Tumor Mutational Load Research Articles

Emerging Trends for Radio-Immunotherapy in Rectal Cancer.

Simple SummaryDespite recent advances in understanding the molecular biology of tumors, obstacles still exist in the treatment of patients affected by rectal cancer. Recent evidence shows that ionizing radiation may have profound immunostimulatory effects, hinting at the possibility of exploiting radiotherapy to boost anti-tumor immunity. A bulk of work in pre-clinical tumor models have highlighted the potential benefit of this approach. Following these results, a few clinical trials are now evaluating the combination of radiotherapy and immune checkpoint inhibition. Remarkably, encouraging safety and toxicity profiles from these studies indicate that radio-immunotherapy combinations could represent a valid opportunity for rectal cancer patients. Yet, the biological and clinical impact of a radio-immunotherapy combination in rectal cancer remains unclear and further studies need to be performed to optimize the effect of these combinations.Rectal cancer is a heterogeneous disease at the genetic and molecular levels, both aspects having major repercussions on the tumor immune contexture. Whilst microsatellite status and tumor mutational load have been associated with response to immunotherapy, presence of tumor-infiltrating lymphocytes is one of the most powerful prognostic and predictive biomarkers. Yet, the majority of rectal cancers are characterized by microsatellite stability, low tumor mutational burden and poor T cell infiltration. Consequently, these tumors do not respond to immunotherapy and treatment largely relies on radiotherapy alone or in combination with chemotherapy followed by radical surgery. Importantly, pre-clinical and clinical studies suggest that radiotherapy can induce a complete reprograming of the tumor microenvironment, potentially sensitizing it for immune checkpoint inhibition. Nonetheless, growing evidence suggest that this synergistic effect strongly depends on radiotherapy dosing, fractionation and timing. Despite ongoing work, information about the radiotherapy regimen required to yield optimal clinical outcome when combined to checkpoint blockade remains largely unavailable. In this review, we describe the molecular and immune heterogeneity of rectal cancer and outline its prognostic value. In addition, we discuss the effect of radiotherapy on the tumor microenvironment, focusing on the mechanisms and benefits of its combination with immune checkpoint inhibitors.

61892 Systemic TLR3-targeting Combinatorial Chemokine Modulation Sensitizes Murine Tumors to PD-1 Blockade

ABSTRACT IMPACT: This work will lead to improved efficacy of immunotherapy directly impacting the survival of patients with hard to treat cancers. OBJECTIVES/GOALS: Immune checkpoint inhibitors (ICI) are most effective against ‘hot’ tumors highly infiltrated with cytotoxic T lymphocytes (CTLs) but have not worked well in poorly infiltrated ‘cold’ tumors. Thus, we are working to achieve a pretreatment regimen that will create a favorable immune profile allowing more effective ?PD-1 therapy. METHODS/STUDY POPULATION: BALB/c or C57BL/6 mice were inoculated with CRC murine cells CT26 or MC38, respectively. Mice were inoculated by two injection types: subcutaneous (SC), for systemic therapy, or intraperitoneal (IP), for local therapy. Tumor-bearing mice were given a two dose course of CKM consisting of IFN-? and rintatolimod via IP injection. Following CKM administration, mice were treated with three doses of ?PD-1 via IP injection. Mice were monitored for the kinetics of tumor growth and survival following treatment. The tumor microenvironment of treated mice was analyzed for production of chemokines, inflammatory cytokines and immune cell infiltration. RESULTS/ANTICIPATED RESULTS: CKM consisting of combination IFN-? and rintatolimod, but neither monotherapy alone, sensitized murine CRC tumors to subsequent ?PD-1 treatment. In both CT26 and MC38 tumor-bearing mice, tumor growth was hindered by CKM plus ?PD-1 treatment, independently on the route of treatment (local or systemic). Mice which experienced complete tumor regression were protected from re-challenge with a dose of tumor cells double that of the initial inoculation. Sensitizing tumors to ?PD-1 did not require intratumoral CKM administration and was observed with systemic application at distant sites. In accordance with these observations we expect that systemic CKM will induce strong increases of total and tumor-specific CTL counts in the tumor tissues as measured by both PCR and flow cytometry. DISCUSSION/SIGNIFICANCE OF FINDINGS: CKM sensitizing cold tumors to ?PD-1 indicates that intratumoral CTLs are an important factor dictating therapeutic effectiveness, independent of other factors such as tumor mutational load. The benefit of the sequential short-term CKM followed by routine ?PD-1 make this strategy feasible for rapid inclusion of into routine immunotherapy plans.

