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Mutations of hMLH1 and hMSH2 in Patients With Suspected Hereditary Nonpolyposis Colorectal Cancer: Correlation With Microsatellite Instability and Abnormalities of Mismatch Repair Protein Expression

From the Departments of Clinica di Oncologia Medica and Anatomia ed Istologia Patologica, University of Ancona, Ancona, Italy.

Address reprint requests to Emilio Porfiri, MD, PhD, Clinica di Oncologia Medica, Ospedale Regionale Torrette, University of Ancona, Via Conca, 60020 Ancona, Italy; email: porfiri{at}popcsi.unian.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The relationship between germ-line mutations of hMSH2 and hMLH1, microsatellite instability (MSI), and loss of DNA mismatch repair (MMR) gene expression were studied to formulate an effective selection protocol for patients with suspected hereditary nonpolyposis colorectal cancer who should be offered genetic testing.

PATIENTS AND METHODS: Patients eligible for germ-line analysis of hMLH1 and hMSH2 were selected. Tumor specimens were obtained to assess MSI and loss of MMR gene expression.

RESULTS: Among 37 patients who participated in the study, two hMSH2 and two hMLH1 missense mutations (11%) were detected, none of which was found in a panel of 60 healthy volunteers. High MSI was found in five tumors (19%) and low MSI in 10 tumors (39%); 12 tumors (46%) were microsatellite stable. Four tumors demonstrated loss of hMLH1, and three tumors demonstrated loss of hMSH2 protein expression.

CONCLUSION: No relationship was found between MMR gene mutations and MSI; low or no MSI was found in the four patients with germ-line mutations, and none of the five patients with high MSI demonstrated abnormalities of MMR genes. On the contrary, loss of hMLH1 or hMSH2 expression was found in the tumors from three of the four patients demonstrating germ-line mutations. These data suggest that germ-line mutations of the MMR gene can occur in people with MSI-negative tumors. Sensitive clinical criteria and the study of MMR gene expression may be useful to identify this subset of patients.

	INTRODUCTION

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HEREDITARY NONPOLYPOSIS colorectal cancer (HNPCC), an inherited syndrome of cancer susceptibility, accounts for approximately 5% of all tumors of the colon and rectum.¹ Families associated with HNPCC are characterized by members with a predisposition to early-onset colorectal cancer and an increased incidence of cancer of other organs, namely tumors of the endometrium, stomach, small bowel, ovary, hepatobiliary tract, renal pelvis, and ureter.^1,2 Germ-line mutations of genes involved in postreplicative DNA mismatch repair (MMR), in particular hMLH1, hMSH2, hPMS1, hPMS2, hMSH3, and hMSH6, are thought to be responsible for HNPCC as they cosegregate with the HNPCC phenotype.³ Cancer of the breast does not belong to the classic HNPCC tumor spectrum; however, experimental evidence has suggested that germ-line MMR gene mutations could also be involved in the development of these tumors.⁴ Cells with a defective DNA MMR demonstrate a mutation rate 100-fold greater than that of normal cells, causing the accumulation of potentially deleterious mutations throughout the genome. These mutations preferentially affect repetitive DNA sequences and are responsible for the occurrence of microsatellite instability (MSI). MSI has been detected in most MMR-deficient tumors and is considered to be the hallmark of HNPCC.^1,5

Although many of the molecular events implicated in HNPCC are understood, controversy still exists regarding the identification of at-risk individuals. In 1990, the Amsterdam Criteria were proposed by the International Collaborative Group on HNPCC (ICG-HNPCC) in order to provide uniformity to collaborative research.⁶ The Amsterdam Criteria, which have been amended to include tumors of the endometrium, small bowel, and urinary tract (Amsterdam Criteria II),⁷ identify families whose members have a high risk for colorectal cancer and who demonstrate a high incidence of germ-line hMLH1 or hMSH2 mutations, ranging from 45% to 86% of cases.⁸ However, germ-line mutations of MMR genes have also been found in patients who do not fulfill the strict Amsterdam Criteria, suggesting that many of these patients may not be recognized and may be excluded from genetic counseling.^8,9

Because colonoscopic surveillance has proven effective in reducing morbidity and mortality in HNPCC mutation carriers,¹⁰ more sensitive, although less specific, criteria have been proposed to select patients who should undergo genetic testing. Among these criteria, the Bethesda Guidelines have been formulated and are used to identify families and patients with suspected HNPCC.¹¹ These patients constitute a numerically relevant group and demonstrate a lower incidence of hMLH1 and hMSH2 germ-line mutations, ranging from 8% to 32%.^9,12 To increase the specificity of patient selection, several authors have included the assessment of MSI in prescreening protocols and have proposed to offer genetic testing only to patients who demonstrate the clinical features of familial colorectal cancer and MSI-positive tumors.¹³ The loss of hMLH1 or hMSH2 protein expression in the tumor tissue has also been linked to the inactivation of MMR genes and to the occurrence of MSI¹⁴; nevertheless, the immunohistochemical study of hMSH2 and hMLH1 has not yet been included in most HNPCC screening protocols.

