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Accuracy of Revised Bethesda Guidelines, Microsatellite Instability, and Immunohistochemistry for the Identification of Patients With Hereditary Nonpolyposis Colorectal Cancer

Accuracy of Revised Bethesda Guidelines, Microsatellite Instability, and Immunohistochemistry for... ContextThe selection of individuals for hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing is challenging. Recently, the National Cancer Institute outlined a new set of recommendations, the revised Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability.ObjectiveTo establish the most effective and efficient strategy for the detection of MSH2/MLH1gene carriers.Design, Setting, and PatientsA prospective, multicenter, nationwide study (the EPICOLON study) in 20 hospitals in the general community in Spain of 1222 patients with newly diagnosed colorectal cancer between November 1, 2000, and October 31, 2001.InterventionsMicrosatellite instability testing and MSH2/MLH1immunostaining in all patients regardless of age, personal or family history, and tumor characteristics. Patients whose tumors exhibited microsatellite instability and/or lack of protein expression underwent MSH2/MLH1germline testing.Main Outcome MeasuresEffectiveness and efficiency of both microsatellite instability testing and immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2/MLH1germline mutations.ResultsTwo hundred eighty-seven patients (23.5%) fulfilled the revised Bethesda guidelines. Ninety-one patients (7.4%) had a mismatch repair deficiency, with tumors exhibiting either microsatellite instability (n = 83) or loss of protein expression (n = 81). Germline testing identified 11 mutations (0.9%) in either MSH2(7 cases) or MLH1(4 cases) genes. Strategies based on either microsatellite instability testing or immunostaining previous selection of patients according to the revised Bethesda guidelines were the most effective (sensitivity, 81.8% and 81.8%; specificity, 98.0% and 98.2%; positive predictive value, 27.3% and 29.0%, respectively) to identify MSH2/MLH1gene carriers. Logistic regression analysis confirmed the revised Bethesda guidelines as the most discriminating set of clinical parameters (odds ratio, 33.3; 95% confidence interval, 4.3-250; P = .001).ConclusionThe revised Bethesda guidelines constitute a useful approach to identify patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and immunostaining are equivalent and highly effective strategies to further select those patients who should be tested for MSH2/MLH1germline mutations.Hereditary nonpolyposis colorectal cancer (HNPCC), also named Lynch syndrome, is an autosomal dominant disorder that accounts for approximately 1% to 3% of all colorectal cancers.HNPCC is caused by germline mutations in DNA mismatch repair genes, mainly MSH2and MLH1.Defects on this pathway lead to changes in the length of nucleotide repeat sequences of tumor DNA, termed microsatellite instability.Although a great advance in the understanding of the molecular basis of HNPCC has taken place in the last decade, optimal selection of individuals for HNPCC genetic testing remains controversial.In 1991, the International Collaborative Group on HNPCC established clinical criteria, known as the Amsterdam criteria, which provided a pivotal definition of this syndrome and were critical in identifying its molecular basis.In response to criticism that the Amsterdam criteria were too stringent, the extended Amsterdam II criteria were developed to include extracolonic HNPCC-associated cancers.The use of the Amsterdam criteria achieved the original purpose of classifying a family as having HNPCC, but its limited sensitivity hampered decisions about which patients should undergo genetic testing.In 1996, an international workshop on HNPCC hosted by the National Cancer Institute outlined a set of recommendations, known as the Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability and/or genetic testing.More recently, a second HNPCC workshop revised these criteria and proposed a new set of recommendations, the revised Bethesda guidelines.As it was previously mentioned, tumor microsatellite instability is a phenotypic indicator of defective DNA mismatch repair.The fact that more than 90% of HNPCC-related cancers exhibit microsatellite instability suggests that screening of tumors for microsatellite instability may be an efficient way of selecting individuals for HNPCC genetic testing.On the other hand, it is well known that most mutations in either MSH2or MLH1genes result in abnormal MSH2 or MLH1 protein expression.As a consequence, immunostaining for these 2 proteins is associated with microsatellite instability,but this association is not without exceptions.Indeed, a mutant protein product can be expressed and detected by immunostaining,whereas germline mutations may occur in patients with microsatellite instability–negative tumors.These conflicting results have precluded the establishment of a unique method for primary screening of mismatch repair deficiency. Specific characteristics of performed studies may have contributed to this fact because most of the studies were performed in selected high-risk populations,different criteria were used to define the microsatellite instability status,immunohistochemical evaluation was not performed systematically,or results were not referred to the presence of germline mutations.Our prospective, multicenter, nation-wide study was aimed at establishing the most effective and efficient strategy for the detection of MSH2or MLH1gene carriers in patients with newly diagnosed colorectal cancers. For this purpose, performance characteristics of both microsatellite instability testing and MSH2/MLH1 protein immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2or MLH1germline mutations. A logistic regression analysis was also performed to determine independent predictors of MSH2/MLH1mutations.METHODSBetween November 1, 2000, and October 31, 2001, all patients with newly diagnosed colorectal cancers in 25 hospitals were included in the EPICOLON study, a clinical epidemiology survey aimed at establishing the incidence of HNPCC in Spain.Twenty of the 25 centers agreed to participate in a nested molecular epidemiology study, which required tissue sample collection. Exclusion criteria were familial adenomatous polyposis, personal history of inflammatory bowel disease, and patient or family refusal to participate in the study. The study was approved by the institutional ethics committee of each participating hospital, and written informed consent was obtained from all patients.Demographic, clinical, and tumor-related characteristics of probands, as well as a detailed family history were obtained using a preestablished questionnaire. Personal parameters at baseline included date and place of birth, sex, smoking history, personal history of neoplasia (ie, HNPCC-related tumors and colorectal adenomas), date of colorectal cancer diagnosis, presenting symptoms, serum carcinoembryonic antigen concentration, presence of synchronous colorectal neoplasms, and treatment. Tumor-related parameters included location, size, pathology TNM stage,degree of differentiation, presence of lymphocytic infiltration, and mucinous carcinoma type (defined by the presence of >50% mucinous carcinoma cells). Synchronous lesions were assessed through colonoscopy before or immediately after surgery, as well as through systematic review of the resected specimen. Pedigrees were traced backward and laterally as far as possible, or at least up to second-degree relatives, regarding cancer history. Age at cancer diagnosis, type, location, and tumor stage of the neoplasm, and current status were registered for each affected family member. As a check on overreporting of disease, all patients were queried about the occurrence of stroke in their relatives to assess possible recall bias. Furthermore, an effort was made to verify reported neoplasia in relatives by obtaining medical records and pathology confirmation.Microsatellite instability testing and immunostaining for DNA mismatch repair proteins were performed in all patients regardless of age, personal or family history, and tumor characteristics. To avoid variability in the quality of results, microsatellite instability testing and immunostaining were centralized in 2 single centers, respectively. Researchers scoring immunostaining were blinded to the microsatellite instability results and vice versa.Tumor Microsatellite Instability AnalysisTissue samples from tumor and healthy colonic mucosa were obtained from each patient, immediately frozen in liquid nitrogen, and stored at −70°C until use. In those cases in which frozen tissue was not available, archival formalin-fixed, paraffin-embedded samples were used. Genomic DNA was isolated using the QiaAmp Tissue Kit (Qiagen, Courtaboeuf, France).Microsatellite instability status was assessed using BAT-26mononucleotide marker based on its high sensitivity.In those cases in which BAT-26was stable but immunostaining revealed lack of