NLRC5/CITA expression correlates with efficient response to checkpoint blockade immunotherapy

Checkpoint blockade-mediated immunotherapy is emerging as an effective treatment modality for multiple cancer types. However, cancer cells frequently evade the immune system, compromising the effectiveness of immunotherapy. It is crucial to develop screening methods to identify the patients who would most benefit from these therapies because of the risk of the side effects and the high cost of treatment. Here we show that expression of the MHC class I transactivator (CITA), NLRC5, is important for efficient responses to anti-CTLA-4 and anti-PD1 checkpoint blockade therapies. Melanoma tumors derived from patients responding to immunotherapy exhibited significantly higher expression of NLRC5 and MHC class I-related genes compared to non-responding patients. In addition, multivariate analysis that included the number of tumor-associated non-synonymous mutations, predicted neo-antigen load and PD-L2 expression was capable of further stratifying responders and non-responders to anti-CTLA4 therapy. Moreover, expression or methylation of NLRC5 together with total somatic mutation number were significantly correlated with increased patient survival. These results suggest that NLRC5 tumor expression, alone or together with tumor mutation load constitutes a valuable predictive biomarker for both prognosis and response to anti-CTLA-4 and potentially anti-PD1 blockade immunotherapy in melanoma patients.

A Combined Epithelial Mesenchymal Transformation and DNA Repair Gene Panel in Colorectal Cancer With Prognostic and Therapeutic Implication.

BackgroundEpithelial mesenchymal transformation (EMT) and DNA repair status represent intrinsic features of colorectal cancer (CRC) and are associated with patient prognosis and treatment responsiveness. We sought to develop a combined EMT and DNA repair gene panel with potential application in patient classification and precise treatment.MethodsWe comprehensively evaluated the EMT and DNA repair patterns of 1,652 CRC patients from four datasets. Unsupervised clustering was used for classification. The clinical features, genetic mutation, tumor mutation load, and chemotherapy as well as immunotherapy sensitivity among different clusters were systematically compared. The least absolute shrinkage and selection operator regression method was used to develop the risk model.ResultsThree distinct CRC clusters were determined. Clustet1 was characterized by down-regulated DNA repair pathways but active epithelial markers and metabolism pathway and had intermediate prognosis. Clustet2 was characterized by down-regulated both epithelial markers and DNA repair pathways and had poor outcome. Clustet3 presented with activation of DNA repair pathway and epithelial markers had favorable prognosis. Clustet1 might benefit form chemotherapy and Clustet3 had a higher response rate to immunotherapy. An EMT and DNA repair risk model related to prognosis and treatment response was developed.ConclusionsThis work developed and validated a combined EMT and DNA repair gene panel for CRC classification, which may be an effective tool for survival prediction and treatment guidance in CRC patients.

Association of tumor mutation burden and epidermal growth factor receptor inhibitor history with survival in patients with metastatic stage III/IV non-small-cell lung cancer: A retrospective study

OBJECTIVES:Lung cancer is the leading cause of cancer-related deaths worldwide. However, factors associated with the survival of patients with advanced non-small-cell lung cancer (NSCLC) who received only hospice care are largely unclear. In this study, we aimed to determine the prognostic factors correlated with survival in patients with advanced NSCLC who had undergone hospice care only.METHODS:A total of 102 patients with recurrent stage III/IV NSCLC after traditional treatment failure were investigated. Survival was measured from the date of enrollment to December 2019 or the time of death. Tumor tissues were collected, and DNA sequencing was performed to identify somatic mutations. Data on clinical factors of patients were collected and analyzed by univariate and multivariate analyses. Overall survival analysis was conducted using the Kaplan-Meier method.RESULTS:The 6-month, 1-year, and 2-year overall survival rates of the 102 patients with metastatic NSCLC were 17.65%, 3.92%, and 0.98%, respectively. The median overall survival of the 102 patients was 3.15 months. Tumor location in the peripheral lung, epidermal growth factor receptor (EGFR) inhibitor history, low tumor mutation load, adenocarcinoma, and poor performance status score were associated with prolonged survival compared with tumor location in the central lung, no EGFR inhibitor history, high tumor mutation load, squamous cell carcinoma, and good performance status score (p=0.045, p=0.003, p=0.045, p=0.021, and p=0.0003, respectively). CONCLUSIONS:EGFR inhibitor treatment history and tumor mutation load are risk factors for the overall survival of patients with stage III/IV NSCLC who have undergone only hospice care. These results provide a critical clinical basis for further study of nontraditional anti-tumor responses induced by EGFR inhibitors.

Targeted next-generation sequencing for cancer-associated gene mutation and copy number detection in 206 patients with non–small-cell lung cancer

ABSTRACT The knowledge of genetic variation in Chinese patients with non–small-cell lung cancer (NSCLC) is still limited. We aimed to profile this genetic variation in 206 Chinese patients with NSCLC using next-generation sequencing. Tumor tissues or whole-blood samples were collected and subjected to whole-exome targeted next-generation sequencing, which included 565 tumor-associated genes, for somatic gene mutation screening and copy number variation (CNV) detection. Potential functions of most commonly mutated genes and genes with CNV were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Atotal of 18,749 mutations were identified using targeted next-generation sequencing, and 85.3% of them were missense mutations. Among the mutation, conversions between pyrimidine and purine were predominant, and C> T/G > A was the most common substitution type. High frequencies of mutations were noted in TP53 (47.6%), EGFR (41.7%), CREBBP (23.1%), KMT2C (16.9%), MUC2 (16.6%), DNMT3A (15.5%), LRP1B (15.5%), MUC4 (15.5%), CDC27 (15.2%), and KRAS (12.8%). EGFR and KRAS mutations were mutually exclusive. The tumor mutation load showed differences depending on gender and tumor type. CNV analysis showed that BCORL1 and ARAF have the highest copy number amplification, whereas KDM6A and RBM10 showed the highest copy number deletion. GO and KEGG analyses indicated that high-frequency mutations and CNV genes were concentrated in tumor-related PI3K-Akt, FoxO, and Ras signaling pathway. Cumulatively, we studied somatic gene mutations involved in NSCLC and predicted their clinical significance in Chinese population. These findings may provide clues for etiology and drug target of NSCLC.

