Predictive Biomarkers For anti-EGFR Therapy Research Articles

Downregulation of miR-506-3p Facilitates EGFR-TKI Resistance through Induction of Sonic Hedgehog Signaling in Non-Small-Cell Lung Cancer Cell Lines.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial–mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.

Role of osteopontin as a predictive biomarker for anti-EGFR therapy in triple-negative breast cancer

ABSTRACTObjective: Effective targeted therapies for patients with triple-negative breast cancer (TNBC) present an unmet clinical need. There is evidence that TNBCs often have increased expression of the epidermal growth factor receptor (EGFR) and of osteopontin (OPN). OPN-mediated signaling can activate EGFR-dependent signaling pathways. Here, we assessed OPN as a potential predictive biomarker for response to anti-EGFR therapy in TNBC.Research design and methods: Using two different TNBC cell lines, MDA-MB-468 and MDA-MB-231, we investigated the impact of stable expression of OPN on efficacy of the EGFR inhibitor erlotinib in vitro.Results: We observed that breast cancer cells engineered to overexpress OPN are more sensitive to growth inhibition by erlotinib than control cells. The level of response was related to the level of OPN expression, possibly due to increased phosphorylation status of EGFR Tyr1068.Conclusions: These results indicate that OPN expression levels are related to sensitivity of TNBC cells to growth inhibition by erlotinib. OPN thus is a promising predictive biomarker for anti-EGFR therapy in breast cancer.

Abstract 3418: Cross-comparison of targeted gene expression technologies for patient stratification

Abstract Colorectal cancer (CRC) is one of the major causes of global cancer mortality. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC, and yet predicting prognosis in clinical practice is still poor. Therefore, a more accurate method for prognosis of CRC patients is needed. Gene expression profiling has shown great promise in predicting prognosis of individual patients in diverse cancers. The development of RNA-sequencing has greatly facilitated identification of biomarkers that can be used to stratify patients for targeted therapies. Despite the decrease in cost of sequencing in last few years, the time and the resources needed for analysis limit its use in clinical trials for patient selection. Targeted gene expression technologies like qPCR and NanoString enable highly customizable assays that can be conveniently performed for patient recruitment. The aim of this study was to investigate potential alternatives for gene profiling using a novel NanoString Plex Set technology. The Plex Set system comes with prepackaged and custom code sets in identifying genetic markers. Up to 8 samples can be pooled to each nCounter cartridge lane, enabling a total of 96 samples per run, thus making the total cost relatively affordable. For this study, gene expression signature was developed using RNA-Seq data where we have profiled 74 CRC samples, 20 of which have matching normal samples. A RAS signature score based on expression profile was calculated for each sample. In order to look for potential gene signatures, differential gene expression analysis was performed between the following groups: (a) samples with high versus those with low RAS signature scores in the 54 CRC, (b) KRAS mutant versus wild-type samples, and (c) tumor versus normal samples in the clinical study. We hypothesized that our genes of interest are most likely significantly differentially expressed in one of these groups. The counts of significantly expressed gene for the groups (a-c) are 1560 and 34, respectively, and we are working on the third case. Therefore, significantly deferentially expressed genes between groups were selected and ranked based on frequency of occurrence. These genes of interest are being analyzed using NanoString Plex Set and qPCR to evaluate the potential of using NanoString Plex Set system for targeted gene expression profiling. Results of these analyses will be presented. Citation Format: Raghavee Venkatramanan, Tuuli Saloranta, Inah Golez, Elliot Swanson, Kimberly Kruse, Vickie Satele, Saman Tahir, Sally Dow, Evan Anderson, Briana Hudson, Spencer Chee, Kerry Deutsch, Steve Anderson, Fang Yin Lo, Anup Madan. Cross-comparison of targeted gene expression technologies for patient stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3418.