MSH2 or MLH1 protein expression, the 4 additional markers of the Bethesda panel (BAT-25, D5S346, D2S123, and D17S250)were also evaluated. Primers were labeled fluorescently and analyzed on a genetic analyzer using GeneScan Analysis software (ABI 310 Genetic Analyzer; Applied Biosystems, Foster City, Calif).Tumor MSH2 and MLH1 Protein ExpressionOne block of formalin-fixed, paraffin-embedded tumor tissue was selected per case. Before immunostaining, antigen retrieval was performed by immersing sections in a 10-M concentration of citrate buffer, pH 6.0, and boiling in a pressure cooker for 5 minutes. Sections were then incubated for 20 minutes at room temperature with mouse monoclonal antibodies against MLH1 protein (clone G168-15, dilution 1:40; PharMingen, San Diego, Calif) and MSH2 protein (clone FE11, dilution 1:35; Oncogene Research Products, Boston, Mass). Ultra-Vision streptavidin-biotin peroxidase detection kit (DAKO, Carpinteria, Calif) was used as secondary detection system. The peroxidase reaction was developed using diaminobenzidine tetrachloride as chromogen. Tumor cells were judged to be negative for protein expression only if they lacked staining in a sample in which healthy colonocytes and stroma cells were stained. If no immunostaining of healthy tissue could be demonstrated, the results were considered ambiguous.MSH2/MLH1Germline Mutation AnalysisPatients found to have tumors with microsatellite instability and/or lack of protein expression of either MSH2 or MLH1 underwent germline genetic testing for MSH2and MLH1genes by both multiple ligation probe amplification analysis and sequencing.Multiple ligation probe amplification was performed using the MLH1/MSH2exon deletion assay (MRC-Holland, Amsterdam, the Netherlands), which allows the detection of genomic rearrangements in these genes.Ligation products were amplified by polymerase chain reaction using a fluorescently labeled primer and analyzed in an ABI 3100 sequencer using GeneScan and Genotyper Analysis software (Applied Biosystems). Peak height of each fragment was compared with those measurements of a control sample and deletions were suspected when peak height was 60% or less of healthy controls. Control DNA samples with known MSH2or MLH1genomic rearrangements were included in each batch of experiments. Multiple ligation probe amplification results were confirmed by reverse transcriptase polymerase chain reaction encompassing contiguous exons of the suspected deleted fragment.Germline mutations in the MSH2and MLH1genes were also sought by direct exon-by-exon sequencing. Amplification products were generated with primers located in the flanking introns approximately 50 base pairs from the respective intron/exon borders to detect all possible splice junction mutations. The sequences were determined on the genetic analyzer (ABI 3100, Applied Biosystems) using fluorescently labeled primers and protocols supplied by the manufacturer.Statistical AnalysisPerformance characteristics of screening strategies based on microsatellite instability testing and/or protein immunostaining, either directly or through previous selection of patients according to the revised Bethesda guidelines, were calculated with respect to the diagnosis of HNPCC associated with MSH2/MLH1germline mutations. Mutations were considered deleterious based on sequencing results, published data, and existing mutation databases. Comparison of paired proportions for sensitivity, specificity, and overall accuracy was performed by the McNemar test.A cost-minimization analysis was also performed to establish the most efficient strategy. For this analysis, costs of microsatellite instability (BAT-26maker), immunostaining (both MSH2 and MLH1), and genetic testing were established at 100 € (US $130), 200 € (US $260), and 2400 € (US $3120), respectively, or 1200 € per gene (US $1560 per gene), according to hospital clinic current billing.To identify parameters associated with MSH2/MLH1gene mutation carriers, univariate and multivariate analyses were performed. Variables evaluated corresponded to any previously suggested predictors including each individual characteristic of the original and revised Bethesda guidelines, as well as those proposed by Aaltonen et alfor screening in general population (any first-degree relative with colorectal cancer or endometrial cancer regardless of age, personal history of colorectal cancer or endometrial cancer, and age at diagnosis of colorectal cancer <50 years in the proband) and Wijnen et alin familial cancer clinics (age at diagnosis of colorectal cancer <50 years within a family, fulfillment of the Amsterdam criteria, and endometrial cancer in ≥1 family member). In addition, combined variables corresponding to each of these 4 sets of recommendations were also evaluated. For the univariate analysis, categorical variables were compared by the Fisher exact test. Variables achieving P<.20 in the univariate analysis were subsequently included in a multivariate analysis using a stepwise forward logistic regression procedure. Two different models were evaluated. First, only combined variables (original and revised Bethesda guidelines, and those proposed by Aaltonen et aland Wijnen et al) were included in the model to identify the most discriminating set of parameters. Second, individual characteristics were added to the previously recognized combined variable to identify other independent predictors of MSH2/MLH1germline mutations that can contribute to refine the model.Continuous variables were expressed as mean (SD). All Pvalues were 2-sided. P<.05 was considered statistically significant. All calculations were performed by using SPSS software version 10.0 (SPSS Inc, Chicago, Ill).RESULTSDuring the study period, 1978 patients with newly diagnosed colorectal cancer were included from 25 centers. One hundred six patients were excluded because the tumor developed in the context of a familial adenomatous polyposis (n = 11) or inflammatory bowel disease (n = 14), the patient did not consent to participate in the study (n = 32), or had an incomplete family history (n = 49). Of the remaining 1872 eligible patients, 1222 patients were diagnosed in 20 centers agreeing to participate in the nested molecular epidemiology study and constituted the basis of our analysis. Characteristics of this subset of patients (Table 1) did not differ from the whole group.Table 1.Characteristics of Patients With Colorectal Cancer (N=1222)CharacteristicNo. (%) of PatientsAge, mean (SD), y70 (11)SexMale731 (59.8)Female491 (40.2)Site of tumor relative to splenic flexureProximal357 (29.2)Distal865 (70.8)TNM tumor stage*I161 (13.2)II510 (41.7)III337 (27.6)IV214 (17.5)Degree of differentiation†Well292 (23.9)Moderate835 (68.3)Poor95 (7.8)Mucinous carcinoma type142 (11.6)Synchronous colorectal cancer71 (5.8)Fulfillment of guidelines‡Amsterdam criteria18 (1.5)Extended Amsterdam II criteria22 (1.8)Original Bethesda guidelines224 (18.3)Revised Bethesda guidelines287 (23.5)*Stage I indicates tumor 1-2 (T1-2), no regional lymph node metastasis, and no metastasis; stage II, T3-4, no regional lymph node metastasis, and no metastasis; stage III, any tumor, node 1-3, and no metastasis; and stage IV, any tumor, any node, and distant metastasis.†Well-differentiated tumors resemble identifiable tissue types and poorly differentiated tumors may only be identifiable by the expression of cell markers or by extremely focal and subtle histologic and/or cytologic findings.‡For an explanation of Amsterdam criteria, see Vasen et al; extended Amsterdam II critera, see Vasen et al; original Bethesda guidelines, see Rodriguez-Bigas et al; and revised Bethesda guidelines, see Umar et al.Proband CharacteristicsDemographic, clinical, and tumor-related characteristics of patients included in the study are shown in Table 1. According to the extended Amsterdam II criteria, 22 patients (1.8%) belonged to families satisfying the clinical definition of HNPCC. On the other hand, 287 patients (23.5%) fulfilled at least 1 criterion of the revised Bethesda guidelines.A total of 91 patients (7.4%) were found to have a mismatch repair deficiency. Of these, 83 tumors (6.8%) showed microsatellite instability and 81 (6.6%) loss of protein expression in either MSH2 (21 cases) or MLH1 (60 cases). Loss of MSH2 or MLH1 expression was found in 73 of the microsatellite instability tumors, whereas expression of both proteins was retained in the remaining 10 tumors with microsatellite instability. In addition, loss of MSH2 or MLH1 expression was found in 8 patients whose tumor did not show microsatellite instability according to the BAT-26marker. Evaluation of the 4 additional markers of the Bethesda panel in these cases confirmed that their tumor was stable. Finally, unequivocal loss of MSH2 or MLH1 was not observed in any of the remaining 1131 tumors not showing microsatellite instability.Germline genetic testing identified 11 mutations (0.9%) in either MSH2(7 cases) or MLH1(4 cases) genes. Characteristics of these patients are shown in Table 2. Three DNA changes corresponded