Salem, Mohamed E.

Abstract Purpose: Routine testing for mismatch repair deficiency (dMMR)/microsatellite instability (MSI) in colorectal cancers is recommended for Lynch syndrome screening, prognosis, and treatment guidance. In the metastatic setting, RAS/BRAF mutations guide treatment decisions. The impact of these mutations on the tumor immune microenvironment (TiME) in MSI/dMMR colorectal cancer is not known. Experimental Design: The study involved retrospective analysis of 448 patients with stage I to IV MSI/dMMR colorectal cancer profiled using next-generation sequencing (Tempus xT DNA sequencing of 595–648 genes at 500x coverage and Tempus xR whole-exome capture RNA sequencing). MSI status was determined by assessing 44 or 239 loci using next-generation sequencing. dMMR was determined using IHC. Tumor mutational burden, tumor neoantigen burden (NTB), PD-L1, immune infiltration, and canonical immune pathways (76 gene set signatures) were analyzed. Results: The median age at diagnosis was 67 years (range, 21–86); 59% of patients were female, and 70% were stage III to IV. Among the 448 patients, 100 (22%) harbored RAS mutations (RASmut), 119 (27%) a BRAFV600E mutation, and 229 (51%) were double wild type. RASmut exhibited lower NTB (median, 12 vs. 15 vs. 16; P = 0.003) and PD-L1 (3.6% vs. 13% vs. 24%; P < 0.001) than BRAFV600E and wild-type tumors, respectively. The RASmut TiME had lower overall inflammation and fewer infiltrating CD8+ T cells than wild-type or BRAFV600E tumors. In contrast, BRAFV600E tumors exhibited hyperproliferative characteristics associated with broad metabolic reprogramming but a similarly inflamed TiME compared with wild-type tumors. Conclusions: Our data suggest that MSI/dMMR colorectal cancers harboring RASmut are less immunogenic, and the TiME contains a lower inflammatory profile than wild-type or BRAFV600E tumors. Further analysis and validation are required to confirm these findings.

e16479 Background: Recent genomic data has demonstrated SBA to be a molecularly unique entity when compared to colon or gastric cancers. The ongoing SWOG 1922 randomized clinical trial is comparing FOLFIRI to ramucirumab/paclitaxel as the second-line treatment for metastatic SBA. We sought to further define the activity of taxane-based therapy in the largest collection of SBA patients treated with this therapy. Methods: A retrospective review of SBA patients treated with taxane-based chemotherapy from 1994 to 2024 at MD Anderson Cancer Center was performed. All patients had pathologic confirmation, received > 1 cycle of therapy, and had documented tumor response evaluation. Response was assessed by the treating physician based on the imaging studies and categorized as response, stable disease or progression. Patient demographics, clinical parameters, molecular data, and taxane treatment was reviewed. Kaplan-Meier survival analysis was performed to calculate time to progression (TTP) and overall survival (OS). Results: 70 SBA patients treated with taxane-based therapy were identified. The median age at diagnosis was 57 years (range 33-80 years), majority being male (59%) with primary tumor located at duodenum (44%), Jejunum (34%), Ileum (16%). The most common presenting metastatic site was peritoneum (39%), followed by liver (31%). The genomic status revealed mutations in the following genes: 62% TP53, 47% KRAS, 24% SMAD4, and 15% APC. Taxane treatment was single agent (nab-paclitaxel, paclitaxel, or docetaxel) in 29%, gemcitabine and taxane combination in 61% and other taxane-based combinations in 10%. Therapy was given in the metastatic setting as 2nd-line in 40%, 3rd-line in 33% and 4th-line or greater in 20%. Response status was response in 24%, stable disease in 26%, and progression in 50%. Median TTP was 3.1 months (95%CI: 2.0-4.2 months), and median OS was 8.7 months (95%CI: 7.4-10.1 months). Median TTP was similar between single agent (2.6 months) and gemcitabine-based combination (2.5 months) (p =0.75). Efficacy did not differ by line of therapy nor primary tumor site. Efficacy differed by TP53 mutation status: TP53 mutated vs wildtype (response 21% vs 43%, p = 0.02; median TTP 2.4m vs 4.9m, p = 0.013; median OS 7.3m vs 10.6m, p = 0.004). Conclusions: This retrospective study of 70 SBA patients confirms the activity of taxane-based therapy demonstrating a response rate of 24% and median TTP of 3.1 months. Though limited by sample size, TP53 mutations may represent a negative predictive marker for taxane-based therapy in SBA patients. These findings support the ongoing SWOG 1922 trial investigating taxane-based therapy as a second line for metastatic SBA.

