Stoppler, Melissa C.

Background: Rates of early-onset colorectal cancer (eoCRC), defined as disease diagnosed at <50 years of age, are increasing. The incidence and spectrum of somatic and pathogenic germline variants (PGV) in this population are not well understood. Methods: This cross-sectional study leveraged Tempus’ clinicogenomic database, including de-identified records of patients diagnosed with CRC between 2000–2022, to analyze and compare eoCRC and average-onset colorectal cancer (aoCRC, disease diagnosed ≥50 years of age) patients. The frequency and spectrum of somatic mutations and PGVs in patients with eoCRC and aoCRC were evaluated and compared. Results: Among 11,006 participants in this study, 57% were male, 76% were white, and 80% had stage 4 disease. Within the total cohort, 2379 had eoCRC and 8627 had aoCRC. Among patients with eoCRC, 4.2% had a tumor with high microsatellite instability and/or deficient mismatch repair (MSI-H/dMMR) and 6.8% with aoCRC had an MSI-H/dMMR tumor (p < 0.001). The most frequent somatic mutations involved TP53, APC, and KRAS, with the most significant difference in BRAF, which was more frequently mutated in aoCRC (9.8% vs. 4.7%, p < 0.0001). In total, 1413 (59.4%) eoCRC and 4898 (56.8%) aoCRC patients had matched normal specimen (blood or saliva) sequencing and a PGV was identified in 6.9% of eoCRC and 5.0% of aoCRC patients. Conclusions: Somatic and germline mutation profiles were similar for eoCRC and aoCRC patients and may not adequately explain differences in tumor behavior and age of disease onset.

496 Background: Incidence of early-onset gastroesophageal adenocarcinoma (eoGEA, ages <50 years) has been increasing in the United States since the 1990s, the etiology of which is still unclear, and limited data exists on molecular drivers. This study evaluates somatic and germline profiles in eoGEA compared to average-onset GEA (aoGEA, ages≥ 50 years). Methods: This is a retrospective, cross-sectional study utilizing data from de-identified records of GEA (esophageal, gastroesophageal junction and gastric adenocarcinomas) patients who underwent somatic NGS testing via the Tempus xT assay (595-648 gene DNA panel) from 12/2017 to 07/2024. This assay assesses somatic tumor mutations (including SNVs, Indels, CNVs, and select SVs), MSI, TMB, and incidentally detected germline SNVs and small indels in matched normal tissue. Clinical characteristics and immunotherapy markers were compared between eoGEA and aoGEA by Wilcoxon Rank Sum or Chi-squared test. Somatic and germline mutations were compared between two age groups with false discovery rate adjustments. Results: We compared the demographic, clinical, and molecular features of a total of 5863 patients with eoGEA (n=785, median age 43) and aoGEA (n=5078, median age 68). Over 80% of patients were diagnosed with stage IV disease. The eoGEA group had a higher proportion of females, non-white, and Hispanic or Latino patients compared to the aoGEA group (p<0.001). Prevalence of MSI-H/ dMMR, and TMB-H was lower in eoGEA vs aoGEA (p<0.001), while PD-L1 expression was similar. Prevalence of somatic CDH1 , CDKN2A , and TP53 SNVs, and FGFR2 and KRAS CNAs were higher in eoGEA vs aoGEA (q<0.001). In a subset of 3286 patients with tumor/normal match testing (eoGEA=464 and aoGEA=2822), eoGEA also had a higher prevalence of germline CDH1 mutations (q<0.001). Conclusions: eoGEA has a unique somatic and germline mutation profile compared to aoGEA; further study evaluating the molecular landscape including epigenetic changes could shed light on underlying mechanisms responsible for the rising incidence of eoGEA. eoGEA (n=785) aoGEA (n=5078) p-value/q-value* Baseline characteristics Median age at diagnosis (IQR) 43 (38, 47) 68 (61,74) <0.001 Gender: male 509 (65%) 3798 (75%) <0.001 Race (white) 280 (69%) 2347 (80%) <0.001 Ethnicity (not hispanic or latino) 206 (60%) 1768 (86% <0.001 Stage IV 495 (85%) 2883 (81%) 0.073 Immunologic markers TMB ≥ 10 25 (3.2%) 519(10%) <0.001 MSI-H 13 (1.7%) 261 (5.1%) <0.001 dMMR 7 (2.2%) 120 (5.9%) 0.007 Somatic mutations profiles * CDH1 126 (16%) 334 (6.6%) <0.001 TP53 515 (66%) 3764 (74%) <0.001 CDKN2A 101 (13%) 1011 (20%) <0.001 KRAS 92 (12%) 931 (18%) <0.001 NOTCH1 12 (1.5%) 228 (4.5%) 0.002 Germline mutation profiles * eoGEA (N=464) aoGEA (N= 2,822) Overall prevalence 46 (9.9%) 207 (7.3%) CDH1 10 (2.2%) 8 (0.3%) 0.001 TP53 3 (0.6%) 0 (0%) 0.039 BRCA2 2 (0.4%) 38 (1.3%) 0.6 BRIP1 4 (0.9%) 10 (0.4%) 0.6

