Immune-related Gene Expression Signatures Research Articles

The tumor microbiome as a predictor of outcomes in patients with metastatic melanoma treated with immune checkpoint inhibitors.

Emerging evidence supports the important role of the tumor microbiome in oncogenesis, cancer immune phenotype, cancer progression, and treatment outcomes in many malignancies. In this study, we investigated the metastatic melanoma tumor microbiome and potential roles in association with clinical outcomes, such as survival, in patients with metastatic disease treated with immune checkpoint inhibitors (ICIs). Baseline tumor samples were collected from 71 patients with metastatic melanoma before treatment with ICIs. Bulk RNA-seq was conducted on the formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tumor samples. Durable clinical benefit (primary clinical endpoint) following ICIs was defined as overall survival >24 months and no change to the primary drug regimen (responders). We processed RNA-seq reads to carefully identify exogenous sequences using the {exotic} tool. The 71 patients with metastatic melanoma ranged in age from 24 to 83 years, 59% were male, and 55% survived >24 months following the initiation of ICI treatment. Exogenous taxa were identified in the tumor RNA-seq, including bacteria, fungi, and viruses. We found differences in gene expression and microbe abundances in immunotherapy responsive versus non-responsive tumors. Responders showed significant enrichment of bacteriophage in the phylum Uroviricota, and non-responders showed enrichment of several bacteria including Campylobacter jejuni. These microbes correlated with immune-related gene expression signatures. Finally, we found that models for predicting prolonged survival with immunotherapy using both microbe abundances and gene expression outperformed models using either dataset alone. Our findings warrant further investigation and potentially support therapeutic strategies to modify the tumor microbiome in order to improve treatment outcomes with ICIs.

Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ

Background and aimDuctal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer with highly variable clinical behavior, but risk stratification is still challenging. We sought to identify immune-related gene expression signatures of pure DCIS associated with different risks of breast cancer recurrence. MethodsA retrospective nested case-control study of 143 pure DCIS was performed including 70 women with subsequent ipsilateral breast event (IBE, in situ or invasive; cases) and 73 DCIS women with no IBE and matched for age, tumor size, treatment, hormone receptors/HER2 status, and follow-up time (controls). RNA was extracted from DCIS samples and subjected to next-generation sequencing gene expression analysis of 395 immune-related genes. Correlations between DCIS immune-related gene expression and IBE were analyzed using weighted Cox regression for nested case-control data. ResultsEight immune-related genes were differentially expressed between cases and controls. MAGEA10 expression (present vs. absent) and high expression levels of IFNA17 and CBLB (Q4 vs. Q1) were observed more frequently in DCIS of women with subsequent IBE, mainly invasive (p-valueFDR < 0.05). Conversely, expression of IL3RA1, TAGAP, TNFAIP8, and high expression levels of CCL2 and LRP1 were associated with a lower risk of IBE (p-valueFDR < 0.05). ConclusionThis exploratory analysis of pure DCIS showed significant differences in immune-related gene expression profiles between women with and with no subsequent IBE, particularly as invasive IBE. These results, after additional validation, could improve risk stratification and management of DCIS patients.

Abstract IA007: Tumor heterogeneity in ovarian clear cell carcinoma

Abstract IA007: Tumor heterogeneity in ovarian clear cell carcinoma

Prognostic and Predictive Value of Immune-Related Gene Expression Signatures vs Tumor-Infiltrating Lymphocytes in Early-Stage ERBB2/HER2-Positive Breast Cancer

Both tumor-infiltrating lymphocytes (TILs) assessment and immune-related gene expression signatures by RNA profiling predict higher pathologic complete response (pCR) and improved event-free survival (EFS) in patients with early-stage ERBB2/HER2-positive breast cancer. However, whether these 2 measures of immune activation provide similar or additive prognostic value is not known. To examine the prognostic ability of TILs and immune-related gene expression signatures, alone and in combination, to predict pCR and EFS in patients with early-stage ERBB2/HER2-positive breast cancer treated in 2 clinical trials. In this prognostic study, a correlative analysis was performed on the Cancer and Leukemia Group B (CALGB) 40601 trial and the PAMELA trial. In the CALGB 40601 trial, 305 patients were randomly assigned to weekly paclitaxel with trastuzumab, lapatinib, or both for 16 weeks. The primary end point was pCR, with a secondary end point of EFS. In the PAMELA trial, 151 patients received neoadjuvant treatment with trastuzumab and lapatinib for 18 weeks. The primary end point was the ability of the HER2-enriched subtype to predict pCR. The studies were conducted from October 2013 to November 2015 (PAMELA) and from December 2008 to February 2012 (CALGB 40601). Data analyses were performed from June 1, 2020, to January 1, 2022. Immune-related gene expression profiling by RNA sequencing and TILs were assessed on 230 CALGB 40601 trial pretreatment tumors and 138 PAMELA trial pretreatment tumors. The association of these biomarkers with pCR (CALGB 40601 and PAMELA) and EFS (CALGB 40601) was studied by logistic regression and Cox analyses. The median age of the patients was 50 years (IQR, 42-50 years), and 305 (100%) were women. Of 202 immune signatures tested, 166 (82.2%) were significantly correlated with TILs. In both trials combined, TILs were significantly associated with pCR (odds ratio, 1.01; 95% CI, 1.01-1.02; P = .02). In addition to TILs, 36 immune signatures were significantly associated with higher pCR rates. Seven of these signatures outperformed TILs for predicting pCR, 6 of which were B-cell related. In a multivariable Cox model adjusted for clinicopathologic factors, including PAM50 intrinsic tumor subtype, the immunoglobulin G signature, but not TILs, was independently associated with EFS (immunoglobulin G signature-adjusted hazard ratio, 0.63; 95% CI, 0.42-0.93; P = .02; TIL-adjusted hazard ratio, 1.00; 95% CI, 0.98-1.02; P = .99). Results of this study suggest that multiple B-cell-related signatures were more strongly associated with pCR and EFS than TILs, which largely represent T cells. When both TILs and gene expression are available, the prognostic value of immune-related signatures appears to be superior.

