Number Of Non-synonymous Mutations Research Articles

Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis invivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Tumor Mutational Burden As a Prognostic Factor in Diffuse Large B-Cell Lymphoma

Background: Tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase (Mb) in the coding regions of a tumor genome, has been associated with improved response to immune checkpoint blockade treatments and more broadly with overall survival (OS) in many cancers. Limited prior studies have found that TMB is higher in diffuse large B-cell lymphoma (DLBCL) compared to many other cancers. However, only two studies have evaluated TMB and prognosis in newly diagnosed DLBCL patients. The prognostic role of TMB in diffuse large B-cell lymphoma (DLBCL) remains unclear as prior studies have been limited to panel sequencing of cancer genes and/or lacked matched germline-tumor samples for filtering. Methods: We characterized TMB from whole exome sequencing (WES) in newly diagnosed DLBCL patients with matched diagnostic tumor-normal samples prior to treatment with standard immunochemotherapy (IC). Tumor DNA was extracted from formalin-fixed, paraffin embedded tissue sections and germline DNA from peripheral blood. We evaluated the association between TMB and event-free (EFS) and OS using Cox regression adjusted for the international prognostic index (IPI) and other factors including cell of origin (COO), MYC double hit (DH) with BLC2 or BCL6 by FISH, and TP53 mutation status. EFS and OS were defined as time from diagnosis to earliest of treatment failure (EFS only), death, or last follow-up; EFS at 24 months (EFS24) was also modeled as a marker of early aggressive DLBCL. An optimal high/low TMB cutpoint was determined based on EFS using maximally selected rank statistics; sensitivity analyses used an 20/80 percentile to define the threshold. We also evaluated the association of driver tumor mutational load (driverTML) with EFS and OS, defined as the total number of mutations in 292 DLBCL driver genes. Results: In the analysis cohort of 394 DLBCL patients, the median age at diagnosis was 65 years (interquartile range: 56-73), 43% were female, 62.2% had IPI 0-2, and 57.4% germinal center B-cell COO. MYC was DH in 7.5% and TP53 was mutated in 16.2% of patients. Through a median follow-up of 7.5 years, there were 141 events and 122 deaths. EFS24 was achieved in 75.1% of the cohort; 5-year EFS was 69% (95% CI 64-74%) and 5-year OS was 75% (95% CI 71-80%). Median TMB in our cohort of DLBCL patients was 3.6 mutations/Mb, which was the fifth highest compared with 32 cancers in The Cancer Genome Atlas. As a continuous variable, increasing TMB (mutation/Mb) was associated with superior EFS (HR=0.89, 95% CI 0.81-0.98) and OS (HR=0.89, 95% CI 0.81-0.99) after adjustment for IPI. Spline analyses confirmed linear associations of TMB with EFS and OS. The optimal modeled cutpoint based on EFS was 4.5 mutations/Mb, with 31% classified as high TMB (>4.5). No significant ( P >.05) differences were observed between high and low TMB patients with clinical (i.e., age, sex, IPI category) or molecular (cell of origin, MYC DH, TP53 mutated) factors. In Cox regression, high-TMB was significantly protective for EFS and for OS compared to low-TMB after adjustment for each clinical factor ( Figure 1), with simultaneous adjustment for all these factors associated with superior EFS (HR=0.52, 95% CI 0.33-0.84) and OS (HR=0.59, 95% CI 0.36-1.00). TMB (high/low) combined with IPI (0-2, 3-5) was significantly ( P =.001) associated with EFS in Kaplan-Meier analysis ( Figure 2). Among patients with IPI 3-5, high TMB was significantly associated with superior EFS (HR=0.47, 95% CI 0.27-0.81) and OS (HR=0.57, 95% CI 0.33-0.99). Among patients with IPI 0-2, we also observed longer EFS (HR=0.62, 95% CI 0.34-1.11) and OS (HR=0.62, 95% CI 0.32-1.20) in high TMB patients, although the associations were not statistically significant ( P >.05). In sensitivity analyses, a 20/80 percentile TMB threshold found similar results for EFS (HR=0.64, 95% CI 0.41-1.03) and OS (HR=0.69, 95% CI 0.42-1.12) as in our main analyses. TMB and driverTML were significantly correlated ( R=0.65, P <10 -16) and we found similar effect sizes for EFS (HR=0.45, 95% CI 0.22-0.91) and OS (HR=0.55, 95% 0.27-1.13) as in our main analyses. Conclusions: In a large and well-defined cohort of IC treated DLBCL patients, we found DLBCL patients with higher TMB had superior EFS and OS after adjustment for other clinical and molecular prognostic factors, and the prognostic association for TMB was robust to threshold and gene set.

Genomic Characterization of Locally Recurrent Prostate Cancer after Radiation Therapy.

