Heterogeneity between primary colon carcinoma and paired lymphatic and hepatic metastases
- Authors:
- Published online on: August 28, 2012 https://doi.org/10.3892/mmr.2012.1051
- Pages: 1057-1068
Abstract
Introduction
Intratumor heterogeneity is one of the recognized characteristics of human tumors, which occurs on multiple levels, including genetic, protein and macroscopic, in a wide range of tumors, including breast, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), prostate, ovarian, pancreatic, gastric, brain and renal clear cell carcinoma (1). Over the past decade, a number of studies have focused on the heterogeneity found in primary tumors and related metastases with the consideration that the evaluation of metastatic rather than primary sites may be of clinical relevance. Numerous reports have evaluated the genetic heterogeneity in primary tumors and corresponding metastases in a range of solid tumors, including breast cancer (2–9), CRC (10–13) and NSCLC (14,15).
In CRC, the heterogeneity of the epidermal growth factor receptor (EGFR) gene status and its downstream signalling proteins, including KRAS, BRAF and PIK3CA mutations, have been identified by comparing primary tumors with corresponding metastases, respectively (10–13). Baldus et al demonstrated particularly high rates of heterogeneity between primary colorectal tumors and lymph node metastases (10). Molinari et al analyzed EGFR gene status and protein expression, KRAS/BRAF mutations and PTEN expression in primary tumors and metastases in 38 metastatic CRC patients, and found EGFR gene deregulation in 69.4% of primary tumors and 80.6% of metastases, and KRAS mutations in 43.2% of primary tumors and 40.5% of metastases (11). However, the heterogeneity in primary CRC and corresponding metastases at the genome-wide gene expression level has not been extensively investigated.
Therefore, in the present study, we investigated the heterogeneity in primary colon carcinoma tissue and its corresponding lymphatic and hepatic metastatic tissues obtained from a female metastatic colon cancer patient, focusing on genome-wide gene expression.
Patients and methods
Patient and tissue samples
Tumor specimens were obtained at initial surgery from a 40-year-old female colon carcinoma patient with lymphatic and hepatic metastases. Samples were snap-frozen and stored in liquid nitrogen until use. Prior written informed consent was obtained from the patient and the study received ethics board approval at the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. The patient had not received chemotherapy or radiation therapy prior to surgery. The histological type was determined according to the WHO criteria. The tumor was diagnosed as mucinous adenocarcinoma (T3N2M1).
Genome-wide gene expression analysis
RNA was extracted from tumor specimens. Total RNA of homogenized tumor samples was prepared with TRIzol RNA extraction reagent (Invitrogen) followed by purification using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. A DNase I (Qiagen) digestion step was included to eliminate genomic DNA. The quality of the total RNA was examined for integrity using RNA LabChips and the Agilent Bioanalyzer 2100 (Agilent Technologies), and the concentration was measured using the Peqlab NanoDrop. Only RNA with an RNA integrity number >6.5 was used for cDNA synthesis. The one-cycle eukaryotic target labeling assay from Affymetrix was used according to manufacturer’s instructions as previously described (16,17). Data analysis was also performed as previously described (16,17).
Statistical analysis
Hierarchical clustering of all microarray experiments was performed based on all 54,675 probe sets represented on the HGU133Plus2.0 array (Affymetrix) (P<0.04) using positive correlation and complete linkage. Gene expression of the primary tumor was compared with the median arrays of the paired lymphatic metastatic and hepatic metastatic tumors from each xenograft model in a paired t-test.
Results
Heterogeneity in primary colon carcinoma and its paired lymphatic and hepatic metastases
Based on the results of the genome-wide gene expression analysis, a paired t-test between the primary colon carcinoma and its paired lymphatic metastasis revealed 190 differentially expressed probe sets with a fold change ≥20. Clustering based on these 190 probe sets demonstrated a clear distinction between primary colon carcinoma and its paired lymphatic metastasis, with the majority of the probe sets (115 of 190) being downregulated in the lymphatic metastasis and only a small number being upregulated (75 of 190) (Table I). A paired t-test between the primary colon carcinoma and its paired hepatic metastasis revealed 150 differentially expressed probe sets with a fold change ≥20. Clustering based on these 150 probe sets demonstrated a clear distinction between primary colon carcinoma and its paired hepatic metastasis, with the majority of probe sets (94 of 150) being downregulated in the hepatic metastasis and only a very small number being upregulated (56 of 150) (Table II). A paired t-test between colon carcinoma lymphatic metastasis and hepatic metastasis revealed 176 differentially expressed probe sets with a fold change of only ≥10. Clustering based on these 176 probe sets demonstrated a clear distinction between colon carcinoma lymphatic metastasis and hepatic metastasis, with the majority of probe sets (114 of 176) being downregulated in the hepatic metastasis and only a very small number being upregulated (62 of 176) (Table III).
 | Table IGenes differentially expressed in the primary colon carcinoma specimen and its paired lymphatic metastasis. |
| Table IIGenes differentially expressed in the primary colon carcinoma specimen and its paired hepatic metastasis. |
| Table IIIGenes differentially expressed in the colon carcinoma lymphatic metastasis specimen and the hepatic metastasis specimen. |
Discussion
The purpose of this study was to investigate the heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases using GeneChip HGU133Plus2.0 expression arrays (Affymetrix). Our results demonstrate that the genome-wide gene expression varies between primary colon carcinoma and its paired lymphatic and hepatic metastases. In our study, we firstly demonstrate that a high degree of heterogeneity in gene expression exists in primary colon carcinoma and corresponding lymphatic and hepatic metastases.
The main purpose of investigating the heterogeneity found in primary tumors and corresponding metastases is to evaluate the effect of such heterogeneity on the efficacy of anticancer therapy and cancer patient prognosis. As previously discussed (1,16,18), the patient-derived tumor tissue (PDTT) xenograft model, which has a sound establishing method and a retained similarity to the corresponding original donor tumors in histological presentation and biological behavior, including protein expression, tumor biomarker status and genomic and genetic status, has the potential to be an ideal strategy to achieve our purpose. In our previous study, we successfully established PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastases using the same tissue samples from three above-mentioned tumor sites (16). The ideal biological characteristics of such PDTT xenograft models, as previously described (16), led to the consideration that such PDTT models may aid in the investigation of the underlying mechanism of heterogeneity-related anticancer therapy response differences in primary colon carcinoma and corresponding lymphatic and hepatic metastases.
In conclusion, in this study, we investigated the heterogeneity in primary colon carcinoma and corresponding lymphatic and hepatic metastases focusing on genome-wide gene expression, and revealed that there were differences in primary colon carcinoma and matched lymphatic and hepatic metastases. In subsequent studies, further investigation is required to ascertain the heterogeneity of functional genes found in primary colon carcinoma and paired lymphatic and hepatic metastases in a larger group of patients with metastatic colon carcinoma, in order to identify specific functional genes related to lymphatic and hepatic metastases.
Acknowledgements
The present study was supported by the State Key Basic Research and Development Program of China (973 Program, Grant No. 2009CB521704), the National High-tech Research and Development Program of China (863 Program, Grant No. 2006AA02A245), the National Natural Science Foundation of China (Grant No. 81000894), the Zhejiang Provincial Science and Technology Projects (Grant No. 2009C13021, 2011C23087), the Science Research Fund of Shaoxing (Grant No. 2011D10013) and the Science Research Fund of Zhuji (Grant No. 2011CC7874).
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