9P Early circulating tumor DNA (ctDNA) kinetics and gene expression analysis to predict treatment outcomes with anti-PD-1 therapy

ctDNA kinetics in the first 6 weeks and immune-related gene expression signatures can predict outcomes in patients (pts) treated with anti-PD-1 therapy. The earliest predictive timepoint in ctDNA kinetics for benefit to this therapy is unknown.

Intratumoral spatial heterogeneity of tumor-infiltrating lymphocytes is a significant factor for precisely stratifying prognostic immune subgroups of microsatellite instability-high colorectal carcinomas

Although the density of tumor-infiltrating lymphocytes (TILs) is known to be linked to prognosis in various cancers, the prognostic impact and immunologic significance of the spatial heterogeneity of TILs have been rarely investigated. In this study, CD3+ and CD8+ TILs were quantified in independent cohorts (discovery, n = 73; and external validation, n = 93) of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) utilizing whole-slide image analysis of CD3/CD8 immunohistochemistry. The Shannon and Simpson indices, which measure intratumoral patch-to-patch evenness of TIL densities, were used to quantitatively assess the spatial heterogeneity of TILs in each case. To uncover immune-related gene expression signatures of spatial heterogeneity-based TIL subgroups of MSI-H CRCs, representative cases were subjected to GeoMx digital spatial profiler (DSP) analysis. As expected, a low density of TILs was significantly associated with poor disease-free survival (DFS) in MSI-H CRCs. The TIL-low tumors were further classified into two subgroups based on the spatial heterogeneity of TILs: TIL-low/heterogeneity-high and TIL-low/heterogeneity-low subgroups. In both discovery and validation cohorts, the TIL-low/heterogeneity-high, TIL-low/heterogeneity-low, and TIL-high subgroups were significantly associated with poor, intermediate, and good DFS, respectively. In the DSP analysis, the TIL-low/heterogeneity-high subgroup showed higher spatial diversity in the expression of immune-related genes than that of the TIL-low/heterogeneity-low subgroup and exhibited upregulation of genes related to immune checkpoints, chemokine/cytokine receptors, and myeloid cells. TIL-low/heterogeneity-high tumors were also enriched with gene sets related to good response to immune checkpoint inhibitor therapy. In conclusion, TIL-low MSI-H CRCs are prognostically heterogeneous and can be divided into prognostically and immunologically distinct subgroups by considering the spatial heterogeneity of TILs. Our data suggest that intratumoral spatial heterogeneity of TILs can be used as a key element for clinically relevant immunologic subtyping of tumors.

