Tumor Mutation Status Research Articles

Prognostic risk analysis related to radioresistance genes in colorectal cancer.

Radiotherapy (RT) is one of the most important treatments for patients with colorectal cancer (CRC). Radioresistance is the crucial cause of poor therapeutic outcomes in colorectal cancer. However, the underlying mechanism of radioresistance in colorectal cancer is still poorly defined. Herein we established a radioresistant colorectal cancer cell line and performed transcriptomics analyses to search for the underlying genes that contribute to radioresistance and investigate its association with the prognosis of CRC patients. The radioresistant cell line was developed from the parental HCT116 cell by a stepwise increased dose of irradiation. Differential gene analysis was performed using cellular transcriptome data to identify genes associated with radioresistance, from which extracellular matrix (ECM) and cell adhesion-related genes were screened. Survival data from a CRC cohort in the TCGA database were used for further model gene screening and validation. The correlation between the risk score model and tumor microenvironment, clinical phenotype, drug treatment sensitivity, and tumor mutation status were also investigated. A total of 493 different expression genes were identified from the radioresistant and wild-type cell line, of which 94 genes were associated with ECM and cell adhesion-related genes. The five model genes TNFRSF13C, CD36, ANGPTL4, LAMB3, and SERPINA1 were identified for CRC radioresistance via screening using the best model. A ROC curve indicated that the AUC of the resulting prognostic model (based on the 5-gene risk score and other clinical parameters, including age, sex, and tumor stages) was 0.79, 0.77, and 0.78 at 1, 2, and 3 years, respectively. The calibration curve showed high agreement between the risk score prediction and actual survival probability. The immune microenvironment, drug treatment sensitivity, and tumor mutation status significantly differed between the high- and low-risk groups. The risk score model built with five radioresistance genes in this study, including TNFRSF13C, CD36, ANGPTL4, LAMB3, and SERPINA1, showed favorable performance in prognosis prediction after radiotherapy for CRC.

Prospective analysis of circulating tumor dna in colorectal cancer liver metastasis: A first look

Background: Colorectal cancer liver metastasis (CCLM) develops in 50% of cases and drives patient outcomes. Liquid biopsy by circulating tumor DNA (ctDNA) is becoming an important diagnostic tool for patients with colorectal cancer. Our study aimed to investigate the feasibility of obtaining ctDNA screening in CCLM patients, its’ potential for evaluating tumor mutational status in these patients and the initial predictive value of this testing.

Self-expanding metal stent placement and pathological alterations among obstructive colorectal cancer cases.

Experimental studies suggest that self-expanding metal stents (SEMSs) enhance the aggressive behavior of obstructive colorectal cancer. The influence of SEMS placement on pathological alterations remains to be elucidated. To determine whether SEMS placement is associated with molecular or pathological features of colorectal carcinoma tissues. Using a nonbiased molecular pathological epidemiology database of patients with obstructive colorectal cancers, we examined the association of SEMS placement with molecular or pathological features, including tumor size, histological type, American Joint Committee on Cancer (AJCC)-pTNM stage, and mutation statuses in colorectal cancer tissues compared with the use of transanal tubes. A multivariable logistic regression model was used to adjust for potential confounders. SEMS placement was significantly associated with venous invasion (P < 0.01), but not with the other features examined, including tumor size, disease stage, mutation status, and lymphatic invasion. In both the univariable and multivariable models with adjustment for potential factors including tumor location, histological type, and AJCC-pT stage, SEMS placement was significantly associated with severe venous invasion (P < 0.01). For the outcome category of severe venous invasion, the multivariable odds ratio for SEMS placement relative to transanal tube placement was 19.4 (95% confidence interval: 5.24-96.2). No significant differences of disease-free survival and overall survival were observed between SEMS and transanal tube groups. SEMS placement might be associated with severe venous invasion in colorectal cancer tissue, providing an impetus for further investigations on the pathological alterations by SEMSs in colorectal cancer development.

A CTLA-4-gátló kezelés lehetőségei melanomában – új remények, új kihívások

The prevalence of melanoma is increasing worldwide, resulting in rising numbers of advanced cases demanding complex management. In the last decade, novel treatment options have become available that can achieve complete regression in patients with metastatic melanoma. Successful basic and clinical research partly focused on the development of inhibitors targeting identified mutant proteins that play a significant role in tumour progression. Nevertheless, the effects of these targeted therapies do not benefit patients without specific mutations. However, enhancing anti-tumour immune response by immune checkpoint inhibitors may be promising independently of tumour mutation status. In this paper, we describe CTLA-4-based treatment options, moreover we also report three cases representing possibilities of their practical application.