Tumor mutational burden on cytological samples: A pilot study.

Immune-checkpoint inhibitors (ICIs) represent an important treatment option for patients who have advanced stage non-small cell lung cancer (NSCLC). Currently, evaluation of the expression level of programmed death-ligand 1 (PD-L1) has proven highly successful as a positive predictive biomarker for ICIs. In addition to PD-L1, other promising predictive biomarkers are emerging, including high tumor mutational burden (TMB-H). However, measuring TMB-H remains challenging for several reasons, among which is the difficulty in obtaining adequate tissue material from NSCLC patients. There are no data in the current literature regarding the possibility of adopting cell blocks (CBs) for TMB evaluation; therefore, our goal was to evaluate the feasibility of analyzing TMB on CBs. For evaluation of differences in run metric parameters, 8 pairs of histological and CB samples from patients with NSCLC were analyzed using the Oncomine Tumor Mutational Load Assay on Ion Torrent S5 GS next-generation sequencing (NGS) platform. Most CBs (6/8, 75.0%) were successfully analyzed by adopting the broad NGS panel approach. CBs provided results similar to those obtained on histological matched specimens in terms of median total reads (7207048.80 vs 7558817.80), median mapped reads (7075753.83 vs 7513822.00), median read lengths (115.50 vs. 113.00), median percentage of reads on-target (97.49% vs. 98.45%), median average reads per amplicon (454.67 vs 476.14), and median uniformity of amplicon coverage (83.52% vs 84.13%). In this pilot study, we demonstrated the technical feasibility of assessing TMB on CBs.

A multiparametric approach to improve the prediction of response to immunotherapy in patients with metastatic NSCLC

BackgroundIt is still unclear how to combine biomarkers to identify patients who will truly benefit from anti-PD-1 agents in NSCLC. This study investigates exosomal mRNA expression of PD-L1 and IFN-γ, PD-L1 polymorphisms, tumor mutational load (TML) in circulating cell-free DNA (cfDNA) and radiomic features as possible predictive markers of response to nivolumab and pembrolizumab in metastatic NSCLC patients.MethodsPatients were enrolled and blood (12 ml) was collected at baseline before receiving anti-PD-1 therapy. Exosome-derived mRNA and cfDNA were extracted to analyse PD-L1 and IFN-γ expression and tumor mutational load (TML) by digital droplet PCR (ddPCR) and next-generation sequencing (NGS), respectively. The PD-L1 single nucleotide polymorphisms (SNPs) c.-14-368 T > C and c.*395G > C, were analysed on genomic DNA by Real-Time PCR. A radiomic analysis was performed on the QUIBIM Precision® V3.0 platform.ResultsThirty-eight patients were enrolled. High baseline IFN-γ was independently associated with shorter median PFS (5.6 months vs. not reached p = 0.0057), and levels of PD-L1 showed an increase at 3 months vs. baseline in patients who progressed (p = 0.01). PD-L1 baseline levels showed significant direct and inverse relationships with radiomic features. Radiomic features also inversely correlated with PD-L1 expression in tumor tissue. In subjects receiving nivolumab, median PFS was shorter in carriers of c.*395GG vs. c.*395GC/CC genotype (2.3 months vs. not reached, p = 0.041). Lastly, responders had higher non-synonymous mutations and more links between co-occurring genetic somatic mutations and ARID1A alterations as well.ConclusionsA combined multiparametric approach may provide a better understanding of the molecular determinants of response to immunotherapy.

Association of clock-like mutational signature with immune checkpoint inhibitor outcome in patients with melanoma and NSCLC

Immune checkpoint inhibitor (ICI) therapy has achieved remarkable clinical benefit in melanoma and non-small cell lung cancer (NSCLC). Tumor mutational signatures are the fingerprints of endogenous and exogenous factors that have acted throughout tumorigenesis and heterogeneity; however, their association with immune response in ICI-treated samples remains unclear. Here, we leveraged whole-exome sequencing (WES)-based mutational profiles combined with clinicopathologic characteristics from melanoma and NSCLC datasets to examine whether tumor genomic features contribute to clinical benefit of ICI treatment. Mutational data acquired from targeted next-generation sequencing (NGS) assays (MSK-IMPACT panels) were also employed for further corroboration. A mutational signature (known as age-related clock-like processing) characterized by enrichment of C>T mutations at NpCpG trinucleotides were identified to be associated with a worse prognosis and lower tumor mutation load (TML) in both WES and targeted NGS immunotherapy cohorts. We also analyzed gene transcriptomic profiles and identified immune regulation-related gene pathways that were significantly altered in samples with different clock-like signature grouping. Leucocyte subset analysis further revealed that clock-like signature was associated with the reduction of cytotoxic cell infiltration and elevation of regulatory T cells. Overall, our work re-annotated that the age-related clock-like signature was associated with worse prognosis and lower immune activity, offering opportunities to stratify patients into optimal immunotherapy plans based on genomic subtyping.