Radiation therapy (RT) is a backbone of treatment for patients with prostate cancer (PCa). However, locally recurrent disease after definitive RT (i.e., radiorecurrent PCa) is not uncommon and is associated with a higher risk of distant metastases and death from PCa. While the genomic landscape of primary PCa is well-characterized, little is known regarding the genomic landscape of radiorecurrent PCa or how this compares to that of primary PCa. We hypothesized that the genomic landscape of radiorecurrent PCa differs significantly from primary PCa and that these differences have clinical relevance. We examined this hypothesis by performing whole exome sequencing (WES) of radiorecurrent PCa. We identified 25 patients with radiorecurrent PCa with available post-RT tissue obtained from biopsy or radical prostatectomy, as well as germline tissue. The tumor and germline tissue for 19 patients successfully underwent WES. We identified genomic variants including single nucleotide variants (SNVs), insertions/deletions, and copy number alterations. Furthermore, we estimated the tumor mutational burden (TMB; number of nonsynonymous mutations per megabase [Mb]) and contribution of individual mutational signatures. We compared our samples to a publicly available large cohort of primary PCa (n = 680) to define genomic alterations unique to radiorecurrent PCa. In the overall cohort of 25 patients, the RT modality included external beam RT (56%), brachytherapy (36%), and combination of both (8%). 40% of patients received upfront androgen deprivation therapy with RT. The median time to local recurrence was 6.5 years. For the 19 radiorecurrent patients with WES data, the median TMB was 2.7 mutations/Mb, which was significantly higher than the median TMB of 0.7 mutation/Mb for primary PCa (P = 0.002 after multivariable adjustment). Radiorecurrent PCa demonstrated an enrichment of short deletions, with a significantly higher deletion/SNV ratio compared to primary PCa (P = 0.006). TP53 was the most frequently mutated gene in radiorecurrent PCa (n = 6), and the TP53 mutation prevalence was significantly higher compared to primary PCa (32% vs 10%, P = 0.016 by Fisher's exact test). TP53 was also determined to be recurrently mutated using MutSigCV (Q = 0.0003). Additionally, 3 samples demonstrated evidence of whole genome doubling. Radiorecurrent PCa has a distinct genomic profile compared to primary PCa, characterized by a higher TMB with an enrichment of short deletions as part of the mutational composition, which may be a scar of nonhomologous end joining subsequent to RT-induced DNA double-stranded breaks. In addition, TP53 mutations may be of functional consequence in radiorecurrent PCa. Further efforts are underway to examine other genomic features apparent in WES data, as well as perform whole transcriptome sequencing to provide complementary insights into radiorecurrent PCa.

Genomic characterization of locally recurrent prostate cancer after radiation therapy.

e17002 Background: Radiation therapy (RT) is a backbone of treatment for patients with prostate cancer (PCa). However, locally recurrent disease after definitive RT (i.e. radiorecurrent PCa) is not uncommon and is associated with a higher risk of distant metastases and death from PCa. While the genomic landscape of primary PCa is well-characterized, little is known regarding the genomic landscape of radiorecurrent PCa or how this compares to that of primary PCa. We hypothesized that the genomic landscape of radiorecurrent PCa differs significantly from primary PCa and that these differences have clinical relevance. We examined this hypothesis by performing whole exome sequencing (WES) of radiorecurrent PCa. Methods: We identified 25 patients with radiorecurrent PCa with available post-RT tissue obtained from biopsy or radical prostatectomy, as well as germline tissue. The tumor and germline tissue for 19 patients successfully underwent WES. We identified genomic variants including single nucleotide variants (SNVs), insertions/deletions, and copy number alterations. Furthermore, we estimated the tumor mutational burden (TMB; number of nonsynonymous mutations per megabase [Mb]) and contribution of individual mutational signatures. We compared our samples to a publicly available large cohort of primary PCa (n = 680) to define genomic alterations unique to radiorecurrent PCa. Results: In the overall cohort of 25 patients, the RT modality included external beam RT (56%), brachytherapy (36%), and combination of both (8%). 40% of patients received upfront androgen deprivation therapy with RT. The median time to local recurrence was 6.5 years. For the 19 radiorecurrent patients with WES data, the median TMB was 2.7 mutations/Mb, which was significantly higher than the median TMB of 0.7 mutation/Mb for primary PCa ( P= 0.002 after multivariable adjustment). Radiorecurrent PCa demonstrated an enrichment of short deletions, with a significantly higher deletion/SNV ratio compared to primary PCa ( P= 0.006). TP53 was the most frequently mutated gene in radiorecurrent PCa (n = 6), and the TP53 mutation prevalence was significantly higher compared to primary PCa (32% vs 10%, P= 0.016 by Fisher’s exact test). TP53 was also determined to be recurrently mutated using MutSigCV (Q = 0.0003). Additionally, 3 samples demonstrated evidence of whole genome doubling. Conclusions: Radiorecurrent PCa has a distinct genomic profile compared to primary PCa, characterized by a higher TMB with an enrichment of short deletions as part of the mutational composition, which may be a scar of nonhomologous end joining subsequent to RT-induced DNA double-stranded breaks. In addition, TP53 mutations may be of functional consequence in radiorecurrent PCa. Further efforts are underway to examine other genomic features apparent in WES data, as well as perform whole transcriptome sequencing to provide complementary insights into radiorecurrent PCa.

Ring stage dormancy of Plasmodium falciparum tolerant to artemisinin and its analogues – A genetically regulated “Sleeping Beauty”

The appearance in 2008 in western Cambodia of Plasmodium falciparum tolerant to artemisinin, defined by longer parasite clearance time following drug administration and in vitro by a slightly higher survival rate of the ring stage after a 3-h treatment with 700 nM artemisinin (or analogues, collectively termed ART), has raised concerns of the possible loss of this frontline antimalarial [used in the form of an artemisinin combination therapy (ACT)], with its low IC50 value against the ring stage and pleiotropic pro-drug/poison property. The key genetic marker of ART tolerance phenotype is a number of non-synonymous mutations in Pfkelch13 propeller domain. This results in defective assembly at the ring stage of a cytostome structure located at cytoplasmic side of the parasite membrane required for invagination of a double-membrane endosome carrying host cytosol haemoglobin to the digestive vacuole. The consequential deprivation of amino acids initiates ring stage parasites bearing the causal mutations in PfK13 (or other key cytostome components) entry into a dormant state (“Sleeping Beauty”), which, after a duration longer than that the short-lived ART, “Sleeping Beauty” ring parasite resumes its normal, but accelerated, development to maintain the 48-h intra-erythrocytic life-cycle. We posit that when ART-tolerant P. falciparum has acquired under ART stress the causative PfK13 mutation (not obligatory if mutations occur in other critical cytostome components), together with other necessary mutations to adjust to the new normalcy and to provide survival competitiveness, ART-tolerant parasite has now evolved into a genetically programmed “Sleeping Beauty”. The onus of preventing the spread of ART-tolerant P. falciparum lies with the efficacy of ACT partner drug, hence the recommendation of a triple ACT (TACT). Nevertheless, attention should also be focussed on understanding the mechanisms of dormancy, such as induction, maintenance and recovery, to enable discovery and development of novel antimalarials targeting this unique parasite stage.