Abstract P1-22-03: Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ

Abstract Background. Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). High variability in clinical outcomes and propensity for invasion among DCIS is reported, but identifying high-risk DCIS remains a major clinical challenge. Therefore, biomarkers to differentiate patients with indolent DCIS from those who would benefit from therapy are warranted. There are recent data on the role of the tumor immune microenvironment in the progression from DCIS to IBC and the risk of recurrences. However, no comprehensive information on the clinical actionability of DCIS immune-biology is available. We hypothesize that immune escape mechanisms might play a critical role in the transition from DCIS to IBC. Here, we sought to establish whether immune-related gene expression signatures of DCIS might identify women at high risk of disease recurrence and/or progression. Methods. We performed a retrospective nested case-control study including women with pure DCIS, diagnosed between 2009 and 2015 at European Institute Oncology (median follow-up 39,5 months) treated with conserving surgery +/- adjuvant therapy (endocrine therapy or radiotherapy). The study group (cases) was composed of women with DCIS and subsequent ipsilateral breast events (IBE, in situ or invasive). Controls were selected in a 1.1 ratio among DCIS without IBE, matched for age, tumor size, treatment, and hormone receptors (HR), and HER2 status. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed according to the International Immuno-Oncology Biomarker Working Group on Breast Cancer guidelines. RNA extracted from formalin-fixed paraffin-embedded blocks was subjected to gene expression analysis using a next-generation sequencing assay (Oncomine™ Immune Response Research Assay) targeting 395 immune-related genes. Samples that achieve run quality parameters (mapped reads &amp;gt;1 million, valid reads &amp;gt;800.000) were further processed with the Affymetrix Transcriptome Analysis Console software to compare the gene expression between cases and controls (ANOVA, gene-level fold change &amp;lt;-2 or &amp;gt;2).Results. A total of 116 patients were included, 58 cases and 58 controls. High sTILs count was significantly associated with high-grade DCIS (p=&amp;lt;0,0001), the presence of necrosis (p=0,0210) and HER2 expression (p=&amp;lt;0,0001) in both groups. No significant association between sTILs count and the probability of relapse was observed. Gene expression data were available for 56 cases and 56 controls that achieved sequencing quality parameters. Overall, five genes were differentially expressed between cases and controls. In particular, cases showed upregulation of IFNA17 (p-value &amp;lt;0,0001; FDR p-value &amp;lt;0.0001), IFNB1 (p-value &amp;lt;0,0001; FDR p-value &amp;lt;0.0001), PECAM-1 (p-value &amp;lt;0,0001; FDR p-value &amp;lt;0.0001) and significant lower expression of CCL2 (p-value &amp;lt;0,0001; FDR p-value &amp;lt;0.0001) as compared to control group. Other genes that were upregulated in DCIS with IBE included FCGR2B, CD3D, CD40LG, while TCF7, CDKN3, ADORA2A were found to be downregulated (p-value &amp;lt;0.05).Conclusion. Quantitative TILs density assessment remains of modest significance in DCIS prognostication in terms of risk of IBE. We showed that pure DCIS displayed significant differences in the expression of immune-related genes between women with and without subsequent breast cancer recurrence regardless of HR and HER2 status. The activation of immune-related pathways might play a part in the development of IBE in patients with a diagnosis of DCIS. The evaluation of immune-related gene expression profiles might improve risk stratification in patients with DCIS. Citation Format: Elena Guerini Rocco, Caterina Fumagalli, Alberto Concardi, Sergio V. Taormina, Aliana Guerrieri-Gonzaga, Federica Corso, Sara Gandini, Bernardo Bonanni, Giuseppe Viale, Massimo Barberis, Nicola Fusco, Lazzeroni Matteo. Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-22-03.

MHC-II Signature Correlates With Anti-Tumor Immunity and Predicts anti-PD-L1 Response of Bladder Cancer.

A large proportion of anti-tumor immunity research is focused on major histocompatibility complex class I (MHC-I) molecules and CD8+ T cells. Despite mounting evidence has shown that CD4+ T cells play a major role in anti-tumor immunity, the role of the MHC-II molecules in tumor immunotherapy has not been thoroughly researched and reported. In this study, we defined a MHC-II signature for the first time by calculating the enrichment score of MHC-II protein binding pathway with a single sample gene set enrichment analysis (ssGSEA) algorithm. To evaluate and validate the predictive value of the MHC class II (MHC-II) signature, we collected the transcriptome, mutation data and matched clinical data of bladder cancer patients from IMvigor210, The Cancer Genome Atlas (TCGA) databases and Gene Expression Omnibus (GEO) databases. Comprehensive analyses of immunome, transcriptome, metabolome, genome and drugome were performed in order to determine the association of MHC-II signature and tumor immunotherapy. We identified that MHC-II signature is an independent and favorable predictor of immune response and the prognosis of bladder cancer treated with immune checkpoint inhibitors (ICIs), one that may be superior to tumor mutation burden. MHC-II signature was significantly associated with increased immune cell infiltration and levels of immune-related gene expression signatures. Additionally, transcriptomic analysis showed immune activation in the high-MHC-II signature subgroup, whereas it showed fatty acid metabolism and glucuronidation in the low-MHC-II signature subgroup. Moreover, exploration of corresponding genomic profiles highlighted the significance of tumor protein p53 (TP53) and fibroblast growth factor receptor 3 (FGFR3) alterations. Our results also allowed for the identification of candidate compounds for combined immunotherapy treatment that may be beneficial for patients with bladder cancer and a high MHC-II signature. In conclusion, this study provides a new perspective on MHC-II signature, as an independent and favorable predictor of immune response and prognosis of bladder cancer treated with ICIs.

Molecular Subtypes and Gene Expression Signatures as Prognostic Features in Fully Resected Clear Cell Renal Cell Carcinoma: A Tailored Approach to Adjuvant Trials.