Alterations in TP53 Are a Potential Biomarker of Bladder Cancer Patients Who Benefit From Immune Checkpoint Inhibition.

In recent years, immune checkpoint inhibitors (ICIs) targeting CTLA-4 or PD1/PDL1 have achieved remarkable success in the treatment of bladder cancer (BLCA), but only a few patients have shown durable clinical benefits. The prognostic role of a mutant form of the tumor suppressor gene TP53 (TP53-MT) in predicting the efficacy of ICIs is highly controversial; therefore, in this study, we obtained data for 210 patients from an immunotherapy cohort, 412 patients from The Cancer Genome Atlas (TCGA)-BLCA cohort and 18 BLCA cell lines from Genomics of Drug Sensitivity in Cancer (GDSC), and we performed integrated bioinformatic analysis to explore the relationships between TP53-MT and clinical benefits derived from ICI treatment and the underlying mechanisms. We conclude that TP53-MT is a potential indicator of a relatively good response to ICIs and associated with prolonged overall survival (OS) (log-rank test, hazard ratio (HR) = 0.65 [95% confidence interval (CI), 0.44-0.99], p = 0.041). Through integrated analysis with several platforms, we found that TP53-MT patients were more likely to benefit from ICIs than wild-type P53 (TP53-WT) patients, which may be the result of 2 major mechanisms. First, the patients with TP53-MT showed stronger tumor antigenicity and tumor antigen presentation, as indicated by a higher tumor mutational load, a higher neoantigen load and increased expression of MHC; second, the antitumor immunity preexisting in tumors was stronger in samples with TP53-MT than in those with TP53-WT, including enrichment of interferon-gamma, positive regulation of TNF secretion pathways and increased expression of some immunostimulatory molecules, such as CXCL9 and CXCL10. This study provided some clues for identifying patients who would potentially benefit from ICIs at the somatic genomic level, developing new indications for targeted second-generation sequencing and promoting the development of precision medicine.

Robust assessment of tumor mutational burden in cytological specimens from lung cancer patients

ObjectivesTumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. Materials and MethodsWe analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%). ResultsAt 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. ConclusionOur results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.

Abstract 6466: Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL)

Abstract Immune dysfunction is a hallmark of CLL/MBL; ~25% individuals with newly diagnosed CLL have hypogammaglobulinemia at diagnosis. In this study of newly diagnosed CLL/MBL, we sought to a) investigate the association between serum immunoglobulins and specific mutations identified by next generation sequencing, and b) determine if serum immunoglobulin levels predict TFT, independent of the CLL-International Prognostic Index (CLL-IPI) and tumor mutational load (TML). We used the Mayo Clinic CLL Resource to identify newly diagnosed CLL/MBL individuals consented within 2 years of diagnosis. Baseline clinical characteristics and the CLL-IPI score was ascertained on all individuals. DNA was extracted from peripheral blood CD5+/CD19+ clonal B-cells; and the entire coding regions of 60 somatically recurring mutated CLL driver genes were enriched using a customized Agilent SureSelect gene panel and sequenced in Illumina Hiseq4000. Serum immunoglobulins were quantitated by radial immunodiffusion using Immunoplates. The relationship between serum immunoglobulins was assessed: a) with individual gene mutations (mutated vs. not); and b) using the TML score (using a value of 0, 1, and 2 or greater [2+] mutated genes). The impact of serum immunoglobulin levels on TFT was analyzed for all individuals. Of 356 individuals identified, 80 (22%) had MBL, median age was 63 years (28-87), 261 (73%) were male, and the CLL-IPI distribution was: low risk (n=149, 42%), intermediate risk (n=112, 32%), and high/very high risk (n=94, 26%). We observed 43%, 39%, and 18% individuals with 0, 1, or 2+ mutated genes, respectively. The most commonly mutated genes were NOTCH1 (13%), SF3B1 (13%), TP53 (8%), ATM (8%), BIRC3 (7%), and NFKBIE (7%). Using standard cutoffs, 135 (38%) individuals had low serum IgG, 73 (21%) had low serum IgA, and 154 (44%) had low serum IgM at diagnosis. There were no differences in individual gene mutation frequencies between individuals with low and normal serum IgG. In contrast, individuals with low serum IgA were more likely to have mutations in NOTCH1 (25% vs 10%) and ATM (14% vs 7%) compared to those with normal IgA, and individuals with low serum IgM were more likely to have mutations in NOTCH1 (19% vs 9%) and BIRC3 (11% vs 4%) compared to those with normal IgM (all p-values <0.05). There were no significant differences in individuals with low serum IgG based on TML scores of 0, 1, and 2+ (31%, 35%, 40%, respectively, p=0.28). However, individuals with low serum IgA and low serum IgM were more likely to have a higher TML (8%, 20%, 36% for IgA, p<0.0001; and 35%, 45%, 54% for IgM, p<0.0001, for TML scores of 0, 1, and 2+, respectively). After a median follow-up of 6.9 years, 157 individuals required CLL therapy. After adjusting for CLL-IPI and TML, low serum IgG, low serum IgA, and low serum IgM (hazard ratio [HR]: 1.8, 2.3, and 2.4, respectively, all p-values <0.05) were independently associated with a shorter TFT. Our study shows that individuals with CLL/MBL who harbor mutations in NOTCH1, ATM and BIRC3 are more likely to have low serum IgA and IgM at diagnosis. Serum IgA and IgM (but not serum IgG) levels decrease with an increase in the TML score. Finally, low serum immunoglobulins can predict TFT, independent of the CLL-IPI and TML, which has important implications for risk stratification in newly diagnosed CLL/MBL. Citation Format: Sameer A. Parikh, Cristine Allmer, Geffen Kleinstern, Nicholas J. Boddicker, Shulan Tian, Cecilia B. De Campos, Laura A. Bruins, Daniel R. o"brien, Brian F. Kabat, Kari G. Rabe, Aaron D. Norman, Huihuang Yan, Xing Li, Timothy G. Call, Wei Ding, Jose F. Leis, Saad J. Kenderian, Celine M. Vachon, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6466.