231 Background: The recent development of KRAS G12C inhibitors underscores the potential to target KRAS mutations. Right-sided and left-sided colon tumors (RT and LT) exhibit different molecular features. We characterize the prevalence of KRAS-variants, interrelation with primary tumor location, and association with immune biomarkers in CRC. Methods: We retrospectively reviewed CRC tumors of all stages (with known sidedness) that underwent NGS with the Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Bivariate analyses were performed to compare KRAS alterations, immune biomarkers, and co-mutations by tumor location. P-values comparing individual co-mutations between groups were adjusted for false discovery (FDR). Results: A total of 3,391 CRC were analyzed (RT: n = 442 [13%], transverse: n = 116 [3%], LT; n = 2,833 [84%]) of which 1486 (44%) tumors harbored KRAS mutations. Overall, KRAS mutations were more frequent in RT compared to transverse tumors and LT (52% vs 41% vs 43%, p<0.001, respectively). The most frequent KRAS mutation variants observed were G12D (29 %), G12V (22%), G13D (16%), and G12C (5.7%). There was no significant difference in the prevalence of KRAS variant types between LT and RT (p=0.5). Significant differences in genomic co-mutations with various KRAS variants were observed in the following genes: TP53, FBXW7, and NF1 (FDR- P<0.05). RT and transverse tumors were more likely have MSI-H and TMB-H (>10 mut/mb) status than LT (MSI-H: 18% vs 22% vs 2.2% and TMB-H (20% vs 22% vs 3%, P<0.001), respectively. CRC tumors harboring G13D variants were more likely to be associated with and MSI-H and TMB-H status (and 7.7% and 8.5%) compared to G12D (2.8 % and 3.9 %), G12V (1.8 % and 2.1%), and G12C (0% and 2.4%); P = 0.003 and 0.001. Conclusions: The most frequent KRAS mutation variants observed in CRC tumors were G12D, G12V, G13D, and G12C. There was no significant difference in the prevalence of KRAS variant types between tumors of the left vs right colon. CRC tumors that harbored G13D variants were significantly more likely to be associated with MSI-H and TMB-H status. [Table: see text]

4023 Background: Metastatic pancreatic cancer (mPC) remains a deadly disease with very limited treatment options. FFX is a standard first-line therapy for mPC with a median overall survival (mOS) of 11.1 months. CPI-613 is a stable intermediate of a lipoate analog that inhibits pyruvate dehydrogenase and α-ketoglutarate dehydrogenase enzymes of the tricarboxylic cycle preferentially within the mitochondria of cancer cells. In a phase I study, CPI-613+mFFX was safe and exhibited promising signal of efficacy. Methods: A global, randomized phase 3 trial was conducted across 73 sites to investigate the efficacy and safety of CPI-613 in combination with mFFX compared to standard dose FFX in treatment-naïve patients with mPC. Treatment was administered in 2-weekly cycles until progression or intolerable toxicity. In the experimental arm, CPI-613 at 500 mg/m2 was given intravenously on days 1 and 3. The doses of irinotecan, oxaliplatin, and 5-fluorouracil in the experimental arm were 65 mg/m2, 140 mg/m2, and 2,400 mg/2, respectively. Primary endpoint was OS. Secondary endpoints were progression-free survival (PFS), overall response rate (ORR), duration of response, pharmacokinetics, patient reported outcomes and safety. Results: 528 patients were randomly assigned (266 in test and 262 in control arm). There were 362 deaths, with a mOS of 11.1 months for CPI-613+mFFX vs. 11.7 months for FFX [hazard ratio (HR), 0.95; 95% CI, 0.77 to 1.18; P = 0.655]; mPFS was 7.8 months vs. 8.0 months respectively [HR, 0.99; 95% CI, 0.76 to 1.29; P = 0.94]; ORR was 39% in the test arm vs. 34% in the control arm [ORR ratio, 1.23 (95% CI, 0.86 to 1.75)]. Grade ≥ 3 treatment-emergent adverse events with ≥ 10% frequency in CPI-613 plus mFFX vs. FFX arm were diarrhea (11.2% vs. 19.6%), hypokalemia (13.1% vs. 14.9%), anemia (13.9% vs. 13.6%), neutropenia (11.2% vs. 14.0%), thrombocytopenia (11.6% vs. 13.6%) and fatigue (10.8% vs. 11.5%). Conclusions: The addition of CPI-613 to mFFX failed to show significant improvements of ORR, PFS or OS. The mFFX in the test arm that had the lowest prospectively tested doses of FFX was without compromise on PFS or OS and may be considered as a reference for future FFX administration. Clinical trial information: NCT03504423.