Trials with adjuvant vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) failed to demonstrate meaningful benefit in clinically high-risk, fully resected clear cell renal cell carcinoma (ccRCC). We evaluated whether the ccrcc1-4 molecular subtypes and gene expression signatures (GES) are associated with outcomes in this setting. We determined molecular subtypes as well as angiogenesis- and immune-related GES through RNA sequencing of 75 fresh frozen (FF) and 62 formalin-fixed, paraffin-embedded (FFPE) tumor samples. We studied disease-free (DFS) and overall survival (OS) and determined correlations among GES and Leibovich score. Angiogenesis-related GES and molecular subtypes were associated with longer DFS and OS across both cohorts, whereas immune-related GES were not. In the FF cohort, molecular subtypes (ccrcc2 & 3 vs. ccrcc1 & 4) were associated with DFS and OS, on bivariable analysis with Leibovich score (HR 0.62, 95%CI 0.39-0.98, P=.04 and HR 0.35, 95%CI 0.19-0.64, P < .001). In the FFPE cohort, molecular subtypes (ccrcc2 & 3 vs. ccrcc1&4) were also associated with DFS (HR 0.53, 95%CI 0.31-0.93, P=.03), but not OS (HR 0.59, 95%CI 0.31-1.13, P=.11) on bivariable analysis with Leibovich score. Leibovich score was significantly inversely correlated with all angiogenesis-related GES (all P < .01), but not correlated with immune-related GES. Molecular subtypes and angiogenesis-related GES are prognostic for DFS and OS in fully resected, localized ccRCC. Favorable ccrcc2 & 3 molecular subtypes with high angiogenesis-related GES, which respond best to VEGFR-TKIs, are at lower risk of relapse but were probably underrepresented in the adjuvant VEGFR-TKI trials since they inversely correlate with Leibovich score. Conversely, immune-related GES are not correlated with Leibovich score and clinically high-risk tumors can display both high and low immune-related GES. Therefore, molecular characterization could guide patient selection for adjuvant treatment.

Neoadjuvant Chemotherapy and Immunotherapy in Luminal B-like Breast Cancer: Results of the Phase II GIADA Trial.

The role of immunotherapy in hormone receptor (HR)-positive, HER2-negative breast cancer is underexplored. The neoadjuvant phase II GIADA trial (NCT04659551, EUDRACT 2016-004665-10) enrolled stage II-IIIA premenopausal patients with Luminal B (LumB)-like breast cancer (HR-positive/HER2-negative, Ki67 ≥ 20%, and/or histologic grade 3). Patients received: three 21-day cycles of epirubicin/cyclophosphamide followed by eight 14-day cycles of nivolumab, triptorelin started concomitantly to chemotherapy, and exemestane started concomitantly to nivolumab. Primary endpoint was pathologic complete response (pCR; ypT0/is, ypN0). A pCR was achieved by 7/43 patients [16.3%; 95% confidence interval (CI), 7.4-34.9]; the rate of residual cancer burden class 0-I was 25.6%. pCR rate was significantly higher for patients with PAM50 Basal breast cancer (4/8, 50%) as compared with other subtypes (LumA 9.1%; LumB 8.3%; P = 0.017). Tumor-infiltrating lymphocytes (TIL), immune-related gene-expression signatures, and specific immune cell subpopulations by multiplex immunofluorescence were significantly associated with pCR. A combined score of Basal subtype and TILs had an AUC of 0.95 (95% CI, 0.89-1.00) for pCR prediction. According to multiplex immunofluorescence, a switch to a more immune-activated tumor microenvironment occurred following exposure to anthracyclines. Most common grade ≥3 treatment-related adverse events (AE) during nivolumab were γ-glutamyltransferase (16.7%), alanine aminotransferase (16.7%), and aspartate aminotransferase (9.5%) increase. Most common immune-related AEs were endocrinopathies (all grades 1-2; including adrenal insufficiency, n = 1). Luminal B-like breast cancers with a Basal molecular subtype and/or a state of immune activation may respond to sequential anthracyclines and anti-PD-1. Our data generate hypotheses that, if validated, could guide immunotherapy development in this context.

A multi-ethnic analysis of immune-related gene expression signatures in patients with ovarian clear cell carcinoma.

Little is known about the immune environment of ovarian clear cell carcinoma (OCCC) and its impact on various ethnic backgrounds. The aim of this OCCC immune-related gene expression signatures (irGES) study was to address the interaction between tumour and immune environment of ethnically-diverse Asian and Caucasian populations and to identify relevant molecular subsets of biological and clinical importance. Our study included 264 women from three different countries (Singapore, Japan, and the UK) and identified four novel immune subtypes (PD1-high, CTLA4-high, antigen-presentation, and pro-angiogenic subtype) with differentially expressed pathways, and gene ontologies using the NanoString nCounter PanCancer Immune Profiling Panel. The PD1-high and CTLA4-high subtypes demonstrated significantly higher PD1, PDL1, and CTLA4 expression, and were associated with poorer clinical outcomes. Mismatch repair (MMR) protein expression, assessed by immunohistochemistry, revealed that about 5% of OCCCs had deficient MMR expression. The prevalence was similar across the three countries and appeared to cluster in the CTLA4-high subtype. Our results suggest that OCCC from women of Asian and Caucasian descent shares significant clinical and molecular similarities. To our knowledge, our study is the first study to include both Asian and Caucasian women with OCCC and helps to shine light on the impact of ethnic differences on the immune microenvironment of OCCC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Molecular underpinnings of glandular tropism in metastatic clear cell renal cell carcinoma: therapeutic implications

