Potential Biomarker For Immunotherapy Research Articles

Characterization of zinc finger protein 536, a neuroendocrine regulator, using pan-cancer analysis

BackgroundPrevious studies suggested that zinc finger protein 536 (ZNF536) was abundant in the central brain and regulated neuronal differentiation. However, the role of ZNF536 in cancer has remained unclear.MethodsZNF536 mutation, copy number alteration, DNA methylation, and RNA expression were explored using public portals. Data from The Cancer Genome Atlas (TCGA) were utilized to analyze pathways and tumor microenvironment (TME), with a focus on prognosis in both TCGA and immunotherapy pan-cancer cohorts. Methylated ZNF536 from small cell lung cancer (SCLC) cell lines were utilized to train with probes for conducting enrichment analysis. Single-cell RNA profile demonstrated the sublocalization and co-expression of ZNF536, and validated its targets by qPCR.ResultsGenetic alterations in ZNF536 were found to be high-frequency and a single sample could harbor different variations. ZNF536 at chromosome 19q12 exerted a bypass effect on CCNE1, supported by CRISPR data. For lung cancer, ZNF536 mutation was associated with longer survival in primary lung adenocarcinoma (LUAD), but its prognosis was poor in metastatic LUAD and SCLC. Importantly, ZNF536 mutation and amplification had opposite prognoses in Stand Up To Cancer-Mark Foundation (SU2C-MARK) LUAD cohort. ZNF536 mutation altered the patterns of genomic alterations in tumors, and had distinct impacts on the signaling pathways and TME compared to ZNF536 amplification. Additionally, ZNF536 expression was predominantly in endocrine tumors and brain tissues. High-dimensional analysis supported this finding and further revealed regulators of ZNF536. Considering that the methylation of ZNF536 was involved in the synaptic pathway associated with neuroendocrine neoplasms, demonstrating both diagnostic and prognostic value. Moreover, we experimentally verified ZNF536 upregulated neuroendocrine markers.ConclusionsOur results showed that ZNF536 alterations in cancer, including variations in copy number, mutation, and methylation. We proved the involvement of ZNF536 in neuroendocrine regulation, and identified highly altered ZNF536 as a potential biomarker for immunotherapy.

Tumor beta2-microglobulin and HLA-A expression is increased by immunotherapy and can predict response to CIT in association with other biomarkers.

Downregulation of MHC class I expression and/or defects in the antigen presentation pathways are commonly reported in human cancers. Numerous studies previously have explored extensively the molecular mechanisms that underlie HLA-class I and Beta2-Microglobulin (B2M) downregulation. However, the techniques presently available to detect expression of MHC class I proteins lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. Furthermore, the dynamics of HLA-class I and B2M expression have not been comprehensively studied as a potential biomarker for immunotherapy. Using novel, validated, immunohistochemistry (IHC)-based methods for quantifying B2M and HLA-A in tumor samples from diverse cancer types, we have determined loss of B2M and HLA-A proteins in 336 archived, primary specimens and 329 biopsies from metastatic patients collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapy candidates as monotherapy or in combination with CPI. Up to 56% of cases with B2M or HLA-A loss were noted in the investigated tumor types. The frequency of loss was dependent on indication and stage of disease and revealed heterogeneous expression patterns across patients. B2M and HLA-A loss was increased in metastatic lesions compared to primary tumors, indicating selection of MHC class I low clones in metastatic and refractory tumor cells. High on-treatment B2M expression correlated with successful clinical outcome (RECIST), while high baseline B2M did not. A treatment-induced increase of B2M expression was noted in most of the patients with low B2M levels at baseline. The triple biomarker combination of B2M, CD8 and PDL1 strongly improved response prediction to cancer immunotherapy. Our results indicate that B2M and HLA-A loss occurs frequently in tumors and is reversed in most instances following immunotherapy which supports the conclusion that MHC class I loss is not the dominant resistance mechanism to CPI treatment. This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by indication, metastatic status, immunophenotype and immunotherapy treatment. Baseline expression levels of B2M on tumors may be of utility as a constituent of a biomarker panel used for selecting patients for immunotherapy clinical trials.

Prognostic implication of a novel lactate score correlating with immunotherapeutic responses in pan-cancer.