Abstract NG13: Metabolomic adaptations and correlates of survival to immune checkpoint blockade

Abstract NG13: Metabolomic adaptations and correlates of survival to immune checkpoint blockade

Abstract 1422: Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors

Abstract INTRODUCTION. miR-155-5p is a well-known oncogenic microRNA frequently overexpressed in human cancers, including breast cancer (BC), but its putative role as biomarker remains controversial. Thus, we aimed at evaluating miR-155-5p levels in a large BC cohort, and its association with clinical parameters. Then, since miR-155-5p was found to be epigenetically controlled by BRCA1, and to increase the tumor mutation load, we analysed miR-155-5p correlation with defects in Homologous Recombination (HR) genes (i.e. HRD), and its capability to alter the response to the first-in-class PARP (Poly-[ADP-ribose]-polymerase-1 (PARP-1)) inhibitor Olaparib (AZD2281). METHODS. We measured miR-155-5p expression in a retrospective cohort of breast cancer patients (CSS cohort, n=283; median follow-up=81 months) by RT-qPCR. Then, we used the TCGA Breast Cancer (TCGA-BC) dataset (n=1095) to validate our results, and evaluate the association between miR-155-5p and mutations in HR genes. Last, we ectopically modulated miR-155-5p in three wtBRCA1/2 cell lines (i.e. MDA-MB-231, MDA-MB-468, MDA-MB-453) and one mutBRCA1 cell line (MDA-MB-436) by using mirVana miR-155-5p mimic or inhibitor, and we measured cell viability before and after treatment with Olaparib or vehicle by PrestoBlue viability reagent. RESULTS. High miR-155-5p levels were associated with unfavourable prognostic indicators including high tumor grade (overall P=0.0007; G2 vs G3 P=0.0053), reduced expression of ER (r= -0.243; P=0.0002) and PgR (r= -0.240; P<0.0001), and high Ki-67 expression (r=0.215; P=0.0005). Consistently, miR-155-5p was differentially expressed across the BC subgroups identified by the surrogate molecular classification in the CSS-cohort (P=0.0008) and by the PAM50 panel in the TCGA-BC dataset (P=0.0001), with the Triple Negative and HER2-amplified tumors showing the highest levels. The analysis of the TCGA BRCA dataset also found higher miR-155 levels in the patient subgroup carrying somatic mutations in HR genes compared to the subgroup without somatic mutations in HR genes (P<0.0001). Last, the in vitro drug testing confirmed that mutBRCA1 MDA-MB-436 cells, exhibiting miR-155-5p endogenous levels 1000-fold higher than the other TNBC cell lines, had a higher sensitivity to a short-term (72hrs) treatment with Olaparib alone (IC50 25.80, 95%CI 24.52-27.15) compared to the other cell lines. Importantly, ectopic overexpression of miR-155-5p in wtBRCA1/2 cell lines, followed by Olaparib exposure, resulted in a significant reduction in viability (p<0.001) compared to Olaparib alone in all cell lines. However, miR-155-5p inhibition in BRCA1mut MDA-MB-436 cell line did not affect the higher sensitivity to PARP inhibition, suggesting that sensitivity to PARPi is not limited to HRD. CONCLUSIONS. Overall, our data point to a role of miR-155-5p in Homologous Recombination Deficiency (HRD), and suggest a putative novel mechanism of synthetic lethality mediated by PARP inhibition and miR-155-5p to promote cancer cell death. Citation Format: Barbara Pasculli, Raffaela Barbano, Andrea Fontana, Tommaso Biagini, Maria Pia Di Viesti, Michelina Rendina, Vanna Maria Valori, Maria Morritti, Sara Bravaccini, Sara Ravaioli, Evaristo Maiello, Paolo Graziano, Roberto Murgo, Massimiliano Copetti, Tommaso Mazza, Vito Michele Fazio, Manel Esteller, Paola Parrella. Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1422.

