Elevated Tumor Mutational Burden Research Articles

Mutational spectrum and precision oncology for biliary tract carcinoma.

Background: The genomic spectrum of biliary tract carcinoma (BTC) has been characterized and is associated with distinct anatomic and etiologic subtypes, yet limited studies have linked genomic alterations with personalized therapies in BTC patients.Methods: This study analyzed 803 patients with BTC:164 with gallbladder cancer, 475 with intrahepatic cholangiocarcinoma (ICC) and 164 with extrahepatic cholangiocarcinoma. We determined genomic alterations, mutational signatures related to etiology and histopathology and prognostic biomarkers. Personalized targeted therapies for patients harboring potentially actionable targets (PATs) were investigated.Results: The median tumor mutation burden (TMB) was 1.23 Mut/Mb, with 4.1% of patients having hypermutated BTCs. Unlike the results obtained from the Western population, the most frequently altered cancer-related genes in our cohort included TP53 (53%), KRAS (26%), ARID1A (18%), LRP1B (14%) and CDKN2A (14%). Germline mutations occurred mostly in DNA damage repair genes. Notably, 35.8% of the ICCs harbored aristolochic acid related signatures and an elevated TMB. TP53 and KRAS mutations and amplified 7q31.2 were demonstrated to negatively affect patient prognosis. Moreover, 19 genes were proposed to be PATs in BTCs, with 25.4% of patients harboring these PATs. Forty-six patients received PAT-matched targeted therapies, achieving a 26.1% objective response rate; the median progression-free survival (PFS) was 5.0 months, with 56.8% of patients obtaining PFS benefits.Conclusions: Extensive genomic diversity and heterogeneity were observed among BTC patients, with contributions according to potential etiology exposures, anatomical subtypes and clinicopathological characteristics. We also demonstrated that patients with refractory BTCs who have PATs can derive considerable benefit from receiving a matched therapy, initiating further prospective clinical trials guided by molecular profiling among this aggressive cancer.

Identification of tumor mutation burden-related hub genes and the underlying mechanism in melanoma.

Background: Tumor mutation burden (TMB) has emerged as an important predictive factor for drug resistance in cancers; however, the specific mechanism underlying TMB function in melanoma remains elusive.Methods: Data on somatic mutations, RNA sequencing (RNA-seq), miRNA sequencing (miRNA-seq), and clinical characteristics for 472 melanoma patients were extracted from the TCGA cohort. RNA-seq data of melanoma cell lines were obtained from the Cancer Cell Line Encyclopedia, and sensitivity of cell lines to therapeutic agents is available in the Cancer Therapeutics Response Portal. TMB was calculated based on somatic mutation data. Differentially expressed gene analysis, weighted gene co-expression network analysis, protein-protein interaction networks, Minimal Common Oncology Data Elements, and survival analysis were leveraged to determine TMB-related hub genes. Competing endogenous RNA (ceRNA) networks were constructed to explore the molecular mechanisms underlying hub gene function. The influence of key genes on drug sensitivity was analyzed to investigate their clinical significance.Results: Elevated TMB levels were significantly correlated with improved survival outcomes. In addition, six tumor-infiltrating immune cells, including naive B cells, regulatory T cells, memory resting CD4 T cells, memory B cells, activated mast cells, and resting NK cells, were significantly overexpressed in the low-TMB group relative to the high-TMB group. Furthermore, we identified FLNC, NEXN, and TNNT3 as TMB-related hub genes, and constructed their ceRNA networks, including five miRNAs (has-miR-590-3p, has-miR-374b-5p, has-miR-3127-5p, has-miR-1913, and has-miR-1291) and 31 lncRNAs (FAM66C, MIAT, NR2F2AS1, etc.). Finally, we observed that TMB-related genes were associated with distinct therapeutic responses to AKT/mTOR pathway inhibitors.Conclusions: We identified three TMB-associated key genes, established their ceRNA networks, and investigated their influence on therapeutic responses, which could provide insights into future precision medicine.

Whole-exome sequencing reveals potential mechanisms of drug resistance to FGFR3-TACC3 targeted therapy and subsequent drug selection: towards a personalized medicine

BackgroundDrug resistance is a major obstacle to effective cancer therapy. In order to detect the change in tumor genomic states under drug selection pressure, we use next-generation sequencing technology to investigate the underlying potential mechanisms of drug resistance.MethodsIn our study, we presented a bladder cancer patient who had been a bona fide responder to first-line gemcitabine plus cisplatin regimen and second-line pazopanib (tyrosine kinase inhibitor (TKI) for FGFR3-TACC3 fusion) but finally had disease progression as an ideal case for showing genomic alteration during drug resistance. We applied whole-exome sequencing and ultra-deep target sequencing to the patient pre- and post- pazopanib resistance. Protein-protein interaction (PPI) network and Gene Ontology (GO) analyses were used to analysis protein interactions and genomic alterations. Patient-derived xenograft (PDX) model was built to test drug sensitivity.ResultsTwelve mutations scattered in 12 genes were identified by WES pre- pazopanib resistance, while 63 mutations in 50 genes arose post- pazopanib resistance. PPI network showed proteins from multiple epigenetic regulator families were involved post- pazopanib resistance, including subunits of chromatin remodeler SWI/SNF complex ARID1A/1B and SMARCA4, histone acetylation writers CREBBP, histone methylation writer NSD1 and erasers KDM6A/5A. GO enrichment analysis showed pazopanib resistance genes were prominently tagged for chromatin modification, transcription, as well as gland development, leaving genes with the best adaptive FGFR TKI-coping mechanisms. In addition, significantly elevated tumor mutational burden suggested possible utility of immunotherapy. Intriguingly, PDX model suggested that, sensitivity to original chemotherapy regimen (cisplatin) was restored in patient tumor post-pazopanib.ConclusionsEpigenetic regulation may play a role in acquired TKI resistance. Our study traced the complete tumor genomic variation course from chemo-resistant but TKI-sensitive to TKI-resistant but chemo-(re) sensitive, revealing the potential complex dynamic drug-driven mechanisms of resistance.