A thorough assessment of lactate-related genes (LRGs) in different types of human cancers is currently lacking. To elucidate the molecular landscape of LRGs, we conducted a comprehensive analysis using genomic, mRNA, and microRNA expression profiles and developed a lactate score model using the least absolute shrinkage and selection operator (LASSO) algorithm. We found that our lactate score could be a prognostic marker instead of LDHA for several cancer patients who possess high-frequency variants in LRGs. The lactate score also demonstrated an association with CD8+ T cells infiltration in multiple cancer types. Furthermore, our findings indicate that the lactate score holds promise as a potential biomarker for immunotherapy in patients with bladder cancer (BLCA) and skin cutaneous melanoma (SKCM). Among the seventeen genes of the lactate score model, PDP1 showed the strongest positive correlation with lactate score and the potential as a standalone biomarker for prognosis. In general, our study has yielded crucial insights into the potential application of the lactate score as a predictive biomarker for both survival outcomes and the response to immunotherapy. By recognizing the prognostic significance of lactate metabolism, we open avenues for further investigations aimed at harnessing the therapeutic potential of lactate.

Integrated multiomic data analysis reveals the clinical significance of TXNIP and contributing to immune microenvironment in triple negative breast cancer

Triple negative breast cancer (TNBC) is a type of breast cancer with the worst clinical outcome. TNBC is not sensitive to typical endocrine therapy and targeted therapy. Thioredoxin interacting protein (TXNIP), known as a tumor suppressor, is related to oxidative stress and energy metabolism. However, the clinical significance of TXNIP in TNBC and mechanism in immunity have not been fully reported. In this study, we found that the expression of TXNIP was downregulated obviously in TNBC tissues and negatively correlated with tumor grade by comprehensive bioinformatics analysis and immunohistochemistry staining of 108 TNBC tissues. Through in vivo and in vitro experiments, we identified TXNIP as a tumor suppressor in TNBC. By bulk mRNA and scRNA analysis, we found that TXNIP could enhance immune response in TNBC and was a potential biomarker for cancer immunity and immunotherapy. We also performed the drug susceptibility analysis to reveal the therapeutic value of TXNIP. In conclusion, our findings demonstrated that TXNIP was a tumor suppressor in TNBC and was involved in tumor malignancy progression. TXNIP was a potential biomarker for immunotherapy and promising molecular therapeutic target.

Exploration of NPC2 as a Potential Biomarker for Immunotherapy Using RNA-seq and Protein Data - A New Hypothesis.

NPC2 is well known as a player for cholesterol transport. However, the biological role of NPC2 in cancer development and therapy is far from clear. Here, we explore the potential role of NPC2 in prognosis and immunotherapy across multiple cancer types by integrating RNA-seq data from TCGA and GTEx, protein data from CPTAC, and multiple web analysis databases. Expression depiction between tumour and normal tissues indicated that NPC2 is overexpressed in the majority of the most common cancer types, including glioblastoma and pancreatic cancer, two cancers mostly difficult to diagnose and treat. Cancer stemness in glioblastoma is negatively associated with NPC2 level. NPC2 expression is positively correlated with immune cell infiltration and the expression of several immune checkpoints. IDH1 mutation in GBM is negatively correlated with NPC2 level, while a positive correlation has been found between TP53 mutation and NPC2 expression in pancreatic cancer. NPC2 is also correlated with levels of serum biomarkers used for diagnosis of pancreatic cancer.

Abstract P5-02-38: Leptin receptor (Ob-R)/leptin axis significantly modulates tumour-infiltrating lymphocytes (TILs) and PD-1 expression in early HER2+ breast cancer (BC) emerging as a new surrogate marker for immunotherapy

