patients. In patients treated with FOLFOX-based chemotherapy, SLFN11 -low patients exhibited numerically better PFS (median PFS 11.2 vs 9.8 months, log-rank p = 0.03, adjusted HR = 0.83, 95% CI 0.65-1.05, p = 0.12) and OS (31.1 vs 26.1 months, log-rank p = 0.07, adjusted HR = 0.84, 95% CI 0.65-1.08, p = 0.16) compared to SLFN11 -high patients. No significant interaction was observed between treatment and SLFN11 expression in terms of PFS ( p = 0.69) and OS ( p = 0.44). In the Caris CODEai cohort, a better OS in SLFN11 -low patients than in SLFN11 -high patients was observed in irinotecan-treated patients ( N = 2906) (median OS, 25.5 vs 21.7 months, HR = 0.90, 95% CI 0.82-1.00, p = 0.048). However, no significant difference in OS between SLFN11 -low and SLFN11-high was observed in oxaliplatin-treated patients ( N = 4428) (median OS, 39.5 vs 35.8 months, HR = 0.98, 95% CI 0.90-1.06, p = 0.56). Conclusions: Low SLFN11 expression showed a potential for better prognosis in mCRC patients receiving irinotecan- and oxaliplatin-containing chemotherapies. These findings are not consistently aligned with preclinical data and clinical evidence in other cancer types previously reported. Further investigation is warranted to better understand the biological role and clinical implication of SLFN11 in mCRC.

592 Background: Deficient mismatch repair/microsatellite instability (MSI-H) in biliary tract cancers (BTC) is rare, estimated to be 1-5%. MSI-H across solid tumors has shown unprecedented durable response to immune checkpoint inhibitors (ICI) regardless of anatomic location. Here we describe the clinical outcomes of unresectable BTC patients with MSI-H treated with ICIs and their conversion to surgery. Methods: We conducted a multicenter, retrospective analysis of patients (pts) with advanced BTC who had been identified with MSI-H by molecular analysis performed between 2017 and 2024 at Mayo Clinic and University Hospitals Seidman Cancer Center. The outcomes of interest were overall survival (OS), defined as time from initiation of first-line systemic treatment to death; Time-to-treatment failure (TTF), defined as time from initiation of treatment to cessation due to any cause; and response. All analyses were done descriptively. Results: We identified 22 MSI-H pts with BTC, 19 with cholangiocarcinoma and 3 with gallbladder cancer. Median age at diagnosis was 61 (range 26-86), 20 (90.9%) were Caucasian, 9 (40.9%) were male gender, and 5 (22.7%) had etiology as Lynch syndrome. Twelve (54.5%) pts had metastatic disease at diagnosis. Twenty pts (90.9%) received first line systemic therapy, of whom 11 (55%) continued onto second line. Among the 20 pts that received first line therapy, median TTF was 7.6 months (95% CI: 4.8-14.3), objective response was observed in 7 (35%) pts, and disease control was observed in 15 pts (75%). Among the 11 pts that continued onto second line systemic treatment, objective response was observed in 4 pts (36%) and disease control was observed in 6 pts (55%). Among the 20 pts that received first line therapy, 19 (95%) ever received ICI +/- chemotherapy as a part of their treatment plan. Two-year OS for pts who ever received ICI was 63% (95% CI: 41%-96%). In total, 4 pts (20%) were restaged to resectable following treatment with an ICI, underwent resection, and currently have no evidence of disease. Conclusions: Administration of ICI during treatment plan in pts with MSI-H confirmed durable response and even downstage pts to resectable stage for curative intent. Testing for MSI-H should be performed upfront to guide treatment decision.