to missense mutations. Although all 3 mutations have been previously reported as deleterious, 1 tumor did not show microsatellite instability, 2 tumors exhibited contradictory immunostaining results, and 1 patient did not fulfill the revised Bethesda guidelines.Table 2.Characteristics of Patients With MSH2/MLH1Germline Mutations (n = 11)CaseFulfillment of GuidelinesMSI StatusIHC Expressionof ProteinGermline MutationAmsterdam II CriteriaRevised Bethesda GuidelinesMSH2MLH1GeneNucleotide ChangeConsequence1YesYesUnstablePresentAbsentMLH1859_860delAAFrameshift at codon 287; stop at codon 3052YesYesUnstablePresentAbsentMLH12136G>AStop at codon 7123YesYesUnstableAbsentPresentMSH2del exon 9-16Out-of-frame deletion of exons 9-164YesYesUnstableAbsentPresentMSH21703insAFrameshift at codon 568; stop at codon 5715NoYesUnstablePresentAbsentMLH1IVS14-1G>AOut-of-frame deletion of exon 156NoYesUnstableAbsentPresentMSH2IVS15-3TTC>CTTSplicing defect, out-of-frame deletion7NoYesUnstableAbsentPresentMSH2688insAFrameshift at codon 230; stop at codon 2328NoYesUnstableAbsentPresentMSH21165C>TStop at codon 3899NoYesUnstableAbsentPresentMLH11852AA>GCLys>Ala at codon 61810NoYesStableAbsentPresentMSH2965G>AGly>Asp at codon 32211NoNoUnstablePresentAbsentMSH2435T>GIle>Met at codon 145Abbreviations: IHC, immunohistochemistry; MSI, microsatellite instability.Performance Characteristics and Efficiency of Screening StrategiesPerformance characteristics of screening strategies for the identification of MSH2/MLH1gene carriers are shown in Table 3. Calculations were performed considering both germline mutations (n=11) and unambiguous pathogenic variants (n=8). Approaches based on either microsatellite instability analysis or MSH2/MLH1 immunostaining had an almost identical effectiveness. Conversely, specificity, overall accuracy, and positive predictive value of these strategies increased when patients were previously selected according to the revised Bethesda guidelines. Finally, combinations of the results obtained in both microsatellite instability testing and protein immunostaining did not provide any additional advantage with respect to the corresponding individual strategies.Table 3.Performance Characteristics of Different Strategies for the Identification of MSH2/MLH1Gene Mutation Carriers*StrategyAny Germline Mutation (n = 11), %Unambiguous Germline Mutations (n = 8), %SensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracySensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracyFulfillment of revised Bethesda guidelines90.977.13.599.977.310077.02.810077.2Presence of MSI90.993.912.299.993.910093.89.610093.9Loss of protein expression81.894.211.499.894.110094.210.110094.2Presence of MSI or loss of protein expression90.993.411.299.993.310093.28.910093.3Presence of MSI and loss of protein expression72.794.811.399.794.610094.811.310094.8Fulfillment of revised Bethesda guidelines andPresence of MSI81.898.0*27.399.897.9*10097.9*24.210097.9*Loss of proteinexpression81.898.2*29.099.898.0*10098.1*25.810098.1*Presence of MSI or loss of proteinexpression81.897.8*25.799.897.7*10097.8*22.910097.8*Presence of MSI and loss of proteinexpression72.798.3*28.699.798.1*10098.4*28.610098.4*Abbreviation: MSI, microsatellite instability.*P<.001 (McNemar test) with respect to the corresponding paired strategy without previous selection of patients according to the revised Bethesda guidelines.Efficiency of all the above mentioned strategies for the identification of MSH2/MLH1-associated HNPCC was evaluated in a cost-minimization analysis. Clinical selection of patients according to the revised Bethesda guidelines followed by either microsatellite instability analysis (11 989 € [US $15 586] per detected mutation) or protein immunostaining (10 644 € [US $13 837] per detected mutation) was more cost-effective than any of these approaches performed directly (32 140 € [US $41 782] and 37 956 € [US $49 343] per detected mutation, respectively). Conversely, performance of both microsatellite instability analysis and protein immunostaining increased the cost (14 900 € [US $19 370] per detected mutation in combination with the revised Bethesda guidelines and 49 020 € [US $63 726] per detected mutation without previous selection of patients according to these clinical criteria) with respect to the corresponding individual strategies.Predictors of MSH2or MLH1Germline MutationsTo further refine clinical criteria for selecting patients who should be submitted to genetic testing, variables associated with MSH2or MLH1germline mutations were identified. Table 4shows parameters significantly associated in the univariate analysis. When only combined variables corresponding to the original set of recommendations were included in the logistic regression analysis, the revised Bethesda guidelines turned out to be the most discriminating set of parameters (odds ratio [OR], 33.3; 95% confidence interval [CI], 4.3-250; P = .001). In that sense, specific characteristics of the revised Bethesda guidelines, which allowed the identification of 2 additional gene carriers with respect to those identified by the original Bethesda guidelines, were criteria number 5 (colorectal cancer diagnosed in ≥2 first-degree or second-degree relatives with HNPCC-related tumors, regardless of age) in 2 cases and number 3 (colorectal cancer with the microsatellite instability–high histology diagnosed in a patient aged <60 years) in 1 case. Furthermore, when individual characteristics were added to the logistic regression model, family history of colorectal cancer diagnosed in at least 1 first-degree relative (OR, 3.6; 95% CI, 1.05-12.7; P = .04) was selected as an independent predictor of MSH2/MLH1germline mutations, along with the revised Bethesda guidelines (OR, 13.7; 95% CI, 1.47-125; P = .02).Table 4.Predictors of MSH2/MLH1Germline Mutations (Univariate Analysis)*CharacteristicNo. (%) of PatientsPValuePositive Predictive Value, %Germline Mutation (n = 11)No Germline Mutation (n = 1211)Colorectal cancer in ≥1 first-degree relative6 (54.5)151 (12.5).0013.8Colorectal or endometrial cancer in ≥1 first-degree relative6 (54.5)180 (14.8).0033.2Personal history of colorectal or endometrial cancer†4 (36.4)90 (7.4).0074.3Colorectal cancer diagnosed at age <50 y3 (27.3)55 (4.5).015.2Colorectal or endometrial cancer in ≥1 first-degree relative, personal history of colorectal or endometrial cancer, or age at diagnosis of proband colorectal cancer <50 y‡§8 (72.7)306 (25.3).0012.5Fulfillment of Amsterdam criteria4 (36.4)14 (1.1)<.00122.2Endometrial cancer in ≥1 family member4 (36.4)63 (5.2).0025.9Colorectal cancer diagnosed at age <50 y within a family, endometrial cancer in ≥1 family member, or fulfillment of Amsterdam criteria‡&par;7 (63.6)156 (12.9)<.0014.3Synchronous, metachronous colorectal, or other HNPCC-related cancer¶5 (45.5)98 (8.1).0014.9Colorectal or other HNPCC-related cancer¶ and/or a colorectal adenoma in ≥1 first-degree relative, 1 of the cancers diagnosed at age <50 y, and the adenoma diagnosed at age <40 y4 (36.4)61 (5.0).0026.2Colorectal or endometrial cancer† diagnosed at age <50 y3 (27.3)78 (6.4).033.7Right-sided colorectal cancer with an undifferentiated pattern (solid/cribriform) on histopathology diagnosed at age <50 y1 (9.1)5 (0.4).0516.7Signet-ring cell-type colorectal cancer diagnosed at age <50 y1 (9.1)14 (1.1).136.7Fulfillment of Bethesda guidelines‡8 (72.7)216 (17.8)<.0013.6Synchronous, metachronous colorectal, or other HNPCC-related tumors,# regardless of age5 (45.5)99 (8.2).0014.8Colorectal cancer with the MSI-high histology** diagnosed at age <60 y3 (27.3)52 (4.3).015.4Colorectal cancer diagnosed in ≥1 first-degree relatives with an HNPCC-related tumor, with ≥1 cancer diagnosed at age <50 y4 (36.4)57 (4.7).0016.6Colorectal cancer diagnosed in ≥2 first- or second-degree relatives with HNPCC-related tumors, regardless of age5 (45.5)83 (6.8).0015.7Fulfillment of revised Bethesda guidelines‡10 (90.9)277 (22.9)<.0013.5Abbreviations: HNPCC, hereditary nonpolyposis colorectal cancer; MSI, microsatellite instability.*When not specified, characteristics are referred to probands.†Patients with endometrial cancer also had colorectal cancer, which qualified inclusion into the study.‡Combinations of individual variables according to the original sets of recommendations.§Criteria suggested by Aaltonen et al.&par;Criteria suggested by Wijnen et al.¶Including endometrial, ovarian, gastric, hepatobiliary, small-bowel, or urinary tract cancer.#Including endometrial, ovarian, gastric, hepatobiliary, small-bowel, urinary tract, pancreatic, and brain cancer, sebaceous gland adenomas, and keratoacanthomas.