Associations of Tissue Tumor Mutational Burden and Mutational Status With Clinical Outcomes With Pembrolizumab Plus Chemotherapy Versus Chemotherapy For Metastatic NSCLC

IntroductionWe evaluated tissue tumor mutational burden (tTMB) and mutations in STK11, KEAP1, and KRAS as biomarkers for outcomes with pembrolizumab plus platinum-based chemotherapy (pembrolizumab-combination) for NSCLC among patients in the phase 3 KEYNOTE-189 (ClinicalTrials.gov, NCT02578680; nonsquamous) and KEYNOTE-407 (ClinicalTrials.gov, NCT02775435; squamous) trials. MethodsThis retrospective exploratory analysis evaluated prevalence of high tTMB and STK11, KEAP1, and KRAS mutations in patients enrolled in KEYNOTE-189 and KEYNOTE-407 and the relationship between these potential biomarkers and clinical outcomes. tTMB and STK11, KEAP1, and KRAS mutation status was assessed using whole-exome sequencing in patients with available tumor and matched normal DNA. The clinical utility of tTMB was assessed using a prespecified cutpoint of 175 mutations/exome. ResultsAmong patients with evaluable data from whole-exome sequencing for evaluation of tTMB (KEYNOTE-189, n = 293; KEYNOTE-407, n = 312) and matched normal DNA, no association was found between continuous tTMB score and overall survival (OS) or progression-free survival for pembrolizumab-combination (Wald test, one-sided p > 0.05) or placebo-combination (Wald test, two-sided p > 0.05) in patients with squamous or nonsquamous histology. Pembrolizumab-combination improved outcomes for patients with tTMB greater than or equal to 175 compared with tTMB less than 175 mutations/exome in KEYNOTE-189 (OS, hazard ratio = 0.64 [95% confidence interval (CI): 0.38‒1.07] and 0.64 [95% CI: 0.42‒0.97], respectively) and KEYNOTE-407 (OS, hazard ratio = 0.74 [95% CI: 0.50‒1.08 and 0.86 [95% CI: 0.57‒1.28], respectively) versus placebo-combination. Treatment outcomes were similar regardless of KEAP1, STK11, or KRAS mutation status. ConclusionsThese findings support pembrolizumab-combination as first-line treatment in patients with metastatic NSCLC and do not suggest the utility of tTMB, STK11, KEAP1, or KRAS mutation status as a biomarker for this regimen.

Neoadjuvant checkpoint inhibitor immunotherapy for resectable mucosal melanoma.

Neoadjuvant checkpoint inhibition (CPI) has recently demonstrated impressive outcomes in patients with stage 3 cutaneous melanoma. However, the safety, efficacy, and outcome of neoadjuvant CPI in patients with mucosal melanoma (MM) are not well studied as MM is a rare melanoma subtype. CPI such as combination nivolumab and ipilimumab achieves response rates of 37-43% in unresectable or metastatic MM but there is limited data regarding the efficacy of these agents in the preoperative setting. We hypothesize that neoadjuvant CPI is a safe and feasible approach for patients with resectable MM. Under an institutionally approved protocol, we identified adult MM patients with resectable disease who received neoadjuvant anti-PD1 +/- anti-CTLA4 between 2015 to 2019 at our institution. Clinical information include age, gender, presence of nodal involvement or satellitosis, functional status, pre-treatment LDH, tumor mutation status, and treatment data was collected. Outcomes include event free survival (EFS), overall survival (OS), objective response rate (ORR), pathologic response rate (PRR), and grade ≥3 toxicities. We identified 36 patients. Median age was 62; 58% were female. Seventy-eight percent of patients received anti-PD1 + anti-CTLA4. Node positive disease or satellite lesions was present at the time of treatment initiation in 47% of patients. Primary sites of disease were anorectal (53%), urogenital (25%), head and neck (17%), and esophageal (6%). A minority of patients did not undergo surgery due to complete response (n=3, 8%) and disease progression (n=6, 17%), respectively. With a median follow up of 37.9 months, the median EFS was 9.2 months with 3-year EFS rate of 29%. Median OS had not been reached and 3-year OS rate was 55%. ORR was 47% and PRR was 35%. EFS was significantly higher for patients with objective response and for patients with pathologic response. OS was significantly higher for patients with pathologic response. Grade 3 toxicities were reported in 39% of patients. Neoadjuvant CPI for resectable MM is a feasible approach with signs of efficacy and an acceptable safety profile. As there is currently no standard approach for resectable MM, this study supports further investigations using neoadjuvant therapy for these patients.