PEG3 mutation is associated with elevated tumor mutation burden and poor prognosis in breast cancer.

Background: Breast cancer is the second most common malignancy in women and considered as a severe health burden. PEG3 mutations have been observed in several cancers. However, the associations of PEG3 mutation with tumor mutation burden (TMB) and prognosis in breast cancer have not been investigated. Methods: In our study, the somatic mutation data of 986 breast cancer patients from The Cancer Genome Atlas (TCGA) were analyzed. Results: It showed that PEG3 had a relatively high mutation rate (2%). After calculated the TMB in PEG3 mutant and PEG3 wild-type groups, we found the TMB value was significantly higher in PEG3 mutant samples than that in PEG3 wild-type samples (P = 5.6e-07), which was independent of the confounding factors including age, stage, mutations of BRCA1, BRCA2 and POLE (odd ratio, 0.45; 95% CI, 0.20–0.98; P=0.044). Survival analysis revealed that PEG3 mutant samples had inferior survival outcome compared with the PEG3 wild-type samples after adjusted for the confounding factors above (hazard ratio, 0.27; 95% CI: 0.12–0.57; P<0.001). Conclusion: These results illustrated that PEG3 mutation was associated with high TMB and inferior prognosis, suggesting PEG3 mutation might play a guiding role in prognosis prediction and immunotherapy selection in breast cancer.

Abstract A27: Microsatellite instability and tumor mutation burden as factors in ovarian clear-cell carcinoma therapy selection

Abstract Background: Microsatellite instability (MSI) and tumor mutation burden (TMB) are being considered as predictive biomarkers for PD-1 and PD-L1 blockade immunotherapy. However, in ovarian clear-cell carcinoma (OCC), which demonstrates characteristic resistance to standard chemotherapy, the extent of MSI and TMB and their subsequent prognostic capability are unresolved. The aim of this study is to investigate TMB and MSI in OCC for improved therapy decision making. Methods: Tumor-normal paired whole-exome data of 55 OCC patients (Itamochi et al., Br J Cancer 2017) were analyzed. Sequence data preprocessing was performed in accordance with GATK best practice workflows using BWA, Picard, the GATK toolkit, and SAMtools. Somatic SNVs and indels were detected by using Mutect and VarScan. Resulting variants were annotated by ANNOVAR, and exonic or splicing variants were extracted. SNVs with allele frequency &amp;gt;= 2.0% were kept and further processed by the combination of the pathogenicity prediction values obtained from SIFT, PolyPhen-2, and PROVEAN; SNVs annotated as deleterious or damaging by at least one of the tools were retained. The TMB of each sample was evaluated by the total number of nonsynonymous SNVs called by the preannotation pipeline. A threshold defining high TMB was derived from the inflection point on the right tail of the TMB distribution; the resulting value was positioned at the 85th percentile of TMB values. MSI was calculated using MSIsensor, with the MSI-high classification threshold set as per the software’s documentation. Results: Among the 55 OCC cases, 11 (20%) had at least one or a combination of either MSI-high status, mismatch repair (MMR) mutations, or TMB-high status. Specifically, three had MSI-high status, five had MMR gene mutations, and eight had TMB-high status. MSI-high samples showed elevated TMB (p= 0.052 Fisher-T). TMB was also elevated in MMR-mutated samples (p=0.018 Fisher-T). Among the three samples with MSI-high status, only one contained a deleterious mutation in MMR gene MSH6. One TMB-high sample had a POLE1 mutation. Critically, discrete- and continuous-valued survival analyses using MSI and TMB as independent variables did not suggest correlation with patient prognosis. Conclusion: Within gynecologic oncology, MSI and TMB are correlated with prognosis in uterine endometrial cancer subtypes, though here they have demonstrated relatively reduced prognostic ability in OCC so far as examined by the samples available for this study. Thus, OCC patients are warranted the consideration of immune checkpoint inhibitor therapy at an earlier stage of treatment, and further studies to improve the resolution between MSI/TMB/MMR and phenotypical aspects should be encouraged. Citation Format: Shiro Takamatsu, Noriomi Matsumura, J.B. Brown, Hiroaki Itamochi, Junzo Hamanishi, Ken Yamaguchi, Toru Sugiyama, Tsukasa Baba, Masaki Mandai. Microsatellite instability and tumor mutation burden as factors in ovarian clear-cell carcinoma therapy selection [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A27.

Somatic alterations of NF1 in colorectal cancer.

