Analysis of gene expression in stage I serous tumors identifies critical pathways altered in ovarian cancer

https://doi.org/10.1016/j.ygyno.2009.04.002Get rights and content

Abstract

Objective

Despite recent advances in the conceptual understanding of the pathogenesis of ovarian cancer, it remains the foremost cause of death from gynecologic malignancies in developed countries. The main reason for such a high rate of mortality is the lack of sensitive and specific biomarkers and imaging techniques for early detection of ovarian cancer. Additional biological insights into early-stage ovarian carcinogenesis are needed to help speed the development of markers for early detection of ovarian cancer. The objective of this study was to characterize differentially expressed genes in high-grade stage I serous carcinoma of the ovary.

Methods

We analyzed gene expression in macrodissected formalin-fixed, paraffin-embedded samples from 5 high-grade stage I serous carcinomas and 5 stage I borderline tumors of the ovary using the Illumina Whole Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation) corresponding to 24,000 genes. Significance Analysis of Microarrays was performed to determine differentially expressed genes in stage I serous carcinoma, and class prediction analysis was performed to determine the predictive value of differentially expressed gene sets to correctly classify serous carcinoma from borderline tumors in 3 independent data sets. Altered transcription factor pathways and biological pathways unique to stage I serous carcinoma were identified through class comparison of differentially expressed genes.

Results

Unsupervised cluster analysis of gene expression correctly classified stage I serous carcinomas from serous borderline tumors. Supervised analysis identified several known, as well as novel, genes differentially expressed in stage I ovarian cancer. Using a differentially expressed gene set, class comparison prediction analysis correctly identified serous carcinomas from serous borderline tumors in 3 independent data sets at over 80% accuracy, sensitivity, and specificity. Pathway analysis demonstrated the significance of p53 and E2F pathways in serous carcinogenesis and significant involvements of cell cycle and immune response pathways in stage I serous epithelial ovarian cancer.

Conclusion

We have identified differentially expressed genes associated with the carcinogenesis of high-grade stage I serous EOC. Furthermore, integrative analysis of biological and transcription pathway data contributed to the confirmation of important biological pathways and discovery of additional unique genes and pathways that may have potential importance in ovarian pathogenesis and biomarker development.

Introduction

Epithelial cancer of the ovary is the most lethal gynecologic malignancy in the United States, with approximately 22,000 new cases and 16,000 deaths occurring annually [1]. Due to the absence of specific signs and symptoms and the lack of effective screening programs, epithelial ovarian cancer (EOC) is diagnosed at advanced stages in most patients, resulting in low overall cure rates. Therefore, there is a critical need to improve our understanding of the biology of early-stage epithelial ovarian cancer in order to rationally design experimental approaches and clinical studies to identify and evaluate biomarkers associated with early-stage disease.

Epithelial ovarian cancer constitutes the majority of ovarian malignancies and is classified into distinct morphologic categories consisting of serous, mucinous, endometrioid, clear cell, transitional, squamous, mixed, and undifferentiated subtypes [2]. Although the precise origin of EOC is not fully understood, distinct precursor lesions and de novo carcinogenesis have been proposed for the major histologic subtypes of EOC [2], [3], [4]. Although Mullerian metaplasia of ovarian surface epithelium and its inclusion glands is generally considered as the origin of EOC [2], accumulating evidence suggests that fallopian tubal carcinomas may provide an alternative primary site of origin for serous EOC, particularly in BRCA mutation-positive women [5], [6], [7], [8]. Thus, EOC may arise as a result of de novo carcinogenesis, tubal carcinoma implants, or progression from borderline tumors [3], [9].

In general, EOC can be classified into two types, based on the two main pathways of tumorigenesis: low grade neoplasms that arise in a stepwise manner from borderline tumors are considered type I, whereas high-grade neoplasms without definable precursor lesions are considered type II [4], [10]. Among high-grade neoplasms of the ovary, serous carcinoma represents the most common type of EOC frequently diagnosed at advanced stages due in part to rapid progression, narrower detection window, and deficiencies in understanding the biology of early-stage, high-grade serous carcinoma.