Abstract 5379: Stratification of colorectal cancer patients based on various sequencing platforms and tumor mutational burden

Abstract Colorectal cancer (CRC) is the third most common type of cancer in the United States. Although chemotherapy, radiation and targeted therapies can improve survival rates, recent studies have shown the potential benefit of immunotherapies to improve outcomes for patients with advanced CRC. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Therefore, to accurately predict patients' response to treatments and improve clinical outcomes, additional prediction and treatment methods are imperative. One of the many efforts to improve prediction for CRC patient's response to the anti-EGFR therapy is the development of gene expression based RAS signature scores for identification of RAS activated tumors independent of mutations in the KRAS gene. There is also considerable effort being placed on combinations of targeted therapy and immunotherapies to improve responses for these cancers. Previously, we have stratified 55 CRC samples by applying a RAS gene signature score which measures MEK pathway functional output independent of tumor genotype. We showed that samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Here we investigate 120 colorectal cancer patients using exome sequencing, targeted panel sequencing, and RNA sequencing. Specifically, we acquired 40 normal-tumor pair samples and performed GATK tumor-normal pair mutation calling. Among these 120 samples, 65 of them are from patients treated with either cetuximab or panitumumab. Continued from our previous study, we stratified these samples based on calculated RAS scores, and showed how the results correlate with clinical outcome. Tumor mutational burden (TMB) can help predict the likelihood that a patient will benefit from certain immunotherapies; however, clinical applicability is limited due to cost and computing requirements of whole-exome sequencing to detect variants with high degree of accuracy. Therefore it would be helpful if TMB can be identified based on targeted sequencing. Here we investigate how TMB calculated based on different sequencing platform correlates with each other and whether targeted sequencing panel can be used instead of exome sequencing for stratifying patients. In addition, we investigated if Illumina's global screening array can be used for TMB analysis. Furthermore, to investigate the potential immune reactivity in these CRC samples, and thereby the potential benefit of immunotherapy, we performed in-silico prediction of neo-antigens and peptide binding affinity between tumor antigens derived from mutations and human HLA alleles. Citation Format: Fang Yin Lo, Claire Olson, Kerry Deutsch, Tuuli Saloranta, Inah Golez, Timothy Yeatman, Steven Anderson, Anup Madan. Stratification of colorectal cancer patients based on various sequencing platforms and tumor mutational burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5379.

Association between miR-31-3p expression and cetuximab efficacy in patients with KRAS wild-type metastatic colorectal cancer: a post-hoc analysis of the New EPOC trial.

BackgroundHigh miR-31-3p expression is associated with inferior outcomes in KRAS wild-type (WT) advanced colorectal cancer patients treated with anti-EGFR therapy. This study evaluated miR-31-3p expression in patients with operable colorectal liver metastases (LM) enrolled in the New EPOC study.MethodsMiR-31-3p expression was measured in primary tumors (PT) from 149 KRAS WT patients including 71 receiving chemotherapy alone (CT) and 78 receiving chemotherapy plus cetuximab (CTX). Each treatment arm was split into tertiles based on miR-31-3p expression levels. MiR-31-3p expression was also measured in LM from 94 patients with tumor tissue available.ResultsThe median progression-free survival for the combined populations with mid or high miR-31-3p expression was shorter in the CTX versus the CT arm (26.7 months versus 12.3 months, HR=2.28 95%CI 1.27; 4.09 p=0.006). Low miR-31-3p expressers had similar outcomes irrespective of treatment (HR=1.06 95%CI 0.43; 2.61 p=0.9). MiR-31-3p expression was correlated between paired PT and LM samples in the CT group but not in the CTX group.ConclusionsPatients with low miR-31-3p expression in the New EPOC study were not harmed by the addition of cetuximab. This supports miR-31-3p as a promising predictive biomarker for anti-EGFR therapy in KRAS WT advanced colorectal cancer.

Abstract 413: Neo-epitope detection and immune infiltrate analysis of colorectal cancer samples