3067 Background: The KEYNOTE-177 trial demonstrated pembrolizumab’s superiority over first-line chemotherapy in patients with MSI-H/dMMR mCRC. However, in a subgroup analysis, patients with KRAS or NRAS mutations did not show the same favorable PFS benefit with PD-1 blockade therapy (HR 1,19; CI 0.68-2,07). The impact of RAS mutations on the immunologic characteristics of the TME of MSI-H/dMMR CRC has not been well characterized. Methods: A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing data (Tempus |xT assay, DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). MSI-H determined by assessment of 239 loci by NGS. Several immune markers were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immune pathways (82 geneset signatures). Results: A total of 463 MSI-H/dMMR CRCs were analyzed, of which 110 (24%) tumors harbored RAS mutations ( RASmut) [ KRAS: 93%, NRAS 6% and HRAS 1%}, while 353 were RAS-wild-type ( RASWT). Compared to MSI-H/dMMR RASWT, MSI-H/dMMR RASmut tumors were more frequently identified in males (53% vs. 38%; P= 0.005), and younger patients (median age: 57 yrs vs. 71 yrs, P< 0.001). Although there were no significant differences in median TMB (40 mut/MB for both, p = 0.9) or frequency of TMB-high status (≥10 mut/MB) between the two groups, RASmuttumors tended to have a lower tumor NTB (16 vs. 12 neoAg/Mb, P <0.001) and lower % CD8 T cell but higher % CD4 T cell infiltration (P < 0.05). Significant differences were observed in genomic alterations co-occurring with RASmut compared to RASWT (e.g., MLH1 (23% vs. 8.8%, P < 0.001), MSH6 (36% vs. 24%, P = 0.017), APC (60% vs. 20%, P< 0.001), ARID1A (54% vs 30%, P< 0.001), PIK3CA (36% vs 19%, P< 0.001), and TP53 (32% vs. 19%, P = 0.014). Pathway enrichment analysis identified 14 differentially expressed pathways among RASmut tumors. Four pathways showed significant upregulation, including Hedgehog, Wnt, TGFβ, and cancer stem cell pathways. Ten pathways of interest showed significant downregulation among RASmut tumors. The majority (9/10) were immune-related, including cytokine signaling [ JAK-STAT, TGFβ, TH1], innate immune [ NK cells], and adaptive immune events (CD8 T cell, Tregs)]. Conclusions: MSI-H/dMMR CRCs harboring RASmut exhibited overall upregulated WNT/SHH pathway activity, coupled with reduced NTB, cytokine signaling, and innate and adaptive immune events. TGFβ is pleiotropic, and different members were associated with variable modulation. These data suggest that MSI-H/dMMR CRCs harboring RAS mutations are less immunogenic and appeared to contain a TME that is less sensitive to immune checkpoint blockade than MSI-H/dMMR RAS wt CRCs.

3066 Background: The BRAFV600E mutation is associated with the hypermethylator phenotype CIMP, which can also lead to the MSI-H phenotype. BRAFV600E mutation and MSI-H/dMMR status seem to be biologically intertwined; however, the impact of coexisting BRAFV600E mutations on the TME and immunometabolomic features of MSI-H/dMMR CRC tumors is not well characterized. Methods: A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing data (Tempus |xT assay: DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). Several immune markers of tumor immunogenicity in BRAF wild-type ( BRAFwt) vs. V600E-mutated (BRAFV600E) tumors were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immuno-metabolomic pathways (82 geneset signatures). Results: A total of 459 MSI-H/dMMR CRC tumors were analyzed, of which 123 (27%) tumors harbored BRAFV600Emutations, and 336 (73%) were BRAFwt. MSI-H/dMMR BRAFV600E tumors were more frequently identified in females (69% vs. 55%; P= 0.01), non-Hispanic or non-Latino (100% vs. 73%; P =0.001), and older patients (median age: 76 yrs vs. 62 yrs; P< 0.001). Compared to BRAFWT, BRAFV600E tumors exhibited significantly higher TMB (Median: 49 mut/MB vs. 36 mut/MB; P < 0.001) and were more frequently associated with TMB-High status (> 10 mut/MB; 100% vs. 95%; P = 0.008); however, no significant differences were observed with tumor NTB, immune score, or T cell infiltration (CD4 or CD8). NK cell infiltration was higher in the BRAFV600Ecohort (< 0.001). When compared to BRAFWT tumors , BRAFV600E tumors harbored a greater frequency of mutations in MSH6(42% vs. 20%), B2M (33% vs. 16%), BRCA2 (31% vs. 12%), ATM (23% vs. 12%), and TP53 (30% vs. 19%) but a lower frequency of MSH2 (3.3% vs. 11%), all P< 0.05. Pathway enrichment analysis identified 10 significantly altered signaling pathways, most of which related to stromal/immune cell signaling and metabolism. Five were upregulated among BRAFV600E tumors: glycerophospholipid, galactose, cyclin-dependent cell signaling; Nucleotide, and TH1 inflammation. Five pathways were downregulated (Wnt, Notch, TH17 inflammation, amino sugar, and cancer stem cell signaling). Conclusions: MSI-H/dMMR BRAFV600E CRCs undergo broad metabolic reprogramming (e.g., glycerophospholipidgalactose, nucleotide). A rise in lipid metabolism, particularly with NK inflammation, suggests that BRAFV600Emutated tumors may be associated with a non-classical immune component. BRAFV600E and BRAFwt CRCs exhibited similar NTB and T cell infiltration, suggesting that both are likely to benefit from immune checkpoint inhibitors.