**Including presence of tumor-infiltrating lymphocytes, Crohn-like lymphocytic reaction, mucinous/signet-ring differentiation, or medullary growth pattern.Table 5summarizes performance characteristics of strategies based on the combination of the 2 independent variables identified in the multivariate analysis. Genetic testing driven by this combination of clinical parameters was not substantially more accurate than when the revised Bethesda guidelines were only used (Table 3).Table 5.Performance Characteristics of Clinical Criteria for the Identification of MSH2/MLH1Gene Mutation Carriers (Multivariate Analysis)*StrategyPerformance Characteristic, %SensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracyFulfillment of clinical criteria10070.32.910070.5Fulfillment of clinical criteria and presence of MSI90.997.928.699.997.9Fulfillment of clinical criteria and loss of protein expression81.898.128.199.897.9Fulfillment of clinical criteria and presence of MSI or loss of protein expression90.997.726.399.997.6Fulfillment of clinical criteria, presence of MSI, and loss of protein expression72.798.328.699.798.1Abbreviation: MSI, microsatellite instability.*Based on the combination of the revised Bethesda guidelinesand family history of colorectal cancer diagnosed in at least 1 first-degree relative.COMMENTOur study represents the first reported attempt to our knowledge to determine the effectiveness of different strategies for identifying MSH2/MLH1gene carriers in the context of the recently proposed revised Bethesda guidelines. The strength of our study relies on the fact that it was performed on a general population basis; it involved the largest number of patients evaluated so far; microsatellite instability analysis and protein immunostaining were performed in a parallel and blinded fashion; results were evaluated according to the presence of germline mutations; and finally, costs were also considered. Results of our study suggest that microsatellite instability testing and protein immunostaining are equivalent strategies in terms of cost-effectiveness, and that when either of these screening methods are performed in patients selected according to the revised Bethesda guidelines, they are highly accurate in identifying patients with MSH2/MLH1-associated HNPCC. However, the relatively low number of identified mutations, as well as the uncertain significance of the 3 missense mutations, may constitute a drawback of the study and, consequently, could have influenced the results. Nonetheless, recalculation of effectiveness and efficiency of the revised Bethesda guidelines, either alone or in combination with microsatellite instability testing or immunostaining, and considering only those unambiguous pathogenic mutations does not modify the conclusions of the study.Because molecular definition of HNPCC was established,identification of MSH2/MLH1gene carriers has become a critical and difficult issue.Currently, several approaches including the use of different sets of clinical criteria and methods for determining mismatch repair deficiency (ie, microsatellite instability testing and immunostaining) are being used and there is no unique and universally accepted strategy.Although it can be anticipated that epidemiological diversity among geographical areas and technical disparity among laboratoriesmay preclude the establishment of a definitive, flawless approach, our results suggest that the revised Bethesda guidelines seem to be the most accurate clinical criteria tested so far for the identification of patients at risk for such an inherited disorder.Indeed, its performance characteristics, either alone or in combination with microsatellite instability testing and/or immunostaining, a highly discriminating predictive value in the logistic regression model with respect to other sets of recommendations, and the lack of benefit of adding other independent predictors in terms of both effectiveness and efficiency warrants the use of these criteria in clinical practice. These results are further reinforced by those obtained in a parallel investigation of our group, in which original and revised Bethesda guidelines were compared directly. In that analysis, the latter provided a more accurate and cost-effective approach than the original criteria (unpublished data).Although current recommendations for HNPCC identification rely on microsatellite instability testing as screening strategy,immunostaining has been proposed as an alternative approach.Because protein immunostaining is often easier to perform than DNA analysis in a clinical setting, the use of this technique appears to offer a relatively convenient and rapid method for the prescreening of tumors with mismatch repair defects.Equivalence of both strategies has been demonstrated in high-risk colorectal cancer populations, in which detection of microsatellite instability or loss of MSH2/MLH1 expression were both useful criteria for selecting patients who should be submitted to genetic testing.In addition, immunostaining is the method of choice to direct the search for germline mutations,and it may help to solve the status of MSH2germline variants of uncertain significance because somatic inactivation of MSH2is a rare event in sporadic microsatellite instability tumors.Finally, results of recent investigations suggest that systematic evaluation of other mismatch repair proteins, such as MHS6and PMS2,may contribute to increase the effectiveness of immunostaining.Despite the high correlation between MSH2or MLH1germline mutations and the absence of protein expression, some conflicting results were observed in our study. Indeed, 2 patients with missense mutations in either MSH2or MLH1exhibited loss of expression of the opposite protein. These contradictory results bring about the issue of determining the pathogenic significance of missense mismatch repair gene mutations. With respect to the MLH1:Lys618Ala variant, although it has been shown to segregate with the HNPCC phenotype,it has also been reported in healthy controls.Regarding the MSH2:Ile145Met variant, it has been reported in other HNPCC families (http://www.insight-group.org), but functional results raised some concerns with respect to its pathogenicity.To overcome this situation, calculations were repeated considering these 3 missense mutations as not proven pathogenic and, although minor variations occurred, the results did not change, thus reinforcing the conclusions of the study.In our study, microsatellite instability testing was systematically performed by analyzing the BAT-26marker alone instead of using the 5-marker panel proposed by the National Cancer Institute.The use of this single marker, in a similar manner as it has been performed in previous investigations,was justified by its high sensitivity, which ranged between 93% and 97%.Indeed, in the great majority of tumors, analysis of mononucleotide repeats BAT-25and BAT-26is sufficient to establish the microsatellite instability status without reference to the germline DNA, because these markers are quasi-monomorphic in white populations.In addition, Loukola et aldemonstrated that the use of BAT-26alone was feasible in screening for individuals with HNPCC because this marker was able to identify all gene mutation carriers. Nevertheless, there is still some controversy with respect to this issue because some studies have suggested that BAT-26can miss some cases with mismatch repair defects (ie, those related to the MSH2gene).With this limitation, we designed our investigation with the hypothesis that simultaneous protein immunostaining would contribute to identify germline mutations in patients with BAT-26stable tumors. However, our results demonstrated that combination of both microsatellite instability testing and protein immunostaining did not provide any advantage with respect to each of these approaches individually. More important, performance of the 4 remaining markers of the Bethesda panel did not contribute to identify any additional gene mutation carrier because all tumors stable for the BAT-26marker and showing loss of protein expression were also stable for the whole panel. Finally, the frequency of MSH2/MLH1germline mutations observed in our study (0.9%) is very similar to that reported in other investigations using different screening strategies; therefore, arguing against the possibility of underestimating the incidence of HNPCC as a result of the screening method used. However, some geographical differences may exist, as noted by the relatively low frequency of MSH2/MLH1gene carriers among patients fulfilling the Amsterdam criteria found in our study, similar to the previously reported studies in Spain.Taking into account all these considerations, evaluation of the BAT-26marker alone seems to be a simple, rapid, and reliable method to screen for microsatellite instability when the final goal is to select patients who should be tested for MSH2/MLH1germline defects.In the last few years, it has been demonstrated that colorectal cancer surveillance of HNPCC gene carriers is effective and considerably less costly than no colorectal cancer surveillance.In addition, screening patients with newly diagnosed colorectal cancer using the original Bethesda guidelines and microsatellite instability testing to drive to subsequent genetic testing for HNPCC is cost-effective, especially if the benefits to the patients’ immediate relatives are considered.However, there is very little information with respect to other