Yonago Acta medica 2003;46:117–124

Mismatch Repair Deficiency in Patients with Double Primary Cancer of the Colorectum and Stomach
Hiroki Takemoto and Masato Makino
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan
We examined the correlation between the expression of mismatch repair (MMR) geneproteins and the development of double primary cancer, and studied clinical implicationsof an MMR deficiency in 15 patients with double primary cancer of the colorectum andstomach, immunohistochemically. The results were compared between the double primarycancer group of 15 patients and the control group consisting of 155 colorectal cancer (CRC)
patients who had never developed other malignant diseases. Patients with hereditary
nonpolyposis colorectal cancer and familial adenomatous polyiposis were excluded fromboth groups. The MMR deficiency in CRC was significantly more frequently detected in
the double primary cancer group than in the control group (46.7% versus 20.6%, P <0.05). Patients with MMR-deficient CRC of the double primary cancer group weresignificantly older, more frequently had poorly differentiated lesions, had less metastases
to the liver and lymph node, and were more advanced in depth of invasion than those of
the control group. We concluded that MMR deficiency might correlate with thedevelopment of double primary cancer of the colorectum and stomach. Patients with
MMR-deficient CRC need periodical and intensive follow-up against the development of
double primary cancer.
Key words: colorectum; double primary cancer; mismatch repair deficiency; stomach

Hereditary nonpolyposis colorectal cancer(HNPCC) is one of the most common predisposition syndromes for cancer caused by germ-line
mutations in DNA mismatch repair (MMR) genes(Leach et al., 1993; Bronner et al., 1994; Nicolaideset al., 1994). This syndrome is characterized bydevelopment at an early age, frequent occurrence
on the right side, histology of poorly differentiatedand mucinous adenocarcinomas and a favorable
prognosis (Vasen et al., 1991; Kunitomo et al.,
1992; Lynch et al., 1993, 1996). Another feature ofHNPCC is the development of multiple primarycolorectal cancer (CRC) (Horii et al., 1994; Brownet al., 1998; Masubuchi et al., 1999; Pedroni et al.,1999). Nearly 5% to 10% of CRC patients develop
a 2nd primary CRC within 10 years after surgicalremoval of the 1st tumor (Tsukuma et al., 1994),
and genetic instability might play an important rolein developing multiple primary CRC: synchronoustumors tend to have more frequent microsatellite
instability (MSI) than metachronous ones (Horii etal., 1994). HNPCC is also characterized by frequently occurring extracolonic tumors, especiallyof the stomach, small intestine, upper urologic tract(renal pelvis and ureter) and endometrium (Vasenet al., 1991; Lynch et al., 1996). Women with endo-Abbreviations: CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; MMR, mismatch repair;
MSI, microsatellite instability117

H. Takemoto and M. Makino

metrial cancer and CRC who had their 1st tumor
diagnosed before age 50 demonstrated a high frequency of MSI (Planck et al., 2002).
Gastric cancer is the most prevalent extracolonic malignancy in double primary cancer withcolon cancer (Duval et al., 2002). Several studies
have reported relations between MSI and gastriccancer: PCR analysis of primary gastric cancer detected one or more MSIs associated with an increased occurrence of remnant gastric cancer (Nakachi etal., 1999). According to Kim et al. (2002), Helicobacter pylori infection might lead to a deficiency ofDNA MMR in gastric epithelial cells, which might
increase the risk of mutation accumulation in gastric mucosa cells and the risk of gastric cancer. But
reports were few on the contribution of MSI in
double primary cancer of the colorectum and stomach.
In the present study, we have immunohistochemically detected an MMR deficiency in double
primary cancer of the colorectum and stomach, andanalyzed clinical implications of the deficiency inthe cancer.

period, a total of 155 CRC patients exclusive of
those with HNPCC and familial adenomatous polyposis, served as controls. The patients developed
no other malignant diseases before the surgery or
during the follow-up period.
After the resection, all specimens were fixedin 10% neutralized formalin and embedded in paraffin. A representative block of each tumor was selected and used to evaluate the expression ofhMLH-1 and hMSH-2.

Immunohistochemistry
The paraffin-embedded sections were dewaxed, rehydrated through a graded alcohol series and washed with distilled water. Antigen retrieval was performed by microwave oven (700 W) for 12 min.
Endogenous peroxidase was blocked by methanol
containing 2% hydrogen peroxide. Tissues were
further blocked by incubation for 30 min with 1%
bovine serum albumin in phosphate-buffered saline. Sections were incubated overnight at 4˚C with
one of the primary antibodies, hMLH-1 (PharMingen,San Diego, CA) at a dilution of 1/100 and hMSH-2(Oncogene Sciences, Cambridge, MA) at a dilutionof 1/200. Antibody binding was detected using aVectastain Elite ABC kit (Vector Laboratories Ltd,Burlingame, CA), based on the biotin-avidin system (manufacturer’s protocol). Sections werestained with a solution of diaminobenzidine andhydrogen peroxide solution, and then counterstained with methyl green. Normal colorectal tissue
adjacent to the cancer was used as a positive control. Loss of expression in the tumor was recorded
when staining was negative in malignant cells, but
positive in adjacent normal epithelial cells (Figs. 1and 2).

Materials and Methods

Patients and specimens
Specimens were 15 lesions each of CRC and gastriccancer removed from 15 patients with double primary cancer in surgery with curative intent between
January 1990 and December 1996 at the First
Department of Surgery, Tottori University Hospital. Lesions of HNPCC and familial adenomatous
polyposis were excluded. All patients were followed-up until December 2001. CRC was clinicopathological determined according to the criteria of
the Japanese Society for Cancer of the Colon andRectum (1998); and gastric cancer, with the rules of
the Japanese Research Society for Gastric Cancer(1999). Double primary cancers of the colorectumand stomach were synchronous in 5 patients andmetachronous in 10 patients.
From patients who had undergone surgicallycurative resection in our hospital during the same

Statistical Analysis
The differences of MMR deficiency between
groups and the associations between clinicopathological factors and hMLH-1 and/or hMSH-2 expression were evaluated by chi-square test. Survival
curves were constructed by Kaplan-Meier’s method
and the differences were examined by log rank test.
118MMR deficiency in double cancerabFig. 1. Immunohistochemical staining of hMLH1 in colorectal cancer (CRC). hMLH1 protein reveals abundantnuclear stainings in CRC cells which are not deficient in mismatch repair (MMR) (a). MMR-deficient CRC cells (b)show negative staining of hMLH1 protein (original magnification, × 200).

Results

P values less than 0.05 were considered statisticallysignificant.

Clinicopathological features of 15 patients withdouble primary cancer of the colorectum and stomach are shown in Table 1. The double primary119