95P Organoid sensitivity for panitumumab based on primary tumor location and mutational status

Drugs targeting the epidermal growth factor receptor (EGFR), such as panitumumab and cetuximab, are standard of care for metastatic colorectal cancer (mCRC) patients with RAS/BRAF-wildtype tumors. Nevertheless, a fraction of these patients does not respond to them. There is a clinical need for an effective biomarker to further select patients for EGFR inhibition. A promising predictive biomarker is in vitro response testing using patient-derived organoids (PDOs).

Focus on pattern recognition receptors to identify prognosis and immune microenvironment in colon cancer.

In 2011, J. Hoffman, and B. Beutler won the Nobel Prize of medicine for the fact that they discovered the pattern recognition receptors (PRRs) and meanwhile described their effect on cell activation from the innate and adaptive immune systems. There are more and more evidences that have proved the obvious effect of PRRs on tumorigenesis progression. Nevertheless, the overall impact of PRR genes on prognosis, tumor microenvironmental characteristics and treatment response in patients with colon adenocarcinoma (COAD) remains unclear. In this research, we systematically assessed 20 PRR genes and comprehensively identified the prognostic value and enrichment degree of PRRs. The unsupervised clustering approach was employed for dividing COAD into 4 PRR subtypes, namely cluster A, cluster B, cluster C and cluster D, which were significantly different in terms of the clinical features, the immune infiltrations, and the functions. Among them, cluster B has better immune activities and functions. Cox and LASSO regression analysis was further applied to identify a prognostic five-PRR-based risk signature. Such signature can well predict patients’ overall survival (OS), together with a good robustness. Confounding parameters were controlled, with results indicating the ability of risk score to independently predict COAD patients’ OS. Besides, a nomogram with a strong reliability was created for enhancing the viability exhibited by the risk score in clinical practice. Also, patients who were classified based on the risk score owned distinguishable immune status and tumor mutation status, response to immunotherapy, as well as sensitivity to chemotherapy. A low risk score, featuring increased tumor stemness index (TSI), human leukocyte antigen (HLA), immune checkpoints, and immune activation, demonstrated a superior immunotherapeutic response. According to the study results, the prognostic PRR-based risk signature could serve as a robust biomarker for predicting the clinical outcomes as well as evaluating therapeutic response for COAD patients.

Identifying the EMT-related signature to stratify prognosis and evaluate the tumor microenvironment in lung adenocarcinoma.

Background: Epithelial-mesenchymal transition (EMT) is a critical process in tumor invasion and metastasis. EMT has been shown to significantly influence the invasion, metastasis, and poor prognosis in lung adenocarcinoma (LUAD). This study aimed to develop a novel EMT-related prognostic model capable of predicting overall survival (OS) in patients with LUAD. Methods: A total of 283 LUAD patients from TCGA RNA-seq dataset were assigned to a training cohort for model building, and 310 LUAD patients from GEO RNA-seq dataset were assigned to a validation cohort. EMT genes were acquired from MsigDB database and then prognosis-related EMT genes were identified by univariate Cox regression. Lasso regression was then performed to determine the genes and the corresponding variables to construct a prognosis risk model from the training cohort. Furthermore, characteristics of the tumor microenvironment (TME), mutation status and chemotherapy responses were analyzed to assess the differences between the two risk groups based on the prognostic model. In addition, RT-qPCR was employed to validate the expression patterns of the 6 genes derived from the risk model. Results: A six-gene EMT signature (PMEPA1, LOXL2, PLOD2, MMP14, SPOCK1 and DCN) was successfully constructed and validated. The signature assigned the LUAD patients into high-risk and low-risk groups. In comparison with the low-risk group, patients in the high-risk group had a significantly lower survival rate. ROC curves and calibration curves for the risk model demonstrated reliable stratification and predictive ability. The risk model was robustly correlated with multiple TME characteristics. Besides, the data showed that patients in the low-risk group had more immune activities, higher stemness scores and cytolytic activity scores and higher TMB. In addition, RT-qPCR results revealed that PMEPA1, LOXL2, PLOD2, MMP14, and SPOCK1 were notably upregulated in LUAD tissues, while DCN was downregulated. Conclusion: Our study successfully developed a novel EMT-related signature to predict prognosis of LUAD patients and guide treatment strategies. The six genes derived from the prediction signature might play a potential role in antitumor immunity and serve as promising therapeutic targets in LUAD.