Abstract PR008: Landscape of actionable mutations and outcomes in metastatic non-small cell lung cancer across age, sex, and race

Abstract Introduction: Lung cancer is the leading cause of cancer-related death due to frequent diagnosis at an advanced stage, especially among underserved populations. Given the marked heterogeneity in disease course for metastatic non-small cell lung cancer (mNSCLC), we sought to further explore its distribution of genomic alterations and outcomes across population subgroups. Methods: Clinicogenomics data were downloaded from the MSK-MET cohort study, one of the largest publicly accessible databases of metastatic cancer featuring 25775 patients in the February 2022 release. Tumor mutation burden (TMB) was quantified as number of non-synonymous mutations per megabase (Mb) sequenced. Categorical variables were compared using the χ2-test. Analysis of overall survival (OS) was performed using Cox proportional hazards regression. Results: Between 2013 and 2020, tumor and matched-normal samples from 4710 adult patients (58% female) with mNSCLC (86% adenocarcinoma) were obtained for genomic characterization. Median age at diagnosis was 67 years (IQR 59–73) and median OS was 38 months (95% CI 34–40 months). Smoking status and treatment history were unavailable. Patients self-identified as non-Hispanic white (76%), Asian (10%), Black (5%), Hispanic (4%), Native American (1%), or none of the above (1%). Compared to patients of other races, Hispanic patients were more likely to be diagnosed with mNSCLC prior to age 50 (17% vs. 7%, P<0.001); Asian patients had a higher frequency of EGFR mutations (56% vs. 22%, P<0.001), lower frequency of KRAS mutations (11% vs. 32%, P<0.001), and longer OS (HR 0.84, 95% CI 0.72–0.98, P=0.026); Black patients had greater TMB (9% vs. 5% with ≥20 mutations per Mb, P=0.019). Every actionable mutation was more common in non-squamous cell carcinoma (SCC) than SCC, except ROS1 fusions (4% vs. 7%, P<0.001). Across and within each race group, men were more likely to possess no actionable mutations compared to women (29% [range 22–67%] vs. 16% [range 5–20%], P<0.05). In multivariable analysis, male sex, diagnosis earlier than age 50 or later than age 70, non-adenocarcinoma histology, greater TMB, and absence of actionable mutations involving EGFR, ALK, MET, or RET were independently associated with worse OS (P<0.05). Conclusions: In this large clinicogenomic analysis of mNSCLC, men were more likely to have no actionable mutations and worse OS. Diagnosis prior to age 50 (most common among Hispanic patients) and greater TMB (most common among Black patients) also independently conferred worse OS. These findings highlight the wide heterogeneity of molecular features and outcomes in mNSCLC across age, sex, and race, underscoring the need to further individualize workup and management. Citation Format: David C. Qian, Conor E. Steuer, Sibo Tian, Jennifer W. Carlisle, Ticiana A. Leal, Madhusmita Behera, Jeffrey D. Bradley, Suresh S. Ramalingam, Kristin A. Higgins. Landscape of actionable mutations and outcomes in metastatic non-small cell lung cancer across age, sex, and race [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR008.

Abstract 2745: The PLANET manufacturing process reproducibly generates high-quality neoantigen-targeted peripheral T cells (NPTs) for adoptive T cell therapy in the TiTAN clinical trial

Abstract Background: Neoantigen (neoAg)-specific T cells can be found in the peripheral blood of patients with solid tumors, and the infiltration of fresh, peripherally derived T cells into tumors has been associated with successful outcomes after checkpoint blockade therapy. We previously described the development of our PLANET™ manufacturing process to create NPTs, based on the empirical identification of neoAgs using the ex vivo ATLAS™ bioassay, for adoptive transfer into patients with cancer. We have initiated the TiTAN™ clinical trial (NCT04596033) to evaluate this candidate immunotherapy, GEN-011, in subjects with checkpoint refractory solid tumors. Here we report the successful manufacturing of NPTs to support the ongoing trial. Methods: Apheresis, FFPE tumor and saliva samples were procured from subjects who consented to participate in the trial. Monocytes and T cells were isolated and cryopreserved. Patient-specific neoAgs against which their T cells were responsive were identified with ATLAS, and up to 30 neoAgs were prioritized for manufacture; pro-tumor Inhibigens™ were excluded. Cryopreserved peripheral blood monocytes and T cells were thawed, monocytes derived into dendritic cells, and then neoAg-specific T cells expanded in the fully closed PLANET manufacturing process. The NPTs were formulated and cryopreserved for administration to patients after release testing. Results: Subjects with five different solid tumor types contributed to these data: CSC, NSCLC, SCLC, SCCHN, and melanoma. A median of 2.1bn monocytes and 6bn T cells were cryopreserved from apheresis products (N=17). The median TMB was 1.8mut/Mb (range 0.01-36.6) with 473 somatic mutations (range 32-8893); the number of non-synonymous mutations ranged from 9 to 767. ATLAS screens identified a mean of 13±4 neoAgs and 11±3 Inhibigens, resulting in an average of 13 (range 2-30) unique neoAgs in each PLANET manufacturing process. Upon conclusion of manufacturing, the mean yield per patient was 1.6±0.3bn NPTs across runs (N=9) completed by the time of data cutoff. Characterization tests revealed the NPTs were consistently 80-90% CD8+ and 10-20% CD4+ T cells, of which 97% (range 64.9-99.8%) were effector memory and 1% (range 0.1-32.3%) central memory. The median purity at release was 98% with 82% viability. By functional assessments, the NPTs retained specificity for 91% (range 82-100%) of their intended neoAg targets (N=4). Administratable doses were successfully manufactured for 100% of patients to date. Conclusions: NPTs can routinely be manufactured in a GMP setting to treat patients with solid tumors. By expanding fresh, non-exhausted NeoAg-specific T cells with known tumor specificity from the periphery, GEN-011 has the potential to provide clinical benefits of TIL with greater accessibility and minimal irrelevant T cells. The TiTAN trial is ongoing. Citation Format: Harshal Zope, Rounak Nande, Manish Jain, Charley Hubbard, Louisa Dowal, James Foti, James Loizeaux, Crystal Cabral, Daniel B. DeOliveira, Guohan Yang, Mercay Reuter, Jessica Baker Flechtner, Raymond Stapleton. The PLANET manufacturing process reproducibly generates high-quality neoantigen-targeted peripheral T cells (NPTs) for adoptive T cell therapy in the TiTAN clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2745.

