Genome Analysis of Latin American Cervical Cancer: Frequent Activation of the PIK3CA Pathway

Abstract

Purpose: Cervical cancer is one of the most common causes of cancer mortality for women living in poverty, causing more than 28,000 deaths annually in Latin America and 266,000 worldwide. To better understand the molecular basis of the disease, we ascertained blood and tumor samples from Guatemala and Venezuela and performed genomic characterization.

Experimental design: We performed human papillomavirus (HPV) typing and identified somatically mutated genes using exome and ultra-deep targeted sequencing with confirmation in samples from Mexico. Copy number changes were also assessed in the exome sequence.

Results: Cervical cancer cases in Guatemala and Venezuela have an average age of diagnosis of 50 years and 5.6 children. Analysis of 675 tumors revealed activation of PIK3CA and other PI3K/AKT pathway genes in 31% of squamous carcinomas and 24% of adeno- and adenosquamous tumors, predominantly at two sites (E542K, E545K) in the helical domain of the PIK3CA gene. This distribution of PIK3CA mutations is distinct from most other cancer types and does not result in the in vitro phosphorylation of AKT. Somatic mutations were more frequent in squamous carcinomas diagnosed after the age of 50 years. Frequent gain of chromosome 3q was found, and low PIK3CA mutation fractions in many tumors suggest that PI3K mutation can be a late event in tumor progression.

Conclusions: PI3K pathway mutation is important to cervical carcinogenesis in Latin America. Therapeutic agents that directly target PI3K could play a role in the therapy of this common malignancy.

Conflict of interest statement

The authors have no conflicts of interest to report.

©2015 American Association for Cancer Research.

Figures

Figure 1

Driver gene mutations in 23 Guatemalan cervical cancers. Genes frequently mutated or amplified in other cancers are indicated, with their gene names at the left and the mutation percentages (%) on the right. The predominant HPV type and pathology is indicated at the top (unlabeled tumors are squamous cell carcinomas). Below the main section, the presence of chromosome rearrangement (>100 chromosome breaks, CHR re), gain of chromosome 3q and loss of 17p are shown.

Figure 2

Copy number changes in cervical tumors. The predicted copy number changes from 23 tumors analyzed by AmpliSeq Exome were combined. Deletions are displayed by red bars to the left of the chromosome ideograms, and gains by blue bars to the right. The height of the bars indicates the combined effect of the CNV. Darker shades of red and blue indicate CNVs above a cutoff of log2 ratio value of above 0.6 and below −1.0, respectively.

Figure 3

The distribution of somatic PIK3CA mutations. A). Mutations in patients from Guatemala (red triangles), Venezuela (black triangles), and Mexico (green triangle) are shown relative to functional domains (ABD, p85 binding domain; RBD, RAS binding domain; C2 domain; Helical domain; and Kinase domain) of the PIK3CA protein. The percentage of mutations detected within each region and the amino acid positions are indicated below. Blue indicates novel mutations from this study and purple indicates mutations without published functional analyses. * indicates the patient with ≥1 PIK3CA mutation. B) Proportion of PIK3CA mutations in cervical cancer in different countries. The domain location of PIK3CA mutations are shown in comparison with the cervical tumors in this study. The brackets denote comparison of helical versus kinase domain in Latin American cervical tumors to breast, endometrial and intestinal tumors (****= P<0.0001). The frequency of PIK3CA mutations is noted above and details are in Supplementary Table 6C) The frequency and location of PIK3CA mutations from colon, breast, endometrial, intestine, ovary, bladder from the literature and the COSMIC Catalogue of Somatic Mutations in Cancer; http://www.sanger.ac.uk/cosmic) database and cervical cancers from this study are displayed (14, 18, 33).

Figure 4. PIK3CA mutation effect on AKT phosphorylation

Expression and analysis of the phosphorylation of AKT in U2OS transfected with empty vector, wild type and mutant PIK3CA constructs. The H1047R constructs showed a significant increase in p110α and Thr308 levels compared with wild type (WT) control, but the 3 helical domain mutations E542K, E545K and E542K/E545K showed a minimal level at Thr308. A H1047R mutant was used as a positive control. Densitometric analysis of specific signals using Image J software, on three independent blots, were quantitated and normalized to actin. One Way AVOVA and Kruskal-Wallis Statistical method was performed using GraphPad Prism version 5 (P