Integrated single-cell RNA-seq analysis identifies immune heterogeneity associated with KRAS/TP53 mutation status and tumor-sideness in colorectal cancers.

BackgroundThe main objective of this study was to analyze the effects of KRAS/TP53 mutation status and tumor sideness on the immune microenvironment of colorectal cancer using integrated scRNA-seq data.MethodsA total of 78 scRNA-seq datasets, comprising 42 treatment-naive colorectal tumors, 13 tumor adjacent tissues and 23 normal mucosa tissues were included. Standardized Seurat procedures were applied to identify cellular components with canonical cell marks. The batch-effect was assessed and corrected using harmony algorithm. The scMetabolism algorithm was used for single-cell metabolic analysis. The results and clinical significance were further validated using immunofluorescent-staining and TCGA-COAD datasets. Immune-infiltration scores of bulk-RNA-seq data were estimated using ssGSEA. The presto-wilcoxauc algorithm was used to identify differentially enriched genes or pathways across different subgroups. Two-sided p-value less than 0.05 was considered statistically significant.ResultsWe refined the landscape of functional immune cell subtypes, especially T cells and myeloid cells, across normal mucosa, tumor adjacent and tumor tissue. The existence and function of two states of exhausted CD8+ T (Tex) subtypes in colorectal cancer, and FOLR2+ LYVE1+ macrophages indicating unfavorable prognosis in colorectal cancer were identified and validated. The diverse tumor mutation status reshaped the immune cell function and immune checkpoint ligands/receptors (ICLs/ICRs) expression pattern. Importantly, the KRAS/TP53 dual mutations significantly reduced the major energy metabolic functions in immune cells, and promoted the cell-to-cell communications towards immunosuppression in colorectal cancers. The results revealed LAG3, CD24-SIGLEC10 and HBEGF-CD9 pathways as potential therapeutic targets for dual mutant colorectal cancers.ConclusionsWe revealed that the immune microenvironment underwent a gradual remodeling with an enrichment of immunosuppressive myeloid cells from normal mucosa to tumor regions in colorectal cancers. Moreover, we revealed the metabolic heterogeneity of tumor-infiltrating immune cells and suggested that the KRAS/TP53 dual mutation may impair antitumor immunity by reducing T and myeloid cell energy metabolism and reshaping cellular interactions toward immunosuppression.

P14.01.B Isolation and characterization of circulating tumor cells in a glioblastoma case with recurrence at distance and correlation with tumor mutational status

Abstract Background Circulating Tumor Cells (CTCs) are considered to be one of the important causes of tumor recurrence and distant metastasis. For many years, glioblastoma (GB) was thought to be restricted to the brain. Nevertheless, a growing body of evidence indicates that, like many other cancers, hematogenic dissemination is a reality. The absence of a procedural uniformity in literature prompted us to develop an innovative and sensitive method to obtain CTCs in GB. Our aim is to define the genetic background of single CTCs compared with the primary GB tumor and its recurrence to assess whether or not their presence in the peripheral circulation correlates with GB migration and dissemination. Material and Methods CTCs were enriched from whole blood of one patient with recurrent GB with Parsortix Cell Separation System and analysed on DEPArray system. After that, CTCs Copy Number Aberrations (CNAs) and sequencing analysis was performed to compare CTCs genetic background with the same patient’s primary and recurrence tissues, analysed by NextSeq 500 (whole exome sequencing). Results We obtained 211 mutations in common between primary and recurrence tumor. Among these, three somatic mutations (c.430 G&amp;gt;A in PRKCB gene, c.815 C&amp;gt;T in TBX1 gene and c.1554 T&amp;gt;G in COG5 gene) were selected to investigate their presence in recurrence CTCs. Almost all of the sorted CTCs (9/13) had at least one of the mutations tested. Conclusion In confirmation of the hypothesis, the CTCs detected in the patient's blood were actually cancer cells deriving from GB tumor.