Loss of p53 function is suggested to be an early molecular event associated with de novo carcinogenesis of type II serous, endometrioid, and clear cell carcinomas of EOC, whereas type I EOCs arising from the progression of borderline tumors frequently contain wild-type p53 and may provide precursor lesions for type I EOC [2], [3], [4], [11]. BRCA dysfunction is also considered to be an early event associated with de novo carcinogenesis of type II serous EOC [2], [3]. Results from recent studies also indicate that alterations in proliferative pathways are often associated with early lesions in serous EOC. For example, gene expression analyses of early-stage disease using frozen specimens indicate deregulation of proliferation pathways [12], [13], [14]. Consistent with the results from these studies [12], immunohistochemical studies of early histological tubal lesions in FFPE specimens also indicate changes in proliferative status, as monitored by MiB staining [6].

High-grade stage I serous carcinomas of the ovary are extremely rare, since most patients with high-grade serous carcinomas present with advanced disease. Better definition of genetic alterations in these early-stage, high-grade serous carcinomas is expected to contribute to better insight into the biology of these early diseases which would, in turn, accelerate the discovery of novel biomarkers for screening and detection of early-stage, high-grade EOC. Successful discoveries of novel biomarkers, such as HE4, have been made using high throughput gene expression platform [15], thus representing a proven approach to the identification of additional biomarkers. Toward this goal, we analyzed gene expression of these rare high-grade stage I serous carcinomas by utilizing a newly developed Whole Genome DASL technology, which allows analysis of gene expression from FFPE specimens. To identify genes and pathways associated with serous carcinogenesis, we compared gene expression between high-grade stage I serous carcinomas and stage I serous borderline tumors. Results from our study demonstrated the feasibility of gene expression analysis from FFPE samples using the Whole Genome DASL technology, identified previously known, as well as unknown, biological pathways, and implicated novel biological pathways in carcinogenesis of EOC.

Section snippets

Samples

A total of 5 serous borderline tumors and 5 high-grade stage I serous carcinoma samples were reviewed by a gynecologic pathologist (D.B.). Samples were collected and used in accordance with the approved institutional review board protocols. One to 8 slides of each ovarian neoplasm were reviewed to confirm the original diagnosis, and all of the slides of the surgical procedure were also reviewed to confirm the absence of extraovarian tumor (to confirm that the cancer was stage I). Areas of

Results

Five stage I high-grade serous carcinomas from the approximately 30 cases in the surgical pathology files of Mayo Clinic were randomly selected for review, as well as 5 randomly selected stage I serous borderline tumors. The original pathologic findings were reviewed and confirmed. Serous borderline tumors showed characteristic features of branching papillae lined by moderately atypical cells compared to high-grade serous carcinomas, which were irregularly papillary or solid with stromal

Discussion

In this study, we described the analysis of gene expression in stage I serous tumors from FFPE samples. Traditionally, these samples contain poor-quality RNA not adequate for expression analysis. However, using newly developed Whole Genome DASL expression profiling, we were able to garner reproducible data and biological insights into the molecular profiles of early-stage serous ovarian cancer.

The DASL (cDNA-mediated annealing, selection, extension and ligation) technology is capable of

Conflict of interest statement

Jian-Bing Fan, Craig April, and Brandy Klotzle are employees and shareholders of Illumina, where whole genome DASL assays were conducted. Other authors declare no conflict of interest.

Acknowledgments

This work was funded by the Ovarian Cancer Research Fund Liz Tilberis Scholars (J.C.), the Wallace and Evelyn Simmers Career Development award (J.C.), and Andersen Foundation Program Project award (L.C.H.). We acknowledged statistical assistance from Ann Oberg and bioinformatics assistance from Yan Asmann.

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