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The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.

Purpose: Immunohistochemistry (IHC) and microsatellite instability (MSI) analysis can be used to identify patients with a possible DNA mismatch repair defect [hereditary nonpolyposis colorectal carcinoma (HNPCC)]. The Bethesda criteria have been proposed to select families for determination of MSI. The aims of this study were to assess the yield of MSI analysis in families suspected for HNPCC, to compare the results of immunohistochemical staining and MSI analysis, and to assess the additional value of PMS2 staining.Experimental Design: Clinical data and tumors were collected from 725 individuals from 631 families with suspected HNPCC. MSI analysis was performed using eight markers including the 5 National Cancer Institute markers. Four immunohistochemical staining antibodies were used (MLH1, MSH2, MSH6 and PMS2).Results: A MSI-H (tumors with instability for >30% of the markers) phenotype in colorectal cancers (CRCs) was observed in 21-49% of families that met the various Bethesda criteria. In families with three cases of CRC diagnosed at age > 50 years, families with a solitary case of CRC diagnosed between ages 45 and 50 years, and families with one CRC case and a first-degree relative with a HNPCCrelated cancer, one diagnosed between ages 45 and 50 years (all Bethesda-negative families), the yield of MSI-H was 10 -26%. Immunohistochemical staining confirmed the MSI results in 93% of the cases. With IHC, adding PMS2 staining led to the identification of an additional 23% of subjects with an hMLH1 germ-line mutation (35 carriers were tested).Conclusions: The Bethesda guidelines for MSI analysis should include families with three or more cases of CRC diagnosed at age > 50 years. The age at diagnosis of CRC in the original guidelines should be raised to 50 years. Routine IHC diagnostics for HNPCC should include PMS2 staining.

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SUMMARY:To evaluate the prognostic impact of human leukocyte antigen class I (HLA-I) expression on immune surveillance in colorectal cancer, we studied 88 curatively resected tumors for HLA-A and HLA-B/C expression and correlated these data to clinical and histopathological parameters. HLA-A was normal (all tumor cells had HLA expression) in 32%, reduced (HLA-negative and -positive tumor cells coexisted) in 56%, or absent (no tumor cells expressed HLA) in 12% of evaluable cases. HLA-B/C was normal in 47%, reduced in 47%, and absent in 7% of the cases. Considering both markers, total HLA-I expression was normal in 27%, reduced in 63%, absent in 7%, and could not be evaluated in 3% of the cases due to absent HLA-A expression in tumor and normal cells. Down-regulation of HLA-A expression significantly correlated with a lower tumor stage (p ϭ 0.005), mucinous tumors (p ϭ 0.05), a lower incidence of recurrences (p ϭ 0.03), and a longer disease-free survival (p ϭ 0.02). Down-regulation of HLA-B/C expression correlated with a lower tumor stage (p Ͻ 0.001) and a longer disease-free survival (p ϭ 0.04). In multivariate analysis, HLA-A down-regulation was the only prognostic factor correlated with a longer disease-free survival (p ϭ 0.02). Six tumors were negative for HLA-A and -B/C and did not recur during follow-up. Therefore, we analyzed microsatellite instability (MSI) in these cases. Three of these six tumors indeed showed down-regulation of MLH-1, MSH-2, or MSH-6, indicating a MSI-high phenotype. Beta-2-microglobulin protein expression was lost in five of six of the HLA-I-negative cases, but frame shift mutations in three repetitive sequences in ␤2-microglobulin were absent. In contrast, loss of MLH-1, MSH-2, and MSH-6-protein expression was only observed in two of nine matched controls with reduced or normal HLA-A and -B/C expression. Our data showed that HLA-I was down-regulated in 72% of colorectal cancers and provided independent prognostic information for a longer disease-free survival. The better prognosis may be caused by elimination of HLA-negative cells by natural killer cells or by an attenuated tumor aggressiveness, as is seen in tumors with a MSI-high phenotype. (Lab Invest 2002, 82:1725-1733.

Background: Abnormalities in Human Leukocyte Antigen (HLA) class I expression are common in colorectal cancer. Since HLA expression is required to activate tumor antigen-specific cytotoxic Tlymphocytes (CTL), HLA class I abnormalities represent a mechanism by which tumors circumvent immune surveillance. Tumors with high microsatellite instability (MSI-H) are believed to face strong selective pressure to evade CTL activity since they produce large amounts of immunogenic peptides. Previous studies identified the prevalence of HLA class I alterations in MSI-H tumors. However, those reports did not compare the frequency of alterations between hereditary and sporadic MSI-H tumors neither the mechanisms that led to HLA class I alterations in each subgroup.

These results suggest that not only HRPT2 but also MEN1 mutations may play a role in sporadic parathyroid cancer formation.