Mingli Yang

A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC.

lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/β-catenin signaling

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

Co-Evolution of Somatic Variation in Primary and Metastatic Colorectal Cancer May Expand Biopsy Indications in the Molecular Era

Introduction Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens. Methods 468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens. Results An average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair). Conclusions Whereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution.

A Composite Gene Expression Signature Optimizes Prediction of Colorectal Cancer Metastasis and Outcome

Purpose: We previously found that an epithelial-to-mesenchymal transition (EMT)–based gene expression signature was highly correlated with the first principal component (PC1) of 326 colorectal cancer tumors and was prognostic. This study was designed to improve these signatures for better prediction of metastasis and outcome. Experimental Design: A total of 468 colorectal cancer tumors including all stages (I–IV) and metastatic lesions were used to develop a new prognostic score (ΔPC1.EMT) by subtracting the EMT signature score from its correlated PC1 signature score. The score was validated on six other independent datasets with a total of 3,697 tumors. Results: ΔPC1.EMT was found to be far more predictive of metastasis and outcome than its parent scores. It performed well in stages I to III, among microsatellite instability subtypes, and across multiple mutation-based subclasses, demonstrating a refined capacity to predict distant metastatic potential even in tumors with a “good” prognosis. For example, in the PETACC-3 clinical trial dataset, it predicted worse overall survival in an adjusted multivariable model for stage III patients (HR standardized by interquartile range [IQR] = 1.50; 95% confidence interval, 1.25–1.81; P = 0.000016, N = 644). The improved performance of ΔPC1.EMT was related to its propensity to identify epithelial-like subpopulations as well as mesenchymal-like subpopulations. Biologically, the signature was correlated positively with RAS signaling but negatively with mitochondrial metabolism. ΔPC1.EMT was a “best of assessed” prognostic score when compared with 10 other known prognostic signatures. Conclusions: The study developed a prognostic signature score with a propensity to detect non-EMT features, including epithelial cancer stem cell–related properties, thereby improving its potential to predict metastasis and poorer outcome in stage I–III patients. Clin Cancer Res; 22(3); 734–45. ©2015 AACR.

Molecular stratification of colorectal cancer populations and its use in directing precision medicine

ABSTRACT Introduction: The intrinsic heterogeneity of colorectal cancer (CRC) makes it difficult to determine which patients will benefit from adjuvant or systemic therapy and which patients will not require further treatment beyond surgical resection. This had led to a generalized ‘one size fits all’ approach to therapy. Such heterogeneity also makes it a great challenge to predict which patients might respond to targeted therapies. Precision medical diagnostics now hold promise to address these new challenges. Areas covered: The authors first review various molecular/genomic classification studies of colorectal cancer, including the prognostic role of MSI (microsatellite instability), CIN (chromosomal instability), and driver genes such as KRAS, NRAS, BRAF, PIK3CA and TP53, as well as our recent finding revealing a new prognostic role of APC and partnering mutations with KRAS and TP53. The authors then discuss prediction of treatment response for either adjuvant therapy or systemic targeted therapy (e.g. anti-EGFR therapy) as well as the effect of tumor sidedness. Expert commentary: There is an urgent need for more precise molecular classification to stratify adjuvant therapy and systemic targeted therapy to ultimately facilitate individualized treatments. Sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC patients.

Abstract 2981: The epithelial to mesenchymal transition (EMT) produces colorectal cancer subpopulations with strikingly different mutation profiles

The epithelial-mesenchymal transition (EMT) has been well-recognized as an important mechanism promoting cancer cell invasion and “stemness”, metastasis and therapeutic resistance. While the EMT-related differential gene expression has been extensively elucidated, genetic understanding of this process in cancer is largely lacking. Using unsupervised analysis, we previously identified an “intrinsic” differential gene expression signature in colorectal cancer (CRC) that was prognostic and highly correlated (p = 10−134) to EMT. Thus, we hypothesized that whole exome sequencing (WES), guided by gene expression phenotype, might identify mutations underpinning the EMT program. We first sorted 2144 CRC tumors based on their assigned degree (scores) of EMT. Ten tumors from the bottom 15% of tumors and nine tumors from the top 15% of tumors were selected, with paired normal control tissues. The WES data were then processed to identify non-synonymous SNVs, indels and LOH. We observed a striking difference in the mean number of variation per tumor (p 400 mutations (4/10, avg. 608) that were subsequently confirmed for microsatellite instability (MSI) status through BAT testing where indels in TGFBR2 were seen often with BRAF mutations (3/4); those with 12-144 mutations (6/10, avg. 65), which still demonstrated significant increases in mutation accounts compared to the high EMT tumors (0-5 mutations, avg. 2.8). Surprisingly, unlike their low EMT counterparts, the high EMT tumors displayed almost no known driver gene mutations (except a TP53 mutation observed in one high EMT sample with p Therefore, our next-generation sequencing analysis has discovered the most mesenchymal-like CRCs as a novel, genetically distinct subpopulation lacking common driver mutations. Citation Format: Mingli Yang, Michael J. Schell, Norman H. Lee, Timothy J. Yeatman. The epithelial to mesenchymal transition (EMT) produces colorectal cancer subpopulations with strikingly different mutation profiles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2981. doi:10.1158/1538-7445.AM2015-2981

PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. Here we describe an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS, a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK. PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.