4066 Background: NF1 encodes neurofibromin, which is a key GTPase-activating protein that downregulates RAS activation. Inactivating mutations in NF1 result in sustained activation of RAS signaling, a key driver for development of colorectal cancer (CRC), and have been suggested to be a potential mechanism of resistance to EGFR inhibition in RAS-wild type (WT) CRC. Little is known about molecular characteristics of NF1-mutated (MT) CRC. Methods: Tumor profiles from 8150 CRC patients (pts) with available NF1 mutation status were retrospectively reviewed. NextGen sequencing by a customized 592-gene panel was performed. Microsatellite instability (MSI) / mismatch repair (MMR) status, tumor mutational burden (TMB) and PD-L1 expression were tested. Molecular profiles between NF1-MT and NF1-WT pts were compared. Results: Out of 8150 pts, 176 (2.2%) had somatic NF1 mutations with pathogenic or presumed pathogenic function. A higher NF1-MT frequency was observed in MSI-H/dMMR vs MSS/pMMR (13.5% vs 1.4%, p &lt; 0.0001), in right-sided vs left sided (2.9% vs 1.8%, p &lt; 0.01), and in RAS-WT vs RAS-MT (3.0% vs 1.4%, p &lt; 0.0001). In MSS/pMMR tumors, no association with sidedness was observed (right: 1.3% vs left: 1.2%, NS). The most prevalent co-mutations with NF-1 were APC (63.2%), ARID1A (57.5%), TP53 (51.5%), KMT2D (32.9%) and KRAS (32.4%) in all cases, and APC (76.2%), TP53 (69.5%), KRAS (38.8%), ARID1A (34.4%) and FBXW7 (21.5%) in MSS/pMMR cases. POLE mutation was observed in 18.4% of NF1-MT/MSS/pMMR pts. Compared to NF1-WT pts, NF1-MT pts had more frequent mutations in ARID1A (All: 57.5% vs 23.3%, p &lt; 0.0001; MSS/pMMR: 34.4% vs 15.2%, p &lt; 0.05), and less frequent mutations in KRAS (All: 32.4% vs 49.0%, p &lt; 0.0001; MSS/pMMR: 38.8% vs 50.3%, p &lt; 0.05). Also, NF1-MT pts had more frequent alterations in homologous recombination pathway compared to NF1-WT pts (All: 39.8% vs 7.5%, p &lt; 0.0001; MSS/pMMR: 17.5% vs 4.4%, p &lt; 0.0001). Mean TMB was significantly greater in NF1-MT than NF1-WT (All: 48.9/Mb vs 10.0/Mb, p &lt; 0.0001; MSS/pMMR: 48.3/Mb vs 8.2/Mb, p &lt; 0.0001). Also, PD-L1 positivity was higher in NF1-MT compared to NF1-WT (All: 12.9% vs 3.6%, p &lt; 0.0001; MSS/pMMR: 7.1% vs 2.6%, p &lt; 0.05). Conclusions: While more frequent than in RAS-MT pts, NF1-MT CRC was a small subset in RAS-WT pts. NF1-MT was associated with alterations in chromatin remodeling and DNA damage response pathways, as well as elevated TMB and PD-L1 expression, which may provide alternative therapeutic strategies beyond EGFR inhibition.

The landscape of DNA damage response (DDR) pathway in colorectal cancer (CRC).

4064 Background: Abnormal DDR is a hallmark of cancer, relating to genome instability, anti-tumor immunity, and sensitivity to chemotherapeutic agents and radiation. We conducted a large-scale investigation to clarify the alteration of DDR pathway in CRC. Methods: Tumor samples from 9321 CRC patients were retrospectively reviewed. Next-Generation Sequencing (NGS) on a custom-designed panel enriching 592 gene targets was performed. Samples with mutations detected in any of 29 DDR-related genes were deemed DDR-mutant (DDR-MT); the rest DDR-wild type (DDR-WT). Microsatellite instability (MSI) status was tested with a combination of immunohistochemistry (IHC), fragment analysis and NGS. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. PD-L1 was tested by IHC (SP142). Consensus molecular subtype (CMS) was developed using RNA sequencing data. Results: Of 9321 cases, 1290 (13.8%) were DDR-MT. DDR-MT frequency was higher in right vs. left sided (20.9% vs 10.8%, p &lt; 0.001) and MSI-H vs. MSS (76.4% vs 9.5%, p &lt; 0.001) cases. In the MSS cases, right-sided had marginally higher frequency of DDR-MT than left-sided (10.6% vs 9.1%, p = 0.055), with much higher frequency of Fanconi anemia pathway alteration in right-sided (1.5% vs 0.7%, p &lt; 0.01). CMS1 subtype had the highest frequency of DDR-MT (34.8%); CMS2 had the lowest (7.1%). DDR-MT cases (vs. DDR-WT) had higher mutation rate of ARID1A (55.0% vs 19.1%, p &lt; 0.0001), PIK3CA (22.6% vs 15.8%, p &lt; 0.0001) and BRAF (20.4% vs 7.3%, p &lt; 0.0001), and lower mutation rate of TP53 (48.2% vs 76.1%, p &lt; 0.0001), APC (60.5% vs 74.5%, p &lt; 0.0001) and KRAS (44.0% vs 49.8%, p &lt; 0.001). Mean TMB was much greater in DDR-MT than DDR-WT (All: 20.9/Mb vs 7.7/Mb, p &lt; 0.0001; MSS: 13.7/Mb vs 7.6/Mb, p &lt; 0.05). PD-L1 positivity was also higher in DDR-MT compared to DDR-WT (All: 10.1% vs 2.7%, p &lt; 0.0001; MSS: 4.8% vs 2.4%, p &lt; 0.0001). Conclusions: Alteration of the DDR pathway was strongly associated with MSI status in CRC. The primary tumor sidedness might also be related, as DDR-MT was more prevalent in right-sided tumors. Elevated TMB and PD-L1 expression in DDR-MT CRC indicate more activated anti-tumor immune profiles compared to DDR-WT, regardless of MSI status, suggesting possible therapeutic benefit from immune checkpoint inhibitors in DDR-MT CRC.

Mucinous Histology, BRCA1/2 Mutations, and Elevated Tumor Mutational Burden in Colorectal Cancer.

Mucinous colorectal carcinomas (MC) constitute 10% of colorectal malignancies. Recently, an increased risk of colorectal cancer has been demonstrated in germline BRCA1/2 mutation carriers. Furthermore, BRCA1/2 germline mutation carriers have exhibited a higher-than-expected frequency of MC tumors. Here, we investigate the relationship between BRCA mutations and mucinous histology in colorectal carcinoma patients, using both an existing cohort of sequenced colorectal tumors and a prospective case-control study comparing MC and conventional adenocarcinoma (AC) patients tested for BRCA mutations. We discovered that MC tumors exhibit a statistically significantly higher incidence of BRCA mutations in addition to a higher average mutation count when compared to AC tumors in the existing cohort. The strongest predictor of the mutation count was mucinous histology, independently of other variables including microsatellite instability. Contrary to our hypothesis, the first association did not recur in the prospective case-control study, likely due to our pathological definition of MC tumors and small sample size. Finally, we observed a higher tumor mutational burden (TMB) in MC tumors compared with AC tumors. We suggest that the association between MC histology, BRCA mutations, and increased TMB may open the door to the utilization of simple tests (such as histopathologic characterization) to detect patients who may benefit from immunotherapy in colorectal cancer.

