Colorectal cancer (CRC) is a leading cause of cancer-associated mortality (
Defects in the DNA mismatch repair (MMR) system may arise sporadically or in patients with hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, may be inherited in an autosomal-dominant manner (
CRC patients with microsatellite stability (MSS) and
A total of 205 patients with CRC (121 males and 84 females; mean age, 62.6 years; range, 25.4–90.1 years) from the Gastrointestinal Department of Cathay General Hospital (Taipei, Taiwan) were enrolled from January 2006 to December 2008 in the current study. Survival data were acquired from 176 patients and others were lost to follow-up due to referral. The mean follow-up time was 17.0±15.6 months (median, 10.5 months). Suspicious growths in patient colonic tissues were sampled with small biopsy forceps inserted through a colonoscope. The tissues were formalin-fixed, paraffin-embedded and cut into slices of 4–5-µm thickness for immunohistochemical staining, or immersed in RNAlater® solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for genomic DNA preparation, according to the manufacturer's protocol. Presence of distant metastasis was routinely confirmed by abdominal computed tomography. In addition, blood samples were collected from each patient to serve as controls when determining the microsatellite status. The study protocol was approved by the Institutional Review Board of Cathay General Hospital, and informed consent was obtained from all patients prior to obtaining tissue samples.
Cells from the human colorectal carcinoma HCT116 [American Type Culture Collection (ATCC) no. CCL-247; MSI) and SW480 (ATCC no. CCL-228; MSS) cell lines were purchased from the ATCC (Manassas, VA, USA) and maintained as recommended by their guidelines (
Sections were dewaxed in xylene and rehydrated through a graded series of ethanol concentrations (100, 95 and 70%) to water. Antigen retrieval was performed by immersing the slides in BD Retrievagen A (pH 6.5 for MLH1 and pH 6.0 for MSH2; BD Biosciences, San Jose, CA, USA) and heating the slides in a microwave oven for 30 min at 95°C. Sections were treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. Sections were washed in PBS and subsequently placed in 20% normal goat serum (G9023; Sigma-Aldrich; Merck Millipore) in PBS for 20 min to reduce non-specific staining. Sections were then incubated with monoclonal antibodies raised against human (h) MLH1 (1:10; clone G168-15; BD Biosciences) for 1 h 32 min at 42°C or against MSH2 (1:25; clone 25D12; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, visualization was performed using the EnVision+Dual Link system-HRP (Dako) according to the manufacturer's protocol, using 3,3′-diaminobenzidine as a chromogen. Finally, slides were counterstained with hematoxylin solution. Both MLH1 and MSH2 were scored as either negative or positive staining. Tissue specimens were analyzed by two independent pathologists blinded to the conditions.
Colonic tissues pathologically diagnosed as CRC were subjected to the assessment of microsatellite status and identification of hotspot mutations of
The components of the reaction for the different genotypes were identical except for the respective allele-specific probes, as described below. The primer sequences were: Forward, 5′-CATGAAGACCTCACAGTAAAAATAGGTGAT-3′ and reverse, 5′-TGGGACCCACTCCATCGA-3′. Allele-specific TaqMan® probes (VIC®-labeled reporter T allele, 5′-CTAGCTACAG[T]GAAATC-3′ and 6-carboxyfluorescein-labeled reporter A allele, 5′-TAGCTACAG[A]GAAATC-3′) (Applied Biosciences) and TaqMan Genotyping Master Mix (Applied Biosciences) were used in a 7300 Real-Time PCR system (Applied Biosciences; Thermo Fisher Scientific, Inc.) (
The association between tumor microsatellite status and patient clinicopathological features was analyzed using a χ2 test. Patient survival time was calculated from the date of complete resection of CRC tumors to the date of last follow-up, with the only patient who succumbed to CRC being excluded from counting towards disease-specific survival (DSS). DSS distributions were estimated using a Kaplan-Meier method and compared using a log-rank test. P<0.05 was considered to indicate a statistically significant difference. All calculations were performed using IBM SPSS Statistics for Windows version 22.0 (IBM SPSS, Armonk, NY, USA).
One patient (age, 58.4 years; female) out of the 205 enrolled patients met the clinical diagnostic criteria for HNPCC (
Significantly different mutation rates of
In the present study, survival data were available for 176 patients with CRC (154 patients with MSS and 22 patients with MSI). Patients with MSS tended to have poorer DSS (57.4±7.0%) compared with patients with MSI, although this difference was not significant (P=0.065;
Due to the clinical significance of microsatellite status and
In addition, distinct expression patterns for KRAS and MSH2 in HCT116 cells treated with 76.9 µM 5-FU for 2 days or 1.4 µM oxaliplatin for 3 days were observed (
It has previously been demonstrated that the microsatellite status of CRC patients responds to specific chemotherapy (
It has been demonstrated that patients with different microsatellite statuses respond differently to chemotherapeutics (
Two first-line chemotherapeutic agents, 5-FU and oxaliplatin, induce a cytotoxic response, and may be used to treat CRC cells through the stable correction of MMR activity (
In conclusion, the microsatellite status and the mutation of
The present study was supported by grants from the Cathay General Hospital (Taipei, Taiwan; grant nos. CGH-MR-9419 and CGH-MR-9701 awarded to C.-L. L.).
colorectal cancer
mismatch repair
hereditary nonpolyposis colorectal cancer
microsatellite instability
mitogen-activated protein kinase pathway
microsatellite stability
disease-specific survival
Immunohistochemical stain of CRC tissue for hMLH1 expression. The left panel presents a positive control for hMLH1 expression. The right panel presents the immunohistochemical staining results of a paraffin-embedded CRC tissue section acquired from a patient with HNPCC (pt 162). Both slides were counterstained with hematoxylin. Bar, 100 µm. HNPCC, hereditary nonpolyposis colorectal cancer; CRC, colorectal cancer; MSI-H, high microsatellite instability; pt, patient; hMLH1, human MutL homolog 1.