532 Background: HCC accounts for 80% of primary liver cancers, and worldwide, is the third common cause of cancer-related death. Two FDA approved 1st line systemic treatment options include atezo/bev and durva ± treme. Herein we report outcomes of these regimens in the 1st line setting via a real-world, multi-institutional retrospective cohort study. Methods: We reviewed clinical outcomes of patients (pts) who initiated 1st line atezo/bev or durva ± treme for unresectable HCC between March 2017 and February 2024, across 5 institutions (Mayo Clinic, University of Alabama, Cedars-Sinai Medical Center, University Hospitals Seidman Cancer Center and University of Michigan). Time to treatment failure (TTF) and overall survival (OS) were analyzed using the Kaplan-Meier method. Best (objective) response was tabulated. Multivariable analyses were performed using Cox models and logistic regression for time-to-event and binary outcomes, respectively, while adjusting for age, sex, race, HCC etiology, Child-Pugh class, and ALBI grade. Results: A total of 433 HCC pts were included; atezo/bev (336), durva ± treme (97). Median age at the start of therapy was 68 years; 77% were male, 82% were Caucasian. Etiologies of HCC were viral hepatitis (38%), MASLD (20%), alcohol (10%), non-viral multifactorial (10%), or unknown (22%). Pts receiving durva ± treme were less likely to have cirrhosis (11% vs. 28%, p<0.01) but similar in terms of macrovascular invasion (63% vs. 58%, p=0.35) compared to pts receiving atezo/bev. Reasons for treatment discontinuation were similar between the two groups, with the most common being disease progression - atezo/bev (56%) and durva ± treme (47%). The differences in clinical outcomes were not statistically significantly and remained consistent after multivariable adjustment. (Table, atezo/bev as the reference group). Outcomes were significantly different between Child-Pugh classes (CP: A,B,C) respectively, OS: 19.0 m, 6.4 m, 1.9 m (p<0.001); TTF: 6.1 m, 2.7 m, 1.3 m (p<0.001). Conclusions: In this real-world retrospective cohort study of unresectable HCC, the differences in overall survival, objective response, and time to treatment failure were not statistically significant between atezo/bev and durva ± treme in the 1st line setting. Outcome Atezo/bev Durva±treme Univariate p-value Multivariable adjusted hazard ratio (95% CI) Multivariable adjusted odds ratio (95% CI) Overall Survival, Median (month) 14.0 14.6 0.77 0.78 (0.54-1.14) --- Time to Treatment Failure, Median (month) 4.9 4.1 0.71 0.95 (0.72-1.26) --- Objective response, % 26.3% 21.1% 0.31 --- 0.78 (0.43-1.39)

285 Background: Immune checkpoint inhibitors (ICIs) have shown limited efficacy in colorectal cancer (CRC) outside of mismatch repair (MMR) deficient/microsatellite instability-high (MSI-H) tumors. Most CRC cases are MMR proficient /microsatellite stable and demonstrate resistance to ICIs, attributed to the tumor microenvironment. Colony Stimulating Factor 1 Receptor (CSF1R), a regulator of tumor-associated macrophages, has emerged as a potential target due to its association with poor prognosis in CRC. Methods: Data from 433 metastatic CRC (mCRC) patients in the CALGB/SWOG 80405 trial were analyzed. CSF1R RNA was extracted from formalin-fixed, paraffin-embedded tumor samples and sequenced. Patients were divided into tertiles of CSF1R expression (high, medium, low). Progression-free survival (PFS) and overall survival (OS) were compared using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. Subgroup analyses were conducted based on liver metastases, treatment type (Bevacizumab, Cetuximab, FOLFOX, FOLFIRI), and MSI status. Results: Low CSF1R expression (CSF1R-L) was associated with significantly better survival outcomes. Median PFS was 12.9 months for CSF1R-L, compared to 11.2 and 9.2 months for CSF1R-M and CSF1R-H (p = 0.003). Median OS was 35.8 months for CSF1R-L versus 32.5 and 24.4 months for CSF1R-M and CSF1R-H (p = 0.00086). Stratification by liver metastases showed that CSF1R-L remained a strong prognostic factor only in patients with liver metastases. There were no significant survival differences based on CSF1R expression in bevacizumab-treated patients. However, in cetuximab-treated patients, CSF1R-L tumors had significantly longer PFS (12.9 vs 8.8 months, p = 0.037) and OS (35.8 vs 21.4 months, p = 0.002) than CSF1R-H tumors. Similarly, in FOLFOX-treated patients, CSF1R-L tumors showed longer PFS and OS (p = 0.018 and p = 0.013, respectively), and in FOLFIRI-treated patients, OS was significantly longer for CSF1R-L tumors (42.2 vs 24.7 months, p = 0.022) than CSF1R-H tumors. Conclusions: Our data suggests that CSF1R expression may serve as a prognostic biomarker in CRC, with lower expression potentially linked to improved survival, particularly in patients with liver metastases. These findings warrant further investigation of CSF1R as a possible prognostic and predictive biomarker, as well as a potential therapeutic target in CRC. Clinical trial information: NCT00265850 .