Abstract P1-06-02: Characterization of gene- and sample-level APOBEC mutagenesis enrichment with respect to intrinsic subtypes, tumor mutational burden, and immune composition in breast cancer

Abstract Introduction: APOlipoprotein B mRNA Editing enzyme Catalytic peptide-like, APOBEC, is a family of innate immune enzymes that catalyze cytosine to uracil deamination in single-stranded DNA, which lead to mutations in preferred trinucleotide contexts. APOBEC mutagenesis portends a worse prognosis in several breast cancer subtypes and associates with increased tumor mutational burden (TMB). Specific mutations enriched with APOBEC mutation signatures and immune phenotypes are not well described. The purpose of our study was to identify genes enriched for APOBEC mutagenesis and determine their relationship to intrinsic subtypes, TMB, and the immune composition of the transcriptome in breast cancers. Methods: We queried TCGA breast cancer database involving 980 breast cancer samples. We used consensus mutation calls from four somatic mutation callers (MuTect2, VarScan2, MuSE, and SomaticSniper). APOBEC mutagenesis scores were calculated for each tumor using a published method (Nik-Zainal et al. 2012) and a modified version thereof. The score was calculated at the gene-level, to identify genes enriched for APOBEC mutagenesis, and the cancer sample-level to investigate the relationship of APOBEC mutagenesis scores with intrinsic subtypes (using PAM50), APOBEC3A and 3B expression levels, and TMB. We also undertook differential expression analysis of CIBERSORT gene signatures and individual genes from the innate immune system, IFN, TGFB, WNT, and immune checkpoints with respect to common somatic mutations in APOBEC enriched genes. Results: The genes with the strongest statistical evidence for enrichment of APOBEC mutagenesis were PIK3CA, TP53, MUC16, and TTN. Among three common somatic mutations in PIK3CA (E542K, E545K, H1047R), the APOBEC score was only observed to be associated with E542K (q=2.47 × 10−2) and E545K (q=9.81 × 10−6) mutations. APOBEC mutagenesis revealed a high degree of enrichment across all breast cancer intrinsic subtypes, 75% in luminal A, 68% in luminal B, 91% in HER2 enriched, 73% in basal, and 89% in normal-like phenotypes. APOBEC mutagenesis scores were observed to significantly associate with APOBEC3A expression (q=1.1 × 10−9) and as a trend of association with APOBEC3B expression (q=1.62 × 10−1). A strong association was observed between APOBEC mutagenesis score and elevated TMB (p&amp;lt;2 × 10−16). No checkpoint genes (i.e. CTLA-4, PD-1, and PD-L1) were observed to be significantly overexpressed among those tumors with E542K and E545K PIK3CA mutations. The CIBERSORT mast cell gene signature (q=1.32 × 10−2) and the individual genes TGFB3 (q=5 × 10−3), ROR (q=7.91 × 10−3), and WNT5A (q=1.92 × 10-2) were overexpressed in breast tumors with E542K PIK3CA mutations. Conclusion: APOBEC mutagenesis patterns are seemingly prevalent across all breast cancer intrinsic subtypes and associate with elevated TMB and mutations in the PIK3CA helical loop domain and TP53 gene in TCGA breast cancer samples. Cancers with PIK3CA mutations in the helical loop domain did not demonstrate checkpoint gene overexpression, but were associated with other immunosuppressive features (e.g. TGFB3 and WNT5A overexpression). Investigation into innate immune cell signaling, mast cell immune signaling, and immunosuppressive expression profiles in APOBEC mutated breast cancers with and without PIK3CA mutations may help to identify novel immune targets to combine with PI3K inhibitors in breast cancer. Citation Format: Jeremy Force, Xiaodi Qin, Dadong Zhang, P. Kelly Marcom, Jeffrey Marks, Mary Love Taylor, Carey Anders, Kouros Owzar, Jichun Xie. Characterization of gene- and sample-level APOBEC mutagenesis enrichment with respect to intrinsic subtypes, tumor mutational burden, and immune composition in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-02.

Distinct genomic landscape in colorectal mucinous carcinoma via comprehensive genomic profiling.

222 Background: Mucinous carcinoma (MC), accounting for 10-15% of colorectal cancer (CRC), has long been associated with an inferior response to treatment. MC as an unfavorable prognostic factor in early stage CRC remains questionable. In this study, we used a large CRC cohort to define the genomic landscape of MC and identify prognostic and clinically actionable information. Methods: The 1226 CRC patient cohort included 997 adenocarcinomas (AC), 129 MC, and 110 adenocarcinomas with mucous composition (AMC). All of the pathological sections were verified by an experienced pathologist. FFPE tumor samples and matched peripheral blood were sequenced using a 450-cancer-related gene panel. Results: MC was significantly associated with right colon (p&lt;0.001) and regional lymph node metastasis (p=0.004) compared to AC. We found a higher mutation rate of BRAF V600E (10.9% vs. 3.3%), PIK3CA (28.7% vs. 19.2%), SMAD4 (34.1% vs. 19.1%), BRCA1/2 (16.3% vs. 6.8%) and homology recombination pathway (40.3% vs. 22.7%), and a lower rate of TP53 (53.5% vs. 79.5%), APC (46.5% vs. 75.1%,), and HER2 amplification (0% vs. 2.1%,) in MC than in AC. MC had a significantly higher proportion of microsatellite instability-high (MSI-H) tumors (22.5% vs. 6.8%, p&lt;0.001). Furthermore, in tumors with MSI stable, POLE mutation was more frequent in MC than in AC (7.0% vs. 2.6%, p=0.094) and resulted in dramatic elevated tumor mutational burden (TMB, range 79.5-591.5 muts/Mb), indicating up to 30% of MC patients may benefit from immunotherapy. MSI-H was associated with better prognosis (5-y DFS, MSI-H 86.7% vs. MSS 56.7%) in stage II/III CRC patients with MC. Importantly, AMC mimicked the genomic features of MC rather than AC. Conclusions: For the first time, comprehensive genomic analysis revealed that MC had distinct molecular features, indicating promising clinically application for both immunotherapy and targeted therapy. Early stage MC had a diverse prognosis due to MSI status and should be interpreted differently. The similarity of molecular profile between AMC and MC suggested the possible usage of MC, but not AC, clinical strategy for AMC.