Abstract Leptin receptor (Ob-R)/leptin axis significantly modulates tumour-infiltrating lymphocytes (TILs) and PD-1 expression in early HER2+ breast cancer (BC) emerging as a new surrogate marker for immunotherapy. Background: There is strong pre-clinical evidence that obesity produces T-cell dysfunction and high PD-1 expression resulting in a paradoxical benefit from immunotherapy. This effect is driven, at least in part, by leptin that exerts its action through binding Ob-R, which is known to be highly expressed in HER2+ BC. TILs correlate with pathological response and long-term outcomes in BC; however the precise mechanism by which these T-cells are activated in and around tumour remains partially unknown. The primary aim of this study was to investigate the role of Ob-R/leptin axis in modulating TILs and PD-1 expression and its effect in pathological response in early HER2+ BC patients who have received neoadjuvant systemic treatment (NST). Methods: Women with HER2+ BC receiving anti-HER2-based NST followed by surgical resection were evaluated. Patient’s height and weight were measured before NST to calculate the body mass index (BMI). Based on the IHC results in diagnostic biopsy, tumors were categorized as HER2+/HR+ and HER2+/HR-. Ob-R expression was routinely measured in the diagnostic biopsy using the BOND RX Research Platform (Leica Biosystems). The Ob-R was classified as over-expressed if there were more than 50% positive cells with weak or strong staining. TILs and PD-1 expression were scored centrally in pre-treatment biopsy. TILs were considered as binary, < 30.0% versus ≥30.0% and PD-1 positive (>1%). Associations with pathological complete response (pCR; ypT0/isN0) were assessed using chi-squared or Wilcoxon test. Results: Of the 74 HER2+ BC patients included in the study, 47 (63.5%) had over-expression of Ob-R, 26 (35.1%) were overweight/obese (BMI ≥25kg/m2), and 42 (56.8%) had pCR status. Ob-R expression was similar regardless of menopausal status, age or HR expression. Patients with Ob-R overexpressed were 21 (80.8%) of 26 with BMI ≥25kg/m2 versus 26 (54.2%) of 48 with BMI < 25Kg/m2 (p=0.023). Tumors with Ob-R overexpressed had significantly higher mean levels of TILs than those with non-overexpressed Ob-R (21.4% [IQR, 7.5-30] vs 12.4% [IQR, 5-10]; p=0.009). Despite higher rates of TILs, the rate of pCR in Ob-R overexpressed tumours (57.4% [27 of 47 patients]) was not higher than in non-overexpressed tumours (55.6% [15 of 27 patients]; p=0.874). This could be due to the fact that Ob-R-overexpressed tumours had a significant higher median PD-1 expression than Ob-R-negative tumours (2% [IQR, 0.5-7.5] vs 0% [IQR, 0-1]; p< 0.001). Finally, no differences were found in terms of Ob-R expression and pathological response by hormone receptor expression. Conclusions: This multidisciplinary clinical study decodes for the first time how obesity, through the OB-R/leptin axis, might activate TILs but apparently dysfunctional as it is not translated into higher pCR; probably due to the presence of exhausted features such as high PD-1 expression. The role of Ob-R together with PD-1 as a potential biomarker for immunotherapy should be further explored. Citation Format: Laura García-Estévez, Adela Fernández, José Palacios, Miguel Sampayo, Isabel Calvo, Eva Díaz, Marta González, Belén Pérez-Míes, Silvia González, José Manuel Pérez-García, Javier Cortés, Gema Moreno-Bueno. Leptin receptor (Ob-R)/leptin axis significantly modulates tumour-infiltrating lymphocytes (TILs) and PD-1 expression in early HER2+ breast cancer (BC) emerging as a new surrogate marker for immunotherapy. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-38.

PIK3CA somatic mutations as potential biomarker for immunotherapy in elder or TP53 mutated gastric cancer patients.

Immune checkpoint inhibitors (ICIs) improve overall survival in patients with advanced gastric cancer (GC). However, the molecular characterization of GC in ICIs responders is unclear. A total of 288 advanced GC patients were included in this study. Next-generation sequencing analysis was performed on tumor tissue and paired blood to screen for somatic mutants in 639 tumor-associated genes. We demonstrated that ARID1A, HER2/3/4, KMT2C/2D, LRP1B, PIK3CA, SPTA1, and TP53 mutations were significantly correlated with high tumor mutation burden (TMB) score, as well as HER2 amplification. For HER2 and PIK3CA mutations types, this relationship was statistically significant with age and TP53 mutation status, which was also found in the CDH1 gene. These results were confirmed by sequencing 873 GC cases in the cBioPortal database. PIK3CA mutations appear to be associated with longer survival in elderly population and TP53 mutant subtypes. For the first time, we found that GC patients ≥60 years old or with TP53 mutated type and PIK3CA mutations were associated with higher TMB and better ICI response. Building upon the age and TP53 mutation status, this study suggested a novel stratification approach to GC patients and explored the correlations between genetic somatic mutations and TMB score.

99P Landscape of homologous recombination repair gene mutations in different molecular subtypes of NSCLC

Lung cancer is one of the malignant tumors with high morbidity and mortality in the world. PARP inhibitors have become a new line of cancer therapy The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer, and several cell line experiments have shown that HR-deficient lung cancers are sensitive to PARP inhibitor. Meanwhile, HHR gene is a potential biomarker for immunotherapy. Herein, we evaluated the characteristics of HRR related gene mutations in Chinese patients with lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC).

