Clinicopathological Features of Non-familial Colorectal Cancer with High-frequency Microsatellite Instability△

Peng Jin,Xiao-ming Meng,Jian-qiu Sheng＊,Zi-tao Wu,Lei Fu,He-juan An,Ying Han,and Shi-rong Li

Department of Gastroenterology,The Military General Hospital of Beijing,Beijing 100700,China

MICROSATELLITE instability (MSI),a kind of abnormality of mismatch repair (MMR) system happening during DNA replication,is characterized by the increase or reduction of DNA simple repetitive sequence.This abnormality is present in more than 90% of the patients with hereditary nonpolyposis colorectal cancer (HNPCC) and about 15% of the patients with non-familial colorectal cancer.Tumors with MSI can be generally classified into two groups:those displaying high-frequency MSI (MSI-H) (MSI in more than 30%-40% of the loci examined) and those with low-frequency MSI (MSI-L) (MSI in less than 30%-40% of the loci examined).1In the patients with non-familial colorectal cancer,most MSI-H is caused by MLH1 promoter methylation,while only a small proportion is due to MMR gene mutation.2In the case of HNPCC,the detection of MSI-H may serve as a method for the preliminary screening.3In addition,since MSI-H colorectal cancer shows resistance to cyclooxygenase-2 (COX-2)-related drugs4,5and can increase the number of tumor-infiltrating lymphocytes (TLs),the detection of MSI-H is strongly associated with the choice of chemotherapy regimes and prognostic judgment,6therefore having important clinical significance.A few studies have suggested that the MSI-H colorectal cancer demonstrates specific clinicopathological features,7,8yet all these studies focus on hereditary/familial colorectal cancer.This study was hence designed to explore the clinicopathological features of MSI-H non-familial colorectal cancer.

PATIENTS AND METHODS

Patients and samples

Altogether 150 patients with colorectal cancer who had no family history were enrolled from the Gastrointestinal Endoscopy Center (The Military General Hospital of Beijing)between June 2006 and June 2008.During colonoscopy,if a tumor was found,its surface was first cleared with water,and then 2 samples were taken at the same site of the tumor for routine pathological examination and DNA extraction.One sample of normal mucous membrane was taken at the same time 10 cm away from the tumor to provide the DNA of normal tissue.All the study approaches have been approved by the Ethics Committee of the hospital.All the patients enrolled into the study have signed written informed consents for their participation.

Detection of MSI

DNA was extracted from the tumor tissues and the corresponding normal tissues with phenol-chloroform method as previously described by Giuffrè et al.9Bethesda standard microsatellite loci includingBAT25,BAT26,D2S123,D5S346,andD17S250were amplified using polymerase chain reaction (PCR) according to the method described by Geng et al.10Primers used in this procedure are as follows:BAT25,FAM-5’-TCGCCTCCAAGAATGTAAGT-3’ (sense),5’-TCTGGATTTTAACTATGGCTC-3’ (anti-sense);BAT26,FAM-5’-TGACTACTTTTGACTTCAGCC-3’ (sense),5’-AACCATTCAACATTTTTAACC-3’ (anti-sense);D2S123,FAM-5’-AAACAGGATGCCTGCCTTTA-3’ (sense),5’-GGACTTTCCACCTATGGGAC-3’ (anti-sense);D5S346,FAM-5’-ACTCACTCTAGTGATAAATCGGG-3’ (sense),5’-AGCAGATAAGACAAGTATTACTAG-3’ (anti-sense);D17S250 FAM-5’-GGAAGAATCAAATAGACAAT-3’ (sense),5’-GCTGGCCATATATATATTTAAACC-3’ (anti-sense).The 5’-ends of sense primers were labeled with carboxyfluorescein-aminohexyl amidite (FAM).PCR products were detected with 6% polyacrylamide gel electrophoresis on ABI Prism 3700 DNA Analyzer (Life Technologies Corporation,Carlsbad,CA,USA).Analysis of the electrophoresis results was performed with Gene-Mapper 3.0 software (Life Technologies Corporation).Of the 5 microsatellite loci,MSI in 2 or more than 2 loci was regarded as MSI-H,MSI in one locus was regarded as MSI-L,and the absence of microsatellite instability was regarded as microsatellite stability (MSS).