OS08.4.A Analysis of melanoma brain metastasis immune microenvironment through multiplex gene expression profiling

Abstract Background Novel immunotherapies based on targeting of specific immune checkpoints enabled a significant improvement of melanoma outcome, but melanoma brain metastases (BM) remain an unmet oncological need with an overall 2-year survival rate lower than 10%. Tumour immune microenvironment has been demonstrated to play a key role in BM establishment and development, but data regarding the specific milieu of melanoma BM is limited. Material and Methods Gene expression profiles of 55 samples of primary melanoma and BM were evaluated using the nCounter PanCancer IO 360 Panel (NanoString Technologies) targeting 770 mRNA involved in tumor immune microenvironment modulation. The case series consisted of 10 primary melanomas and their 10 matched BM, 25 unmatched BM, and 10 locally advanced control melanomas without evidence of BM after &amp;gt;5 year follow up. Results Among BM samples, most patients (25/45) were males and median age at BM diagnosis was 61,2 years with a median time to BM development of 2,1 years. Median OS from BM diagnosis was 1,3 years. Several genes resulted significantly downregulated in BM compared to primary melanomas, including SERPINB5 (p&amp;lt;0.001), ARG1 (p=0.0067), S100A8 (p&amp;lt;0.001), S100A9 (p&amp;lt;0.001), S100A12 (p=0.0037), IL1RN (p=0.0012), CCL21 (p=0.0012), CCL22 (p=0.0012) and CCL13 (p=0.037) and SELE (p=0.026); conversely, C7 was upregulated (p&amp;lt;0.001). Downregulated signatures in BM involved those associated with multiple immune cell populations, including neutrophils, dendritic cells, mast cells and Treg, as well as inflammatory chemokines, the CTLA4 immune checkpoint and ARG1 enzyme function; conversely, MAGEs-related signature was upregulated. Comparison between primary melanomas which developed BM and those which did not showed a significant overexpression of RRM2 (p=0.0247) and TNFRSF1A (p=0.032) genes in the latter group and an upregulation of the PD-1 pathway. Analysis according to tumour mutational status showed an upregulation of signatures associated with inflammatory chemokines, dendritic and myeloid cells and neutrophils. No differences were observed according to time to BM development and survival from BM diagnosis. Conclusion Our findings show that melanoma BM harbor distinct immunosuppressive mechanisms compared to primary tumors: this data elicits the importance of investigating the heterogeneity of BM microenvironment. Genes and pathways selectively overexpressed or downregulated in melanoma BM should be validated to be possibly considered as novel therapeutic targets.

801P Neoadjuvant checkpoint inhibitor immunotherapy (IMT) for resectable mucosal melanoma (MM)

Neoadjuvant checkpoint inhibition (CPI) has recently demonstrated impressive outcomes in patients (pts) with stage 3 cutaneous melanoma. However, the safety, efficacy, and outcome of neoadjuvant CPI in pts with mucosal melanoma (MM) are not well studied as MM is a rare melanoma subtype. CPI such as combination nivolumab and ipilimumab achieves response rates of 37-43% in unresectable or metastatic MM but there is limited data regarding the efficacy of these agents in the preoperative setting. We hypothesize that neoadjuvant CPI is a safe and feasible approach for pts with resectable MM. Under an institutionally approved research protocol, we identified adult MM pts with resectable disease who received neoadjuvant anti-PD1 +/- anti-CTLA4 between 2015 to 2019 at our institution. Clinical information include age, gender, presence of nodal involvement or satellitosis, functional status, pre-treatment LDH, tumor mutation status, and treatment data was collected. Outcomes include event free survival (EFS), overall survival (OS), objective response rate (ORR), pathologic response rate (PRR), and grade ≥3 toxicities were assessed. We identified 36 pts. Median age was 62; 58% were female. 78% of pts got anti-PD1 + anti-CTLA4. Node-positive disease or satellite lesions was present in 47% of pts. Primary sites of disease were anorectal (53%), urogenital (25%), head and neck (17%), and esophageal (6%). A minority of pts did not undergo surgery due to complete response (n=3, 8%) and disease progression (n=6, 17%), respectively. With a median follow up of 37.9 months, the median EFS was 9.2mo with 3-year EFS rate of 29%. Median OS had not been reached and 3-year OS rate was 55%. ORR was 47% and PRR was 35%. EFS was significantly higher for pts with objective response and for pts with pathologic response. OS was significantly higher for pts with pathologic response. Grade 3 toxicities were reported in 39% of pts. Neoadjuvant CPI for resectable MM is a feasible approach with signs of efficacy and an acceptable safety profile. As there is currently no standard approach for resectable MM, this study supports further investigations using neoadjuvant therapy for these pts.