APC mutations as a predictive marker of endometrial cancer immunotherapy: a retrospective cohort study

The APC regulator of WNT signalling pathway gene (previously known as adenomatous polyposis coli gene; APC), located on 5q22.2, is a chromatin remodelling-related gene and a typical tumour suppressor. Patients with high expression of PD-L1 or a high tumour mutational burden have been reported to benefit from immunotherapy in endometrial cancer. The objective of this study was to show that APC is a new biomarker target for the diagnosis and treatment of endometrial cancer by analysing the correlation of APC mutations with PD-L1 expression or tumour mutational burden. We did an integrative analysis of a commercially available panel (Burning Rock Biotech, Guangzhou, China) including 520 cancer-related genes on 99 tumour samples from an endometrial cancer cohort in China and DNA-seq data from The Cancer Genome Atlas to identify new gene mutations as endometrial cancer immunotherapy markers. To discover the effect of gene mutations on endometrial cancer, we assessed the correlation between gene mutations and tumour immune microenvironment, and explored the immune microenvironment in endometrial cancer, including tumour mutational burden, PD-L1 expression, and lymphocytic infiltration. We found that the relevant mutated genes were related to the chromatin state and generated a discovery set including 12 mutated genes that significantly correlated with PD-L1 expression and tumour mutational burden. We pinpointed the APC gene, which was mutated in 18 (18%) of 99 tumour samples, as a new potential biomarker for immunotherapy. Further analysis showed that tumours with an APC mutation had a high tumour mutational burden, increased expression of PD-L1, and increased lymphocytic infiltration. By assessing the relationship between immunotherapy biomarkers and APC, we verified that APC has inactive mutations (eg, missense mutations, truncating mutations, and a mixture of missense and truncating mutations) in endometrial cancer, which might affect the immune response, including PD-L1 expression, microsatellite instability, and lymphocytic infiltrate. From The Cancer Genome Atlas data, we also found that patients with the APC mutation had longer overall survival. Our study shows that APC could have an important role in enhancing the response to endometrial cancer treatment, particularly immunotherapy. The study was supported by the National Natural Science Foundation of China (grant numbers 81472427, 32070583, 81672574, and 81702547), the Shanghai Health System Outstanding Talents Program (grant number 2018YQ23), the Shanghai New Frontier Technology Project (grant number SHDC12015110), and the Shanghai Municipal Medical and Health Discipline Construction Projects (grant number 2017ZZ02015).

Pan-Cancer Analysis of the Oncogenic and Immunological Role of RCN3: A Potential Biomarker for Prognosis and Immunotherapy.

Despite emerging publications have elucidated a functional association between RCN3 and tumors, no evidence about a pan-cancer analysis of RCN3 is available. Our study first conducted a comprehensive assessment of its expression profiles, prognosis value, immune infiltration, and relevant cellular pathways via bioinformatics techniques based on the public database of TCGA (The Cancer Genome Atlas). RCN3 is highly expressed in most tumors, and it is associated with poor prognosis. Kaplan-Meier analysis and Cox regression analysis suggested that the high expression of RCN3 was associated with poor overall survival (OS) in pan-cancer, Cox regression analysis also indicated high RCN3 expression was correlated with disease-specific survival (DSS) and progression-free interval (PFI) in most tumors. We observed a regulation function of RCN3 at genetic and epigenetic levels through CNA and DNA methylation using cBioPortal database. Based on Gene Set Enrichment Analysis, we first identified related pathways of RCN3 and its potential biological functions in pan-cancer, RCN3 was implicated in oncogenic pathways, and was related to extracellular matrix and immune regulation. We found that RCN3 positively correlated with the levels of infiltrating cells such as TAMs and CAFs, but negatively correlated with CD8+ T-cells by analyzing immune cell infiltration data we downloaded from published work and online databases, further investigation of the correlation between immunosuppressive genes, chemokines, chemokines receptors, and high RCN3 expression showed a significant positive association in the vast majority of TCGA cancer types. These results indicated its role as an immune regulatory in cancers and suggested that RCN3 is a potential biomarker for immunotherapy. Also, we found that expression of RCN3 was much higher in CRC tissues than in normal tissues with a higher expression level of RCN3 closely correlating to advanced American Joint Committee on Cancer (AJCC) stage, poor differentiation, increased tumor size, and poor prognosis of CRC. Biological function experiments showed that RCN3 regulated CRC cells’ proliferation and metastasis ability. Upregulation of RCN3 in CRC cells increased the expression of immune related factor, including TGFβ1, IL-10, and IL-6. Thus, our pan-cancer analysis offers a deep understanding of potential oncogenic roles of RCN3 in different cancers.

Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Pancreatic Cancer: Systematic Review and Still-Open Questions.

Simple SummaryTumor mutational burden (TMB) represents the number of mutations per megabase (muts/Mb) harbored by tumor cells in a given neoplasm, and can be determined with next-generation sequencing. High values are an indicator of potential response to immunotherapy. With this systematic review, we assessed its role in pancreatic ductal adenocarcinoma (PDAC). Our main findings can be summarized as: (i) high-TMB can be found in about 1% of PDAC; (ii) it is associated with mucinous/colloid and medullary histology; (iii) high-TMB PDAC frequently harbor other actionable alterations, with microsatellite instability as the most common; (iv) immunotherapy has shown promising results in high-TMB PDAC. Tumor mutational burden (TMB) is a numeric index that expresses the number of mutations per megabase (muts/Mb) harbored by tumor cells in a neoplasm. TMB can be determined using different approaches based on next-generation sequencing. In the case of high values, it indicates a potential response to immunotherapy. In this systematic review, we assessed the potential predictive role of high-TMB in pancreatic ductal adenocarcinoma (PDAC), as well as the histo-molecular features of high-TMB PDAC. High-TMB appeared as a rare but not-negligible molecular feature in PDAC, being present in about 1.1% of cases. This genetic condition was closely associated with mucinous/colloid and medullary histology (p < 0.01). PDAC with high-TMB frequently harbored other actionable alterations, with microsatellite instability/defective mismatch repair as the most common. Immunotherapy has shown promising results in high-TMB PDAC, but the sample size of high-TMB PDAC treated so far is quite small. This study highlights interesting peculiarities of PDAC harboring high-TMB and may represent a reliable starting point for the assessment of TMB in the clinical management of patients affected by pancreatic cancer.

Antiplexin D1 Antibodies Relate to Small Fiber Neuropathy and Induce Neuropathic Pain in Animals.

ObjectivesTo assess the prevalence of antiplexin D1 antibodies (plexin D1-immunoglobulin G [IgG]) in small fiber neuropathy (SFN) and the effects of these antibodies in vivo.MethodsWe developed an ELISA for plexin D1-IgG using a recombinant extracellular domain of human plexin D1 containing the major epitope and sera from 58 subjects previously studied with a standard tissue-based indirect immunofluorescence assay (TBA). We screened 63 patients with probable SFN and 55 healthy controls (HCs) for serum plexin D1-IgG using ELISA. The results were confirmed by TBA. IgG from 3 plexin D1-IgG-positive patients, 2 plexin D1-IgG-negative inflammatory disease controls, and 2 HCs was intrathecally injected into mice, which were assessed for mechanical and thermal hypersensitivity 24 and 48 hours after injection.ResultsThe ELISA had 75% sensitivity and 100% specificity using the TBA as a standard, and the coincidence rate of ELISA to TBA was 96.6% (56/58). The frequency of plexin D1-IgG was higher in patients with SFN than in HCs (12.7% [8/63] vs 0.0% [0/55], p = 0.007). Purified IgG from all 3 plexin D1-IgG-positive patients, but not 2 plexin D1-IgG-negative patients, induced significant mechanical and/or thermal hypersensitivity compared with IgG from HCs. In mice injected with plexin D1-IgG-positive but not D1-IgG-negative patient IgG, phosphorylated extracellular signal-regulated protein kinase immunoreactivity, an activation marker, was confined to small dorsal root ganglion neurons and was significantly more abundant than in mice injected with HC IgG.ConclusionsPlexin D1-IgG is pathogenic but with low prevalence and is a potential biomarker for immunotherapy in SFN.

P0548 - CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma

P0548 - CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma

CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma.

The introduction of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of metastatic renal cell carcinoma (mRCC) [1]. The expression of programmed death-ligand 1 (PD-L1) as assessed by immunohistochemistry (IHC) has been extensively studied as predictive biomarker for patients receiving ICIs. However, results in clinical RCC trials concerning the predictive value of PD-L1 expression were highly controversial [2]. In the Checkmate 214 trial [3], patients with PD-L1 positivity were found to benefit most from ICI combination. On the contrary, PD-L1 expression was not found to be predictive for nivolumab in the 2nd line setting [4]. Thus, all ICIs (combination and monotherapy) were approved in mRCC, irrespective of PD-L1 status.