strategies.In that sense, no study has evaluated the efficiency of the revised Bethesda guidelines or compared microsatellite instability testing and protein immunostaining in terms of cost-effectiveness for the identification of MSH2/MLH1germline mutations. Our results suggest that clinical selection of patients according to the revised Bethesda guidelines along with either microsatellite instability testing or immunostaining was more efficient than any of these approaches separately. However, because of the noteworthy variations in costs of medical procedures among countries and health systems as well as the minimal difference observed, the superiority of immunostaining vs microsatellite instability testing cannot be clearly established.In conclusion, our results demonstrate that the revised Bethesda guidelines constitute a very useful approach to select patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and protein immunostaining are equivalent and highly cost-effective strategies to further select those patients who should be tested for MSH2/MLH1germline mutations. Considering this equivalence and that immunostaining is more available than DNA analysis in a clinical setting, the use of immunohistochemistry may help identify a larger proportion of patients with HNPCC.Corresponding Author:Antoni Castells, MD, Department of Gastroenterology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain (castells@clinic.ub.es).Author Contributions:Dr Castells had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Piñol, Castells, Andreu, Llor, Payá.Acquisition of data: Piñol, Castells, Andreu, Llor, Xicola, Rodriguez-Moranta, Jover, Bessa.Analysis and interpretation of data: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Rodríguez-Moranta, Jover, Bessa.Drafting of the manuscript: Piñol, Castells, Andreu.Critical revision of the manuscript for important intellectual content: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Llor, Xicola, Rodríguez-Moranta, Payá, Jover, Bessa.Statistical analysis: Castells.Obtained funding: Castells, Llor.Administrative, technical, or material support: Andreu, Llor, Bessa.Study supervision: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Llor, Xicola, Rodríguez-Moranta, Jover, Bessa.Histopathological advice: Alenda, Payá.Financial Disclosures:None reported.Gastrointestinal Oncology Group of the Spanish Gastroenterological Association Investigators and Study OrganizationWriting Committee and Working Group:Antoni Castells (co-chair), Montserrat Andreu (co-chair), and Virgínia Piñol.Participants:Hospital 12 de Octubre, Madrid, Spain: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, Alberto Ibáñez; Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francisco Rodríguez-Moranta, Francesc Balaguer, Antonio Soriano, Rosa Cuadrado, Maria Pellisé, Rosa Miquel, J. Ignasi Elizalde, Josep M. Piqué; Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, Javier Ortego; Hospital Cristal-Piñor, Complexo Hospitalario de Ourense, Ourense: Joaquin Cubiella (local coordinator), Mª Soledad Díez, Mercedes Salgado, Eloy Sánchez, Mariano Vega; Hospital del Mar, Barcelona: Montserrat Andreu (local coordinator), Xavier Bessa, Agustín Panadés, Asumpta Munné, Felipe Bory, Miguel Nieto, Agustín Seoane; Hospital Donosti, San Sebastián: Luis Bujanda (local coordinator), Juan Ignacio Arenas, Isabel Montalvo, Julio Torrado, Ángel Cosme; Hospital General Universitario de Alicante, Alicante: Artemio Payá (local coordinator), Rodrigo Jover, Juan Carlos Penalva, Cristina Alenda; Hospital General de Granollers, Granollers: Joaquim Rigau (local coordinator), Ángel Serrano, Anna Giménez; Hospital General de Vic, Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, Ramon Barniol; Hospital General Universitario de Guadalajara, Guadalajara: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, Jaime Sánchez; Hospital General Universitario de Valencia, Valencia: Enrique Medina (local coordinator), Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, Elisa Orti; Hospital do Meixoeiro, Vigo: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, Eugenia Sanchez; Hospital San Eloy, Baracaldo: Luis Bujanda (local coordinator), Carmen Muñoz, María del Mar Ramírez, Araceli Sánchez; Hospital Universitari Germans Trias i Pujol, Badalona: Xavier Llor (local coordinator), Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, Elisenda Pons, José M. Hernández, Miquel A. Gassull; Hospital Universitari Mútua de Terrassa, Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, Maria Esteve; Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, Joan Viñas; Hospital Universitario de Canarias, Tenerife: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, Antonio Martín; Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, Teresa Sala; Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator), María del Valle García, Patricia López, Fernando López, Rosa Ortega, Javier Briceño, Javier Padillo; Fundació Hospital Son Llatzer, Palma de Mallorca: Àngels Vilella (local coordinator), Carlos Dolz, Hernan Andreu.Funding/Support:This study was supported by grants FIS 01/0104-01, 01/0104-02, and 01/0104-03 from the Fondo de Investigación Sanitaria; RC03/02 and RC03/10 from the Instituto de Salud Carlos III; SAF 04-07190 from the Ministerio de Ciencia y Tecnología; and from Merck, Sharp and Dhome, Spain. Dr Piñol received a research grant from the Institut d’Investigacions Biomèdiques August Pi i Sunyer; Dr Llor a contract from Programa Ramon y Cajal (Ministerio de Ciencia y Tecnología); and Dr Rodríguez-Moranta from the Hospital Clínic and Instituto de Salud Carlos III.Role of the Sponsor:The funding sources had no involvement in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.Acknowledgment:We thank Josep M. Piqué, MD, for his critical review and helpful comments on the manuscript, as well as his enthusiastic and continuous support to the EPICOLON project.REFERENCESAKRustgiHereditary gastrointestinal polyposis and nonpolyposis syndromes.N Engl J Med1994331169417027969362JPTerdimanJRGumJrPGConradEfficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.Gastroenterology2001120213011208710FMGiardielloJDBrensingerGMPetersenAGA technical review on hereditary colorectal cancer and genetic testing.Gastroenterology200112119821311438509AUmarJIRisingerETHawkJCBarrettTesting guidelines for hereditary non-polyposis colorectal cancer.Nat Rev Cancer2004415315814964310HFVasenJPMecklinPMKhanHTLynchThe International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC).Dis Colon Rectum1991344244252022152HFVasenPWatsonJPMecklinHTLynchNew clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC.Gastroenterology19991161453145610348829MARodriguez-BigasCRBolandSRHamiltonA National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst199789175817629392616AUmarCRBolandJPTerdimanRevised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst20049626126814970275CRBolandSNThibodeauSRHamiltonA National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res199858524852579823339LAAaltonenRSalovaaraPKristoIncidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease.N Engl J Med1998338148114879593786RSalovaaraALoukolaPKristoPopulation-based molecular detection of hereditary nonpolyposis colorectal cancer.J Clin Oncol2000182193220010829038CLambertiRKruseCRuelfsMicrosatellite instability—a useful diagnostic tool to select patients at high risk for hereditary non-polyposis colorectal cancer: a study in different groups of patients with colorectal cancer.Gut19994483984310323887WSSamowitzKCurtinHHLinThe colon cancer burden of genetically defined hereditary nonpolyposis colon cancer.Gastroenterology200112183083811606497JTWijnenHFVasenPMKhanClinical findings with implications for genetic testing in families with clustering of colorectal cancer.N Engl J Med19983395115189709044SNThibodeauAJFrenchJMCunninghamMicrosatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.Cancer Res199858171317189563488SNThibodeauAJFrenchPCRocheAltered expression of hMSH2and hMLH1in tumors with microsatellite instability and genetic alterations in mismatch repair genes.Cancer Res199656483648408895729JMCunninghamCYKimERChristensenThe frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas.Am J Hum Genet20016978079011524701WDietmaierSWallingerTBockerFKullmannRFishelJRuschoffDiagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res199757474947569354436NMLindorLJBurgartOLeontovichImmunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors.J Clin Oncol2002201043104811844828MChristensenNKatballeFWikmanAntibody-based screening for hereditary nonpolyposis colorectal carcinoma compared with microsatellite analysis and sequencing.Cancer2002952422243012436451Ade la ChapelleMicrosatellite instability phenotype of tumors: genotyping or immunohistochemistry? the jury is still out.J Clin Oncol20022089789911844809SSWahlbergJSchmeitsGThomasEvaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2and MLH1mutations in hereditary nonpolyposis colon cancer families.Cancer Res2002623485349212067992MScartozziFBianchiSRosatiMutations of hMLH1and