Background Glandular metastases (GM) have been associated with improved survival in metastatic clear cell renal cell carcinoma (m-ccRCC). We aimed to molecularly characterize m-ccRCC with GM. Material and methods We performed a retrospective cohort study on all m-ccRCC patients with available tissue at our institution, diagnosed with metastatic disease from 2000 to 2019. We determined previously described angiogenesis- and immune-related gene expression signatures (GES) and ccrcc molecular subtypes through whole transcriptome RNA sequencing of primary tumors and metastases. We tested differences in GES and molecular subtypes across groups and studied overall (OS) and progression-free survival (PFS) using Kaplan–Meier survival analysis and Cox regression models. Results Primary tumors of patients who developed GM (n = 55) had higher IMmotion Angio (p < 0.001) and JAVELIN Angio (p = 0.003) GES as well as a higher proportion of angiogenic ccrcc2 molecular subtypes (p = 0.008) than primary tumors of patients with non-GM (n = 128). Metastatic lesions in glandular organs (n = 32) also had higher IMmotion Angio (p = 0.008) and JAVELIN Angio (p = 0.02) GES and were more frequently of the ccrcc2 molecular subtype (p = 0.03), compared to metastatic lesions in non-glandular organs in patients who did not develop any GM (n = 231), but not compared to metastatic lesions in non-glandular organs in patients who also developed GM (n = 18). Patients with GM had better OS (HR 0.49, p < 0.001) and PFS on first-line vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) (HR 0.64, p = 0.045) than patients with non-GM. PFS on first- or any-line immuno-oncology (IO) was not different. IMmotion Angio, JAVELIN Angio GES, and ccrcc2 molecular subtype were associated with better OS and PFS on first-line VEGFR-TKIs, but not PFS on first or any-line IO. Conclusions Patients with m-ccRCC who develop GM are molecularly characterized by heightened angiogenesis, translating into better prognosis and better outcomes on VEGFR-TKIs, but not IO. Based on these findings, VEGFR-TKIs should be included in the first-line treatment of m-ccRCC patients with GM.

Immune-related gene expression signatures in colorectal cancer.

The immune system is crucial in regulating colorectal cancer (CRC) tumorigenesis. Identification of immune-related transcriptomic signatures derived from the peripheral blood of patients with CRC would provide insights into CRC pathogenesis, and suggest novel clues to potential immunotherapy strategies for the disease. The present study collected blood samples from 59 patients with CRC and 62 healthy control patients and performed whole blood gene expression profiling using microarray hybridization. Immune-related gene expression signatures for CRC were identified from immune gene datasets, and an algorithmic predictive model was constructed for distinguishing CRC from controls. Model performance was characterized using an area under the receiver operating characteristic curve (ROC AUC). Functional categories for CRC-specific gene expression signatures were determined using gene set enrichment analyses. A Kaplan-Meier plotter survival analysis was also performed for CRC-specific immune genes in order to characterize the association between gene expression and CRC prognosis. The present study identified five CRC-specific immune genes [protein phosphatase 3 regulatory subunit Bα (PPP3R1), amyloid β precursor protein, cathepsin H, proteasome activator subunit 4 and DEAD-Box Helicase 3 X-Linked]. A predictive model based on this five-gene panel showed good discriminatory power (independent test set sensitivity, 83.3%; specificity, 94.7%, accuracy, 89.2%; ROC AUC, 0.96). The candidate genes were involved in pathways associated with ‘adaptive immune responses’, ‘innate immune responses’ and ‘cytokine signaling’. The survival analysis found that a high level of PPP3R1 expression was associated with a poor CRC prognosis. The present study identified five CRC-specific immune genes that were potential diagnostic biomarkers for CRC. The biological function analysis indicated a close association between CRC pathogenesis and the immune system, and may reveal more information about the immunogenic and pathogenic mechanisms driving CRC in the future. Overall, the association between PPP3R1 expression and survival of patients with CRC revealed potential new targets for CRC immunotherapy.

Abstract PD9-03: Genomic analysis of the CALGB 40603 (Alliance) neoadjuvant trial in TNBC identifies immune features associated with pathological complete response and event-free survival