Pathological analysis of tumor tissue

The pathological features of the sample tumor tissues were observed with the observer blind to MSI status.According to the criteria previously proposed,8the histological grades (well differentiated,moderately differentiated,and poorly differentiated) of the tumors were determined based on the findings in the sample tissues.The presence of mucin in more than 50% of tumor area was regarded as an indicator of mucinous differentiation.8The tumors which presented two or more kinds of different differentiation grades,structural features,and growth patterns were recognized as with histological heterogeneity.7Crohn’s-like reaction was recorded when at least three lymphoid aggregates were discerned on the edge of tumor growth in each section.8

Immunohistochemical assay

Immunohistochemical staining was carried out according to the method described by Young et al11except that we used different anti-CD4 and anti-CD8 monoclonal antibodies and DAB color reagent kit (all from Zhongshan Goldenbrige Biotechnology Co.,Ltd.,Beijing,China).After immunohistochemical staining,normal intestinal mucosa in the tumor tissue slices was referred to as internal control to compare the expressions of CD4 and CD8 between tumor cells and normal cells.In the visual field with 200×magnification (Olympus BX40 microscope,Olympus,Tokyo,Japan),the presence of less than 10 CD4+lymphocytes was recorded as negative,and the presence of more than 10 as positive.12As for CD8,the absence of CD8+lymphocytes was regarded as negative,while the presence as positive.11

Statistical analysis

Statistical analysis was performed with SPSS 13.0 software.Chi-square test was used for analysis of enumeration data.Regression coefficient and regression equation were produced by stepwise logistic regression analysis.

RESULTS

General information of the patients

Of the enrolled 150 patients with non-familial colorectal cancer,73 were men and 77 were women,with a mean age of about 53.2 years (range,23-85 years).The mean age of the patients with MSI-H colorectal cancer was 51 years.Fifty-four patients had right-sided colon cancer and the other ninety-six had left-sided colon cancer.

MSI frequency and its clinicopathological distribution

Among the 150 patients,MSI-H was detected in 20 cases(13.33%),MSI-L was detected in 9 (6.00%),and MSS in the other 121 (80.67%).

Of the 53 patients under fifty,MSI-H occurred in 11 cases (20.8%).As regards the distribution of MSI-H across sex,women accounted for 70.0% of the patients with MSI-H while men accounted for 30.0%.In the 130 patients with no MSI-H,48.5% were female and 51.5% were male.When taking tumor location into consideration,MSI-H occurred in 29.6% of the patients with right-sided colon cancer while in only 4.2% of those with left-sided colon cancer.

According to pathological findings,25.8% poorly differentiated tumor tissues exhibited MSI-H,while only 10.0% well and moderately differentiated tumor tissues showed MSI-H.Mucinous differentiation was noticed in 30.0% cases of MSI-H colorectal cancer,and in 8.46%cases of non-MSI-H colorectal cancer.Histological heterogeneity occurred in 55.0% of MSI-H colorectal cancer,and 10.0% of non-MSI-H colorectal cancer.The proportion of Crohn’s-like reaction in MSI-H colorectal cancer and non-MSI-H colorectal cancer was 55.0% and 22.3%,respectively.

TLs (including CD4+and CD8+lymphocytes) occurred in 70.0% of MSI-H colorectal cancer and in 16.9% of non-MSI-H colorectal cancer.MSI-H occurred in 38.9% of colorectal cancer with positive TLs and in 5.3% of colorectal cancer with negative TLs.

ThePvalue of chi-square test and respective sensitivity and specificity of each clinicopathological feature for identifying MSI-H were shown in Table 1.

Immunohistochemical findings

We noted positive result of tumor-infiltrating lymphocyte CD4+in 5 patients with MSI-H and 8 patients with MSS,but negative in patients with MSI-L.As for another tumorinfiltrating lymphocyte,CD8+,it was observed in 9 patients with MSI-H,2 patients with MSI-L,and 12 patients with MSS.

Table 1.Clinicopathological features of patients with or without MSI-H (n=150)

Stepwise logistic regression equation

Eight clinicopathological features underwent logistic stepwise regression analysis,including diagnostic age less than 50,being female,right-sided colon cancer,poor differentiation,mucinous differentiation,histological heterogeneity,Crohn’s-like reaction,and the presence of TLs,and a regression equation was obtained to identify the microsatellite status of colorectal cancer.According to the analysis results produced by SPSS 13.0 software,poor differentiation,histological heterogeneity,Crohn’s-like reaction,and TLs were singled out as independent factors to identify MSI-H non-familial colorectal cancer.The obtained equation was as follows:P=A/A+1,A=exp (α+β1 poor differentiation+β2 histological heterogeneity+β3 Crohn’s-like reaction+β4 TLs).In the equation,α=－1.872,EXP (α)=0.154;β1=2.292;EXP (β1)=9.893;β2=3.205,EXP (β2)=24.649;β3=3.183,EXP (β3)=24.116;β4=4.407,EXP(β4)=82.043.When the calculatedPvalue was less than 0.05,the tumor was suggested to be MSI-H.The logistic regression equation showed an overall sensitivity of 70.0%,specificity of 99.2%,and accuracy of 95.3% in predicting MSI-H non-familial colorectal cancer.