The mutational pattern of homologous recombination (HR)-associated genes and its relevance to the immunotherapeutic response in gastric cancer.

Objective:Currently, there is an urgent need to identify immunotherapeutic biomarkers to increase the benefit of immune checkpoint inhibitors (ICIs) for patients with gastric cancer (GC). Homologous recombination deficiency (HRD) can modify the tumor immune microenvironment by increasing the presence of tumor-infiltrating lymphocytes and therefore might serve as a biomarker of immunotherapeutic response. We aimed to analyze the mutational pattern of HR-associated genes in Chinese patients with GC and its relevance to the tumor immune profile and clinical immunotherapeutic response.Methods:A panel of 543 cancer-associated genes was used to analyze genomic profiles in a cohort comprising 484 Chinese patients with GC. Correlations between HR gene mutations and tumor immunity or clinical outcomes were identified via bioinformatic analysis using 2 GC genomic datasets (TCGA and MSK-IMPACT).Results:Fifty-one of the 484 (10.54%) patients carried at least one somatic mutation in an HR gene; ATM (16/484, 3.31%) was among the most frequently mutated HR genes in the Chinese cohort. Mutations in HR genes were associated with elevated tumor mutational burden, enhanced immune activity, and microsatellite instability status. In the MSK-IMPACT cohort comprising 49 patients with stomach adenocarcinoma or gastroesophageal junction adenocarcinoma treated with ICIs, patients with HR-mut GC (n = 12) had significantly better overall survival than those with HR-wt GC (n = 37) (log-rank test, P < 0.05).Conclusions:Our data suggest that detection of somatic mutations in HR genes might aid in identifying patients who might benefit from immune checkpoint blockade therapy.

PATH-41. LONGITUDINAL MATCHED MUTATIONAL ANALYSIS IN RECURRENT AND MULTIFOCAL GLIOMAS

Abstract Re-resection of recurrent gliomas and multifocal gliomas are relatively uncommon resulting in limited understanding of the molecular profile of gliomas in those settings. Here we present a comprehensive mutational analysis via next generation sequencing (NGS) of 20 gliomas from 9 patients treated at our institution: one recurrent oligodendroglioma, one secondary glioblastoma (GBM), five locally recurrent primary GBMs, and two multifocal recurrent primary GBMs. Tumor mutational burden (TMB) range was 1.8–60.6 mutations/megabase (mt/Mb). Elevated TMB was seen in the initial (49.2 mt/Mb) and recurrent (60.6 mt/Mb) secondary GBM samples and in the distal recurrent GBM sample (50.9 mt/Mb) of one of the two patients with multifocal recurrent disease, suggesting a temozolomide-induced hypermutated state. Interestingly, the elevated TMB in the patient of multifocal recurrence occurred 5 years after completing 12 cycles of TMZ and was seen in the distal but not local focus of disease recurrence where TMB remained at 1.8 mt/Mb. The patient with multifocal (right frontal and right temporal) GBM at diagnosis developed recurrence in the temporal location only. NGS of the 2 resected tumors revealed 4 common somatic mutations, 3 mutations unique to the right frontal lesion and 8 mutations unique to the right temporal lesion. Additionally, our oligodendroglioma who was heavily treated in the six years prior to his last recurrence retained 4/6 of the initial somatic mutations and acquired 5 new ones upon the last recurrence. This analysis further highlights glioma spatiotemporal heterogeneity and the linear and divergent evolution upon disease recurrence and in the multifocal pre-treatment and recurrent settings. We also show that extensive oncogenic alterations can occur upon glioma recurrence, which is a major therapeutic barrier in managing recurrent disease, a barrier that can render the initial genomic profile of the disease, the basis of molecularly targeted therapy in the majority of cases, less meaningful.

Tumor Mutational Burden as a Predictive Biomarker for Response to Immune Checkpoint Inhibitors: A Review of Current Evidence.

Treatment with immune checkpoint inhibitors (ICPIs) extends survival in a proportion of patients across multiple cancers. Tumor mutational burden (TMB)-the number of somatic mutations per DNA megabase (Mb)-has emerged as a proxy for neoantigen burden that is an independent biomarker associated with ICPI outcomes. Based on findings from recent studies, TMB can be reliably estimated using validated algorithms from next-generation sequencing assays that interrogate a sufficiently large subset of the exome as an alternative to whole-exome sequencing. Biological processes contributing to elevated TMB can result from exposure to cigarette smoke and ultraviolet radiation, from deleterious mutations in mismatch repair leading to microsatellite instability, or from mutations in the DNA repair machinery. A variety of clinical studies have shown that patients with higher TMB experience longer survival and greater response rates following treatment with ICPIs compared with those who have lower TMB levels; this includes a prospective randomized clinical trial that found a TMB threshold of ≥10 mutations per Mb to be predictive of longer progression-free survival in patients with non-small cell lung cancer. Multiple trials are underway to validate the predictive values of TMB across cancer types and in patients treated with other immunotherapies. Here we review the rationale, algorithm development methodology, and existing clinical data supporting the use of TMB as a predictive biomarker for treatment with ICPIs. We discuss emerging roles for TMB and its potential future value for stratifying patients according to their likelihood of ICPI treatment response. IMPLICATIONS FOR PRACTICE: Tumor mutational burden (TMB) is a newly established independent predictor of immune checkpoint inhibitor (ICPI) treatment outcome across multiple tumor types. Certain next-generation sequencing-based techniques allow TMB to be reliably estimated from a subset of the exome without the use of whole-exome sequencing, thus facilitating the adoption of TMB assessment in community oncology settings. Analyses of multiple clinical trials across several cancer types have demonstrated that TMB stratifies patients who are receiving ICPIs by response rate and survival. TMB, alongside other genomic biomarkers, may provide complementary information in selecting patients for ICPI-based therapies.