ASXL transcriptional regulator 1 (ASXL1) is one of the three most frequently mutated genes in age-related clonal hematopoiesis (CH), alongside DNA methyltransferase 3 alpha (DNMT3A) and Tet methylcytosine dioxygenase 2 ( TET2) . CH can progress to myeloid malignancies including chronic monomyelocytic leukemia (CMML) and is also strongly associated with inflammatory cardiovascular disease and all-cause mortality in humans. DNMT3A and TET2 regulate DNA methylation and demethylation pathways, respectively, and loss-of-function mutations in these genes reduce DNA methylation in heterochromatin, allowing derepression of silenced elements in heterochromatin. In contrast, the mechanisms that connect mutant ASXL1 and CH are not yet fully understood. CH/CMML-associated ASXL1 mutations encode C-terminally truncated proteins that enhance the deubiquitinase activity of the ASXL-BAP1 “PR-DUB” deubiquitinase complex, which removes monoubiquitin from H2AK119Ub. Here, we show that ASXL1 mutant proteins interact with the euchromatic histone lysine methyltransferases 1 and 2 (EHMT1–EHMT2) complex, which generates H3K9me1 and me2, the latter a repressive modification in constitutive heterochromatin. Compared to cells from age-matched wild-type mice, we found that expanded myeloid cells from old (≥18-mo-old) Asxl1tm/+ mice, a heterozygous knock-in mouse model of CH, display genome-wide decreases of H3K9me2, H3K9me3, and H2AK119Ub as well as an associated increase in expression of transposable elements (TEs) and satellite repeats. Increased TE expression was also observed in monocytes from ASXL1 -mutant CMML patients compared to monocytes from healthy controls. Our data suggest that mutant ASXL1 proteins compromise the integrity of both constitutive and facultative heterochromatin in an age-dependent manner by reducing the levels of H3K9me2/3 and H2AK119Ub. This increase in TE expression correlated with increased expression of nearby genes, including many interferon-inducible (inflammation-associated) genes (ISGs).

Abstract Background Atezolizumab plus bevacizumab (A/B) received FDA approval as the first-line therapy for patients with advanced hepatocellular carcinoma (HCC) in 2020. However, optimal subsequent treatment options are unclear. Here, we describe clinical outcomes of advanced HCC patients following first-line treatment with A/B. Patients and Methods We conducted a multi-site analysis of patients with HCC treated with first-line A/B between January 2018 and December 2022 at Mayo Clinic. This study cohort included all patients receiving second-line systemic therapy after A/B. Median overall survival (OS) and time-to-treatment discontinuation (TTD) were estimated using the Kaplan-Meier method. Child Pugh (CP) scores are also described at diagnosis, prior to first line, and prior to second-line therapy. Results Of the 342 patients who received A/B, 107 (31.3%) received second-line treatments including anti-VEGF therapy or immune checkpoint inhibitor (ICI) and were included in the final analysis. Median OS for all patients was 11.1 months from initiation of second-line therapy. Median OS was 10.7 months (95% CI: 7.2-12.8) and 15.7 months (95%CI: 6.8-NE) for those receiving anti-VEGF inhibitors and ICI ( P = .50). Median TTD for second-line therapies was 2.4 months (95% CI: 1.7-3.3) and 2.6 months (95% CI: 1.5-5.1) for anti-VEGF inhibitors and ICI, respectively (P = .87). In multivariate analyses, CP was significantly associated with survival. Conclusion Following first-line A/B treatment, there is no statistically significant difference in survival between ICI and anti-VEGF therapy, nor in time to treatment discontinuation. CP score remains an important prognostic tool.