External validation of the VIGex gene-expression signature (GES) as a novel predictive biomarker for immune checkpoint treatment (ICT).

2510 Background: VIGex is a 12- gene GES classifier initially developed on the Nanostring platform and validated for RNA-seq. VIGex classifies samples into Hot, intermediate-Cold (I-Cold) and Cold subgroups. The Hot subgroup as defined by VIGex has been associated with better (PFS) in patients (pts) treated on phase 1 ICT trials at Vall D’Hebron Hospital (VH) (ESMO2020). We investigated the performance of VIGex in pts treated with Pembrolizumab (P) in the INSPIRE clinical trial (NCT02644369) at Princess Margaret Cancer Centre (PM) and compared VIGex with other predictive ICT biomarkers. Methods: Pts with advanced solid tumors were treated with P 200 mg IV Q3wks. RNA-seq from baseline biopsies was performed using the Illumina NextSeq550 platform. Tumor RNA-seq data were transferred from PM to VH and classified by the VIGex algorithm blinded to clinical data. Bespoke circulating tumor DNA (ctDNA) was assayed at baseline (B) and start of cycle 3 (C3) using a pt-specific amplicon-based NGS assay (Signatera). Tumor mutational burden (TMB) was defined as the number of non-synonymous mutations per megabase and PD-L1 was assessed by immunohistochemistry (22C3). Hot subgroup (HOT) was compared to I-Cold + Cold (COLD). We defined 4 groups based on the combination of VIGex subgroups and the change in ctDNA at cycle 3 from baseline (ΔctDNA). Survival times were calculated with the Kaplan–Meier method and Cox proportional-hazard models were constructed. Results: Out of 76 pts, median age was 55y (range 21-81y), M:F 31:45, all ECOG 0-1, 16 High-grade serous ovarian, 12 triple negative breast, 12 head and neck, 10 melanoma and 26 other. Median no. of P cycles was 3 (range 1–35); follow up was 14m (range 1-67); Median PFS 10.9m and median overall survival (OS) 14m. Overall response rate (RECIST 1.1) was 24% in HOT and 10% in COLD (p = 0.22 two-sided Fisher's exact test). The HOT subgroup was significantly associated with higher OS and PFS when included in a multivariate model adjusted by tumor histology, TMB and PD-L1 (HR 0.43; 95%CI 0.23-0.81; p = 0.009) and (HR: 0.48; 95%CI 0.25-0.95; p = 0.036) respectively. A total of 57 pts had both VIGex and ΔctDNA data. The addition of ΔctDNA further improved the predictive performance of VIGex for OS (Table). Conclusions: VIGex maintained its predictive power for ICT outcomes when applied to an independent external dataset using RNA-seq. The predictive information provided by VIGex was independent of PD-L1 and TMB. Our data indicates that the addition of ΔctDNA to baseline VIGex may refine prediction for ICT outcomes. [Table: see text]

Abstract P1-19-03: Phase II trial of durvalumab and tremelimumab in the hormone receptor-positive metastatic breast cancer with high tumor mutational burden selected by whole exome sequencing: Korean cancer study group trial (KCSG BR17-04)

Abstract Background: Hormone-receptor (HR) positive breast cancer is the main subtype of breast cancer. Although overall survival of HR-positive metastatic breast cancer (MBC) patients has improved by various therapies including endocrine therapies, CDK4/6 inhibitors, and cytotoxic chemotherapy, it is still considered incurable. Immune checkpoint inhibitors have rarely been clinically tested in HR-positive breast cancer, despite proving anti-cancer activity in early and metastatic triple-negative breast cancer in various trials. We evaluated efficacy and safety of combined durvalumab and tremelimumab in the HR-positive MBC, which was enriched with high tumor mutational burden (TMB). Methods: HR-positive MBC patients who received prior 1 or more lines of therapy in metastatic setting were prescreened with whole exome sequencing (WES) using metastatic or recurred tumor biopsies. Criterion of high TMB was defined as upper 30%. In the beginning, the criterion of high TMB was 2.1 mutations per Mb, based on the retrospective WES database in Yonsei Cancer Center and this criterion was recalculated every 30 cases. Patients who met upper 30% of TMB were treated with combined durvalumab (1500mg every 4 weeks upto 13 doses) and tremelimumab (75mg every 4 weeks upto 4 doses). Response was evaluated every 2 cycles using RECIST 1.1 and toxicity was evaluated using NCI-CTCAE 4.03. Tumor-infiltrating lymphocyte (TIL) and PD-L1 expression were also analyzed to investigate a correlation with TMB. Results: Biopsies of recurrent or metastatic tumors were taken from a total of 119 patients for WES assay. A median turn-around-time of TMB data was 30.0 days (range, 16~67). Of these 119 patients, a median number of nonsynonymous mutations was 2.0 per Mb (range, 0~21.7) with upper 30% criterion of 3.1. High TMB showed a trend toward old age (P=0.074) and single positivity of estrogen receptor (ER) or progesterone receptor (PR) compared to positivity of both ER and PR (P=0.055). TMB was positively correlated with TILs (r=0.289, P=0.005). Thirty patients with high TMB received study treatment with a median 2 cycles (range, 1~13). A median prior lines of therapies in metastatic setting was 4 (range, 1~9). The objective response and clinical benefit rates were 6.3% (2 PRs of 30) and 20% (2 PRs plus 4 SDs of 30). There was one treatment-related mortality due to pneumonitis. Immune-related adverse events included endocrinopathy (n=3; hypothyroidism in 2, hyperthyroidism in 1), enteritis (n=2), skin rash (n=2), pneumonitis (n=1), and so on. Biomarker analyses are underway. Conclusions: WES-based TMB using metastatic tumor biopsy was a feasible platform to prescreen HR-positive MBC patients. Combined durvalumab and tremelimumab showed a modest activity and good tolerability in heavily treated, HR-positive MBC with high TMB. Citation Format: Yong Wha Moon, Eunyoung Kim, Min Hwan Kim, Gun Min Kim, Seul-Gi Kim, YeeSoo Chae, Jieun Lee, Jae Ho Jeong, Kyung-Hun Lee, Han Jo Kim, Joo Young Jung, Su-Jin Koh, Kyoung Eun Lee, Hee-Jun Kim, Kyong Hwa Park, Seungtaek Lim, Yeon Hee Park, Tae Hoen Kim, Sewha Kim, Yohan Yang, Sangwoo Kim, Joohyuk Sohn. Phase II trial of durvalumab and tremelimumab in the hormone receptor-positive metastatic breast cancer with high tumor mutational burden selected by whole exome sequencing: Korean cancer study group trial (KCSG BR17-04) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-19-03.