Abstract Purpose: CALGB (now part of the Alliance for Clinical Trials in Oncology) 40603 was a randomized Phase II study investigating pathological complete response rates (pCR) in triple-negative breast cancer (TNBC) patients receiving neoadjuvant weekly paclitaxel followed by doxorubicin and cyclophosphamide +/- bevacizumab and/or carboplatin for which 5-year event-free survival (EFS) results are now available. This is a valuable resource to characterize clinical and genomic features associated with response and survival. Methods: Clinical parameters and pre-treatment tumor biopsy RNA-sequencing (RNAseq) from 295 TNBC patients were analyzed to identify features associated with pCR and/or EFS. A panel of 689 previously published gene expression signatures were evaluated to provide insights regarding potential associations between clinical endpoints and genomically determined cell types and signaling pathway activities. Additionally, B cell receptor (BCR) and T cell receptor (TCR) sequences were examined, and repertoire abundance, richness, and diversity measures were calculated to investigate correlations with pCR and EFS status. Univariate Mann-Whitney U-Tests were used for continuous variables, and Fisher’s test was used for categorical features, with unadjusted p-values &amp;lt; 0.05 designated as significant. Results: While the addition of bevacizumab and carboplatin each significantly improved pCR rates, neither improved EFS. We examined outcomes according to race and no differences were seen for either pCR rate or EFS. 131 features, including high proliferation and multiple interferon signatures were significantly associated with pCR, but not with EFS. Alternatively, 69 features, including clinical factors for T stage and node status, were prognostic for EFS, but not significantly associated with pCR. Nevertheless, pCR itself was the strongest predictor of EFS, and was the only feature significantly associated with EFS after adjusting for multiple comparisons (Benjamini-Hochberg False Discovery Rate = 6.7e-3). In total, 52 genomic features were significantly correlated with both pCR and EFS, 44 of which were features of the immune microenvironment. Immune associated features included signatures of T cells, B cells and NK cells, immune checkpoint pathways (PD-1, PD-L1, CTLA4), and antigen presentation (dendritic cells, MHC-I, MHC-II). In particular, low BCR evenness, which is a measure of uniformity of unique BCR sequence abundance, was strongly associated with both pCR and EFS, suggesting that an antigen-specific adaptive immune response with clonally selected B cells is occurring in patients that have improved response and survival. Furthermore, a multivariate Cox Proportional Hazards model assessing BCR evenness along with age, T stage, N stage, grade and pCR found BCR evenness to be an independent prognostic feature for EFS in TNBC. Conclusions: Evidence of distinct predictors of pCR and EFS in TNBC patients treated with neoadjuvant chemotherapy suggests that, while pCR is still the strongest prognostic feature, high expression of many immune related gene expression signatures in pretreatment tumor samples are promising biomarkers of improved EFS. In addition to the important role of T cells in an anti-tumor response, these data show high IgG expression and evidence of B cell clonal selection associates with improved response and survival, supporting an important role for B cells in the adaptive response that portends a long-term benefit of chemotherapy in TNBC. Support: U10CA180821, U10CA180882, U24CA196171, P50-CA58223, Genentech, and The Breast Cancer Research Foundation. https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifier: NCT00861705 Citation Format: Jonathan H Shepherd, Terry Hyslop, Cheng Fan, Aranzazu Fernandez Martinez, Joel Parker, Katherine Hoadley, Zhiyuan Hu, Yun Li, Matthew Soloway, Patricia Spears, Ann Partridge, William Sikov, Lisa A Carey, Charles M Perou. Genomic analysis of the CALGB 40603 (Alliance) neoadjuvant trial in TNBC identifies immune features associated with pathological complete response and event-free survival [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-03.

An immune-related gene expression signature predicts brain metastasis in lung adenocarcinoma patients after surgery: gene expression profile and immunohistochemical analyses.

BackgroundLung adenocarcinoma (LUAD) with brain metastasis (BM) occurs frequently and has a poor prognosis. In this study, we aimed to assess the correlation between gene expression signatures and the development of BM after surgical resection of LUAD.MethodsWe analyzed the immune-related gene expression profiles of 72 LUADs with and without BM after surgery and verified them using NanoString method and immunohistochemistry (IHC). We matched the Tumor, Node, Metastasis (TNM) stage in the groups with and without BM to minimize the effect of TNM stage. Pathway enrichment studies were also performed.ResultsIn the NanoString results, we identified 11 upregulated immune-related gene signature that correlated specifically with BM in the discovery and validation sets [area under the curve (AUC) =0.750 and 0.787, respectively]. The discovery set achieved 94% sensitivity and 62% specificity and the validation set displayed 100% sensitivity and 50% specificity. Eight out of the 11 genes were verified by IHC and had profiles similar to the gene expression profile results (AUC =0.844 for the discovery set and AUC =0.795 for the validation set). Subgroup analysis revealed that 11 immune-related gene signature enabled prediction of BM at all TNM stages. There were no differences in the 11 immune-related gene expression signatures between the primary LUAD samples and the matched brain samples. Pathway enrichment analysis revealed that the cytokine-cytokine receptor interaction pathway was closely correlated with BM.ConclusionsThe 11 identified immune-related gene expression signatures may be potentially clinically useful predictors for BM and can provide patient-specific treatment options.

EPHA5 mutation predicts the durable clinical benefit of immune checkpoint inhibitors in patients with lung adenocarcinoma.

Immune checkpoint inhibitor (ICI) therapy has shown remarkable clinical benefit in lung adenocarcinoma (LUAD) patients. Genomic mutations may be applicable to predict the response to ICIs. Eph receptor A5 (EPHA5) is frequently mutated in breast cancer, lung cancer, and other tumors; however, its association with outcome in patients who receive immunotherapy remains unknown. In this study, we report that EPHA5 mutations were associated with increased tumor mutation burden (TMB), neoantigen load, levels of immune-related gene expression signatures, and enhanced tumor-infiltrating lymphocytes (TILs) in LUAD. LUAD patients with EPHA5 mutations in the immunotherapy cohort achieved a longer progression-free survival (PFS) time than patients with wild-type EPHA5. Immune response pathways were among the top enriched pathways in samples with EPHA5 mutations. In addition, patients with EPHA5 mutations tended to be more sensitive to certain targeted molecular inhibitors, including serdemetan, lox2, and PF1-1. Collectively, our results suggest that identifying mutations in the EPHA5 gene may provide insight into the genome-wide mutational burden and may serve as a biomarker to predict the immune response of patients with LUAD.