To formulate an effective selection procedure to identify patients with suspected HNPCC who should be offered genetic testing, we studied the relationship between germ-line mutations of hMLH1 and hMSH2, MSI, and MMR gene expression in a panel of 37 unrelated patients whose personal or familial cancer history fulfilled at least one of the Bethesda Guidelines, amended to include breast cancer among the HNPCC-related malignancies.

	PATIENTS AND METHODS

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Patients
Thirty-seven unrelated patients who met at least one of the Bethesda Guidelines amended to include breast cancer among the HNPCC tumor spectrum took part in the study (Table 1). Nineteen patients were men and 18 were women; the median age at diagnosis was 40 years (range, 23 to 66 years). Patients were selected among those referred to our department from June 1997 to June 2001. The purpose of the study was explained, and informed consent was obtained from all participating patients. Personal and familial cancer history, including site and type of cancer as well as age at diagnosis, was collected from the proband. Pedigrees were traced back for at least three generations and laterally to second- and third-degree relatives. Cancer diagnoses and deaths in relatives were confirmed by medical or pathologic records. According to age at diagnosis and to family and personal cancer history, the patients were divided into four groups, as follows: (1) patients fulfilling the Amsterdam Criteria (two patients); (2) patients fulfilling the Amsterdam Criteria II (three patients); (3) patients who met two of the Bethesda Guidelines (nine patients); and (4) patients who met one of the Bethesda Guidelines (23 patients).

Mutation Analysis
Total RNA and genomic DNA were purified from peripheral-blood lymphocytes by the Rneasy Mini Kit and the QIAamp DNA Blood Mini Kit (both from Qiagen, Hilden, Germany) according to the instructions of the manufacturer. cDNA was prepared with 2 µg of total RNA in a reaction containing 20 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Paisley, Scotland) and 5.4 ng of random hexamers, ribonuclease inhibitor, and dNTPs. The entire open reading frame of hMLH1 and of hMSH2 was amplified from cDNA in three overlapping fragments of approximately 1,000 base pairs each (primer sequences are available from the corresponding author). Genomic DNA was used to amplify via polymerase chain reaction (PCR) the 5'- and 3'-untranslated regions of both hMLH1 and hMSH2 and to amplify specific exons when required by the study protocol. PCR products were purified with Microcon YM-100 filters (Millipore, Malsheim, France) and sequenced by an automated ABI PRISM 310 sequencing apparatus (Applera Co, Norwalk, CT). Sequencing reactions were carried out by the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applera) according to the protocol suggested by the manufacturer. To optimize sequencing results, additional forward and reverse primers were generated so that approximately 450 base pairs were studied in each sequencing reaction. All germ-line mutations identified during the study were confirmed by sequencing the corresponding exon that was PCR amplified from genomic DNA.

MSI Analysis
Tumor DNA was purified from formalin-fixed, paraffin-embedded tumor tissue with the QIAamp Mini Kit (Qiagen) according to the protocol suggested by the supplier. Tumor samples were microdissected under the supervision of a pathologist (I.B.) to ensure that only areas enriched in cancer cells (> 80% of all cells) were used for tumor DNA preparation. To assess MSI status, we used a panel of 12 microsatellite markers, as follows: two poly(A) repeats, BAT25 and BAT26; eight CA repeats, D2S123, MFD41, MFD27, D3S1611, D18S58, D18S34, D18S55, and D18S61; and two tetranucleotide repeats, VWA31 and GABRB1. Six of these markers were among those recommended for the assessment of MSI in tumors (Table 2). Microsatellite sequences were PCR amplified from tumor and lymphocyte DNA (sequences of PCR primers are available from the corresponding author).