Tumor Mutational Burden in Histiocytic Neoplasms

▪BackgroundHistiocytic disorders are rare hematologic neoplasms characterized by their high clinical heterogeneity. Evidence of constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in a number of these disorders has led to the increasing use of BRAF- and MEK-inhibitors as a therapeutic strategy. Response to these therapies is not universal and additional effective treatment options are required. Immune checkpoint inhibitors have proven effective for a wide array of malignancies. Tumor mutational burden (TMB), the total number of somatic pathogenic variants per coding region in a tumor genome, is a clinical biomarker associated with response to immunotherapy. We have previously reported preliminary findings of low TMB in a small cohort of patients with histiocytic neoplasms (Goyal G et al. Blood 2019). In this study, our aim was to confirm the findings in a larger clinical cohort using next generation sequencing studies.MethodsA retrospective review of adult patients consecutively seen at Mayo Clinic from 2017 to April 2021 and diagnosis of a histiocytic neoplasm was performed. Electronic records were queried for demographic and laboratory data of all patients who consented to undergo tumor-tissue next-generation sequencing with the Tempus-xT ® or xO ® assay (Chicago, IL), throughout the course of their evaluation. TMB was reported as the number of nonsynonymous mutations per coding area of a tumor genome. TMB across groups was compared via an independent-samples T-test or via analysis of variance and post hoc subgroup testing via Turkey's Test as required.ResultsSeventy-three patients were included in the study. Individual diagnoses included: Erdheim-Chester disease (ECD) in 31.5% (n=23), including 3 patients with overlap features of ECD and other histiocytoses; Rosai Dorfman disease (RDD) in 28.8% (n=21); Langerhans cell histiocytosis in 21.9% (n=16), histiocytic sarcoma (HS) in 5.5% (n=4), and other histiocytic neoplasms in 12.3% (n=10) including adult xanthogranuloma, xanthoma disseminatum, undifferentiated histiocytoses, and one case of Langerhans cell sarcoma.Median tumor percentage on analyzed samples was 30% (range 10%-90%) and was ≤20% in 29 samples. TMB was <1.0 mutations per megabase (mpMb) in 42.5% (n=31) and >5.0 mpMb in only 6 cases. Median TMB was 1.58 (range 0 - 5.26) for ECD, 1.08 (range 0 - 6.3) for LCH, 0.2 (range 0 - 5.8) for RDD, 3.19 (range 1.67 - 6.8) for HS, and 1.6 (range 0 - 7.5) for other histiocytic neoplasms (Figure 1). No clear association between increasing TMB and number of systems with histiocytic infiltration was observed (Figure 2). A significant increase in TMB was observed among the 5 cases of sarcoma as compared to the rest of the cohort (p=0.014). When not considering sarcomas, no differences were observed in TMB between patients with ECD, LCH, RDD or other histiocytoses (F=0.775; p=0.512).ConclusionIn this large cohort of histiocytic disorders, TMB was low compared to that historically seen in other neoplasms though this is likely influenced by tumor purity ≤20% in 29 cases. Only 6 tumors had TMB reports with mpMb above the threshold thought to be associated with an increased likelihood of response to PD-1/PDL1-targeted therapies (>5 mpMb). Our results suggest a low likelihood of response among histiocytic disorders using immune checkpoint inhibitors. Further exploration among malignant histiocytoses (HS and Langerhans cell sarcoma) is warranted given our findings of higher TMB compared with other histiocytic neoplasms. [Display omitted] DisclosuresBennani: Kymera: Other: Advisory Board; Vividion: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Verastem: Other: Advisory Board.

Abstract 471: Reference materials for tumor mutational burden transcriptome profiling

Abstract Immune Checkpoint Inhibitor (ICI) therapies continue to revolutionize treatment of advanced malignancies. However, identification of individuals who respond to ICI treatment remains challenging. Along with programmed death ligand 1(PD-L1 aka CD274) expression and microsatellite instability, tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase of genome, has emerged as a promising biomarker for differentiating responders from non-responders, especially in non-small cell lung cancer. Although promising, TMB is not a perfect biomarker for ICI efficacy with some individuals responding conversely to expectations. The imperfection of TMB as a prognostic biomarker may result from it being a proxy measurement of potential neoantigens. Indeed, all mutations that contribute to TMB will not be expressed as neoantigens and not all neoantigens will illicit immune responses. In refining ICI response prediction, tumor abundance of nonsynonymous mutations has aided in neoantigen prediction. In order to begin development of quality reference materials for neoantigen prediction, we have characterized the transcriptomes of four tumor cell lines that were previously used to formulate TMB reference materials. Sequencing libraries were prepared with the TruSeq Stranded Total RNA with Illumina Ribo-Zero Plus rRNA Depletion Kit and Invitrogen Collibri Stranded RNA Library Prep Kit from Illumina with Collibri H/M/R rRNA Depletion Kit. The resultant transcriptome libraries were sequenced on an Illumina NextSeq2000 platform and relative transcript counts were determined. Broad ranges of expression were observed among transcripts containing nonsynonymous mutations in each of the tumor cell lines tested. These datasets will serve as benchmarks for others attempting to combine TMB and tumor abundance in predicting neoantigen potential. Citation Format: Matthew G. Butler, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Reference materials for tumor mutational burden transcriptome profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 471.