Abstract B46: Gene expression in colorectal liver metastases: Distinct immune signatures and opportunities for immune modulating therapy

Abstract Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer death in the Western world. Up to 50% of CRC patients develop metastatic disease and the liver is the most common site. The recently identified consensus molecular subtypes (CMS1-4) based on analyses of primary CRC have prognostic and therapeutic implications, but it is unclear whether these molecular subtypes are valid for metastatic disease. The tumor microenvironment plays a major role in the dynamic shaping of the tumor phenotype and the clinical impact of the immune contexture within given CMS subgroups is emerging. In this study we performed gene expression profiling of a CLM cohort for patient stratification. Surgically resected CLMs (n=44) were analyzed by genome-wide transcription profiling. Clustering approaches were applied to define subgroups. Differential gene expression analyses were performed to reveal altered expression patterns in subgroups selected by clinicopathologic parameters. Functional annotation of gene signatures was conducted. Validation in public data sets and by proteomic analyses (RPPA) is ongoing. Quantification of T-cell subsets by immunohistochemistry was scored. The CMS classifier tool revealed the cohort to be highly enriched for CMS2 and thus of limited stratification utility. In contrast, intrinsic subgroups were revealed by alternative computational approaches. Distinct immune profiles were associated with the subgroups identified. Unsupervised clustering based on high-variance genes identified a 55-gene cluster that split the cohort near evenly. Functional annotation of the 55 genes identified one subgroup to display dysregulated cholesterol/lipid homeostasis with reciprocal links to immune regulatory genes. Supervised clustering using EMT/mesenchymal signatures identified a mesenchymal subgroup (20% of the samples). Functional annotation suggested coexistence of immunosuppression with the mesenchymal phenotype. Elevated expression of immune checkpoint molecules including TIM-3, and M2 macrophage markers correlated with the EMT/mesenchymal signature. The subgroup showed relative high density of T-cell subset infiltration (CD3, CD4, CD8, FOXP3). In differential gene expression analysis, CLMs exposed to neoadjuvant chemotherapy showed activation of interferon gamma (IFNG) signaling and genes linked to immunogenic cell death. Co-expression of immune regulatory genes suggested counterbalance of the immune responses triggered by IFNG. Immune-related gene expression signatures were associated with the CLM subgroups identified. These observations underline the integration and importance of the immune interactome in CLM. The coordinated expression of immunosuppressive factors suggests not only a rationale for immune therapy, but also that development of immune-modulating strategies may be required to increase efficacy of current therapy for selected CLM patients. Citation Format: Vigdis Nygaard, Vegar Johansen Dagenborg, Sheraz Yaqub, Åsmund Avdem Fretland, Olga Østrup, Laxmi Siwal-Pandit, Krzysztof Grzyb, Anne-Lise Børresen-Dale, Gunhild Mari Mælandsmo, Anne Hansen Ree, Bjørn Edwin, Kjersti Flatmark. Gene expression in colorectal liver metastases: Distinct immune signatures and opportunities for immune modulating therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B46.

Screening and validating the immune-related gene expression signatures in peripheral blood mononuclear cells of nonischaemic cardiomyopathy.

Background: Mountainous evidence has revealed that an activated immune response in the heart can provoke left ventricular dysfunction and result in adverse cardiac remodelling. This study was designed to explore the potential mechanisms and identify core biomarkers in peripheral blood mononuclear cells (PBMCs) of nonischaemic cardiomyopathy (NICM). Methods: GSE9128, particularly, the samples of PBMCs from 3 control individuals and 4 NICM patients, was downloaded from the GEO database to identify differentially expressed genes (DEGs). DAVID was employed to perform gene ontology (GO) and Kyoto PBMC of Genes and Genomes (KEGG) analyses. Meanwhile, a protein-protein interaction (PPI) network was constructed to display the interactions among these DEGs. The expression of the top 5 upregulated and downregulated DEGs was validated in a NICM mice model induced by isoproterenol via real-time PCR. Results: In this study, a total of 371 DEGs (fold change > 2), consisting of 288 upregulated DEGs and 83 downregulated DEGs, were captured in the PBMCs. GO and KEGG analysis demonstrated that these DEGs were particularly enriched in inflammatory response, immune response, extracellular exosome and cell differentiation. Additionally, the hub genes with the 15 highest connectivity degrees were also identified, namely, JUN, MYC, HSP90AA1, PCNA, CREB1, IL1B, IL8, SMARCA2, TLR4, RB1, RANBP2, EGR1, PTGS2, ENO1 and XPO1. Finally, our in vitro experiment not only validated the mRNA expression levels of the top 5 upregulated and downregulated DEGs in mice but also further clarified their expression in subtypes of PBMCs. Conclusion: Our study unveiled potential biomarkers and molecular mechanisms in NICM, which could provide a non-invasive strategy for the diagnosis and treatment of NICM.