1166P Social determinants of lung cancer in East London

In England more deprived groups are more likely to acquire lung cancer and have a higher associated mortality despite adjusting for disease stage at diagnosis and comorbidities [1]. More deprived populations are more frequently exposed to risk factors for disease development such as smoking and air pollution [2]. St Bartholomew’s Hospital serves a diverse population from East London. We aim to understand if there are associations between clinico-pathological features of lung cancer and levels of deprivation in our region. Electronic patient records were reviewed for patients diagnosed with lung adenocarcinoma at our centre over 1 year between January and December 2021. Deprivation deciles were obtained via the English Indices for Deprivation 2019 [3]. 170 patients were identified, of which 75.3% (n=128) had an oncogenic driver mutation. Median age was 69 years with 45.3% females (n=77). The most frequently identified mutation was KRAS (n=62, 36%), followed by EGFR (n=18, 10.6%). 6.5% of patients (n=11) had 2 or more mutations. EGFR was associated with higher deprivation in the living environment subdomain (p=0.046), measuring exposure to poor quality air and housing, road traffic accidents and access to central heating. No other significant relationships between deprivation score and tumour mutational status were identified. Lung cancer patients at our centre are distributed along a deprivation gradient, comparable to the local population makeup. Considering EGFR’s association with never-smoking populations, these findings are consistent with the hypothesis that these populations are also exposed to important oncogenic factors beyond smoking tobacco [2] and that a drive to reduce the burden of cancer must not be limited to smoking cessation alone.

Necroptosis-Related Prognostic Signature and Nomogram Model for Predicting the Overall Survival of Patients with Lung Cancer.

Background Necroptosis is a type of programmed cell death mode and it serves an important role in the tumorigenesis and tumor metastasis. The purpose of this study is to develop a prognostic model based on necroptosis-related genes and nomogram for predicting the overall survival of patients with lung cancer. Method Differentially expressed necroptosis-related genes (NRDs) between lung cancer and normal samples were identified. Univariate and LASSO regression analyses were performed to establish a risk score (RS) model, followed by validation within TCGA and GSE37745. The correlation between RS model and tumor microenvironment, mutation status, or drug susceptibility was analyzed. By combining clinical factors, nomogram was developed to predict 1-, 3-, and 5-year survival probability of an individual. The biological function involved by different risk groups was conducted by GSEA. Results A RS model containing six NRDs (FLNC, PLK1, ID1, MYO1C, SERTAD1, and LEF1) was constructed, and patients were divieded into low-risk (LR) and high-risk (HR) groups. Patients in HR group were associated with shorter survival time than those in the LR group; this model had better prognostic performance. Nomogram based on necroptosis score, T stage, and stage had been confirmed to predict survival of patients. The number of resting NK cells and M0 macrophages was higher in HR group. In addition, higher tumor mutational burden and drug sensitivity were observed in the HR group. Patients in HR group were involved in p53 signaling pathway and cell cycle. Conclusion This study constructed a robust six-NRDs signature and established a prognostic nomogram for survival prediction of lung cancer.

Prognostic attributes of immune signatures in soft tissue sarcomas show differential dependencies on tumor mutational burden.

BackgroundCellular and intrinsic markers of sarcoma immunogenicity are poorly understood. To gain insight into whether tumor–immune interactions correlate with clinical aggressiveness, the authors examined the prognostic significance of immune gene signatures in combination with tumor mutational burden (TMB) and cancer–testis antigen (CTA) expression.MethodsRNA sequencing and clinical data of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between published immune gene signatures and patient overall survival (OS) in the contexts of TMB, as computed from whole‐exome sequencing data, and CTA gene expression. Multivariate Cox proportional hazards regression models and log‐rank tests were used to assess survival associations.ResultsImmune signature scores that reflected in part the intratumoral abundance of cytotoxic T cells showed significant positive associations with OS. However, the prognostic power of the T‐cell signatures was highly dependent on TMB‐high status, consistent with protective effects of tumor‐infiltrating T cells in tumors with elevated antigenicity. In TMB‐low tumors, a signature of infiltrating plasma B cells was significantly and positively associated with OS, independent of T‐cell signature status. Although tumor subtypes based on differential expression patterns of CTA genes showed different survival associations within leiomyosarcoma and myxofibrosarcoma histologies, neither CTA nor histologic subtype interacted with the T‐cell–survival association.ConclusionsSignatures of T‐cell and plasma B‐cell infiltrates were associated with a survival benefit in soft tissue sarcomas. TMB, but not CTA expression, influenced the prognostic power of T‐cell–associated, but not plasma B‐cell–associated, survival.Lay summary Clinical data and RNA analysis of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between five published gene immune cell expression signatures and survival in the context of tumor mutations.Activated T cells had a significant positive association with patient survival.Although high tumor mutation burden was associated with good survival, the prognostic power of T‐cell signatures was highly dependent on tumor mutational status, consistent with protective effects of tumor‐infiltrating T cells in tumors with high levels of antigens.In low tumor mutation‐bearing tumors, plasma B cells were positively associated with survival.