Abstract PO-029: USP14 is a potential target to improve the efficacy of radiotherapy in combination with immune-checkpoint inhibitors in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) represents ~85% of lung cancer cases and has a 5-year survival rate of ~23%. Thus, research aimed at determining more effective treatment strategies is critical. Immune checkpoint inhibitors (ICis), e.g. anti-PD-L1 antibodies, have transformed cancer treatment in recent years. Clinical trial studies have indicated that radiotherapy enhances the curative effect of ICis. However, most patients do not respond to immune-radiotherapy combinations. While best known to induce DNA double strand break (DSB)-mediated cytotoxicity, ionizing radiation (IR) induces an increase of cytosolic DNA and activates the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) signaling pathway that leads to the type 1 interferon (IFN) response, essential to achieve anti-tumor immunity in combination with ICis. Homologous recombination (HR) DNA repair deficiency is an important determinant of radiation response that can also increase the immune recognition of cancers by increasing the number of non-synonymous mutations, as well as by increasing the cGAS/STING-mediated type1 IFN response. We have shown an important role for ubiquitin specific protease (USP14) in regulating DNA repair (1, 2,3). Here, we identify a novel role of USP14 in regulating HR and the cGAS/STING pathway in response to IR in NSCLC. USP14 is a proteasomal deubiquitinase that we show to have a prominent role in NSCLC. Knockdown of USP14 (shUSP14) or its pharmacological inhibition (IU1 or VLX1570) led to: (i) radio-sensitization, as shown by reduced clonogenic survival in NSCLC cell lines, and delayed growth of NSCLC tumor xenografts in nude mice; increased (ii) HR-deficiency; (iii) chromosomal aberrations and micronuclei formation; as well as (iv) cGAS puncta in response to IR. Further, USP14 interacts with and deubiquitinates cGAS in IR-treated NSCLC cells. USP14-mediated deubiquitination of cGAS has been shown to inhibit cGAS protein turn-over and increase the levels of cGAS in response to virus infection. Unexpectedly, however, in response to IR shUSP14 NSCLC cells have significantly higher cytoplasmic cGAS puncta and type 1 IFN response. Overall, these findings identify USP14 as a potential target to enhance sensitization to immune-radiotherapy by both regulation of HR and direct regulation of cGAS ubiquitination, leading to type 1 IFN response. Understanding the mechanisms that regulate DSB repair and cGAS/STING pathways holds potential for developing strategies to enhance the effectiveness of immune-radiotherapy combinations in NSCLC.

#84: Evolutionary Pressure from APOBEC Causes an Underrepresentation of TC Motifs in Human Polyomavirus

Abstract Background Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) is a family of enzymes found in mammals that deaminate cytidine to uridine on ssDNA, facilitating a C-to-T mutation. Less commonly, APOBEC effectively mutates C-to-G as well. APOBEC has the potential to mutate the viral genome, rendering the virus nonproductive. The enzyme targets certain mutational motifs, also known as hotspots: most APOBECs deaminate at TC motifs. We hypothesize that human polyomaviruses (hPyV) have an under-representation of TC hotspots in their genomes to avoid APOBEC targeting. Specifically, we analyzed the degree of TC motif under-representation in five conserved genes of hPyV, each of which is expressed in either the early or late phase of the viral life cycle. Methods We utilized a statistical tool, the Cytidine Deaminase Under-representation Reporter (CDUR) that can be used to analyze sequences and determine whether these sequences have mutated under the evolutionary pressure of APOBEC based on the targeted hotspot. This tool uses a permutation test to determine whether the number of hotspots at a given coding sequence is significantly more or significantly less than expected under pseudo-random biological sequence evolution. The same analysis was used to determine whether the number of nonsynonymous mutations that might occur at those hotspots is significantly more or significantly less than expected by chance. Results TC motifs in early genes were more underrepresented compared with late genes in most hPyV species. Most early genes were susceptible to nonsynonymous mutations at the TC motifs. Those species which did not fit into this paradigm showed a similar degree of under-representation and susceptibility to nonsynonymous mutations at AC motifs. We also find a large over-representation of TG hotspots, but not TT hotspots. Conclusions hPyV may have evolved a reduced number of hotspots to subvert APOBEC and evade host immune defenses. Under-representation of TC motifs is more prevalent in early genes. Early genes include the small T and large T antigens, essential for viral proliferation. In addition, the large T antigen was particularly susceptible to mutation at TC motifs. This underscores the evolutionary pressure imposed on hPyV by APOBEC to evade host immunity. It is possible that the over-representation of TG motifs in hPyV is due to specific host DNA-repair mechanisms, but this requires further investigation.

Number of nonsynonymous genomic variants may correlate with prognosis in advanced pancreatic cancer.