Immunohistochemical Analysis
Immunohistochemical investigation of hMLH1 and hMSH2 protein expression was performed on paraffin-embedded tissue sections. Five-micron-thick sections containing tumor tissue and normal colonic mucosa (internal control) were removed from the paraffin and rehydrated with xylene and alcohol. Endogenous peroxidase was blocked by dipping the sections in 3% aqueous H₂O₂ for 10 minutes, and antigen retrieval was performed with a 10-minute microwave treatment in 10 mmol/L citrate buffer, pH 6.00. After antigen retrieval, sections were incubated overnight at 4°C with a mouse monoclonal antibody to the hMLH1 protein (clone G168-728, 1:50 dilution; PharMingen, San Diego, CA) or to the hMSH2 protein (clone FE11, 1:100 dilution; Oncogene Research Products, Cambridge, MA) and lightly counterstained with hematoxylin. Immunostaining was performed by the avidin-biotin peroxidase complex technique; diaminobenzidine was used as a chromogen. The normal staining pattern for both hMLH1 and hMSH2 was assessed, and tumors were considered negative for hMSH2 or hMLH1 expression when there was a complete absence of nuclear staining of tumor cells in the presence of an internal positive control represented by normal epithelial cells, stromal cells, or lymphocytes.¹⁵

	RESULTS

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hMLH1 and hMSH2 Gene Mutations
We detected two hMLH1 and two hMSH2 germ-line mutations among the 37 patients who participated in the study; one mutation affected a patient who met two of the Bethesda Guidelines, and the other three mutations were found in patients fulfilling only one of the Bethesda Guidelines (Table 1). All four mutations were missense. One hMSH2 mutation was located in exon 7 and resulted in a Arg-Ser amino acid substitution at codon 359 (AT). This mutation was found in patient no. 8, who developed rectal cancer at the age of 30 years and had two first-degree relatives with stomach cancer, one of whom was diagnosed at the age of 42 years. The other hMSH2 mutation was found in patient no. 27, who developed colon cancer at 42 years of age; this patient’s family history was negative for cancer. This patient harbored an exon 3 mutation that caused an Asp-His change at codon 167 (GC). Patient no. 15 demonstrated a hMLH1 mutation affecting exon 17 and resulting in a Tyr-Cys change at codon 646 (AG). This patient developed breast cancer at the age of 48 years and rectal cancer at the age of 72 years. The other hMLH1 mutation was found in patient no. 24, who was diagnosed with rectal cancer at age 39 years and demonstrated a double base change in exon 16, which resulted in a Lys-Ala amino acid substitution at codon 618 (AAGC). The five patients who met the Amsterdam Criteria or Amsterdam Criteria II did not demonstrate any pathogenic mutation of hMLH1 and hMSH2, or sequence change of uncertain significance.

We also detected several known polymorphisms of hMLH1 and hMSH2 (Table 3). These variants are thought not to have any causative role in HNPCC and are among those listed in the ICG-HNPCC mutation database (http//www.nfdht.nl). The most frequent polymorphism of hMLH1 was the exon 8 AG base change at nucleotide 655, resulting in a Ile219Val amino acid substitution (allele frequency, 32%). The intronic change CG at 211+9 was the most common variant detected in hMSH2 (allele frequency, 50%).

The microsatellite markers that were investigated displayed a variable incidence of alteration. The highest frequency of instability was detected in D18S55, which was altered in 21% of the tumors. D2S123, D18S58, and MFD27 demonstrated MSI in 19% of the tumors (Table 2). One marker, D3S1611, was stable in all the tumor samples analyzed, and another marker, BAT26, demonstrated instability in only one tumor (Table 2). This marker was included in the reference panel of microsatellite loci suggested for the study of MSI in tumors and has been used by many authors for MSI assessment.^5,13

Immunohistochemical Analysis
Slides for the immunohistochemical study of hMLH1 and hMSH2 expression were available from 28 patients. Loss of expression of hMLH1 was detected in four patients, two of whom demonstrated a germ-line hMLH1 mutation. Loss of nuclear staining for hMSH2 was found in three patients, one of whom harbored a germ-line hMSH2 mutation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
We studied the correlation between germ-line mutations of hMLH1 and hMSH2, MSI, and abnormalities of MMR gene expression in 37 unrelated patients who met at least one of the Bethesda Guidelines, amended to include breast cancer among the HNPCC-related tumors. We detected four missense mutations in four patients, representing 11% of the study population. One of these genetic abnormalities, the hMLH1 Lys618Ala mutation, has been previously described in HNPCC patients and has been demonstrated to cause loss of DNA MMR activity.¹⁶ The remaining hMLH1 Tyr646Cys mutation and the two hMSH2 mutations Asp167His and Arg359Ser are novel; they are not listed in the ICG-HNPCC mutation database. Furthermore, we did not detect these genetic abnormalities in our reference population of 60 healthy individuals. The pathogenic effects of these mutations have not yet been established; it is possible that they represent rare polymorphisms or genetic changes associated with an attenuated phenotype. To ascertain their biologic role, it will be necessary to assess their frequency in the general population and their relationship with the HNPCC phenotype. In addition, their biochemical consequences should also be estimated because the predicted amino acid changes could affect DNA MMR functions. With regard to this, the hMLH1 Tyr646Cys mutation occurs in a region critical for the interaction of hMLH1 with hPMS2 and causes the substitution of a polar tyrosine residue with a nonpolar cysteine.¹⁷ It may be hypothesized that such amino acid substitution alters the assembly of the MMR complex, thereby affecting its functions. This mutation was found in a patient with rectal and breast cancer who was admitted to the study. This patient had two first-degree relatives (father and daughter) who had died as a result of gastric cancer at the age of 72 years and of brain cancer at the age of 45 years, respectively; however, we could not obtain tissue samples from them to perform germ-line analysis.