Abstract 2409: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR mutated cancers

Abstract Cancer metastasis process involves dysregulated oncogenic kinase signaling, but the precise mechanism by which this orchestrates metabolic networks and signal cascades for metastasis is largely unclear. Here we report that pharmacological inhibition of a glutaminolytic enzyme glutamate dehydrogenase 1 (GDH1) and ribosomal S6 kinase 2 (RSK2) synergistically attenuates cell invasion, anoikis resistance, and immune escape more evidently in tumors harboring EGFR activating or EGFR inhibitor-resistant mutations. Mechanistically, GDH1 is activated by EGFR through phosphorylation at tyrosine 135, and together with RSK2 enhances transcriptional activity of CREB via CaMKIV signaling fostering metastasis. Moreover, GDH1, RSK2, and CREB phosphorylation positively correlate with EGFR mutation status and metastatic cancer progression in primary patient tumors. Our finding reveals a crosstalk between kinase, metabolic, and transcription machinery in metastasis and offers an alternative combinatorial therapeutic strategy to target EGFR therapy-resistant metastatic cancers. Citation Format: JiHoon Kang, Jung Seok Hwang, Austin Boese, Courteney Malin, Sumin Kang. EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR mutated cancers [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 2409.

Abstract 3400: Predictive ability of circulating tumor DNA by Guardant360 in poziotinib-treated patients with NSCLC harboring HER2 exon 20 insertion mutations

Abstract Introduction: Detection of ctDNA in plasma samples permits temporal assessment of tumor mutation status during treatment. Poziotinib is an oral pan-HER TKI that has been demonstrated to be efficacious in NSCLC patients harboring HER2 exon 20 insertion mutations. We assessed serial plasma samples for changes in tumor genotype in both treatment naive and second line patients to evaluate correlation with clinical response. Methods: For NSCLC patients, HER2 exon 20 insertion mutations identified by tumor tissue based NGS were required for entry into the ZENITH20 study. Plasma samples were collected prior to treatment and at C3D1 (8 weeks post treatment 16mg poziotinib QD). The Guardant360® 74-gene liquid biopsy assay, which assessed changes in ctDNA and, subsequently, mean variant allele fraction (mVAF), was utilized for analysis of samples. The Guardant360 Response™ Molecular Response (MR) algorithm was calculated as a ratio of mVAF of oncogenic alterations at baseline compared to poziotinib treatment at C3D1. Results: 23 patients with tumor tissue confirmed NSCLC harboring HER2 exon 20 insertion mutations were studied. 22 of 23 (96%) had baseline plasma samples with detectable ctDNA. 21 of 22 samples had detectable HER2 exon 20 insertion mutations, resulting in a concordance of 95% versus tissue based NGS. The most prevalent HER2 exon 20 insertion alteration was the A775_G776ins YVMA variant, found in 50% of the baseline blood and tumor samples using both methods (100% concordance). 16 of 17 patients had both baseline and C3D1 samples, permitting assessment of temporal response. 15 of the 16 (94%) patients demonstrated a decrease in mVAF at C3D1 compared to the mVAF at baseline. 12 of 15 patients demonstrated an &amp;gt;50% reduction in mVAF at C3D1, with 7 of the 12 patients showing &amp;gt;95% reduction in mVAF at C3D1 with clinical outcomes of 5 PRs, 1 non-CR/non-PD and 1 SD. Interestingly, 3 of these patients showed complete clearance of ctDNA target HER2 exon 20 insertions at the C3D1 timepoint. Conclusions: Baseline plasma ctDNA genotyping correlated with tumor tissue based NGS in an NSCLC patient population with HER2 mutations. Poziotinib treatment resulted in mVAF reduction, which correlated with clinical response per RECIST1.1. Assessment of longitudinal changes in ctDNA during drug therapy may potentially be used to predict patient response and possibly tumor resistance. Further evaluation in larger cohorts and longer duration of treatment is required to help elucidate the impact of these findings. Citation Format: Arunthi Thiagalingam, Sribalaji Lakshmikanthan, Allysia J. Mak, Scott A. Shell, Sharon Leu, Rocky Washington, Lyndah Dreiling, Gajanan Bhat, Francois Lebel, John A. Barrett. Predictive ability of circulating tumor DNA by Guardant360 in poziotinib-treated patients with NSCLC harboring HER2 exon 20 insertion mutations [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 3400.