Abstract 1996: Tumor mutational burden is affected by next-generation sequencing enrichment method in highly deaminated samples from solid tumors

Abstract Background: Tumor mutational burden (TMB) is currently under investigation as a biomarker for predicting response to anti-cancer immunotherapy. TMB can be defined as the number of somatic nonsynonymous mutations per Mb in a cancer genome, therefore DNA artifacts could lead to inaccurate measurements. Cytosine deamination (C:G>T:A) is a well-known phenomenon in formalin-fixed, paraffin-embedded (FFPE) samples, producing errors in next-generation sequencing (NGS). Herein, we measured TMB in FFPE samples using two comprehensive NGS panels for routine diagnostics implementation. Methodology: TMB assessment was performed on FFPE samples by amplicon-based target enrichment on Thermo Fisher's Oncomine™ Tumor Mutation Load (OTML; n=9) and/or hybrid capture-based on Illumina's TruSight™ Oncology 500 Assay (TSO500; n=8), according to manufacturer's instructions. TMB values were compared for uracil-DNA glycosylase (UDG) treatment prior to PCR amplification and enrichment method. Samples were also compared according to their previous results in a clinically-certified NGS and an in-house microsatellite instability (MSI) assay. TMB >=10 mutations/Mb was considered high, and MSI >=20% was considered unstable. Results and Discussion: The concordance between TMB values for OTML and TSO500 was 75% (n=4) and 62.5% (n=8) with and without UDG treatment, respectively. UDG reduced artifactual C:G>T:A for both enrichment approaches, reinforcing its presence in FFPE DNA; however, there was no alterations on TMB results for both methods (TSO500 n=5; OTML n=7). C:G>T:A variants could be successfully filtered by low allelic frequency (<5%) or low quality (<q20) in non-treated samples. For OTML, a high baseline noise was observed in samples with high deamination scores (>60), leading to TMB overestimation (>350), which could not be corrected even after UDG treatment and manufacture's bioinformatic adjustment. Interestingly, the same samples presented low TMB values for TSO500. Samples with low deamination rates were not influenced by UDG treatment or bioinformatics adjustment regardless of enrichment method. TMB values from a certified NGS was completely concordant for TSO500 (n=4) but only 25% for OTML (n=4). MSI was fully concordant between certified NGS and TSO500 (n=4) and between in-house method and TSO500 (n=4) (value not analyzed by OTML assay). No QC parameter predicted differences between high and low deaminated samples. Conclusions: NGS enrichment methods strongly influenced TMB assessment. Hybrid-based capture showed better performance than amplicon-based. UDG treatment and higher allelic frequency cutoff could minimize false positives at variant calling level, although no significant effect could be observed for general TMB values. Future experiments should cover other alternatives to distinct FFPE DNA damages. Citation Format: Michele A. Pereira, Feliciana L. Marinho, Joice P. Silva, Renato Puga, Maíra C. Freire, Mariana R. Monteiro, Rafael L. Guedes, Luiz H. Araujo. Tumor mutational burden is affected by next-generation sequencing enrichment method in highly deaminated samples from solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1996.

Abstract A15: Uninflamed immunologic microenvironment of muscle-invasive bladder cancer associates with activating FGFR3 gene alterations

Abstract Background and Objective: Muscle-invasive bladder cancer (MIBC) is a complex disease for which perioperative chemotherapy followed by radical cystectomy is a standard treatment. In recent years new therapy options for advanced urothelial tumors have emerged, representing a personalized medicine approach for patients. For example, targeting fibroblast growth factor receptors (FGFR) as well as restoring antitumor activity using PD-1/PD-L1 inhibitors are currently being evaluated in clinical trials and show promising results. Our present goal in this study was to assess the relationship of FGFR3-gene alterations, immune cell types, gene expression of checkpoint inhibitors like PD-L1/PD-1, and immune-related genes in MIBC to gain insight into significant differential expression. Materials and Methods: We analyzed the TCGA cohort (n=407). Tumors were identified with activating FGFR3-gene alterations (FGFR3-altered: mutation, amplification, fusion, gain). A comparison was performed between tumors with FGFR3-altered or wild-type tumors and tumor mutational burden (TMB), neoantigen load, immune checkpoint, and immune-related gene expression as well as specific immune cell populations using the CIBERSORT-algorithm. Results: Activating FGFR3-altered tumors showed significantly less TMB and neoantigen load compared to wild-type cases (p=0.037 and p=0.04). Tumors with high expression of PD-1, PD-L1, CTLA4, and IDO1 were predominately not FGFR3-altered (p<0.0001). Additionally, expression of immune-related genes like CD3Z, CD8A, FOXP3 and amounts of specific immune cell populations were significantly inversely associated with an FGFR3-altered status. Conclusion: Strikingly, our analysis demonstrates that activating FGFR3 gene alterations in MIBC show low immune checkpoint and immune-related gene expression as well as low amounts of immune cells, demonstrating association with an uninflamed tumor microenvironment. Therefore, combination therapies targeting FGFR3 and, e.g., PD-1/PD-L1 might not be effective. It will be essential to validate these results in another MIBC cohort as well as unravel the role of aberrant signaling of FGFR3. Citation Format: Veronika Weyerer, Robert Stoehr, Pamela Strissel, Reiner Strick, Christian Bolenz, Arndt Hartmann, Philipp Erben, Markus Eckstein. Uninflamed immunologic microenvironment of muscle-invasive bladder cancer associates with activating FGFR3 gene alterations [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A15.