Discovery of a potential biomarker for immunotherapy of melanoma: PLAC1 as an emerging target

Background Melanoma has increased in incidence worldwide prompting investigators to search for new biomarkers for targeted immunotherapy of this disease. Placenta specific 1 (PLAC1) is a new member of cancer-testis antigens with widespread expression in many types of cancer. Here, we aimed to study for the first time the expression pattern of PLAC1 in skin cancer samples including cutaneous melanoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC) in comparison to normal skin and nevus tissues and potential therapeutic effect of anti-PLAC1 antibody in melanoma cancer cell lines in vitro. Materials and methods Polyclonal and monoclonal antibodies were applied for immunohistochemical profiling of PLAC1 expression using tissue microarray. The cytotoxic action of anti-PLAC1 antibody alone or as an antibody drug conjugate (with anti-neoplastic agent SN38) was investigated in melanoma cell lines. Results We observed that 100% (39 of 39) of melanoma tissues highly expressed PLAC1 with both cytoplasmic and surface expression pattern. Investigation of PLAC1 expression in BCC (n = 110) samples showed negative results. Cancer cells in SCC samples (n = 66) showed very weak staining. Normal skin tissues and nevus samples including congenital melanocytic nevus failed to express PLAC1. Anti-PLAC1-SN38 exerted a specific pattern of cytotoxicity in a dose- and time-dependent manner in melanoma cells expressing surface PLAC1. Conclusions Our findings re-inforce the concept of re-expression of embryonic/placental tissue antigens in cancer and highlight the possibility of melanoma targeted therapy by employing anti-PLAC1 antibodies. The data presented here should lead to the future research on targeted immunotherapy of patients with melanoma.

1132P DNA damage repair mutation burden (DDRMB) was significantly associated with TMB and predicted prognosis of immunotherapy in melanoma

Tumor mutation burden (TMB) was emerging biomarker and was associated with prognosis of immunotherapy. Mutations in DNA damage repair (DDR) pathway hampers DNA damage repair and would lead to accumulation of mutations, which might increase TMB. Although there were sporadic reports about association between mutation in certain DDR genes (such as TP53, BRCA1/2, BER) and tumor prognosis, the association between DNA damage repair mutation burden (DDRMB), and TMB and prognosis of immunotherapy was not well evaluated yet. We retrospectively analyzed the whole exome sequencing (WES) results of 110 and 64 patients from 2 published prospective studies in melanoma and their prognosis of immunotherapy. 201 genes were defined as DDR genes. Correlation between DDRMB and TMB was analyzed. Difference in prognosis was conveyed by Kaplan-Meier analysis. Difference of TMB between groups were conveyed by T test. Strong correlation between DDRMB and TMB was revealed (R2=0.8798). TMB in patients with DDR mutations was significantly higher than others (P<0.001). Patients were divided into 4 subgroups with ≥ 3, 2, 1 and 0 DDR mutations. Pairwise comparison among each group indicated that groups with more DDR mutations contained significantly higher TMB than groups with less mutations (P<0.001). The overall survival (OS) in patients with DDR mutation was significantly higher than patients without DDR mutation (P=0.05). Besides, subgroups with ≥ 3 DDR mutations showed numerically longer OS than patient with less mutations. DDR mutation burden was highly correlated with TMB. Melanoma patients with higher DDRMB bears higher TMB, and showed worse OS in immunotherapy. DDRMB might be a potential biomarker for immunotherapy in melanoma. Larger trials are needed for further validation.

The mutation of homologous recombination repair genetics is a potential biomarker for immunotherapy in microsatellite stable colon cancer.