hMSH2in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.J Clin Oncol2002201203120811870161SSalahshorKKoelbleCRubioALindblomMicrosatellite instability and hMLH1and hMSH2expression analysis in familial and sporadic colorectal cancer.Lab Invest20018153554111304573RWardCMeldrumRWilliamsImpact of microsatellite testing and mismatch repair protein expression on the clinical interpretation of genetic testing in hereditary non-polyposis colorectal cancer.J Cancer Res Clin Oncol200212840341112200596AEDe JongMVan PuijenbroekYHendriksMicrosatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer.Clin Cancer Res20041097298014871975TFurukawaFKonishiKShitohMKojimaHNagaiTTsukamotoEvaluation of screening strategy for detecting hereditary nonpolyposis colorectal carcinoma.Cancer20029491192011920458VPiñolMAndreuACastellsAPayáXBessaRJoverFrequency of hereditary non-polyposis colorectal cancer and other colorectal cancer familial forms in Spain: a multicenter, prospective, nation-wide study.Eur J Gastroenterol Hepatol200416394515095851American Joint Committee on CancerManual for Staging of Cancer.5th ed. Philadelphia, Pa: JB Lippincott; 1997JMHoangPHCottuBThuilleRJSalmonGThomasRHamelinBAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res1997573003039000572ALoukolaKEklinPLaihoMicrosatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC).Cancer Res2001614545454911389088IGonzalez-GarciaVMorenoMNavarroStandardized approach for microsatellite instability detection in colorectal carcinomas.J Natl Cancer Inst20009254454910749909XPZhouJMHoangYJLiDetermination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites.Genes Chromosomes Cancer1998211011079491320JJGilleFBHogervorstGPalsGenomic deletions of MSH2and MLH1in colorectal cancer families detected by a novel mutation detection approach.Br J Cancer20028789289712373605YIonovMAPeinadoSMalkhosyanDShibataMPeruchoUbiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature19933635585618505985AHemminkiPPeltomakiJPMecklinLoss of the wild type MLH1gene is a feature of hereditary nonpolyposis colorectal cancer.Nat Genet199484054107894494NPapadopoulosNCNicolaidesYFWeiMutation of a mutl homolog in hereditary colon cancer.Science1994263162516298128251BLiuRParsonsNPapadopoulosAnalysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.Nat Med199621691748574961WMullerLJBurgartRKrause-PaulusThe reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer: results of an international collaborative study.Fam Cancer20011879214574003SSyngalEAFoxCEngRDKolodnerJEGarberSensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2and MLH1.J Med Genet20003764164510978352American Gastroenterological AssociationAmerican Gastroenterological Association medical position statement: hereditary colorectal cancer and genetic testing.Gastroenterology200112119519711438508PPeltomakiHFVasenMutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study.Gastroenterology1997113114611589322509TLiuPTannergardPHackmanMissense mutations in hMLH1associated with colorectal cancer.Hum Genet199910543744110598809JWijnenPMKhanHVasenHereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.Am J Hum Genet1997613293359311737RKariolaROtwayKELonnqvistTwo mismatch repair gene mutations found in a colon cancer patient: which one is pathogenic?Hum Genet200311210510912522549MRavnik-GlavacUPotocnikDGlavacIncidence of germline hMLH1and hMSH2mutations (HNPCC patients) among newly diagnosed colorectal cancers in a Slovenian population.J Med Genet20003753353610970186APercesepeFBorghiMMenigattiMolecular screening for hereditary nonpolyposis colorectal cancer: a prospective, population-based study.J Clin Oncol2001193944395011579115TCaldesJGodinoMde la HoyaPrevalence of germline mutations of MLH1and MSH2in hereditary nonpolyposis colorectal cancer families from Spain.Int J Cancer20029877477911920650MPalicioJBalmanaSGonzalezMismatch repair gene analysis in Catalonian families with colorectal cancer [letter].J Med Genet200239E2912070261HFVasenMvan BallegooijenEBuskensA cost-effectiveness analysis of colorectal screening of hereditary nonpolyposis colorectal carcinoma gene carriers.Cancer199882163216379576281SDRamseyLClarkeREtzioniMHigashiKBerryNUrbanCost-effectiveness of microsatellite instability screening as a method for detecting hereditary nonpolyposis colorectal cancer.Ann Intern Med200113557758811601929CMReyesBAAllenJPTerdimanLSWilsonComparison of selection strategies for genetic testing of patients with hereditary nonpolyposis colorectal carcinoma: effectiveness and cost-effectiveness.Cancer2002951848185612404277 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA American Medical Association

Accuracy of Revised Bethesda Guidelines, Microsatellite Instability, and Immunohistochemistry for the Identification of Patients With Hereditary Nonpolyposis Colorectal Cancer

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American Medical Association
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Copyright 2005 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
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0098-7484
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1538-3598
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10.1001/jama.293.16.1986
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15855432
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Abstract

ContextThe selection of individuals for hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing is challenging. Recently, the National Cancer Institute outlined a new set of recommendations, the revised Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability.ObjectiveTo establish the most effective and efficient strategy for the detection of MSH2/MLH1gene carriers.Design, Setting, and PatientsA prospective, multicenter, nationwide study (the EPICOLON study) in 20 hospitals in the general community in Spain of 1222 patients with newly diagnosed colorectal cancer between November 1, 2000, and October 31, 2001.InterventionsMicrosatellite instability testing and MSH2/MLH1immunostaining in all patients regardless of age, personal or family history, and tumor characteristics. Patients whose tumors exhibited microsatellite instability and/or lack of protein expression underwent MSH2/MLH1germline testing.Main Outcome MeasuresEffectiveness and efficiency of both microsatellite instability testing and immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2/MLH1germline mutations.ResultsTwo hundred eighty-seven patients (23.5%) fulfilled the revised Bethesda guidelines. Ninety-one patients (7.4%) had a mismatch repair deficiency, with tumors exhibiting either microsatellite instability (n = 83) or loss of protein expression (n = 81). Germline testing identified 11 mutations (0.9%) in either MSH2(7 cases) or MLH1(4 cases) genes. Strategies based on either microsatellite instability testing or immunostaining previous selection of patients according to the revised Bethesda guidelines were the most effective (sensitivity, 81.8% and 81.8%; specificity, 98.0% and 98.2%; positive predictive value, 27.3% and 29.0%, respectively) to identify MSH2/MLH1gene carriers. Logistic regression analysis confirmed the revised Bethesda guidelines as the most discriminating set of clinical parameters (odds ratio, 33.3; 95% confidence interval, 4.3-250; P = .001).ConclusionThe revised Bethesda guidelines constitute a useful approach to identify patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and immunostaining are equivalent and highly effective strategies to further select those patients who should be tested for MSH2/MLH1germline mutations.Hereditary nonpolyposis colorectal cancer (HNPCC), also named Lynch syndrome, is an autosomal dominant disorder that accounts for approximately 1% to 3% of all colorectal cancers.HNPCC is caused by germline mutations in DNA mismatch repair genes, mainly MSH2and MLH1.Defects on this pathway lead to changes in the length of nucleotide repeat sequences of tumor DNA, termed microsatellite instability.Although a great advance in the understanding of the molecular basis of HNPCC has taken place in the last decade, optimal selection of individuals for HNPCC genetic testing remains controversial.In 1991, the International Collaborative Group on HNPCC established clinical criteria, known as the Amsterdam criteria, which provided a pivotal definition of this syndrome and were critical in identifying its molecular basis.In response to criticism that the Amsterdam criteria were too stringent, the extended Amsterdam II criteria were developed to include extracolonic HNPCC-associated cancers.The use of the Amsterdam criteria achieved the original purpose of classifying a family as having HNPCC, but its limited sensitivity hampered decisions about which patients should undergo genetic testing.In 1996, an international workshop on HNPCC hosted by the National Cancer Institute outlined a set of recommendations, known as the Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability and/or genetic testing.More recently, a second HNPCC workshop revised these criteria and proposed a new