Abstract IA24: Predictive biomarkers of response to neoadjuvant therapy

Abstract The addition of cisplatin-based neoadjuvant chemotherapy prior to radical cystectomy provides a modest overall survival benefit for patients with muscle-invasive bladder cancer (MIBC). Moreover, the extent of pathologic response at cystectomy correlates with survival, and patients who achieve a complete pathologic response following neoadjuvant chemotherapy have significantly improved survival compared to patients with residual MIBC. Clinical features alone are unable to predict which patients will respond to neoadjuvant chemotherapy, and there is an urgent clinical need to identify genomic biomarkers to guide neoadjuvant therapy for MIBC. The Cancer Genome Atlas (TCGA) and related efforts have defined the genomic landscape of MIBC and have identified recurrent genomic alterations including mutations, chromosomal translocations, and copy number alterations. Additionally, gene expression profiling has defined molecular subtypes of MIBC that have distinct clinical and genomic features. These genomic data are being harnessed to understand the association among genomic features, response to neoadjuvant therapy, and clinical outcomes. Alterations in DNA damage and repair (DDR) genes have emerged as a biomarker of response to neoadjuvant cisplatin-based chemotherapy in several studies. Loss-of-function mutations in the nucleotide excision repair gene ERCC2 as well as in related DDR genes including ATM, FANCC, and RB1 have been associated with complete pathologic response to cisplatin-based chemotherapy and improved survival in MIBC patients receiving cisplatin-based chemotherapy. Functional studies have confirmed that many of the clinically identified DDR gene mutations confer loss of DNA repair capacity and drive cisplatin sensitivity in preclinical bladder systems. These data have informed the design of several ongoing biomarker-driven clinical trials that seek to identify a subset of patients with DDR-altered tumors who may be cured with cisplatin-based chemotherapy alone. In addition to driving response to DNA damaging-based chemotherapy, DDR gene alterations have also recently been associated with improved response to immune checkpoint inhibition in patients with metastatic urothelial cancer. The mechanism underlying this association remains incompletely understood; however, DDR pathway alterations can lead to increased tumor mutational burden and neoantigen load, which have each been associated with improved response to anti-PD1/PD-L1 agents. Immune checkpoint inhibitors have transformed the management of metastatic bladder cancer, and these agents are now being investigated in numerous clinical trials for patients with localized bladder cancer. However, only a subset of patients benefit from anti-PD1/PD-L1 therapy; therefore, identifying reliable predictive biomarkers of immunotherapy response in bladder cancer remains a critical need. Gene expression-based analyses have defined molecular subtypes that exhibit distinct clinical properties and therapeutic vulnerabilities. Tumors with basal-like gene expression properties appear to respond more robustly to neoadjuvant chemotherapy than luminal-like tumors. However, the specific genetic/epigenetic drivers of chemotherapy response among basal tumors are incompletely understood. Recently, gene expression subtypes were also found to correlate with response to immune checkpoint inhibition, including robust responses among a small but unique subset of patients with a neuronal tumor subtype. It is likely that applying similar genomic approaches to additional cohorts may provide further insights into the genomic drivers of therapeutic response and resistance to neoadjuvant therapies. Citation Format: Kent W. Mouw. Predictive biomarkers of response to neoadjuvant therapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA24.

Abstract PR04: Tumor immune microenvironment drives prognostic relevance correlating with bladder cancer subtypes