Abstract Introduction Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H), defined as TMB ≥ 10 mut/Mb, independent of deficient mismatch repair (dMMR) and microsatellite instability high (MSI-H) status. Methods De-identified records of patients with colorectal cancer (CRC) profiled with the Tempus xT assay (DNA-seq of 595-648 genes at 500×) were identified from the Tempus Database. Results Among 9136 CRC samples profiled, the frequency of POLE/POLD1 genomic alterations was 2.4% (n = 217). Copy number loss was the most common genomic alteration (64%, n = 138) of POLE/POLD1, followed by copy number amplifications (18%, n = 40) and short variant mutations (18%, n = 39). The POLE/POLD1 mutated group presented with a higher frequency of TMB-H phenotype relative to wild type (WT; 22% vs. 9%, P &lt; .001), with a median TMB of 127 mut/Mb in the TMB-H POLE/POLD1 subset. The TMB showed a dramatic contrast between POLE/POLD1 short variant mutations as compared to the group with copy number alterations, with a TMB of 159 mut/Mb vs 15 mut/Mb, respectively. Thus, the short variant mutations represented the so-called ultra-hypermutated phenotype. The POLE/POLD1 mutated group, as compared to WT, exhibited a higher rate of coexisting mutations, including APC, ALK, ATM, BRCA2, and RET mutations. Conclusion Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers (ie, TMB, MMR, and MSI-H) and molecular co-alterations. Those with short variant mutations represented 18% of the POLE/POLD1 cohort and 0.4% of the total cohort examined. This group of patients had a median TMB of 159 mut/Mb (range 34-488), representing the ultra-hypermutated phenotype. This group of patients is important to identify given the potential for exceptional response to immune checkpoint inhibitors.

Background: The predictive and prognostic role of BRAF alterations has been evaluated in colorectal cancer (CRC); however, BRAF alterations have not been fully characterized in non-CRC gastrointestinal (GI) malignancies. In the present study, we report the frequency and spectrum of BRAF alterations among patients with non-CRC GI malignancies. Methods: Patients with CRC and non-CRC GI malignancies who underwent somatic tumor profiling via a tissue-based or liquid-based assay were included in this study. Gain-of-function BRAF alterations were defined as pathogenic/likely pathogenic somatic short variants (SVs), copy number amplifications ≥8, or fusions (RNA or DNA). Results: Among 51,560 patients with somatic profiling, 40% had CRC and 60% had non-CRC GI malignancies. BRAF GOF alterations were seen more frequently in CRC (8.9%) compared to non-CRC GI malignancies (2.2%) (p < 0.001). Non-CRC GI malignancies with the highest prevalence of BRAF GOF alterations were bile duct cancers (4.1%) and small intestine cancers (4.0%). Among BRAF GOF alterations, class II (28% vs. 6.8%, p < 0.001) and class III (23% vs. 14%, p < 0.001) were more common in non-CRC GI malignancies. Among class II alterations, rates of BRAF amplifications (3.1% vs. 0.3%, p < 0.001) and BRAF fusions (12% vs. 2.2%, p < 0.001) were higher in non-CRC GI malignancies compared to CRC. Conclusions: Non-CRC GI malignancies demonstrate a distinct BRAF alteration profile compared to CRC, with a higher frequency of class II and III mutations, and more specifically, a higher incidence of BRAF fusions. Future studies should evaluate clinical implications for the management of non-CRC GI patients with BRAF alterations, especially BRAF fusions.