DISCUSSION

The detection of MSI-H not only may serve as an approach for the preliminary screening of HNPCC,but also is conducive to selecting appropriate chemotherapy regimes and making prognostic judgment,hence the rapid and effective detection of MSI-H phenotype is important for treating colorectal cancer.It has been indicated that MSI-H colorectal cancer has pathological features different from those of non-MSI-H colorectal cancer,7,8suggesting the possibility of identifying MSI-H colorectal cancer based on clinicopathological findings.The present study analyzed the clinicopathological features of Chinese patients with MSI-H non-familial colorectal cancer,shedding some light on clinical practice concerning colorectal cancer.

In this study,the mean age of patients with MSI-H non-familial colorectal cancer was 51 years,younger than the 74 years previously reported by Halvarsson et al,13but slightly older than the 50 years recommended by emendatory Bethesda Guidelines,suggesting that the age of onset of MSI-H colorectal cancer might be different between Chinese and western populations.In the patients with MSI-H colorectal cancer,the ratio of women was 70%,the same as that previously reported by Halvarsson et al,13yet chi-square test indicated no statistical significance in the ratio of MSI-H colorectal cancer to non-MSI-H colorectal cancer in women.In the 20 patients with MSI-H colorectal cancer,16 patients (80%) had right-sided colon cancer,similar to the percentage (74%-87%) reported by Jenkins et al8and Halvarsson et al,13demonstrating that MSI-H may occur more often in right-sided colon cancer.Among the eight clinicopathological features undergoing stepwise logistic regression analysis,the result flagged four as independent risk factors of MSI-H,namely poor differentiation,histological heterogeneity,Crohn’s-like reaction,and TLs.The regression coefficients of these four features were 2.292,3.205,3.183,and 4.407,respectively,and their probabilities to separately identify MSI-H were 60.4%,79.1%,78.8%,and 92.7%,respectively.The logistic regression equation could play a role in the identification of microsatellite status,the choice of chemotherapy regimes,and prognostic judgment.

It is worth pointing out that TLs have been considered to be a predictor of good prognosis of MSI-H colorectal cancer.The sensitivity of 5 TLs/10 high power field (HPF)was reported at 93% and 81% in two previous studies,respectively,and the specificity of 5 TLs/10HPF at 62% and 60%,respectively,in the identification of MSI-H colorectal cancer.14,15Although the above-mentioned two studies employed HE staining in visualizing TLs,different from the immunohistochemical approach applied in our study,the results obtained were similar,indicating the consistency of the results produced by the two different methods and further demonstrating that TLs have important significance in the identification of MSI-H colorectal cancer.

In addition,it has been demonstrated that there are two different pathogenic pathways in MSI-H non-familial colorectal cancer and MSI-H HNPCC,namely the serrated adenoma-carcinoma pathway of DNA methylation and BRAF gene mutation in MSI-H non-familial colorectal cancer,and the traditional adenoma-carcinoma pathway of MMR gene,adenomatous polyposis coli(APC),β-catenin,and K-rasmutation in MSI-H HNPCC.16,17Comparison between the two types of colorectal cancer revealed that female,poor differentiation,mucinous differentiation,histological heterogeneity,and serrated adenoma around cancer tissue are more common and the age at diagnosis older in MSI-H non-familial colorectal cancer,while Crohn’s-like reaction,TLs,and traditional adenoma around cancer tissue are more common in MSI-H HNPCC.17It is noteworthy that MSI-H HNPCC is more like common type of colorectal cancer,while MSI-H non-familial colorectal cancer possesses specific features.Although the detections of MLH1 promoter methylation and BRAF gene mutation could help to distinguish the two types of colorectal cancer,clinicopathological features may serve as a powerful supplementary tool.

The results of this study suggest that MSI-H nonfamilial colorectal cancer demonstrates specific clinicopathological features.Effective identification of MSI-H nonfamilial colorectal cancer could therefore be realized on the basis of logistic regression equation of these features.

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