NPM1 mutations (NPM1+) are found in about 30% of adult AML. While NPM1+ are generally associated with favorable risk, prior studies have shown that NPM1+ older patients, defined as ≥ 65 years, have significantly worse outcomes. The updated European Leukemia Net 2022 (ELN22) now classifies NPM1+ AML patients with FLT3-ITD (ITD+) and MDS-associated mutations (MDSm+) as adverse-risk, while NPM1+/ITD-/MDSm+ patients remain favorable-risk unless they have an adverse-risk karyotype. A recent report (Chan et al., Blood Adv 2024) found MDSm+ conferred worse OS only in patients ≥ 60 years; however another report (Othman et al., Blood 2024) did not find MDSm+ were significantly associated with adverse outcomes in a larger cohort of NPM1+ patients. To further assess this question, we examined the mutational landscape in a large cohort of pre-treatment, diagnostic specimens from 456 patients enrolled in SWOG clinical trials (SWOG-9031, SWOG-9333, S0106, and S0112) and 126 patients from the UW/FHCC Hematopoietic Repository (Total N=582). Patients were treated with curative intent as per standard practice or clinical trials. Out of the 582 patients, 191 were NPM1+ (33%), of which 148 patients had cytogenetic and long-term outcome data. Kaplan-Meier and cumulative incidence estimates were estimated. C-statistics and AUC for Cox, cause-specific and logistic regression models for overall survival (OS), event-free survival (EFS), time to relapse (TTR, calculated in n=106 who achieved complete remission [CR], death in remission analyzed as a competing risk) and CR were calculated for age, ELN17, and ELN22, as well as ELN17 and ELN22 adjusting for age. ELN17 favorable-risk patients reclassified as adverse-risk (N=5 of 108) demonstrated significantly shorter EFS (HR=2.50, p=0.05) and CR rate (OR=0.20, p=0.024), although with similar OS (HR 1.30, p=0.66) and TTR (HR=1.20, p=0.86). ELN17 favorable-risk patients reclassified as ELN22 intermediate-risk (N=23 of 108) did not show a significant difference in OS (HR 1.47, p=0.20), EFS (HR 1.35, p=0.12), CR rate (OR 0.75, p=0.46), or TTR (HR=1.64, p=0.11) compared to ELN22 favorable-risk. For ELN17 intermediate-risk (N=40), 5 patients were reclassified as ELN22 adverse-risk (12.5%) and had significantly shorter EFS (HR 2.89, p=0.037) compared to ELN22 intermediate risk, although there was not a significant difference in CR rate (OR 0.31, p=0.23) or OS (HR 0.81, p=0.67). We next compared the prognostic significance of age relative to ELN risk classification. Age was associated with higher C-statistics (OS=0.65, TTR=0.57 and EFS=0.60) and AUC (CR=0.64) compared to both ELN17 (OS=0.56, TTR=0.54, EFS=0.54, CR=0.54) or ELN22 (OS=0.57, TTR=0.57, EFS=0.57, CR=0.58). The combination of age and ELN22 resulted in the highest C-statistics for OS, TTR, and EFS (OS=0.68, TTR=0.60, EFS=0.64). To further assess the impact of MDSm on risk-stratification of NPM1+ AML, we performed multivariable analyses adjusting for FLT3-ITD, MDSm, and age in patients without adverse-risk disease. These analyses showed MDSm did not have a significant association with OS when controlling for age or FLT3-ITD. We also compared ELN22 favorable-risk patients with and without MDSm (MDSm+ N=18; MDSm- N=61). Compared with the MDSm-, there was a trend toward an inferior OS (HR 1.85, p=0.054) for MDSm+ patients but not EFS (HR 1.38, p=0.3) or TTR (HR 1.2, p=0.64). When stratified by age (&lt;65, N=53 vs. ≥65, N=26), MDSm+ in younger patients did not show a difference for OS (HR 0.96, p=0.96), EFS (HR 0.63, p=0.53) or TTR (HR 0.4, p=0.37). Older ELN22 favorable-risk patients (≥65) had similarly poor OS (MDSm- HR 3.33 and MDSm+ 3.27, respectively, p=0.001 for both) and EFS (MDSm- HR 3.11 and MDSm+ 2.39, p=0.001 and 0.01) compared to younger MDSm- patients. In our study, the ELN22 guidelines incrementally improve risk stratification for EFS in NPM1+ patients. However, age remains the most prognostic factor, and patients ≥ 65 years have similar adverse clinical outcomes, whether classified as favorable risk by ELN17, ELN22, or incorporating MDSm. Moreover, MDSm do not confer a worse prognosis in NPM1+ patients when accounting for age or FLT3-ITD status. These findings differ from a recent report by Chan et al. and are in keeping with the report by Othman et al. Therefore, further understanding of the underlying biology of age and its effect on NPM1+ AML is needed to improve outcomes for older patients.