759 Background: BRAF V600E mutations are present in 5-10% of patients with advanced colorectal cancer (CRC) and associated with poor prognosis. Recently, there was tumor agnostic FDA approval of dabrafenib + trametinib for BRAF V600E mutated solid tumors. However, the frequency of BRAF alterations ( BRAF alt), especially non-V600E, in other gastrointestinal (GI) cancers are not well described. This study characterizes BRAF alt in CRC vs other GI cancers (non-CRC). Methods: De-identified records from 51,560 patients diagnosed with GI cancers were retrospectively analyzed from the Tempus database. The molecular landscape including presence of pathogenic/likely pathogenic BRAF alt was evaluated by panel-based tissue DNA, RNA or ctDNA NGS sequencing (Tempus Labs, Inc.). The frequency of BRAF alt, co-mutations, MSI, TMB and MMR were compared between CRC and (non-CRC) by Chi-squared/Fisher’s Exact or Wilcoxon rank-sum tests. False-discovery rate correction was used for multiple testing. Results: BRAF alt were identified in 8.9% of CRC and 2.2% of non-CRC cohort. BRAF alt frequency varied by tumor type with the highest observed in colon (11%), jejunum (9.6%), intrahepatic bile duct (5.3%), rectal (4.5%), and ampulla of vater (4.5%) and lower in esophageal (0.9%) and gastric (1.6%). BRAF alt CRC had higher frequency of females (57% vs 45%, p <0.001) and older age at diagnosis (67 vs 66 yrs, p=0.029) compared to non-CRC group. Race was also significantly different, with white patients being more predominant in both groups (85% CRC,77% non-CRC). Among the BRAF alt, fusions (12% vs 2.2%, p <0.001) and amplifications (3.1% vs 0.3%, p <0.001) were higher in non-CRC vs CRC (Table). BRAF V600E was most common in all GI tumors, but higher in CRC vs non-CRC (75% vs 27%, q<0.001). Mutations higher in non-CRC vs CRC (q<0.009) included class II (G469A, K601E, BRAF-SND1, and BRAF amplification) and class III (D594N & N581S) BRAF alt. BRAF co-occurring mutations in CRC were TP53 (70%), APC (42%), RNF43 (30%), SMAD4 (22%), and PIK3CA (21%), and in non-CRC: TP53 (52%), CDKN2A (32%), CDKN2B (20%) SMAD4 (17%) and MTAP (16%). Among tissue samples, MSI-H (30% vs 4.%, p <0.001) and TMB-H ≥10 mut/MB (32% vs 6.7%, p <0.001) were more frequent in BRAF alt CRC vs non-CRC. Conclusions: Meaningful differences were found between the groups in terms of demographics, frequency, and type of BRAF alt and co-occurring mutations. In particular, certain BRAF fusions were present in a higher proportion of other GI cancers and could be an important therapeutic target for this patient population.[Table: see text]

3536 Background: Early-onset colorectal cancer (eoCRC, initial CRC diagnosis at age < 50 years) has been increasing in the past two decades especially in Western countries. This study evaluates somatic and germline profiles in eoCRC compared to average-onset CRC (aoCRC, initial CRC diagnosis at age ≥ 50 years). Methods: This is a retrospective, cross-sectional study utilizing data from de-identified records of colorectal cancer patients tested with the Tempus xT assay from 2017 to 2022. Briefly, the assay is a targeted panel that detects single nucleotide variants, insertions and/or deletions, and copy number variants in 598-648 genes, as well as chromosomal rearrangements in 22 genes with high sensitivity and specificity. Baseline characteristics and immunologic markers were compared between eoCRC and aoCRC by Wilcoxon Rank Sum or Chi-squared test (reporting p-values). Somatic and germline mutations were compared between two age groups with false discover rate adjustments (reporting q-values). Results: In this study, 2,379 eoCRC and 8,627 aoCRC patients were included, with the majority diagnosed with stage IV disease. Additionally, germline alterations were assessed on a subset of 6,311 patients with tumor/normal match testing (eoCRC = 1,413 and aoCRC = 4,898). Left-sided primaries were more common in eoCRC (85% left/rectum in eoCRC vs. 75% left/rectum in aoCRC (p < 0.001), and rates of microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) were lower compared to aoCRC (4.2% vs. 6.8%, p < 0.001). eoCRC has a unique somatic mutation profile compared to aoCRC. A higher prevalence of germline mutations was observed in eoCRC overall (6.9% vs. 5%, p = 0.006); however, no statistically significant differences were observed in individual germline genes compared to aoCRC, likely due to relatively small numbers. Conclusions: eoCRC has a unique mutational profile and presence of germline mutations in 6.9% of eoCRC, indicating a potential role for universal germline testing in CRC. [Table: see text]