92PD - Comprehensive pan-cancer analysis of KRAS genomic alterations (GA) including potentially targetable subsets

BackgroundKRAS GA are common oncogenic drivers. Effective systemic treatment of KRAS altered cancers has been largely elusive; however covalent inhibitors of G12C (AMG510, MRTX849) and SHP2 inhibitors (TNO155, RMC-4630) have recently entered the clinic in multiple tumor types. MethodsHybrid capture-based comprehensive genomic profiling (CGP) was performed on tumor samples from 213,312 unique patients with solid or hematological malignancies. Tumor mutational burden (TMB) was determined on 0.8-1.1 Mbp of sequenced DNA. PD-L1 expression was determined by IHC (22C3 or SP142 antibodies) for 17% of cases. ResultsKRAS GA were detected in 22% (n=46,182) of cases: 88% mutations (m; >99% substitutions and <0.2% indels), 8.3% amplification (a) and 3.8% both. KRASm were most common in pancreatic (83%, 8,063/9,723) gastrointestinal (GI; 39%, 13,507/35,019) and lung adenocarcinoma (LUAD; 35%, 9,159/25,968). KRASa (median 10 copies, range 6-421) was most common in esophageal adenocarcinomas (17%, 673/3,920) and testicular germ cell tumors (25%, 46/185). Co-KRASm+a was most common in pulmonary sarcomatoid carcinoma (PSC; 7.4%, 26/353). Median TMB was 3.5, 6.1, and 4.3 mut/Mb in cases with KRASm, KRASa, or KRASm+a. 124 unique KRASm were identified, most commonly G12D/V/C and G13D (26%, 21%, 14%, 6.9% of KRAS GA). G12D resulted from transitions (primarily G>A), and G12V (primarily G>T) and G12C (exclusively G>T) from transversions. G12C was most frequent in LUAD (14%, 3,613/25,968) and PSC (11%, 40/353) but uncommon in pancreatic (1.7%, 161/9,723) and GI (2.7%, 941/35,019) where G12D/V were most common. Tobacco signature (TS; 20% vs 4.5% vs 3.5%), elevated TMB (median 7.0 vs 3.5 vs 3.5 mut/Mb) and PD-L1 positivity (56% vs 29% vs 31%) were all significantly associated with G12C vs non-G12C KRASm and non-KRASm cancers (all p<0.0001). In LUAD, TS was present in 25% G12C, 19% non-G12C KRASm and 15% non-KRASm (all p<0.0001). ConclusionsDiverse KRAS GA are frequent across cancers and subtypes are associated with different solid malignancies. G12C is most common in LUAD and associated with smoking, elevated TMB and PD-L1 positivity. As multiple trials of novel therapeutic agents are currently enrolling for KRAS GA, CGP to identify these alterations is needed. Legal entity responsible for the studyThe authors. FundingFoundation Medicine. DisclosureS.I. Ou: Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Novartis; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Pfizer; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Roche; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony: Genentech; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Research grant/Funding (institution): Ariad; Research grant/Funding (institution): Ignyta; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony: Takeda; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): AstraZeneca; Research grant/Funding (institution): MedImmune; Research grant/Funding (institution): Clovis; Research grant/Funding (institution): Peregrine; Research grant/Funding (institution): GlaxoSmithKline; Research grant/Funding (institution): Astellas; Research grant/Funding (institution): TP Therapeutics; Research grant/Funding (institution): Blueprint Medicines. E.S. Sokol: Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche. R. Madison: Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche. J. Chung: Shareholder/Stockholder/Stock options: Roche; Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine. J.S. Ross: Leadership role, Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche. V.A. Miller: Leadership role, Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche; Advisory/Consultancy: Revolution Medicines. B.M. Alexander: Leadership role, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche. S.M. Ali: Shareholder/Stockholder/Stock options, Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche; Advisory/Consultancy: Revolution Medicines. A.B. Schrock: Full/Part-time employment: Foundation Medicine; Shareholder/Stockholder/Stock options: Roche. S.S. Ramalingam: Advisory/Consultancy, Research grant/Funding (institution): AstraZeneca; Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Brystol Myers Squib; Advisory/Consultancy: Genentech/Roche; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy, Research grant/Funding (institution): Tesaro; Advisory/Consultancy: Takeda; Advisory/Consultancy: Nektar.

Lymphovascular Invasion Is Associated With Mutational Burden and PD-L1 in Resected Lung Cancer

High tumor mutational burden (TMB) and programmed death ligand 1 (PD-L1) expression are leading biomarkers in metastatic non-small cell lung cancer (NSCLC) and predict favorable response to checkpoint inhibitors. We sought to identify clinicopathologic characteristics associated with elevated TMB and PD-L1 expression among patients who underwent resection for NSCLC. NSCLC patients undergoing primary resection (2016-2018) were prospectively enrolled in an immunogenomic profiling project. Multiplex immunofluorescence quantified densities (cells/mm2) of CD3+, CD3+CD8+, CD3+CD8+PD-1+, malignant cells (MCs), MCsPD-L1+, CD68+, CD68+PD-L1+, and CD20+ cells. Whole-exome sequencing quantified TMB (mutations/megabase). TMB and MCsPD-L1+ were dichotomized according to the median of each. A total of 55 patients completed multiplex immunofluorescence and whole-exome sequencing profiling. In this sample, 41.8% (23 of 55) had pathologic stage I disease. Median TMB and MCsPD-L1+ were 3.91 and 0.62 cells/mm2, respectively. TMB was higher among smokers (P= .001) and tumors with lymphovascular invasion (LVI) (P= .051). TMB was positively correlated with densities of MCsPD-L1+ (r= 0.293, P= .030), CD68+PD-L1+ (r= 0.289, P= .033), and CD20+ (r= 0.310, P= .043) cells. The density of MCsPD-L1+ was associated with increased CD3+CD8+ (r= 0.319, P= .018) and CD68+PD-L1+ (r= 0.371, P= .005) cells. Patients with PD-L1HighTMBHigh tumors (30.9%, 17 of 55) had higher intratumoral densities of CD3+, CD3+CD8+, CD68+, CD68+PD-L1+, and CD20+ cells. On multivariable analysis LVI was associated with synchronous elevated TMB and PD-L1 expression (odds ratio 3.53, P= .039). NSCLC tumors with elevated TMB and PD-L1 expression are associated with LVI and increased intratumoral immune cell infiltration. These findings may potentially improve patient selection for checkpoint inhibitor therapy trials in the adjuvant setting.