432 Background: Pancreatic cancer is associated with poor outcomes at any stage. A very small number of patients - approximately 3% of those with metastatic disease - experience long-term survival through 5 years but the biologic mechanisms underlying the benefit observed with these “exceptional responders” are incompletely understood. We explored potential genomic differences between exceptional responders and typical responders to treatment for advanced pancreatic cancer that could confer a more favorable biology. Methods: We included consecutive exceptional responders and matched controls (MCs) with advanced pancreatic cancer in a 1:3 ratio from the Cleveland Clinic from April, 2013 – August, 2017. ERs were defined as patients with overall survival (OS) &gt; 18 months for metastatic disease and &gt; 24 months for locally advanced disease. Controls were matched by age, gender, stage and type of chemotherapy given and experienced OS at or below median survival for their stages. Clinical variables including demographics, comorbidities, stage, histology and treatment history were collected. Next generation sequencing (NGS) was performed for DNA sequencing of 648 genes and tumor mutation burden (TMB) on available tissue. The study population initially comprised of 14 exceptional responders and 42 MCs. However, only 4 exceptional responders and 6 MCs were included for analysis due to insufficient tissue for NGS for the remaining patients. Descriptive statistics were used for statistical analysis. Differences in survival outcomes were assessed using the Kaplan-Meier method and log-rank test. Results: The median ages for the exceptional responders and MCs were 69 and 67.5 years, respectively. Both groups were at least 75% male and white. Of the exceptional responders, 50% had pancreatic tail primaries compared to 0% of the MCs. There were no differences between groups in first-line chemotherapy used. Exceptional responders had significantly fewer non-synonymous mutations compared to MCs (2.25 vs. 5.17, p = 0.014). Mutation count &lt; 3 was associated with significantly better progression-free survival (PFS) and OS as shown in the Table. TMB did not differ between exceptional responders and MCs (4.88 vs. 5.70 Muts/Mb, p = 0.39). Of the exceptional responders, 50% had alterations in BAGE2 versus 0% of MCs. Conversely, 50% of MCs had alterations in LRP1B, CUL4B or APC vs. 0% of exceptional responders. Conclusions: Having a lower number of non-synonymous mutations may correlate with exceptional response to systemic therapy and therefore with improved survival in pancreatic cancer. Pancreatic tail primary may also be associated with improved outcomes. These findings, though limited by small sample size, should encourage future study into genomic signatures of exceptional response. [Table: see text]

Co-mutations of KRAS/BRAF and TP53 or the high tumor mutation burden to predict survival in patients with biliary tract carcinoma.

e16650 Background: Biliary tract carcinoma (BTC), including cholangiocarcinoma and gallbladder carcinoma, is the second most common type of hepatobiliary cancer. Patients with BTC always show poor prognosis, here we revealed the molecular landscape of BTC in the Chinese population and evaluated the role of different mutations in informing prognosis. Methods: Formalin-Fixed Paraffin-Embedded (FFPE) or freshly-sampled tumor tissues from 59 BTC patients were conducted next-generation sequencing of 620 genes related to oncogenesis. Tumor mutation burden (TMB) value represents the number of non-synonymous mutations per mega base pairs in each sample. Kaplan-Meier survival curves were generated and compared using the log-rank test. Results: Altogether, 59 patients have mutations mainly in TP53, Ras/Raf, PI3K, CDK signaling pathways and SWI/SNF complex. The most frequently mutated gene was TP53(53%), followed by KRAS(23%), ARID1A(17%), ATM(12%), CDKN2A(10%), SMAD4(8%), BRCA2(8%), STK11(7%), BRAF(5%), IDH1(5%) and FGFR3 (3%). Noticeably, only one patient with FGFR2 fusion was detected. The Median TMB of these patients is 2.80 Muts/Mbp (0-36.52 Muts/Mbp). Existing data showed that KRAS/BRAF alterations were associated with a worse overall survival (OS) (median OS 166d vs. 294d, p= 0.063). Further analysis indicated that RAS/BRAF mutations were often co-current with TP53 alternations. And patients with coaltered RAS/BRAF and TP53 demonstrated the worst prognosis (media OS 123d vs. 294d, p= 0.087). In addition, a higher TMB ( &gt; 2.80 Muts/Mb) was also associated with a worse survival (median OS 174d vs. 355d, p= 0.085). Conclusions: We identified KRAS/BRAF, or co-mutations with TP53 and high TMB could predict poor prognosis in BTC patients. These findings will be useful for clinical decision making in patients with refractory biliary tract cancer and for risk stratification of patients in future clinical studies.

An increase in serum choline levels to predict progression-free survival (PFS) in patients (pts) with advanced cancers receiving pembrolizumab.

3102 Background: Recent work from our laboratory demonstrated that T cell-derived acetylcholine induces vasodilation and increases T cell migration to infected tissues in response to viral infection (Cox et al. Science 2019). Choline acetyltransferase catalyzes the production of acetylcholine from choline and acetyl-CoA, however acetylcholine is challenging to quantify due to its extremely short half-life while choline is stable. This study is the first reported attempt to correlate serum choline levels in patients (pts) with advanced solid tumors receiving pembrolizumab with treatment outcomes. Methods: Blood samples were collected pre-treatment in 106 pts treated with pembrolizumab 200 mg IV Q3W in the investigator-initiated INSPIRE study (NCT02644369). Of these, 81 pts had on-treatment blood samples collected at week 7 (pre-cycle 3). Serum choline was analyzed with an HPLC-tandem mass spectrometry assay. PD-L1 staining was performed in baseline tumor tissues using 22C3 antibody and scored using modified proportion score. Tumor mutational burden (TMB) was calculated based on number of nonsynonymous mutations detected using whole exome sequencing. Multivariable Cox models were used to assess the impact of choline on PFS and OS, while adjusting for cohort, PD-L1 expression and TMB. Results: This pan-cancer group of 106 pts (median age 55, 62% females) comprised of 5 cohorts: squamous cell carcinoma of the head and neck = 19 pts, triple negative breast cancer = 22, high grade serous ovarian cancer = 21, melanoma = 19, mixed solid tumors = 32. With a median follow-up of 11 months, the median PFS = 1.9 months and median OS = 13.9 months for the entire cohort. In univariable analysis adjusted by cohort, baseline serum choline levels in 106 pts did not correlate with PFS or OS. However, an increase in serum choline level at week 7 compared to pre-treatment (D choline) in 81 pts was significantly associated with a better PFS (aHR 0.49, 95% CI 0.28-0.85, p = 0.01), and a trend towards a better OS (aHR 0.57, 95% CI 0.32-1.03, p = 0.064). In multivariable analysis, D choline remains significantly associated with an improved PFS (p = 0.0087) after adjustment for cohort, PD-L1 and TMB. Conclusions: This is the first exploratory report of serum choline levels in pan-cancer pts receiving pembrolizumab. The association between improved PFS and D choline suggests a possible role for the cholingeric system in the regulation of antitumor immunity. Further nonclinical and clinical studies are required to validate this finding.

