Gene Expression Analysis of Sporadic Early-Onset Rectal Adenocarcinoma

Research Article

Gastrointest Cancer Res Ther. 2016; 1(1): 1005.

Gene Expression Analysis of Sporadic Early-Onset Rectal Adenocarcinoma

Nfonsam V1,2*, Xu W³, Koblinski J² and Jandova J1,2

¹UA Cancer Center, 1515 N Campbell Avenue, Tucson, AZ 85724, USA

²UA Department of Surgery, Division of Surgical Oncology, 1501 N Campbell Avenue, Tucson, AZ 85724, USA

³NanoString Technologies, 530 Fairview Avenue North, Seattle, WA 98109, USA

*Corresponding author: Valentine Nfonsam, University of Arizona, Department of Surgery, Division of Surgical Oncology, 1501 N Campbell Avenue, AHSC 4334, Tucson, AZ 85724, USA

Received: June 24, 2016; Accepted: August 01, 2016; Published: August 03, 2016

Abstract

Background: Overall declines in incidence of rectal cancer (RC) in patients older than 50 years have been mostly attributed to improvement in treatment modalities and introduction of age-based screening. Recent studies, however, have shown a rise in the incidence of RC in patients younger than 50 years. The etiology of early-onset (EO) RC is not well understood. The aim of this study is to elucidate the molecular features of (EO) RC and show its uniqueness compared to late-onset (LO) disease.

Methods: Two cohorts of patients with sporadic RC were identified. Tumors and matching non-involved tissues from six (EO) RC patients (< 50 years) and six (LO) RC patients (>65 years) were obtained from Pathology archives. Deparaffinized tissues were macro-dissected from FFPE sections, RNA isolated and used for expression profiling of 770 cancer related genes representing 13 canonical pathways. Statistical analysis was performed using the Gene Expression R-script module within the nCounter software v2.6. A gene was considered to be above background if the average count for the target gene was greater than the average counts for the eight negative control genes and if the P value of the t-test was less than 0.05.

Results: When we compared rectal tumors to non-involved rectal tissues, changes in expression levels of 171 genes were statistically significant in earlyonset group and 151 genes in late-onset group. Further comparative gene expression analysis between early- and late-onset rectal tumors normalized to their matching non-involved tissues revealed that changes in expression of 65 genes were unique to early-onset rectal tumors with 16 genes being up- and 49 genes down-regulated using the cutoff criteria of expression levels difference >2 fold and p-value <0.01. At the pathway level, MAPK signaling was the most deregulated pathway in early-onset rectal tumors compared to PI3K-AKT signaling pathway being the most deregulated in late-onset rectal tumors.

Conclusions: Results of this study suggest that sporadic early-onset rectal cancer is characterized by distinct molecular events compared to late-onset disease.

Keywords: Sporadic early- and late-onset rectal cancer; Gene expression profiling; Pathway analysis

Introduction

Colorectal cancer (CRC) remains the third most commonly diagnosed cancer and the third most frequent cause of cancer related death in the United States with approximately one-third of patients with CRC having their cancer limited to the rectum [1-4]. Overall, CRC incidence and mortality rates are higher in men than in women [5] with higher percentage (31%) of rectal tumors being diagnosed in men compared to women (24%) [6]. In 2015, 23,200 new rectal cancer cases were predicted to be diagnosed in men and 16,410 in women in the United States [7]. There are also disparities in the age at which CRC is diagnosed with the median age at diagnosis being lower for rectal cancer (63 years in men and 65 years in women) and higher for colon cancer (69 years in men and 73 years in women) [8].

Even though there has been a significant progress in reducing the overall incidence of CRC over the past two decades, recent studies have reported an alarming increase in incidence within a population of CRC patients younger than 50 years [9-11]. Several studies have indicated a rise in colon cancer in younger patients; however, to the best of our knowledge, few have specifically addressed the incidence of cancer involving the rectum [10-13]. Meyer et al, in their 2010 study, analyzed the Surveillance Epidemiology and End Results (SEER) Program Cancer Registries data for incidence of rectal cancer between 1973 and 2005 and showed a significant increase in incidence of rectal and recto-sigmoid cancer in patients aged <40 years [13]. Our 2011 study demonstrated that the incidence of rectal cancer is continuously increasing in every age group (5-year intervals) from 20 years to 49 years, with the most impressive increase seen within the age group 40-44 years [11]. Our comprehensive 15-year analysis of the Arizona Cancer Registry revealed an alarming 225% increase in incidence of rectal cancer in the age group 30-34 years [14].

Overall declines in incidence of CRC in patients older than 50 years have been mostly attributed to improved treatment modalities and the introduction of age-based screening using fecal occult blood testing, sigmoidoscopy, or colonoscopy [15].

Biomarkers have been shown to be promising for cancer screening and diagnosis [16]. However, despite numerous significant technological and methodological advances, CRC research has not yielded a novel molecular biomarker or biomarker panel suitable for population-wide screening purposes [17]. Cytogenetic alterations such as microsatellite instability (MSI), chromosomal instability (CIN), and the CpG island methylator phenotype (CIMP) have been considered as potential CRC molecular markers as they can help with diagnostic, prognostic, and predictive treatment response information [18-20]. Molecular tests are expected to provide genetic information about the malignancy in progression. Intense research efforts aiming at identifying molecular markers (DNA, RNA, or protein) to develop novel, noninvasive biomarker detection methods for CRC in blood and stool are underway. Molecular markers can be used to assess the risk of malignancy, its aggressiveness over the time, and the probability that a patient will respond to a particular treatment what can result in helping physicians to make personalized treatment decisions. Even though there have been some promising advances in the use of biomarkers for detection of CRC, comprehensive molecular studies using larger cohorts of patients are necessary to determine if a single marker or a group of biomarkers can serve as first line test to diagnose CRC. The current conventional molecular tests used for evaluation of CRC patients include microsatellite instability (MSI) analysis and BRAF and KRAS mutation analysis. Innovative tumorbased molecular tests include CIMP, RNA expression, miRNAs and EGRF pathway biomarkers to predict response to anti-EGFR treatment in CRC patients with wild type KRAS [17].

The aim of this study was to perform a comprehensive genomic and pathway analysis of early- and late-onset rectal tumors in an attempt to answer questions about the etiology and uniqueness of early-onset rectal cancer and also look at possibilities for targeted and personalized treatments in this cohort of patients.

Materials and Methods

Patient samples

De-identified FFPE rectal cancer samples and matching noninvolved rectal tissues from a group of patients younger than 50 years (early-onset group) and a group of patients older than 65 years (lateonset group) were obtained from the University of Arizona Pathology archives. Patients with Lynch syndrome, familial adenomatous polyposis (FAP) and inflammatory bowel disease (IBD) were excluded. Patient samples were matched by stage of disease, gender and pathology. There was almost equal representation of males (n=2 for EO and n=3 for LO group) and females (n=4 for EO and n=3 for LO group), equal representation of stage II (n=2 for each age group) and stage III (n=4 for each age group) samples with the pathology of moderately differentiated adenocarcinoma for all samples (Table 1).