Abstract Background Disease-free survival (DFS) with a 3-year median follow-up (3-year DFS) was validated as a surrogate for overall survival (OS) with a 5-year median follow-up (5-year OS) in adjuvant chemotherapy colon cancer (CC) trials. Recent data show further improvements in OS and survival after recurrence in patients who received adjuvant FOLFOX. Hence, reevaluation of the association between DFS and OS and determination of the optimal follow-up duration of OS to aid its utility in future adjuvant trials are needed. Methods Individual patient data from 9 randomized studies conducted between 1998 and 2009 were included; 3 trials tested biologics. Trial-level surrogacy examining the correlation of treatment effect estimates of 3-year DFS with 5 to 6.5-year OS was evaluated using both linear regression (RWLS2) and Copula bivariate (RCopula2) models and reported with 95% confidence intervals (CIs). For R2, a value closer to 1 indicates a stronger correlation. Results Data from a total of 18 396 patients were analyzed (median age = 59 years; 54.0% male), with 54.1% having low-risk tumors (T1-3 and N1), 31.6% KRAS mutated, 12.3% BRAF mutated, and 12.4% microsatellite instability high or deficient mismatch repair tumors. Trial-level correlation between 3-year DFS and 5-year OS remained strong (RWLS2 = 0.82, 95% CI = 0.67 to 0.98; RCopula2 = 0.92, 95% CI = 0.83 to 1.00) and increased as the median follow-up of OS extended. Analyses limited to trials that tested biologics showed consistent results. Conclusions Three-year DFS remains a validated surrogate endpoint for 5-year OS in adjuvant CC trials. The correlation was likely strengthened with 6 years of follow-up for OS.

3502 Background: DFS with 3 years median follow-up (3yDFS) was validated as a surrogate for OS with 5 years median follow-up (5yOS) in adjuvant chemotherapy CC trials prior. Recent data showed improved survival after recurrence and OS, over time, in pts who received adjuvant FOLFOX. Hence, re-evaluation of the association between DFS and OS, as well as the optimal follow-up of OS to aid its utility in future adjuvant trials is needed. Methods: Individual patient data from 8 randomized adjuvant studies conducted from 1998-2009 were included; 3 trials tested anti-VEGF or anti-EGFR agents. Trial-level surrogacy examining the correlation of treatment effect estimates (i.e. hazard ratios) of 3yDFS and 5y to 8yOS was evaluated using both linear regression (R2WLS) and Copula bivariate (R2Copula) models. For the R2, a value closer to 1 indicates a stronger correlation. Prespecified criteria for surrogacy required either R2WLS or R2Copula ≥ 0.80 and neither < 0.7, with lower-bound 95% Confidence Interval (CI) > 0.60. The rank correlation coefficient (ρ) quantified the individual-level surrogacy. Results: Total of 18,886 pts were analyzed, with median age 60, 54% male, 83% stage III, 59% > 12 nodes examined. Median follow-up for survival ranged from 5 to 10 years across trials. Trial level correlation between 3yDFS and OS remained strong (R2WLS ≥0.74; R2Copula ≥ 0.89) and increased as the median follow-up of OS extended longer (see table). Analyses limited to stage III pts and/or trials tested biologics showed consistent results. Conclusions: 3yDFS remains a validated surrogate endpoint for 5yOS in adjuvant trials in CC pts per prespecified criteria. The correlation was strengthened with more than 6 years of follow-up for OS. [Table: see text]