MSI is a widely accepted marker of genetic instability and of inactivation of the DNA MMR system.⁵ In agreement with this assumption, several authors have proposed that MSI assessment should be used to select patients who should undergo genetic testing for germ-line mutations of MMR genes.^5,13 In our study, MSI-H was found in five tumors, none of which was associated with germ-line mutations of hMLH1 or hMSH2. It is possible that these patients harbored a germ-line mutation in another gene, such as hMSH3, hMSH6, hPMS1, and hPMS2, although germ-line alterations of these genes are found only in a small percentage of HNPCC patients,¹ and, as in the case of hMSH6, they are not always associated with MSI.¹⁸ The study of MSI in the four tumors from the patients with germ-line mutations demonstrated a MSI-L profile in three tumors and a MSS profile in one tumor. Such a low level of MSI may suggest that these mutations were not implicated in the familial susceptibility to colon cancer. However, it has been demonstrated that certain hMLH1 missense mutations do not correlate with MSI, even though they retain a definite role in the HNPCC syndrome.¹⁶ The hMLH1 Lys618Ala mutation represents a paradigmatic example because it inactivates DNA MMR but does not associate with MSI.^16,19

It has been proposed that tumors from patients carrying a germ-line hMLH1 or hMSH2 mutation should test negative for expression of the corresponding proteins because somatic inactivation of the wild-type copy of the gene is required for tumor development.¹⁴ Our data are consistent with this hypothesis; the two patients with hMLH1 germ-line mutations demonstrated loss of hMLH1 protein expression in the tumor, and one patient with a hMSH2 mutation demonstrated loss of hMSH2 expression. Nevertheless, in one case, we observed a negative nuclear staining for hMLH1 in a patient with a hMSH2 mutation, and in a further three patients, we did not find germ-line mutations associated with the loss of expression of MMR genes. In these cases, we hypothesize that a somatic inactivation of both alleles occurred during tumorigenesis.²⁰ We did not find a correlation between MSI and MMR gene expression because low or no MSI was found in six tumors that tested negative for hMLH1 or hMSH2 immunohistochemistry, whereas normal expression levels of MMR genes was present in three tumors with high MSI.

The question of selection criteria usually arises when genetic testing is to be performed in large population samples. Strict selection criteria, such as the Amsterdam Criteria, have proven insufficiently sensitive, although highly specific, in identifying mutation carriers; thus, more sensitive, although less specific, criteria have been proposed.²¹ MSI assessment has also been used to prescreen patients with suspected HNPCC, and a correlation between occurrence of MSI and the presence of MMR gene germ-line mutations has been reported, especially in patients with a family history fulfilling the Amsterdam Criteria.^13,22 However, our data suggest that germ-line MMR gene mutations may occur in the absence of significant MSI, particularly in the case of missense mutations linked to an attenuated phenotype.¹⁶ Although the pathogenic role of these mutations remains to be ascertained, MSI assessment and the use of stringent selection criteria could be insufficient to identify these patients, who are also candidates for genetic counseling. We propose that sensitive clinical criteria, such as the Bethesda Guidelines, and the immunohistochemical study of MMR gene expression could constitute effective tools to identify HNPCC patients with MSI-negative MMR mutations.

	ACKNOWLEDGMENTS

 
Supported by grants from the Associazione Italiana per la Ricerca sul Cancro and the Ministero per l’Universita’ e la Ricerca Scientifica e Tecnologica. F.B. and S.R. are fellows of Fondazione Italiana per la Ricerca sul Cancro.

We thank Franca Saccucci, BSc, from the Istituto di Biologia e Genetica, University of Ancona, Ancona, Italy, for providing a reference library of genomic DNA from healthy volunteers.

	NOTES

 
M.S. and F.B. contributed equally to this study.

	REFERENCES

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Submitted August 23, 2001; accepted November 14, 2001.

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