set of recommendations, the revised Bethesda guidelines.As it was previously mentioned, tumor microsatellite instability is a phenotypic indicator of defective DNA mismatch repair.The fact that more than 90% of HNPCC-related cancers exhibit microsatellite instability suggests that screening of tumors for microsatellite instability may be an efficient way of selecting individuals for HNPCC genetic testing.On the other hand, it is well known that most mutations in either MSH2or MLH1genes result in abnormal MSH2 or MLH1 protein expression.As a consequence, immunostaining for these 2 proteins is associated with microsatellite instability,but this association is not without exceptions.Indeed, a mutant protein product can be expressed and detected by immunostaining,whereas germline mutations may occur in patients with microsatellite instability–negative tumors.These conflicting results have precluded the establishment of a unique method for primary screening of mismatch repair deficiency. Specific characteristics of performed studies may have contributed to this fact because most of the studies were performed in selected high-risk populations,different criteria were used to define the microsatellite instability status,immunohistochemical evaluation was not performed systematically,or results were not referred to the presence of germline mutations.Our prospective, multicenter, nation-wide study was aimed at establishing the most effective and efficient strategy for the detection of MSH2or MLH1gene carriers in patients with newly diagnosed colorectal cancers. For this purpose, performance characteristics of both microsatellite instability testing and MSH2/MLH1 protein immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2or MLH1germline mutations. A logistic regression analysis was also performed to determine independent predictors of MSH2/MLH1mutations.METHODSBetween November 1, 2000, and October 31, 2001, all patients with newly diagnosed colorectal cancers in 25 hospitals were included in the EPICOLON study, a clinical epidemiology survey aimed at establishing the incidence of HNPCC in Spain.Twenty of the 25 centers agreed to participate in a nested molecular epidemiology study, which required tissue sample collection. Exclusion criteria were familial adenomatous polyposis, personal history of inflammatory bowel disease, and patient or family refusal to participate in the study. The study was approved by the institutional ethics committee of each participating hospital, and written informed consent was obtained from all patients.Demographic, clinical, and tumor-related characteristics of probands, as well as a detailed family history were obtained using a preestablished questionnaire. Personal parameters at baseline included date and place of birth, sex, smoking history, personal history of neoplasia (ie, HNPCC-related tumors and colorectal adenomas), date of colorectal cancer diagnosis, presenting symptoms, serum carcinoembryonic antigen concentration, presence of synchronous colorectal neoplasms, and treatment. Tumor-related parameters included location, size, pathology TNM stage,degree of differentiation, presence of lymphocytic infiltration, and mucinous carcinoma type (defined by the presence of >50% mucinous carcinoma cells). Synchronous lesions were assessed through colonoscopy before or immediately after surgery, as well as through systematic review of the resected specimen. Pedigrees were traced backward and laterally as far as possible, or at least up to second-degree relatives, regarding cancer history. Age at cancer diagnosis, type, location, and tumor stage of the neoplasm, and current status were registered for each affected family member. As a check on overreporting of disease, all patients were queried about the occurrence of stroke in their relatives to assess possible recall bias. Furthermore, an effort was made to verify reported neoplasia in relatives by obtaining medical records and pathology confirmation.Microsatellite instability testing and immunostaining for DNA mismatch repair proteins were performed in all patients regardless of age, personal or family history, and tumor characteristics. To avoid variability in the quality of results, microsatellite instability testing and immunostaining were centralized in 2 single centers, respectively. Researchers scoring immunostaining were blinded to the microsatellite instability results and vice versa.Tumor Microsatellite Instability AnalysisTissue samples from tumor and healthy colonic mucosa were obtained from each patient, immediately frozen in liquid nitrogen, and stored at −70°C until use. In those cases in which frozen tissue was not available, archival formalin-fixed, paraffin-embedded samples were used. Genomic DNA was isolated using the QiaAmp Tissue Kit (Qiagen, Courtaboeuf, France).Microsatellite instability status was assessed using BAT-26mononucleotide marker based on its high sensitivity.In those cases in which BAT-26was stable but immunostaining revealed lack of MSH2 or MLH1 protein expression, the 4 additional markers of the Bethesda panel (BAT-25, D5S346, D2S123, and D17S250)were also evaluated. Primers were labeled fluorescently and analyzed on a genetic analyzer using GeneScan Analysis software (ABI 310 Genetic Analyzer; Applied Biosystems, Foster City, Calif).Tumor MSH2 and MLH1 Protein ExpressionOne block of formalin-fixed, paraffin-embedded tumor tissue was selected per case. Before immunostaining, antigen retrieval was performed by immersing sections in a 10-M concentration of citrate buffer, pH 6.0, and boiling in a pressure cooker for 5 minutes. Sections were then incubated for 20 minutes at room temperature with mouse monoclonal antibodies against MLH1 protein (clone G168-15, dilution 1:40; PharMingen, San Diego, Calif) and MSH2 protein (clone FE11, dilution 1:35; Oncogene Research Products, Boston, Mass). Ultra-Vision streptavidin-biotin peroxidase detection kit (DAKO, Carpinteria, Calif) was used as secondary detection system. The peroxidase reaction was developed using diaminobenzidine tetrachloride as chromogen. Tumor cells were judged to be negative for protein expression only if they lacked staining in a sample in which healthy colonocytes and stroma cells were stained. If no immunostaining of healthy tissue could be demonstrated, the results were considered ambiguous.MSH2/MLH1Germline Mutation AnalysisPatients found to have tumors with microsatellite instability and/or lack of protein expression of either MSH2 or MLH1 underwent germline genetic testing for MSH2and MLH1genes by both multiple ligation probe amplification analysis and sequencing.Multiple ligation probe amplification was performed using the MLH1/MSH2exon deletion assay (MRC-Holland, Amsterdam, the Netherlands), which allows the detection of genomic rearrangements in these genes.Ligation products were amplified by polymerase chain reaction using a fluorescently labeled primer and analyzed in an ABI 3100 sequencer using GeneScan and Genotyper Analysis software (Applied Biosystems). Peak height of each fragment was compared with those measurements of a control sample and deletions were suspected when peak height was 60% or less of healthy controls. Control DNA samples with known MSH2or MLH1genomic rearrangements were included in each batch of experiments. Multiple ligation probe amplification results were confirmed by reverse transcriptase polymerase chain reaction encompassing contiguous exons of the suspected deleted fragment.Germline mutations in the MSH2and MLH1genes were also sought by direct exon-by-exon sequencing. Amplification products were generated with primers located in the flanking introns approximately 50 base pairs from the respective intron/exon borders to detect all possible splice junction mutations. The sequences were determined on the genetic analyzer (ABI 3100, Applied Biosystems) using fluorescently labeled primers and protocols supplied by the manufacturer.Statistical AnalysisPerformance characteristics of screening strategies based on microsatellite instability testing and/or protein immunostaining, either directly or through previous selection of patients according to the revised Bethesda guidelines, were calculated with respect to the diagnosis of HNPCC associated with MSH2/MLH1germline mutations. Mutations were considered deleterious based on sequencing results, published data, and existing mutation databases. Comparison of paired proportions for sensitivity, specificity, and overall accuracy was performed by the McNemar test.A cost-minimization analysis was also performed to establish the most efficient strategy. For this analysis, costs of microsatellite instability (BAT-26maker), immunostaining (both MSH2 and MLH1), and genetic testing were established at 100 € (US $130), 200 € (US $260), and 2400 € (US $3120), respectively, or 1200 € per gene (US $1560 per gene), according to hospital clinic current billing.To identify parameters associated with MSH2/MLH1gene mutation carriers, univariate and multivariate analyses were performed. Variables evaluated corresponded