Abstract Background: Muscle-invasive bladder cancer (MIBC) represents approximately two thirds of invasive urothelial bladder cancers (UBC) and has high morbidity and mortality. Despite intensive efforts to improve patient treatment and outcome, two thirds of patients with UBC will have a recurrence or disease progression within 5 years. We conducted this study to gain further insights in the immunologic tumor microenvironment (TIME). Material and Methods: Stromal tumor-infiltrating lymphocytes (sTILs) were scored continuously on HE slides in a cohort of 135 patients with MIBC treated by radical cystectomy (adjuvant chemotherapy n= 34) according to current recommendations (Salgado et al., 2015). In parallel, we assessed intrinsic subtypes by 21-gene Nanostring signature adapted from the MDACC-subtyping approach. Tertiary lymph structures were assessed by whole slide immunohistochemistry of CD3, CD8, CD68, and CD79a. Spatial immune profiling was carried out on regionally (tumor center, invasive margin) designed TMAs by CD3, CD8, CD56 (NK-cells), CD68, PD-1, and PD-L1 and revealed spatial organized immune phenotypes. Results were validated in 407 MIBC of the TCGA cohort by hierarchical clustering analysis, immune cell population analysis via CIBERSORT, and sTIL-scoring on digitalized HE-slides. Furthermore, tumor mutational burden, neoantigen load, and mutational patterns as well as mutational signatures were correlated with immune phenotypes in the TCGA cohort. Results: We demonstrate that quantity and spatial distribution of sTILs within the tumor immune microenvironment (TIME) predict stages of tumor inflammation, subtypes, and patient survival and correlate with expression of immune checkpoints in an analysis of 542 MIBC. High sTILs indicate an inflamed subtype with 80% 5-year disease-specific survival. A lack of immune infiltrates identifies an uninflamed subtype with a survival rate of less than 25%. A separate immune-evading phenotype with upregulated immune checkpoints was associated with poor survival. Within the TIME are tertiary lymph node structures (TLS), which can mediate antitumor activity via active immune cells. High TLS amounts and close tumor distance correlated significantly with an inflamed phenotype and favorable survival. The uninflamed and evasion phenotypes showed lowest TLS numbers and farthest tumor distances and shortest survival. High inflammation also correlated with increased neoantigen load, high TMB, and specific mutational patterns (TCGA-MSig1, TCGA-MSig3/4). Patients treated with adjuvant chemotherapy showed a favorable prognosis dependent on high sTILs. Conclusion: Determination of sTILs and tumor subtypes may stratify therapy success and patient survival. Considering sTILs can easily be quantified using simple morphologic parameters such as hematoxylin-eosin, sTILs can be implemented for predicting patient survival and outcome after adjuvant platinum-containing chemotherapy in a routine manner. This abstract is also being presented as Poster A03. Citation Format: Markus Eckstein, Carolin Pfannstiel, Katherine B. Chiappinelli, Danijel Sikic, Sven Wach, Ralph M. Wirtz, Adrian Wullweber, Helge Taubert, Johannes Breyer, Wolfgang Otto, Thomas Worst, Maximilian Burger, Bernd Wullich, Christian Bolenz, Nicole Fuhrich, Carol Geppert, Veronika Weyerer, Robert Stoehr, Simone Bertz, Bastian Keck, Franziska Erlmeier, Philipp Erben, Arndt Hartmann, Pamela Strissel, Reiner Strick. Tumor immune microenvironment drives prognostic relevance correlating with bladder cancer subtypes [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR04.

Somatic mutations in genes associated with mismatch repair predict survival in patients with metastatic cancer receiving immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have emerged as one of the most promising therapeutic options for patients with advanced cancer. The aim of the present study was to investigate the prognostic value of somatic mutations in mismatch repair (MMR) genes in metastatic cancers after ICI treatment, as well as their association with tumor mutational burden (TMB). Information regarding gene mutations in mismatch repair and the survival time of patients with advanced cancer following ICI treatment was collected from the cBioPortal database. The prognostic value of somatic mutations in MMR genes and the association between the mutation status and TMB score were analyzed among multiple types of cancer. Somatic mutation frequency in the MMR genes was identified to be 7% among all patients, which varied across different types of cancer. Somatic mutations in the MMR genes were associated with improved overall survival time in all tested patients (P=0.004). Following stratification by type of ICI treatment, a significant association was observed between somatic mutations in the MMR genes and overall survival time in patients treated with cytotoxic T-lymphocyte-associated protein 4 inhibitors (P=0.01). In addition, marked but non-significant association between somatic mutations in the MMR genes and overall survival time was revealed in patients administered with programmed death-1/programmed death-ligand-1 inhibitors (P=0.09). Multivariate Cox proportional hazards regression analysis demonstrated that somatic mutations in MMR genes were significantly associated with overall survival time (hazard ratio, 0.683; 95% confidence interval, 0.497-0.938; P=0.01). Patients with somatic mutations in the MMR genes demonstrated higher TMB compared with those not harboring mutations (P<0.01). The results of the present study suggested that somatic mutations in the MMR genes may be used as a prognostic marker of a positive outcome in patients with metastatic cancer receiving ICI treatment, since somatic mutations in the MMR genes may be one of the main factors affecting the tumor mutation load.

Epigenetic Mechanisms of Resistance to Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICIs) have demonstrated to be highly efficient in treating solid tumors; however, many patients have limited benefits in terms of response and survival. This rapidly led to the investigation of combination therapies to enhance response rates. Moreover, predictive biomarkers were assessed to better select patients. Although PD-L1 expression remains the only validated marker in clinics, molecular profiling has brought valuable information, showing that the tumor mutation load and microsatellite instability (MSI) status were associated to higher response rates in nearly all cancer types. Moreover, in lung cancer, EGFR and MET mutations, oncogene fusions or STK11 inactivating mutations were associated with low response rates. Cancer progression towards invasive phenotypes that impede immune surveillance relies on complex regulatory networks and cell interactions within the tumor microenvironment. Epigenetic modifications, such as the alteration of histone patterns, chromatin structure, DNA methylation status at specific promoters and changes in microRNA levels, may alter the cell phenotype and reshape the tumor microenvironment, allowing cells to grow and escape from immune surveillance. The objective of this review is to make an update on the identified epigenetic changes that target immune surveillance and, ultimately, ICI responses, such as histone marks, DNA methylation and miR signatures. Translational studies or clinical trials, when available, and potential epigenetic biomarkers will be discussed as perspectives in the context of combination treatment strategies to enhance ICI responses in patients with solid tumors.