Mauer, Elizabeth

Abstract Background The association of germline pathogenic and likely pathogenic variants (GPVs) in hereditary breast cancer genes with underlying tumor biology and clinical outcomes remains incompletely understood. This study characterized differences in somatic alterations and intrinsic subtypes between sporadic and hereditary breast cancers associated with GPVs in ATM, BRCA1, BRCA2, CHEK2, or PALB2. Methods This retrospective cohort study included women with breast cancer and an ATM, BRCA1, BRCA2, CHEK2, or PALB2 GPV who had undergone tumor sequencing and whole transcriptome RNA expression analysis. Clinicopathological features, intrinsic subtypes, somatic alterations, and survival were compared by GPV status and immunohistochemistry-defined subtype and to sporadic cases. All statistical significance tests were 2-sided. Results The 4988 women with breast cancer in this study included 98 BRCA1, 126 BRCA2, 74 PALB2, 54 ATM, and 83 CHEK2 GPVs. Compared with sporadic cases, hormone receptor–positive/(ERBB2) formerly HER2–negative tumors in BRCA1 GPVs were statistically significantly enriched for basal subtype (45.5% vs 11.4%; P < .001), while CHEK2 carriers had a higher prevalence of luminal A subtype (80.4% vs 60.3%; P = .006). In hormone receptor–positive/(ERBB2)-negative breast cancers, BRCA1 GPVs were enriched for TP53 alterations (84.6% vs 29.8%; q < 0.001), ATM GPVs with FGFR1 alterations (35.4% vs 12.7%; q = 0.04), and BRCA2 GPVs with APC alterations (10.1% vs 1.5%; q = 0.004). Conversely, BRCA2 GPVs were inversely associated with PIK3CA alterations (13.0% vs 34.1%; q = 0.005) and CHEK2 GPVs with TP53 alterations (8.0% vs 29.8%; q = 0.02). Conclusions The GPVs in BRCA1, BRCA2, ATM, CHEK2, and PALB2 are associated with distinct intrinsic breast cancer subtypes and somatic genomic alterations. These findings may enhance precision in risk stratification and guide personalized treatment strategies.

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.

Abstract Purpose: Germline alterations in homologous recombination repair (gHRR) genes affect the pathogenesis, treatment options, and survival of patients with cancer. However, distinct gHRR gene alterations may differentially affect treatment response and oncogenic signaling. In this study, we interrogated genomic and transcriptomic data and assessed clinical outcomes of patients with gHRR mutations across four BRCA-associated cancers (breast, ovarian, pancreatic, and prostate cancers) to identify therapeutic vulnerabilities. Experimental Design: We assessed 24,309 patients undergoing matched tumor/normal next-generation DNA and RNA sequencing. Annotated gHRR gene variants [germline BRCA1, germline BRCA2, germline PALB2, germline ATM (gATM), and germline CHEK2] were analyzed. HRs were used to assess survival outcomes comparing germline versus sporadic groups. Somatic alterations and their frequencies were compared across gHRR-altered groups. Differential gene expression and gene set enrichment analysis were used to compare transcriptomic profiles. Results: Somatic TP53 mutations were depleted in gATM carriers (P < 0.05) across all four BRCA-associated cancers by up to 2.5-fold. Tumors with germline BRCA1/2 mutations were associated with improved survival in patients with ovarian cancer and had consistent enrichment of TP53 mutations in all four cancers. gATM mutations displayed elevated p53 transcriptional activity in all four cancers, with significance reached in breast and prostate cancers (P < 0.01). In breast, ovarian, and prostate cancers, gATM tumors demonstrated significantly increased inflammatory pathways (P < 0.001). Finally, using gene dependency data, we found that cell lines that were highly dependent on ATM were co-dependent on canonical p53 function. Conclusions: gATM-associated cancers seem to require intact p53 activity and this synthetic essentiality may be used to guide targeted therapies that perturb canonical TP53 function.

Abstract Purpose: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. Experimental Design: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. Results: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. Conclusions: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

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]

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]

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.

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.