260 Background: NGS to identify HRR mutations (mut) is indicated in advanced prostate cancer (aPC); samples for testing may be tissue or plasma. In a large real-world (RW) database, we determined concordance between plasma ctDNA and primary tumor tissue (PT) and/or metastatic tissue (MT) for BRCA1, BRCA2, and ATM mut in PC patients (pts) who received both LB and tissue NGS any time during standard of care (SOC) management. The study objective was to assess the utility of LB to detect actionable mut in these HRR genes and demonstrate the utility of combined LB and tissue testing. Methods: We examined de-identified data from pts who had NGS using both Tempus xF (105 genes-LB) and Tempus xT (648 genes-tissue) during SOC for aPC. Analyses included 1) PT and LB and 2) MT and LB. The results from the pts’ earliest PT or MT and earliest LB were used for paired analyses. The prevalence of a pathogenic/likely pathogenic germline and/or somatic mut in BRCA1, BRCA2, or ATM was reported as N (%), 95% CI. The sensitivity of the LB to identify observed HRR mut in tissue was also reported as N (%), 95% CI. Concordance between pairs was evaluated by Cohen’s kappa statistic with 95% CI. Results: HRR results are show for 1074 PC pts who had NGS performed on PT and LB and 451 PC pts with NGS on both MT and LB. In the PT and LB analysis, HRR mut were found in 6.2% (95% CI: 4.9%-7.9%) of LB and 8.8% (95% CI: 7.2%-11%) of PT. The sensitivity of LB to detect HRR mut in the PT was 53% (95% CI: 43%-64%). Cohen’s kappa statistic was 0.59, 95% CI=0.50-0.68, between PT and LB. In the MT and LB analysis, HRR mut were found in 10% (95% CI 7.8%-14%) of LB and 10% (7.6%-13%) of MT. The sensitivity of LB to detect HRR mut in the MT was 70% (95% CI: 54%-82%). Cohen’s kappa statistic was 0.65, 95% CI=0.54-0.77, between MT and LB. Conclusions: In this large RW dataset, plasma ctDNA-based analysis of BRCA1, BRCA2, and ATM mut showed greater concordance between LB and MT than LB and PT, and may be used as an initial tool for HRR mut detection. When LB results are negative, further exploration with tissue-based testing may identify HRR mut to guide clinical decisions. [Table: see text]

199 Background: The progression of neoplasia in colorectal cancer (CRC) has been well characterized, with the evolution typically involving mutations in APC, KRAS, and p53, among others. POLE/POLD1 genes encode for proteins essential in enzymatic DNA polymerase function, yet POLE/POLD1 mutations in tumors are poorly characterized. Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H) independent of deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H). Studies have shown associations between this hypermutated phenotype and susceptibility to immune checkpoint inhibition, which may be attributable to neoantigen creation. As such, these tumors are a clinically relevant phenotype requiring further investigation. Here, we characterized POLE/POLD1 alterations in a large, real-world cohort of patients with CRC. Methods: We retrospectively analyzed de-identified records of 9,136 primary CRC patients profiled with the Tempus xT assay. The assay includes DNA-seq of 595-648 genes at 500x to evaluate single-nucleotide variants, insertions/deletions, and copy number changes. Immunological markers analyzed included tumor mutational burden (TMB), MSI, and dMMR. MSI-H was determined by the assay through assessment of 239 loci. dMMR was determined by immunohistochemistry. Results: A total of 217 patient tumors harbored somatic POLE/POLD1 mutations (n = 203 POLE, n = 13 POLD1, and n = 1 POLE and POLD1; none germline), with no differences noted in baseline demographics including gender and age. Among the POLE/POLD1-mutant cohort, POLE copy number losses accounted for most mutations (n = 127, 59%), with short variants and copy number amplifications accounting for 35% (n = 76). The remainder of tumors exhibited POLD1 copy number changes (n = 14), including one with both POLE and POLD1 copy number loss. POLE/POLD1-mutated tumors presented with a lower frequency of MSI-H compared to wild-type (1.8% vs 6.1%, P= 0.018). Conversely, there was a higher frequency of TMB-high cases ( > 10 mut/Mb) for POLE/POLD1-mutated compared to wild-type tumors (22% vs 9.9%, P< 0.001). Differences between POLE/POLD1-mutated and wild-type tumors were also observed among many co-mutated genes, including APC (80% vs 65%, P< 0.001), ALK (31% vs 11%, P< 0.001), ATM (12% vs 3.6%, P< 0.001), BRCA2 (12% vs. 3.2%), and RET (24% vs 8.9%, P< 0.001). Conclusions: Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers and molecular co-alterations. POLE/POLD1 mutations in CRC have been previously described to present with a hypermutated phenotype; however, 78% of tumors exhibited low TMB despite POLE/POLD1 mutations. Thus, these results have identified POLE/POLD1-mutated tumors as a unique genomic subpopulation, and further studies are needed to better characterize POLE/POLD1 mutations in CRC.