4076 Background: Patients with microsatellite instability-high (MSI-H) colonic adenocarcinoma (COAD) are always immunotherapy-sensitive for the use immune checkpoint inhibitors, however, the vast majority of COAD patients (85%) are microsatellite stable (MSS). Homologous recombination deficiency (HRD) is demonstrated to be a response predictor to immunotherapies in gynecologic cancers, while limited studies were reported in colon cancer. We focus herein on the mutational pattern of HRR related genes in a large Chinese COAD cohort and further analyze the relationship between HRR-gene mutations and clinical response to immunotherapy in MSS COAD. Methods: The genomic profiling of Genecast cohort which is consisted of 406 Chinese patients with COAD were analyzed in a panel of 543 cancer related genes via next generation sequencing (NGS). The correlation between HRR-gene mutations and tumor immunity or clinical outcome using two COAD genomics datasets (TCGA and MSK-COAD) by the bioinformatic approach. Results: In Genecast Cohort, seventy of 406 (17.2%) patients were identified genomic alterations in HRR-gene, the most frequently mutated genes were ATM (9%), BRCA2 (4%), ATR (3%), RAD50 (3%) and BRIP1 (3%). In MSK-COAD cohort (treated with immune checkpoint inhibitors), HRR-mut group (n = 34) had a significantly better OS than HRR-wt group (n = 50) (log-rank test, P = 0.0087). From the analysis of TCGA cohort, we found that mutations of HRR-gene could increase immune activity in MSS COAD, including increased cytotoxic cells infiltration ( P = 0.035), increased exhausted CD8 T cells infiltration ( P = 0.0098) and higher IFN-g score ( P = 0.03). In contrary, similar results were not found in MSI-H COAD (all P &gt; 0.05). Conclusions: Mutations of HRR-gene could significantly increase immune activity in patients with MSS COAD, implying the feasibility of using HRR-mut as a response predictor of immunotherapy in MSS-COAD.

The study of pleural effusion supernatant cell-free tumor DNA in tumor mutational burden assessment of advanced lung cancer

Objective: To evaluate the feasibility of cell-free tumor DNA in pleural effusion supernatant for assessing the tumor mutational burden (TMB) of advanced lung cancers. Methods: From December 2016 to August 2018, 34 lung cancer patients (19 males and 15 females) with pleural effusion were enrolled at Zhongshan Hospital, Fudan University. The median age of the patients was 65 (range, 34-85) years. Before systemic or local antitumor therapy, tumor specific mutations in tumor tissue, pleural effusion supernatant, pleural effusion sediment, and plasma samples from these patients were examined using targeted next-generation sequencing, and TMB levels were calculated respectively. Subgroup analysis was based on smoking history and driver mutation status. Statistical differences were determined using SPSS 16.0 software, and individual groups were compared using the one-way analysis of variance (ANOVA) and LSD-t test. Results: The median TMB level of pleural effusion supernatant was 6.23 mutations/Mb, similar to that of tumor tissue (6.23 vs 6.86 mutations/Mb, t=1.174, P=0.245), but significantly higher than that of pleural effusion sediment (2.49 mutations/Mb, t=3.044, P=0.003) and plasma (2.49 mutations/Mb, t=2.464, P=0.016). Compared with tumor tissue in TMB assessment, pleural effusion supernatant had a positive percentage agreement of 52% (9/17), and a negative percentage agreement of 65% (11/17). Subgroup analysis showed that the TMB level was higher in smokers (n=11) than that in non-smokers (n=23, 14.4 vs 5.4 mutations/Mb, t=3.238, P=0.003). Conclusion: For advanced lung cancer patients with pleural effusion, pleural effusion supernatant is a promising substitute to tumor tissue for TMB assessment, which is a potential biomarker for immunotherapy.

Abstract 4011: Notch 1/2/3 mutation as a potential biomarker for immunotherapy in smokers with non-small cell lung cancer via increasing tumor mutational burden