Cell-free Circulating Tumor DNA Variant Allele Frequency Associates with Survival in Metastatic Cancer.

Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis. We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival. cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level <3.5 g/dL, number of nonvisceral metastatic sites >0 and number of prior therapies >4 were independent predictors of worse OS. Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

Abstract 2514: Inter-test variability in tumor mutational burden (TMB) quantification and identification of TMB thresholds

Abstract Introduction: Tumor mutational burden (TMB) is a possible predictive biomarker for response to immune checkpoint inhibitors (ICI). However, there is significant heterogeneity in the calculation of TMB and the thresholds that have been identified to associate with clinical benefit. We analyzed TMB and clinical outcomes across next generation sequencing (NGS) panels to address inter-panel heterogeneity and to identify consistent TMB thresholds. Methods: Non-small cell lung cancer sequencing data generated from three validated clinical platforms (OncoPanel n = 1155, MSK-IMPACT n = 1520, and Foundation Medicine n = 3377) and whole exome sequencing from The Cancer Genome Atlas (TCGA, n = 1144) were obtained. Tumor mutational burden (TMB) was calculated across cohorts as the number of nonsynonymous mutations per length of genome sequenced. The distribution of TMB was plotted for each cohort and compared to TCGA. For the subset of patients treated with ICI (OncoPanel n = 296; MSK-IMPACT,n = 227), durable clinical benefit (DCB) was defined as responsive/stable disease lasting ≥ 6 months and was associated with TMB. Results: There was high concordance across sequencing panels in the mean number of mutations per individual gene (OncoPanel vs TCGA R2 = 0.8914; Foundation vs TCGA R2 = 0.9025; MSK-IMPACT vs TCGA R2 = 0.8579). Absolute TMB values were higher in NGS panels compared to TCGA, likely due to enrichment for oncogenes and differences in germline filtration. Harmonization between NGS panels and WES was attempted by applying a linear transformation of panel TMB values to WES TMB values. Linear transformation constants were variable at the highest and lowest TMB values, and in low TMB subgroups such as EGFR mutants and never smokers. Consistent with prior studies, TMB was higher in patients with DCB compared to those with no durable benefit (NDB) (Oncopanel p-value=0.003; MSK-IMPACT p-value=0.006). Increasing TMB thresholds were associated with increase in DCB rate, likely due to enrichment of the highest TMB responders; DCB rates did not linearly associate with TMB at lower TMB values. Conclusion: There were significant differences in TMB quantification among NGS and TCGA panels, particularly at lower TMBs. Categorization, rather than fitting absolute values, may be an approach for harmonizing distinct panels. In these cohorts, the relationship between benefit of ICI and TMB was not consistent across the TMB spectrum but accentuated in the highest TMB patients. These findings may have implications for the application of TMB as a biomarker in clinical practice. Citation Format: Natalie Vokes, Elizabeth Jimenez Alguilar, Renato Umeton, Anika Adeni, Lynette Sholl, Matthew Hellmann, Hira Rizvi, Mark Awad, Eliezer Van Allen. Inter-test variability in tumor mutational burden (TMB) quantification and identification of TMB thresholds [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 2514.

Prognostic impact of mutation profiling in Chinese patients with colorectal liver metastases.

e15157 Background: To investigate mutation profiling in Chinese patients with colorectal liver metastases and explore new prognostic biomarkers. Methods: We investigated 197 patients who underwent surgery for colorectal liver metastases at Beijing Cancer Hospital between January 2015 and February 2017. Tumor DNA was isolated from FFPE tissues from liver metastatic lesions. Mutation spectrum was obtained by targeted deep sequencing of 620 selected genes. Tumor mutational burden (TMB) was defined as the number of non-synonymous mutations per 1Mbp and divided into quartiles. MSI status was tested with next-generation sequencing. Studies of impacts of mutation status on disease-free survival (DFS) and overall survival (OS) were performed with Kaplan-Meier analysis. Results: Mutation significance analysis confirmed enrichment in colorectal cancer driver gene mutations of TP53, APC, KRAS, SMAD4, SOX9, FBXW7 and PIK3CA in 72%, 61%, 29%, 14%, 10%, 10% and 9% of liver metastases, with no BRAF mutation found in any of these patients. Additionally, we observed frequent gene-level copy number variations, including amplifications of MYC, BRCA2, ERBB2, CCND2 and deletion of APC. Among 197 patients, only three showed MSI-high, who also exhibited high TMBs. Moreover, KRAS, MXRA5, and SETD2 mutations were significantly associated with shorter DFS ( KRAS mt vs wt: 8 months vs 11 months, p = 0.0086; MXRA5 mt vs wt: 6 months vs 11 months, p = 0.0178; SETD2 mt vs wt: 8 months vs 11 months, p = 0.0381), but KMT2A mutation associated with longer DFS (mt vs wt: 31.5 months vs 10 months, p = 0.0409). SPEN, GNAS, CTNNB1, SETD2 and FANCM mutations were significantly associated with poor OS ( SPEN mt vs wt: 23 months vs 30 months, p = 0.0047; GNAS mt vs wt: 24.5 months vs 30 months, p = 0.0386; CTNNB1 mt vs wt: 20 months vs 30 months, p = 0.0001; SETD2 mt vs wt: 20 months vs 30 months, p = 0.0026; FANCM mt vs wt: 13 months vs 30 months, p = 0.0007). Conclusions: Our study has found a novel panel of prognostic biomarkers in colorectal liver metastases. The clinical significance of mutations of MXRA5, SETD2, KMT2A, SPEN, GNAS, CTNNB1, FANCM needs to be confirmed on a larger scale.