A molecular signature for the prediction of recurrence in colorectal cancer
Lisha Wang
0
1
Xiaohan Shen
0
1
Zhimin Wang
Xiuying Xiao
Ping Wei
0
1
Qifeng Wang
0
1
Fei Ren
0
1
Yiqin Wang
0
1
Zebing Liu
0
1
Weiqi Sheng
0
1
Wei Huang
Xiaoyan Zhou
0
1
Xiang Du
0
1
0
Department of Oncology, Shanghai Medical College, Fudan University
,
Shanghai 200032
,
China
1
Department of Pathology, Fudan University Shanghai Cancer Center
,
Shanghai 200032
,
China
Background: Several clinical and pathological factors have an impact on the prognosis of colorectal cancer (CRC), but they are not yet adequate for risk assessment. We aimed to identify a molecular signature that can reliably identify CRC patients at high risk for recurrence. Results: Two hundred eighty-one CRC samples (stage II/III) were included in this study. A two-step gene expression profiling study was conducted. First, gene expression measurements from 81 fresh frozen CRC samples were obtained using Affymetrix Human Genome U133 Plus 2.0 Arrays. Second, a focused gene expression assay, including prognostic genes and genes of interest from literature reviews, was performed using 200 fresh frozen samples and a Taqman low-density array (TLDA) analysis. An optimal 31-gene expression classifier for the prediction of recurrence among patients with stage II/III CRC was developed using logistic regression analysis. This gene expression signature classified 58.5% of patients as low-risk and 41.5% as high-risk (P < 0.001). The signature was the strongest independent prognostic factor in the multivariate analysis. The five-year relapse-free survival (RFS) rates for the low-risk patients and the high-risk patients were 88.5% and 41.3% (P < 0.001), respectively. Conclusion: We identified a 31-gene expression signature that is closely associated with the clinical outcome of stage II/III CRC patients.
-
Introduction
Colorectal cancer (CRC) is the third most common type
of cancer, with a worldwide annual incidence of over 1.2
million cases and a mortality rate of approximately 50%
[1,2]. The growing awareness that CRC is a
heterogeneous disease with respect to clinical behavior and
prognosis translates into an urgent need for robust molecular
subclassifiers in addition to the current parameters. To
date, some clinical and pathologic features, such as
intestinal perforation/obstruction, adjacent organ
involvement (T4), high tumor grade, lymphatic/vascular invasion
and inadequate sampling of lymph nodes, can identify a
minority of CRC patients who are at a high risk of
recurrence [3,4]. However, these prognostic factors are all
relatively weak.
To address this issue, the prognostic potential of
molecular markers, including chromosome and
microsatellite instability (MSI) and the mutation status of the
KRAS or BRAF genes, has been systematically
investigated in CRC [5-8]. Thus far, only KRAS mutation
analysis has been used in clinical practice as a predictive
marker of the effect of EGFR antibodies in metastatic
disease [5,9]. Analyses of other known critical CRC
molecular markers are not currently recommended for
screening or for prognostic prediction because they
require further validation.
With the recent advent of microarray technology, risk
assessment for CRC has been improved by the use of
gene expression profiling. DNA microarray technology
can measure thousands of mRNA transcripts at once
and may be able to describe the complex biology of a
tumor more accurately than single markers [10,11]. In
the current study, we used gene expression analysis data
from recurrent and non-recurrent patients with CRC to
identify differentially expressed probes. To further
validate gene expression, we selected 48 genes that could be
assayed using a TaqMan low-density array (TLDA), a
real time quantitative PCR (RT-qPCR) based technology,
using fresh frozen CRC tissues.
Patients and methods
Patients and tumor samples
Samples were prospectively collected between 2007 and
2009 at Fudan University Shanghai Cancer Center. The
inclusion criteria were as follows: primary sporadic
colorectal adenocarcinoma (excluding familial adenomatous
polyposis (FAP) and hereditary nonpolyposis colorectal
cancer (HNPCC)), 18 to 75 years of age, no preoperative
chemotherapy and radiotherapy, and similar
postoperative chemotherapy regimens. The patients were staged
according to the American Joint Committee on Cancer/
International Union against Cancer (AJCC/UICC) TNM
staging system- seventh edition (2010). Histologic
grading (differentiation) was based on the WHO
classification of tumors of the digestive system-fourth edition
(2010). This study was approved by the Ethical
Committee of our Cancer Center, and written informed
consent was obtained from each patient.
Microarray gene expression profiling
Tumor tissues were taken from 81 patients with CRC,
rapidly frozen in RNAlater, and stored at 80C until
processing. All samples were visually inspected by two
pathologists, who confirmed the presence of tumor cells
(70%) in all samples. RNA was isolated using Trizol
(Life Technologies, Car (...truncated)