PTEN Gene Expression and Mutations in the PIK3CA Gene as Predictors of Clinical Benefit to Anti-Epidermal Growth Factor Receptor Antibody Therapy in Patients With KRAS Wild-Type Metastatic Colorectal Cancer

Abstract

Purpose

To identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor–directed antibody therapy in patients with metastatic colorectal cancer.

Patients and Methods

Seventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy and with available tumor tissue were included. Tumor tissue was tested by pyrosequencing for mutations at known hot spots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes. PTEN promoter methylation status was analyzed by methylation-specific polymerase chain reaction, and expression was determined by immunohistochemistry (IHC). Forty-four patients had 4 weeks of therapy and were considered for clinical correlates.

Results

Consistent with previous studies, KRAS gene mutations were associated with a shorter progression-free survival (PFS) and overall survival (OS). Among the patients with wild-type KRAS, preservation of PTEN expression and PIK3CA wild-type status was associated with improved OS (median OS, 80.4 vs. 32.5 weeks; hazard ratio, 0.33; P = .0008) and a trend toward improved PFS (median PFS, 24.8 vs. 15.2 weeks; hazard ratio, 0.51; P = .06), compared with PTEN-negative or PIK3CA-mutant tumors. PTEN methylation was more common in the metastatic samples than in the primary samples (P = .02). The simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity (P = .026).

Conclusion

In addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of a lack of benefit to anti-EGFR therapy in metastatic colorectal cancer. PTEN promoter methylation and mutation status was predictive of PTEN expression and may be used as an alternative means of predicting response to EGFR-targeted therapy.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States, with more than 52,000 deaths in 2010.¹ The median survival of patients with metastatic CRC has improved to 24-30 months,2, 3 largely due to the availability of newer therapeutic options, including the epidermal growth factor receptor (EGFR) targeted monoclonal antibodies (mAb), cetuximab (Erbitux; Eli Lilly, Indianapolis, IN) and panitumumab (Vectibix; Amgen Inc, Thousand Oaks, CA).4, 5 Response rates (RR) to anti-EGFR mAb range from 10% to 22% when used alone or in combination with irinotecan.6, 7 Activation of the EGFR pathway occurs after ligand binding, which leads to EGFR phosphorylation and oligodimerization at the cell membrane; this, in turn, triggers downstream signaling events, including activation of the Ras/Raf/mitogen-activated protein kinase (MAPK), and the phosphoinositide-3-kinase-(PI3K)-PTEN-AKT-mTOR pathways.8, 9 Anti-EGFR mAb inhibit these signaling pathways by inhibiting ligand binding.

The ability to predict the likelihood of clinical benefit to these agents is an area of intense research. It is well established that mutations in the KRAS gene predict for a lack of response to anti-EGFR mAb.10, 11, 12, 13 BRAF gene mutation is considered a prognostic marker, however, its predictive nature is uncertain.14, 15, 16, 17, 18 Among the patients with wild-type (WT) KRAS, multiple studies that evaluated EGFR-based therapy documented highly variable RR that ranged from 17% to 60%.10, 11, 12, 19, 20, 21, 22, 23 This heterogeneity suggests that there may be other predictive variables, besides KRAS, that determine responsiveness to EGFR antibodies.

Our group was one of the first to demonstrate, in vitro, that mutations in the PI3K signaling pathway also predict response to cetuximab.²⁴ We observed that, in CRC cell lines, activating PIK3CA mutations or loss of PTEN gene expression predicted resistance to cetuximab. Since then, several clinical reports followed, with conflicting results, with some reports that suggest a predictive role of this pathway and others refuting this finding.24, 25, 26, 27, 28, 29, 30 Notably, a recent report suggested that mutations in exon 20 of PIK3CA specifically predicted for resistance to cetuximab.³⁰

Importantly, multiple components of the PI3K signaling pathway have been shown to be mutated in colon cancer, including mutations in PIK3CA, PTEN, PIK3R1 (regulatory subunit of the PIK3CA gene, p85α³¹), and AKT1. Furthermore, loss of PTEN expression in CRC has been shown to be mediated by promoter hypermethylation.32, 33 In this study, we performed a comprehensive investigation of all components of the PI3K signaling pathway previously shown to be mutated or methylated in CRC and linked perturbations in the pathway with anti-EGFR mAb response. Mutation status of KRAS and BRAF also was performed as a means of validating our findings with previous reports.