606 Background: Between 3-5% of CRC patients have BRCA1/2 pathogenic mutations. This study aims to identify associations between BRCA1 and BRCA2 mutations and clinical characteristics in CRC. Methods: A total of 6396 CRC tumor samples were tested with Next-Generation Sequencing (NGS) on a 592-gene panel, pathogenic or presumed pathogenic variants were counted as mutations (mt). Microsatellite instability (MSI) and tumor mutational burden (TMB) were tested by NGS. Statistical correlations were investigated using ANOVA, Chi-square and t-test. Results: Among tumors sampled, 53% derived from male patients and median age was 60 years. BRCA1 mt were detected in 1.1% (n = 72) of tumors, while BRCA2 in 2.8% (n = 179). BRCA1 mt were more frequent in women (W;65%) than men (M;35%) (p = 0.0019) while no relationship with sex was seen for BRCA2 mt (42% F vs. 58% M). No significant associations with age were noticed. Majority of pathogenic mt in BRCA1 (52%; n = 34) and BRCA2 (62%; n = 103) occurred in MSI-High (MSI-H) cases. MSI-H pts had more frameshift mt in both BRCA1/2 than MSS pts. MSS cases had lower rates of BRCA1 and 2 pathogenic mt (44% and 37%, respectively). Right-sided tumors were significantly associated with BRCA1 (p = 0.0056) and BRCA2 (p < 0.0001) mt in MSI-H cases only. BRCA1/2 mt were associated with higher TMB in all CRCs, including MSI-H and MSS cases (p < 0.001). POLE mt (n = 31) were associated with higher BRCA1/2 mt rates (9.6%, 55% respectively). Among MSS cases with POLE wild-type status, BRCA1 (p = 0.0269) and BRCA2 (p = 0.0151) mt were associated with high TMB and combining both BRCA1/2 mutations led to an even higher TMB (3.6%; p = 0.001). Conclusions: This is the first study to show that BRCA1/2 mutations are more frequent in MSI-H, and independently associated with higher TMB, pathogenic POLE mutations, and right-sided tumors in MSI-H CRCs. Given their relationship with TMB, the presence of BRCA1/2 mutations may be potential predictive biomarkers for checkpoint or PARP inhibitors in CRC, a finding that should be prospectively evaluated.

60 Background: DDR mutations are associated with higher neoantigen load and tumor infiltrating lymphocytes, and are a potential biomarker for immunotherapy. We investigated the association of DDR mutations in gastric (GC), esophageal (EC), and gastroesophageal junction (GEJ) cancers with MSI, PD-L1, and TMB, known predictors for immune checkpoint inhibitors. Methods: 20 DDR mutations were tested by Next-Generation Sequencing (NGS) with a 592-gene panel on a total of 1935 (709 EC; 831 GC; 355 GEJ) cancers. TMB was assessed by NGS, MSI by NGS or fragment analysis, and PD-L1 by IHC (22c3 for CPS or SP142). Results: GC had the highest DDR mutation rate compared to EC and GEJ (27% vs. 20%, p = 0.0005 and 17%, p = 0.0002, respectively). MSI-High (MSI-H) was significantly more common in the DDR mutated cohort (DDR-M) compared to non-mutated cancers (18% vs. 1%; p < 0.0001). TMB-High (≥ 10 mutations/megabase [mt/MB]) was higher in DDR-M (35% vs. 21%; p < 0.0001); in DDR-M cohort, GC had the highest TMB compared to DDR-M EC and GEJ (mean: 13.8 vs. 9.4 vs. 10 mt/MB, respectively; p < 0.0001). DDR mutations were more frequent in the PD-L1 combined positive score (CPS) ≥ 50 group than CPS 0 (42.9% vs. 24%; p = 0.037) and CPS 1-9 (42.9% vs. 20.6%; p = 0.005). ARID1A, ATRX, BRCA2, and PTEN were the most prevalent DDR mutations in MSI-H (87%, 31%, 25%, 24%, respectively); ARID1A, ATRX, BRCA2, and PTEN in TMB-High (47%, 7.7%, 6.7%, 6.8%) and ARID1A, BRCA2, RAD50, and WRN in PD-L1 high (CPS ≥ 10) (48.5% vs. 5.2% vs. 2.5% vs. 3.4%). Conclusions: MSI-H, TMB-high and high PD-L1 expression were significantly more prevalent in the DDR-M cohort compared to non-DDR-mutated cancers, most pronounced in GC. Alterations in ARID1A, ATRX, BRCA2, and PTEN were correlated with MSI-H and TMB-high while ARID1A, BRCA2, RAD50, and WRN were correlated with increased PD-L1 expression. Our findings may help identify patients for tailored immunotherapy approaches in future clinical trials.