Disease-specific survival of non-HNPCC patients according to MSS. Disease-specific survival was defined as the time from surgery to either CRC-associated mortality or end of follow-up. Survival probabilities were estimated by the Kaplan-Meier method and compared using the log-rank test according to MSS in non-HNPCC patients. Patients with available survival data were stratified into two groups: MSI (n=22) and MSS (n=154) (P=0.065). CRC, colorectal cancer; MSI, microsatellite instability; MSS, microsatellite stability; HNPCC, hereditary nonpolyposis colorectal cancer.
Disease-specific survival of 154 MSS patients according to
Relative protein quantitation in different cellular compartments of SW480 cells following treatment with chemotherapy agents. Determination of KRAS expression under (A) 5-FU and (B) oxaliplatin treatment. Determination of MSH2 expression under (C) 5-FU and (D) oxaliplatin treatment. SW480 cells were treated with 17.5 µM 5-FU or 150 µM oxaliplatin. The different cellular fractions (cytoplasmic and nuclear) were separately harvested. A 10-µg sample of each fraction was electrophoresed, and each protein band was quantified by densitometry using image processing FluorChem FC2 software. Relative protein levels were determined by normalizing their expression to that of α-tubulin (for the cytoplasmic fraction) or lamin A/C (for the nuclear fraction) (**P<0.001). Cyto, cytoplasm; Nu, nucleus; DMSO, dimethyl sulfoxide; 5-FU, 5-fluorouracil; MSH2, MutS protein homolog 2.
Relative protein quantitation in different cellular compartments of HCT116 cells under treatment with chemotherapeutic agents. (A) Determination of KRAS expression under 5-FU treatment. (B) Determination of KRAS expression under oxaliplatin treatment. (C) Determination of MSH2 expression under 5-FU treatment. (D) Determination of MSH2 expression under oxaliplatin treatment. HCT116 cells were treated with 76.9 µM 5-FU or 1.4 µM oxaliplatin. Different cellular fractions (cytoplasmic and nuclear) were separately harvested. A 10-µg sample of each fraction was electrophoresed, and each protein band was quantified by densitometry using image processing FluorChem FC2 software. Relative protein levels were determined by normalizing their expression to that of α-tubulin (for the cytoplasmic fraction) or lamin A/C (for the nuclear fraction) (*P<0.05, **P<0.001). Cyto, cytoplasm; Nu, nucleus; DMSO, dimethyl sulfoxide; 5-FU, 5-fluorouracil; MSH2, MutS protein homolog 2.
Clinicopathological characteristics of patients.
Feature | Number of patients |
---|---|
Age, years | |
≤62.6 | 100 |
>62.6 | 105 |
Gender | |
Male | 121 |
Female | 84 |
AJCC staging | |
I+II | 73 |
III+IV | 88 |
Microsatellite status | |
MSS | 179 |
MSI | 26 |
Wild type | 141 |
Mutant | 64 |
Differentiation | |
Well/moderate | 157 |
Poor | 15 |
Tumor location | |
Right | 48 |
Left | 91 |
Rectum | 66 |
Tumor size, cm | |
≤4.3 | 89 |
>4.3 | 82 |
The number of cases that were assessed in each category was dependent on the number of available cases. All information on female patients includes clinical information regarding the aforementioned patient with hereditary nonpolyposis colorectal cancer. AJCC, American joint committee on cancer; MSS, microsatellite stability; MSI, microsatellite instability.
High percentage of patients with MSS in different CRC groups.
Feature | Percentage, % (n) | P-value |
---|---|---|
Microsatellite status vs. |
||
MSS | 79.7 (51/64) | 0.027 |
MSI | 20.3 (13/64) | |
Microsatellite status vs. distal/rectal sites | ||
MSS | 90.4 (142/157) | 0.015 |
MSI | 9.6 (15/157) |
MSS, microsatellite stability; MSI, microsatellite instability.
Mutation rate of
Feature | P-value | Odds ratio | 95% CI | |
---|---|---|---|---|
Age, years |
||||
≤62.6 | 23.0 (23/100) | 0.013 | 2.15 | 1.17–3.94 |
>62.6 | 39.0 (41/105) | |||
Differentiation |
||||
Well/moderate | 27.4 (43/157) | 0.036 | 3.03 | 1.04–8.86 |
Poor | 53.3 (8/15) |
Mean age, 62.6 years.
The number of assessed cases was dependent on the number of available cases. CI, confidence interval.