Metagenomic analysis reveals a rich bacterial content in high-risk prostate tumors from African men.

BackgroundInflammation is a hallmark of prostate cancer (PCa), yet no pathogenic agent has been identified. Men from Africa are at increased risk for both aggressive prostate disease and infection. We hypothesize that pathogenic microbes may be contributing, at least in part, to high‐risk PCa presentation within Africa and in turn the observed ethnic disparity.MethodsHere we reveal through metagenomic analysis of host‐derived whole‐genome sequencing data, the microbial content within prostate tumor tissue from 22 men. What is unique about this study is that patients were separated by ethnicity, African vs European, and environments, Africa vs Australia.ResultsWe identified 23 common bacterial genera between the African, Australian, and Chinese prostate tumor samples, while nonbacterial microbes were notably absent. While the most abundant genera across all samples included: Escherichia, Propionibacterium, and Pseudomonas, the core prostate tumor microbiota was enriched for Proteobacteria. We observed a significant increase in the richness of the bacterial communities within the African vs Australian samples (t = 4.6‐5.5; P = .0004‐.001), largely driven by eight predominant genera. Considering core human gut microbiota, African prostate tissue samples appear enriched for Escherichia and Acidovorax, with an abundance of Eubacterium associated with host tumor hypermutation.ConclusionsOur study provides suggestive evidence for the presence of a core, bacteria‐rich, prostate microbiome. While unable to exclude for fecal contamination, the observed increased bacterial content and richness within the African vs non‐African samples, together with elevated tumor mutational burden, suggests the possibility that bacterially‐driven oncogenic transformation within the prostate microenvironment may be contributing to aggressive disease presentation in Africa.

A Novel Next-Generation Sequencing Approach to Detecting Microsatellite Instability and Pan-Tumor Characterization of 1000 Microsatellite Instability–High Cases in 67,000 Patient Samples

Microsatellite instability (MSI) is an important biomarker for predicting response to immune checkpoint inhibitor therapy, as emphasized by the recent checkpoint inhibitor approval for MSI-high (MSI-H) solid tumors. Herein, we describe and validate a novel method for determining MSI status from a next-generation sequencing comprehensive genomic profiling assay using formalin-fixed, paraffin-embedded samples. This method is 97% (65/67) concordant with current standards, PCR and immunohistochemistry. We further apply this method to >67,000 patient tumor samples to identify genes and pathways that are enriched in MSI-stable or MSI-H tumor groups. Data show that although rare in tumors other than colorectal and endometrial carcinomas, MSI-H samples are present in many tumor types. Furthermore, the large sample set revealed that MSI-H tumors selectively share alterations in genes across multiple common pathways, including WNT, phosphatidylinositol 3-kinase, and NOTCH. Last, MSI is sufficient, but not necessary, for a tumor to have elevated tumor mutation burden. Therefore, MSI can be determined from comprehensive genomic profiling with high accuracy, allowing for efficient MSI-H detection across all tumor types, especially those in which routine use of immunohistochemistry or PCR-based assays would be impractical because of a rare incidence of MSI. MSI-H tumors are enriched in alterations in specific signaling pathways, providing a rationale for investigating directed immune checkpoint inhibitor therapies in combination with pathway-targeted therapies.

Abstract 746: Genomic alteration of Chinese dMMR/MSI-H gastric cancer

Abstract Background: Gastric cancer is a high incidence malignancy often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. A small fraction of gastric cancer with dMMR/MSI-H has extraordinary response to the immune checkpoint inhibitor treatment attributing to the immunogenicity generated by the elevated tumor mutation burden. At the meantime, the extensive genomic variants also cause profound impact on many other cancer related genes. Thus the understanding of dMMR/MSI-H gastric cancer genomic profiling is urgent for exploring clinical strategy to this cohort. Methods: Formalin Fixed Paraffin Embedded (FFPE) samples of 30 Chinese gastric cancer patients were collected for 450 gene panel based next-generation sequencing (NGS) assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangement were assessed. Microsatellite instability (MSI) status and tumor mutational burden (TMB) were also calculated by NGS algorithm. Results: There were 15 males (50%) and 15 females (50%) diagnosed as gastric cancer with a median age of 67 years old. The TMB value of this cohort ranged from 29 to 118 muts/Mb, with the median value of 58 muts/Mb. The most frequent genomic alterations in Chinese MSI-H gastric cancer patients were revealed as KMT2D (50%), RNF43 (50%), CIC (50%), PIK3CA (50%), KMT2C (43%), ACVR2A (40%), TP53 (30%), ATM (33%), and ERBB3 (33%). Truncation has been found the most frequent gene alteration among these top mutations due to the deficiency of mismatch repair system caused reading frame shift. Epigenetic modifier genes with functions of chromatin remodeling and histone methylation have been found extensively mutated in MSI-H gastric cancer. Among eighteen (60%=18/30) patients carrying truncations in ARID1A whose coding protein participates in chromatin remodeling, seven of them possess loss-of-function biallelic truncations. Moderate frequency of mutation also occur at other genes in chromatin remodeling such as ARID1B (23%), PBRM1 (17%) and SMARCA4 (26%). About 77% patients in this cohort carry at least one truncation in genes belonging to the histone lysine methyltransferase 2 (KMT2) family such as KMT2A, KMT2C and KMT2D. Mutations of B2M gene which are commonly associated with resistance to immune checkpoint inhibitor have been found in 23% MSI-H gastric cancer. About 66% of the patients in this cohort carrying mutations in FBXW7, PIK3CA, PTEN, STK11 and TSC may benefit from the mTOR inhibitors. Conclusions: In general, dMMR/MSI-H gastric cancers possess elevated and dispersive TMB value. Epigenetic modifier genes involving in chromatin remodeling and histone methylation mutate frequently in MSI-H gastric cancer. About 66% of the patients in this cohort may benefit from the mTOR inhibitors. Citation Format: Jia Wei, Jian Wang, Xiangshan Fan, Yue Wang, Yao Fu, Kai Wang, Nandie Wu, Qin Liu, Yang Yang, Weifeng Wang, Baorui Liu. Genomic alteration of Chinese dMMR/MSI-H gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 746.