Abstract Colorectal cancer (CRC) is the third most common type of cancer in the United States. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Previously we have stratified 55 CRC samples by applying a RAS gene signature score which measures MEK pathway functional output independent of tumor genotype. We showed that samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Here, we further investigate the potential immune reactivity in these CRC samples, and thereby the potential benefit of immunotherapy, by evaluating the tumor neo-epitope burden, and the quality of immune cell infiltration based on exome-seq and RNA-seq analysis. In the 53 samples that were sequenced, 779 unique non-synonymous mutations were detected by exome-seq. These 779 mutations spanned across 263 genes. The majority of these mutations are not shared between samples (~ 5% of the mutations were shared by more than 2 samples). Several driver gene mutations were identified in this study, including KRAS, TP53, PIK3CA, APC and HER2. HLA prediction based on Exome-Seq and RNA-Seq data shows that ~86.7% of the alleles predicted to be present in 53 samples were concordant between the two RNA-seq datasets. The predicted alleles based on exome-seq and RNA-seq results have 67-69% concordance. Prediction of neo-epitopes show that HLA-binding neo-epitopes are more frequent than TCR-binding ones, and that most neo-epitopes are private and not shared between samples. A more in-depth analysis of the tumor microenvironment was performed using the RNA-seq data. The epithelial, stromal and immune content of the tumors was comparable to tumors from TCGA CRC data. Next, the immune cell compartment was further stratified into 7 different immune cell types using signatures specific to CD8 andCD4 T, T-regulatory, NK, B-cells, Macrophages and Myeloid derived suppressor cells (MDSC). The immune make up of colorectal cancer is dominated by macrophages and MDSCs. Interestingly, both granulocytic G- and monocytic M-MDSCs are present together, supporting the idea that MDSCs confer an immune suppressive microenvironment in this cancer. Significantly, high MDSC infiltrated tumors showed upregulated expression of pro-tumorigenic insulin-like growth factor pathway genes. Additionally, tumors with lower burden of MDSC showed signature of complement activation suggesting innate cell-mediated anti-tumorigenic mechanisms of tumor control in CRC. These analyses provide potential biomarkers to stratify CRC patients based on their immune reactivity and predict response to cancer immunotherapy drugs. Citation Format: Fang Yin Lo, Nitin Mandloi, Timothy Yeatman, Kiran Paul, Ashwini Patil, Steven Anderson, Ravi Gupta, Anup Madan. Neo-epitope detection and immune infiltrate analysis of colorectal cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2017-413

Abstract 3946: Stratification of metastatic colorectal cancer patients using DNA and RNA sequencing and in-silico prediction of tumor antigens for consideration in immunotherapy

Abstract Colorectal cancer (CRC) is the third most common type of cancer in the United States. Although chemotherapy, radiation and targeted therapies can improve survival rates, recent studies have shown the potential benefit of immunotherapies to improve outcomes for patients with advanced CRC. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Therefore, to accurately predict patients’ response to treatments and improve clinical outcomes, additional prediction and treatment methods are imperative. One of the many efforts to improve prediction for CRC patient's response to the anti-EGFR therapy is the development of gene expression based RAS signature scores for identification of RAS activated tumors independent of mutations in the KRAS gene. Recently there have been major advances in immunotherapeutic approaches in a wide variety of cancers. In solid tumors such as melanoma and colon cancers, immune checkpoints have been shown to improve clinical outcomes. There is considerable effort being placed on combinations of targeted therapy and immunotherapies to improve responses for these cancers. Similarly, since no single treatment can apply to all CRC patients, we aim to stratify patients using a combination of three methods: 1. RAS signature score based on the expression profile of 18 genes. This RAS signature score enables measurements of mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway functional output independent of tumor genotype. 2. Expression profile of immune checkpoint inhibitor target genes, such as PD1 and PDL1, and 3. In-silico prediction of neo-antigens and peptide binding affinity between tumor antigens derived from mutations and human HLA alleles. 55 FFPE samples were selected from a cohort of 468 samples with matching FF samples. These 55 samples have about 1:1:1 ratio of high, medium and low RAS scores. Here we showed our ability to obtain RAS signature scores with concordant results using different platforms including RNA-seq, targeted RNA-seq, Nanostring and Affymetrix microarray. Samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Interestingly, expression of PD-L1 was significantly lower in tumor samples harboring mutations of genes such as MET, PTEN, NRAS, FBXW7, and GNAS. Kruskal-Wallis test showed that the expression of PD-L1 was significantly lower in samples with higher RAS signature scores (p<0.05). Combined with further prediction of tumor antigen derived from genes with missense mutations, we provide a combinatorial method for stratifying metastatic CRC patients. Citation Format: FangYin Lo, Sharon Austin, Kellie Howard, Mollie McWhorter, Heather Collins, Amanda Leonti, Lindsey Maassel, Christopher Subia, Tuuli Saloranta, Nicole Christopherson, Kathryn Shiji, Shradha Patil, Saman Tahir, Sally Dow, Evan Anderson, Jon Oblad, Kerry Deutsch, Timothy Yeatman, Steven Anderson, Anup Madan. Stratification of metastatic colorectal cancer patients using DNA and RNA sequencing and in-silico prediction of tumor antigens for consideration in immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3946.