Improved response prediction to immune checkpoint inhibition by combining TMB and WGS-based genomic features in NSCLC.

e21077 Background: The appearance of immune checkpoint inhibitors (ICIs) has revolutionized treatment strategies in advanced NSCLC. The clinical efficacy and indications for ICIs are currently estimated using the immunohistochemistry (IHC) profile of programmed death-ligand 1 (PD-L1) protein. Recent findings indicate that tumor mutational burden (TMB) is a unique feature that may improve response prediction to ICIs across multiple cancer types [1]. Nevertheless, only a minority of NSCLC patients will benefit from ICIs. Therefore, unraveling the genomics of NSCLC and the subsequent discovery of novel predictive biomarkers remains a high unmet need. Whole-genome sequencing (WGS) bounded with AI may identify biomarkers and composed signatures which will better than TMB or PD-L1 predict response to ICIs. Methods: To develop the PD1Dx classifier with the highest predictive value for ICI response, we combined WGS data analyses with machine learning approaches. Consistently, we divided WGS data of 57 NSCLCs derived from the Hartwig Medical Foundation (HMF) database into training (n = 39) and hold-out (n = 18) sets. Patients treated with pembrolizumab or nivolumab alone were labeled based solely on clinical metadata provided by the repository, reaching a total of 22 responders and 35 non-responders. Results: Recursive Feature Elimination using SHAP importance (ShapRFECV) selected 15 top predictive genomic features, based on which the final random forest model was built. The random forest model achieved the best performance, with an accuracy of 0.72, a recall of 0.73, and a precision of 0.80. Using the same learning method and dataset, we built a model based on a single TMB feature whose performance determined by accuracy, recall and precision was 0.67, 0.64, and 0.78, respectively. Apart from TMB, we also identified other predictive features, among the 15 top of which were, e.g., features related to transcriptional strand bias and C &gt; A/T single nucleotide variants correlating with mismatch repair. Conclusions: Our results confirm the predictive value of features other than TMB alone in predicting ICI responses in NSCLC. Further investigation is required to validate biomarkers of sensitivity and resistance to ICIs. Krieger T, Pearson I, Bell J, Doherty J, Robbins P. Targeted literature review on use of tumor mutational burden status and programmed cell death ligand 1 expression to predict outcomes of checkpoint inhibitor treatment. Diagn Pathol 2020;15:6.

Integration of molecular cancer classification and NGS to identify patients with metastatic cancer eligible for BRAF inhibitors.

e15118 Background: Melanoma, non-small cell lung cancer (NSCLC), colorectal adenocarcinoma and thyroid tumors frequently harbor BRAF mutations and are tumor types eligible for treatment with FDA-approved BRAF inhibitors. Although there are many ongoing clinical trials with BRAF inhibitors in various advanced tumor types, currently eligibility requires both tumor type identification and BRAF mutation confirmation. The 92-gene assay (CancerTYPE ID) is a gene expression-based classifier of 50 tumor types and subtypes for metastatic patients with diagnostic ambiguity. Multimodal biomarker testing, including NGS, IHC and FISH, enables identification of actionable biomarkers to guide targeted therapy selection. Here, a database of metastatic cases integrating tumor type with biomarker analysis was used to identify patients eligible for BRAF inhibitor treatment. Methods: MOSAIC (Molecular Synergy to Advance Individualized Cancer Care) is an IRB-approved, de-identified database of diagnostically ambiguous cases submitted for CancerTYPE ID testing with tumor type-guided multimodal biomarker testing (Neogenomics). In this study, metastatic cancers of various tumor types with BRAF mutations were identified. Results: CancerTYPE ID determined 27 different tumor types in 2929 (92.5%) of 3168 cases. BRAF mutations were identified in 87 cases across 18 tumor types and subtypes. Out of the 87 cases, 22 (25.3%) had BRAF mutations in a tumor type eligible for FDA-approved BRAF inhibitor treatment, including melanoma, NSCLC, colorectal adenocarcinoma and thyroid cancer. Another 26 (29.9%) BRAF-mutated cases were detected in a tumor type with ongoing BRAF inhibitor clinical trials, including lymphoma, pancreaticobiliary and ovarian cancer (Table). Conclusions: This analysis identified patients with metastatic cancer with actionable BRAF mutations eligible for FDA-approved therapy, and patients who may be eligible for investigational BRAF inhibitors based on tumor type and mutation status. Notably, cancer type identification is critical given that FDA-approved BRAF inhibitor treatment regimens vary based on the tumor-specific indication. These findings support the clinical utility of molecular tumor type classification by CancerTYPE ID combined with biomarker profiling to identify additional treatment options for patients with metastatic cancer.[Table: see text]