Abstract Background: Smoking status have been discovered to be associated with the response of anti-PD-1/PD-L1 treatment in advanced non-small cell lung cancer (NSCLC), on account of the smoking-induced higher tumor mutational burden (TMB). However, merely partial smokers with NSCLC exhibit high TMB and respond to anti-PD-1/PD-L1 therapy. The underlying signalling pathway that involved in the accumulation of mutational burden and response to immunotherapy in smokers with NSCLC has not been clarified. Methods: Data from Rizvi study (n = 240, nivolumab, tissue-sample NGS) and OAK study (n = 642, atezolizumab vs. docetaxel, blood-sample NGS) was retrieved and analysed as training cohorts, followed with validation in our independent NSCLC cohort (n = 44, anti-PD-1/PD-L1, blood-sample NGS). In survival analysis, Kaplan-Meier curves were compared by log-rank test, and the hazard ratio (HR) was determined through a multivariable Cox regression model. Results: In Rizvi cohort, mutation of Notch 1/2/3 was associated with higher TMB in smokers with NSCLC (P &amp;lt; 0.001) not in non-smokers with NSCLC (P = 0.908). Notch 1/2/3mut was associated with prolonged progression-free survival (PFS) compared with Notch 1/2/3wt in smokers with NSCLC (P = 0.015; HR, 0.51, 95% CI, 0.30-0.88), while not in non-smokers with NSCLC (P = 0.73; HR, 0.83, 95% CI, 0.30-2.35). Consistent results were observed in OAK study that Notch 1/2/3mut was associated with higher TMB (P &amp;lt; 0.001) and longer PFS exclusively in current smokers with NSCLC treated with immunotherapy (P = 0.018; HR, 0.28, 95% CI, 0.10-0.81). No association of Notch 1/2/3 status and PFS was observed in smokers with NSCLC treated with chemotherapy (P = 0.80; HR, 0.92, 95% CI, 0.47-1.78). We further validated these results in our independent cohort. Similar upregulation of TMB was observed in the Notch1/2/3-mutated smokers with NSCLC, and Notch1/2/3mut improved DCR (100% vs. 41.2%, P = 0.065) and significantly prolonged PFS (P = 0.046; adjusted HR, 0.12, 95% CI, 0.01-0.96). Conclusions: Notch1/2/3 mutation may serve as a novel predictor of response to anti-PD-1/PD-L1 treatment in smokers with NSCLC via upregulating TMB. Citation Format: Xiaohua Hong, Aijun Shen, Yu Xu, Chengcheng Li, Guoqiang Wang, Li Liu, Chuang Qi. Notch 1/2/3 mutation as a potential biomarker for immunotherapy in smokers with non-small cell lung cancer via increasing tumor mutational burden [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 4011.

Assessment of Blood Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Patients With Non–Small Cell Lung Cancer With Use of a Next-Generation Sequencing Cancer Gene Panel

Tumor mutational burden (TMB), as measured by whole-exome sequencing (WES) or a cancer gene panel (CGP), is associated with immunotherapy responses. However, whether TMB estimated by circulating tumor DNA in blood (bTMB) is associated with clinical outcomes of immunotherapy remains to be explored. To explore the optimal gene panel size and algorithm to design a CGP for TMB estimation, evaluate the panel reliability, and further validate the feasibility of bTMB as a clinical actionable biomarker for immunotherapy. In this cohort study, a CGP named NCC-GP150 was designed and virtually validated using The Cancer Genome Atlas database. The correlation between bTMB estimated by NCC-GP150 and tissue TMB (tTMB) measured by WES was evaluated in matched blood and tissue samples from 48 patients with advanced NSCLC. An independent cohort of 50 patients with advanced NSCLC was used to identify the utility of bTMB estimated by NCC-GP150 in distinguishing patients who would benefit from anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (anti-PD-L1) therapy. The study was performed from July 19, 2016, to April 20, 2018. Assessment of the Spearman correlation coefficient between bTMB estimated by NCC-GP150 and tTMB calculated by WES. Evaluation of the association of bTMB level with progression-free survival and response to anti-PD-1 and anti-PD-L1 therapy. This study used 2 independent cohorts of patients with NSCLC (cohort 1: 48 patients; mean [SD] age, 60 [13] years; 15 [31.2%] female; cohort 2: 50 patients; mean [SD] age, 58 [8] years; 15 [30.0%] female). A CGP, including 150 genes, demonstrated stable correlations with WES for TMB estimation (median r2 = 0.91; interquartile range, 0.89-0.92), especially when synonymous mutations were included (median r2 = 0.92; interquartile range, 0.91-0.93), whereas TMB estimated by the NCC-GP150 panel found higher correlations with TMB estimated by WES than most of the randomly sampled 150-gene panels. Blood TMB estimated by NCC-GP150 correlated well with the matched tTMB calculated by WES (Spearman correlation = 0.62). In the anti-PD-1 and anti-PD-L1 treatment cohort, a bTMB of 6 or higher was associated with superior progression-free survival (hazard ratio, 0.39; 95% CI, 0.18-0.84; log-rank P = .01) and objective response rates (bTMB ≥6: 39.3%; 95% CI, 23.9%-56.5%; bTMB <6: 9.1%; 95% CI, 1.6%-25.9%; P = .02). The findings suggest that established NCC-GP150 with an optimized gene panel size and algorithm is feasible for bTMB estimation, which may serve as a potential biomarker of clinical benefit in patients with NSCLC treated with anti-PD-1 and anti-PD-L1 agents.