P-236 Other RAS mutations incidence in CRCm in routine clinical practice: a center experience

Introduction: Patients with metastatic colorectal cancer that harbors KRAS mutations in exon 2 do not benefit from anti-epidermal growth factor receptor (EGFR) therapy. Other activating RAS mutations also are negative predictive biomarkers for anti-EGFR therapy, as recently shown in a subanalysis of the PRIME study. Methods: From June 2013 we began to make the determination of other mutations of RAS. The determination of the mutational status was performed using a validated analytical method for determining KRAS mutations (exons 2, 3 and 4) and NRAS (exons 2,3 and 4) in formalin-fixed, paraffin-embedded specimens. It was used cobas® KRAS mutation test (Roche), which is a real-time PCR test intended for the identification of mutations in codons 12, 13 and 61 of the gene. Pyrosequencing of the NRAS exon 1 (codon 12 and 13), exon 2 (codons 60 and 61) and exon 4 (codon 117 and 146) of NRAS and KRAS was performed using the TheraScreen® NRAS Pyro kit (Qiagen). We would like to thank the staff of the “BioBanco del Hospital Universitario Virgen de las Nieves (Granada)” for all their support in carrying out some of the techniques studied. Results: From June 2013 to February 2015, 314 patients were analyzed. Of these, 134 patients (43%) had mutations in exon 2 (codons 12/13) of KRAS. 16 patients (5%) had an invalid result. From the 164 (52%) patients with wild type KRAS exon 2, 132 patients (80%) were able to analyze other RAS mutations. A total of 12 patients (7.3%) with nonmutated KRAS exon 2 had other RAS mutations: 6 patients in the KRAS exon 3 (codon 61), 1 patient NRAS exon 2 (codon 12/13), 4 patients NRAS exon 3 (codon 61) and 1 patient in NRAS exon 4 (codon 117/146). Conclusion: RAS mutations, in addition to KRAS exon 2 mutations, predict a lack of response to anti-EGFR therapy in patients with metastatic colorectal cancer. Approximately 20% of KRAS exon 2 wild-type tumors harbored one of the new RAS mutations, as reported in various retrospective studies. In our series other RAS mutations were detected in 7,3%, lower than published so far.

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology

Emerging Roles of MicroRNAs in EGFR-Targeted Therapies for Lung Cancer.

Lung cancer is a leading cause of cancer mortality worldwide. Several molecular pathways underlying mechanisms of this disease have been partly elucidated, among which the epidermal growth factor receptor (EGFR) pathway is one of the well-known signaling cascades that plays a critical role in tumorigenesis. Dysregulation of the EGFR signaling is frequently found in lung cancer. The strategies to effectively inhibit EGFR signaling pathway have been mounted for developing anticancer therapeutic agents. However, most anti-EGFR-targeted agents fail to repress cancer progression because of developing drug-resistance. Therefore, studies of the mechanisms underpinning the resistance toward anti-EGFR agents may provide important findings for lung cancer treatment using anti-EGFR therapies. Recently, increasing numbers of miRNAs are correlated with the drug resistance of lung cancer cells to anti-EGFR agents, indicating that miRNAs may serve as novel targets and/or promising predictive biomarkers for anti-EGFR therapy. In this paper, we summarize the emerging role of miRNAs as regulators to modulate the EGFR signaling and the resistance of lung cancer cells to anti-EGFR therapy. We also highlight the evidence supporting the use of miRNAs as biomarkers for response to anti-EGFR agents and as novel therapeutic targets to circumvent the resistance of lung cancer cells to EGFR inhibitors.

Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review.

BackgroundKRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear.MethodsWe utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI.ResultsKRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation].ConclusionsTumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted.

RAS mutations distribution in a 108 patients series of colorectal cancer

Background As described previously in high impact studies,1 patients with other activating RAS out of codon 2 of kras mutations may also be negative predictive biomarkers for anti-EGFR therapy. Here we present the description of a new series in which has been performed the analysis of mutations in KRAS and NRAS genes. Methods Samples of 108 patients with colorectal cancer were used. DNA were extracted from formalin fixed paraffin embedded samples using COBAS DNA sample preparation kit (Roche). Mutation status was determined by targeted pyrosequencing with N/K RAS Pyro Kit (Qiagen). Results Sixty-four out of 108 samples were wild type (WT) both for KRAS and NRAS. Thirty-five patients presented mutation on KRAS (27 in codon 12, 4 in 13, 1 in 117, and 3 in 146). The rest of the patients (9) presented alterations in NRAS (1 in codon 12, 3 in 13, and 5 in 61). Conclusion Our results closely resemble those published to date,2 but due to small sample size are not fully comparable. Further studies must be done to determine the real distribution of the new RAS mutations.