Characteristics of mismatch repair deficiency in sarcomas

Due to the efficacy of immune checkpoint inhibitor therapy in tumors with deficient mismatch repair, there has been a surge in demand for mismatch repair deficiency testing in various tumor types. Mismatch repair deficiency is not known to play a significant role in the pathogenesis of sarcomas, and the utility of testing these tumor types is not established. This study aimed to determine the frequency, pattern, and clinicopathologic correlates of mismatch repair deficiency in sarcomas. Three hundred and four sarcomas were profiled using a genomic platform that employs massively parallel sequencing to interrogate 447 cancer-associated genes. Mismatch repair status was evaluated by determining the number of small insertion/deletion events occurring in homopolymer regions per megabase of exonic sequence data across all genes. Molecular characteristics of mismatch repair-deficient sarcomas were compared to mismatch repair-deficient carcinomas (n = 70) also identified using the sequencing panel. Seven sarcomas (2.3%) were classified as mismatch repair-deficient: four unclassified sarcomas, and one each of pleomorphic rhabdomyosarcoma, epithelioid leiomyosarcoma and malignant PEComa. One patient had an established diagnosis of Lynch syndrome. In the remaining patients, the mismatch repair gene mutation was confirmed or suspected to be somatic. Mismatch repair immunohistochemistry confirmed the mismatch repair-deficiency status of all cases with alterations in the tested proteins. As expected, mismatch repair-deficient sarcomas showed a significantly elevated tumor mutation burden relative to mismatch repair-proficient sarcomas (median 16 versus 4.6, p < 0.001). However, in comparison to mismatch repair-deficient carcinomas, mismatch repair-deficient sarcomas showed a lower tumor mutation burden (median 28 versus 16, p = 0.006) and a significantly greater degree of chromosomal instability. Among mismatch repair-deficient sarcomas, PD-L1 was variably expressed on tumor-associated macrophages but not on tumor cells. Three patients received pembrolizumab: two progressed and one has stable disease with five months follow-up. Mismatch repair deficiency in histologically classifiable sarcomas is rare (1%) and is more common in unclassified sarcomas (10%). Additional study is required to determine the predictive role of mismatch repair-deficiency in sarcomas for immunotherapy.

Frequency of BRCA mutation in biliary tract cancer and its correlation with tumor mutational burden (TMB) and microsatellite instability (MSI).

4085 Background: Biliary tract cancers constitute ~3% of cancers worldwide with incidence increasing, especially for intrahepatic cholangiocarcinoma (IHC). The prognosis of these tumors remains dismal and novel treatment strategies are needed to improve overall survival. BRCA mutations occur in biliary tract cancers but their frequency in distinct sites of biliary tract cancer is unknown. Moreover, no data are available correlating BRCA mutation with immunogenic markers such as TMB, MSI, or PD-L1 expression. Methods: Tumor samples from 1288 primary biliary tract cancers, comprising IHC (n = 746), extrahepatic cholangiocarcinoma (EHC) (n = 189), gallbladder (GBC) (n=353) were profiled at Caris Life Sciences, Phoenix, AZ. Testing included NextGen SEQ (MiSeq on 47 genes, NextSeq on 592 genes) and PD-L1 IHC (SP142). TMB was calculated based on somatic nonsynonymous missense mutations, and MSI was evaluated by NGS of known MSI loci. Results: BRCA mutations were detected in 3.6% (N = 46) of samples ( BRCA1 0.6%, BRCA2 3%), no differences were seen based on the site of the tumor. In GBC and IHC BRCA2 mutations (4.0% and 2.7%) were more frequent than BRCA1 (0.3% and 0.4, p &lt; 0.05) while in EHC, similar frequency was observed ( BRCA1: 2.1%; BRCA2: 2.6%). There was no significant association with gender or age. In BRCA-mutant biliary tract cancer the most frequently mutated genes were TP53 (55.6%), ARID1A (52.2%) and KRAS (26.1%), KMT2D/C (20%, 13%) and CDKN2A(13%). Overall, BRCA mutations were associated with a higher rate of MSI-H (19.5% vs 1.7%, p = 0.001) and higher TMB in both MSI-H and MSS tumors (p&lt;0.05). When investigated separately, BRCA association with elevated TMB was seen in IHC and EHC, but not in GBC. No correlation was seen with PD-L1 expression. TP53, KMT2D/C, RB1, PTEN, KDM6A mutations and FGFR1 amplifications were significantly higher in BRCA mutated tumors (p &lt; 0.05). Conclusions: BRCA mutations are found in a significant subgroup of biliary tract tumors and are associated with an immunogenic tumor profile. These data provide rationale for trials testing PARP inhibitors in combination with immunotherapy and targeted therapies in patients with BRCA-mutant biliary tract cancers that are MSS.