Digital Immune-Related Gene Expression Signatures In High-Grade Serous Ovarian Carcinoma: Developing Prediction Models For Platinum Response.

PurposeResponse to platinum-based therapy is a major prognostic factor in high-grade serous ovarian cancer (HGSOC). While the exact mechanisms of platinum-resistance remain unclear, evidence is accumulating for a connection between the organism’s immune-response and response to platinum. However, predictive tools are missing. This study was performed to examine the putative role of the genetic tumor immune-microenvironment in mediating differential chemotherapy response in HGSOC patients.Patients and methodsExpression profiling of 770 immune-related genes was performed in tumor tissues from 23 HGSOC cases. Tumors were screened for prognostic and predictive biomarkers using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel. As validation cohort, gene expression data (RNA Seq) of 303 patients with epithelial ovarian carcinoma (EOC) were retrieved from the The Cancer Genome Atlas (TCGA) database. Different scoring-systems were computed for prediction of risk-of-resistance to cisplatin, disease-free survival (DFS) and overall survival (OS).ResultsValidated on the TCGA-dataset, the developed scores identified 11 significantly differentially expressed genes (p <0.01**) associated with platinum response. HSD11B1 was highly significantly associated with lower risk of recurrence and 7 targets were found highly significantly influencing OS time (p <0.01**).ConclusionOur results suggest that response to platinum-based therapy and DFS in ovarian HGSOC is associated with distinct gene-expression patterns related to the tumor immune-system. We generated predictive scoring systems which proved valid when applied to a set of 303 EOC patients.

999PD - Immune-related gene expression signatures (irGES) in patients (pts) with ovarian clear cell carcinomas (OCCC): A multicenter analysis

BackgroundWe previously showed that OCCC can be classified into 4 molecular subgroups based on their irGES profiles with distinct clinicopathological characteristics and prognostic outcomes. A combined analysis of samples from 3 independent centres was performed to investigate the prognostic relevance of the irGES profiles in a larger cohort of OCCC patients. MethodsImmune-related gene profiling was performed on 255 FFPE OCCC samples collected from 3 centres (NUH, Singapore, Saitama, Japan, and Edinburgh, UK) using the nanoString nCounter PanCancer Immune Profiling Panel. Unsupervised hierarchical clustering analysis was performed and correlated with clinical outcome. MMR protein levels were assessed by immunohistochemistry. ResultsTotal of 255/264 samples from 3 independent cohorts were successfully profiled. Median age at diagnosis was 57 yrs. 137 (53.7%) were stage 1 (Singapore 46.9%, Saitama 65.8%, Edinburgh 50.6%) with 113 (44.3%) Stages 2-4 (Singapore 47.9%, Saitama 34.2%, Edinburgh 49.4%) and 5 (1.9%) missing data. 68.8%, 80.3% and 80.7% of pts from Singapore, Saitama and Edinburgh respectively, received adjuvant treatment. Median PFS for OCCC pts from Singapore, Saitama and Edinburgh were 27, 29.2 and 31.9 mths, respectively. Median OS were 33.5, 35.1 and 41.6 mths respectively for pts from Singapore, Saitama and Edinburgh. Based on irGES, 4 distinct molecular subgroups of OCCCs were consistently identified and reproducible. The PD-1 high subgroup was observed to have poorer 3yr OS rate (52% vs 57% vs 73% vs 74%, p=0.05) and a trend towards poorer 3 year PFS rate (42% vs 58.7% vs 51% vs 65%, p=0.12) compared to the CTLA4, ProA and AP subgroups respectively. A similar trend was observed when pts were stratified based on stage and whether they received adjuvant treatment. MMR expression levels were noted to be deficient in 6.3% and 5.1% of pts from Singapore and Saitama, respectively and were not significantly associated with any irGES group. ConclusionsClinical outcomes, based on irGES profiles, appeared to be similar across Asian and Caucasian OCCC pts. 4 molecular subgroups based on their irGES profiles were consistently identified between 3 independent cohorts with distinct prognostic outcomes. Legal entity responsible for the studyThe authors. FundingNUHS Clinician Scientist Programme-Residency (NCSP-R) Grant NMRC -Centre grant scheme Yong Siew Yoon fellowship grant Clinician Scientist Award (IMAC) CSA-NMRC grant Nicola Murray Foundation. DisclosureC. Gourley: Honoraria (self), Advisory / Consultancy: Roche, AstraZeneca, Tesaro, Nucana, Clovis, Foundation One, Sierra Oncology, Cor2Ed; Research grant / Funding (self): AstraZeneca, Novartis, Aprea, Nucana, Tesaro; Licensing / Royalties: PCT/US2012/040805, PCT/GB2013/053202, 1409479.1, 1409476.7 and 1409478.3. D.S. Tan: Honoraria (self): Roche, Bayer, MSD, Genmab, AstraZeneca, Merck Serono, Tessa Therapeutics, Novartis; Research grant / Funding (institution): Bayer, Karyopharm, AstraZeneca. R. Huang: Spouse / Financial dependant: ACT genomics Singapore. All other authors have declared no conflicts of interest.