to any previously suggested predictors including each individual characteristic of the original and revised Bethesda guidelines, as well as those proposed by Aaltonen et alfor screening in general population (any first-degree relative with colorectal cancer or endometrial cancer regardless of age, personal history of colorectal cancer or endometrial cancer, and age at diagnosis of colorectal cancer <50 years in the proband) and Wijnen et alin familial cancer clinics (age at diagnosis of colorectal cancer <50 years within a family, fulfillment of the Amsterdam criteria, and endometrial cancer in ≥1 family member). In addition, combined variables corresponding to each of these 4 sets of recommendations were also evaluated. For the univariate analysis, categorical variables were compared by the Fisher exact test. Variables achieving P<.20 in the univariate analysis were subsequently included in a multivariate analysis using a stepwise forward logistic regression procedure. Two different models were evaluated. First, only combined variables (original and revised Bethesda guidelines, and those proposed by Aaltonen et aland Wijnen et al) were included in the model to identify the most discriminating set of parameters. Second, individual characteristics were added to the previously recognized combined variable to identify other independent predictors of MSH2/MLH1germline mutations that can contribute to refine the model.Continuous variables were expressed as mean (SD). All Pvalues were 2-sided. P<.05 was considered statistically significant. All calculations were performed by using SPSS software version 10.0 (SPSS Inc, Chicago, Ill).RESULTSDuring the study period, 1978 patients with newly diagnosed colorectal cancer were included from 25 centers. One hundred six patients were excluded because the tumor developed in the context of a familial adenomatous polyposis (n = 11) or inflammatory bowel disease (n = 14), the patient did not consent to participate in the study (n = 32), or had an incomplete family history (n = 49). Of the remaining 1872 eligible patients, 1222 patients were diagnosed in 20 centers agreeing to participate in the nested molecular epidemiology study and constituted the basis of our analysis. Characteristics of this subset of patients (Table 1) did not differ from the whole group.Table 1.Characteristics of Patients With Colorectal Cancer (N=1222)CharacteristicNo. (%) of PatientsAge, mean (SD), y70 (11)SexMale731 (59.8)Female491 (40.2)Site of tumor relative to splenic flexureProximal357 (29.2)Distal865 (70.8)TNM tumor stage*I161 (13.2)II510 (41.7)III337 (27.6)IV214 (17.5)Degree of differentiation†Well292 (23.9)Moderate835 (68.3)Poor95 (7.8)Mucinous carcinoma type142 (11.6)Synchronous colorectal cancer71 (5.8)Fulfillment of guidelines‡Amsterdam criteria18 (1.5)Extended Amsterdam II criteria22 (1.8)Original Bethesda guidelines224 (18.3)Revised Bethesda guidelines287 (23.5)*Stage I indicates tumor 1-2 (T1-2), no regional lymph node metastasis, and no metastasis; stage II, T3-4, no regional lymph node metastasis, and no metastasis; stage III, any tumor, node 1-3, and no metastasis; and stage IV, any tumor, any node, and distant metastasis.†Well-differentiated tumors resemble identifiable tissue types and poorly differentiated tumors may only be identifiable by the expression of cell markers or by extremely focal and subtle histologic and/or cytologic findings.‡For an explanation of Amsterdam criteria, see Vasen et al; extended Amsterdam II critera, see Vasen et al; original Bethesda guidelines, see Rodriguez-Bigas et al; and revised Bethesda guidelines, see Umar et al.Proband CharacteristicsDemographic, clinical, and tumor-related characteristics of patients included in the study are shown in Table 1. According to the extended Amsterdam II criteria, 22 patients (1.8%) belonged to families satisfying the clinical definition of HNPCC. On the other hand, 287 patients (23.5%) fulfilled at least 1 criterion of the revised Bethesda guidelines.A total of 91 patients (7.4%) were found to have a mismatch repair deficiency. Of these, 83 tumors (6.8%) showed microsatellite instability and 81 (6.6%) loss of protein expression in either MSH2 (21 cases) or MLH1 (60 cases). Loss of MSH2 or MLH1 expression was found in 73 of the microsatellite instability tumors, whereas expression of both proteins was retained in the remaining 10 tumors with microsatellite instability. In addition, loss of MSH2 or MLH1 expression was found in 8 patients whose tumor did not show microsatellite instability according to the BAT-26marker. Evaluation of the 4 additional markers of the Bethesda panel in these cases confirmed that their tumor was stable. Finally, unequivocal loss of MSH2 or MLH1 was not observed in any of the remaining 1131 tumors not showing microsatellite instability.Germline genetic testing identified 11 mutations (0.9%) in either MSH2(7 cases) or MLH1(4 cases) genes. Characteristics of these patients are shown in Table 2. Three DNA changes corresponded to missense mutations. Although all 3 mutations have been previously reported as deleterious, 1 tumor did not show microsatellite instability, 2 tumors exhibited contradictory immunostaining results, and 1 patient did not fulfill the revised Bethesda guidelines.Table 2.Characteristics of Patients With MSH2/MLH1Germline Mutations (n = 11)CaseFulfillment of GuidelinesMSI StatusIHC Expressionof ProteinGermline MutationAmsterdam II CriteriaRevised Bethesda GuidelinesMSH2MLH1GeneNucleotide ChangeConsequence1YesYesUnstablePresentAbsentMLH1859_860delAAFrameshift at codon 287; stop at codon 3052YesYesUnstablePresentAbsentMLH12136G>AStop at codon 7123YesYesUnstableAbsentPresentMSH2del exon 9-16Out-of-frame deletion of exons 9-164YesYesUnstableAbsentPresentMSH21703insAFrameshift at codon 568; stop at codon 5715NoYesUnstablePresentAbsentMLH1IVS14-1G>AOut-of-frame deletion of exon 156NoYesUnstableAbsentPresentMSH2IVS15-3TTC>CTTSplicing defect, out-of-frame deletion7NoYesUnstableAbsentPresentMSH2688insAFrameshift at codon 230; stop at codon 2328NoYesUnstableAbsentPresentMSH21165C>TStop at codon 3899NoYesUnstableAbsentPresentMLH11852AA>GCLys>Ala at codon 61810NoYesStableAbsentPresentMSH2965G>AGly>Asp at codon 32211NoNoUnstablePresentAbsentMSH2435T>GIle>Met at codon 145Abbreviations: IHC, immunohistochemistry; MSI, microsatellite instability.Performance Characteristics and Efficiency of Screening StrategiesPerformance characteristics of screening strategies for the identification of MSH2/MLH1gene carriers are shown in Table 3. Calculations were performed considering both germline mutations (n=11) and unambiguous pathogenic variants (n=8). Approaches based on either microsatellite instability analysis or MSH2/MLH1 immunostaining had an almost identical effectiveness. Conversely, specificity, overall accuracy, and positive predictive value of these strategies increased when patients were previously selected according to the revised Bethesda guidelines. Finally, combinations of the results obtained in both microsatellite instability testing and protein immunostaining did not provide any additional advantage with respect to the corresponding individual strategies.Table 3.Performance Characteristics of Different Strategies for the Identification of MSH2/MLH1Gene Mutation Carriers*StrategyAny Germline Mutation (n = 11), %Unambiguous Germline Mutations (n = 8), %SensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracySensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracyFulfillment of revised Bethesda guidelines90.977.13.599.977.310077.02.810077.2Presence of MSI90.993.912.299.993.910093.89.610093.9Loss of protein expression81.894.211.499.894.110094.210.110094.2Presence of MSI or loss of protein expression90.993.411.299.993.310093.28.910093.3Presence of MSI and loss of protein expression72.794.811.399.794.610094.811.310094.8Fulfillment of revised Bethesda guidelines andPresence of MSI81.898.0*27.399.897.9*10097.9*24.210097.9*Loss of proteinexpression81.898.2*29.099.898.0*10098.1*25.810098.1*Presence of MSI or loss of proteinexpression81.897.8*25.799.897.7*10097.8*22.910097.8*Presence of MSI and loss of proteinexpression72.798.3*28.699.798.1*10098.4*28.610098.4*Abbreviation: MSI, microsatellite instability.*P<.001 (McNemar test) with respect to the corresponding paired strategy without previous selection of patients according to the revised Bethesda guidelines.Efficiency of all the above mentioned strategies for the identification of MSH2/MLH1-associated HNPCC was evaluated in a cost-minimization analysis. Clinical selection of patients according to the revised Bethesda guidelines followed by either microsatellite instability analysis (11 989 € [US $15 586] per detected mutation) or protein immunostaining (10 644 € [US $13 837] per detected mutation) was more cost-effective than any of these approaches performed directly (32 140 € [US $41 782] and 37 956 € [US $49 343] per detected mutation, respectively). Conversely, performance of both microsatellite instability analysis and protein immunostaining increased the cost (14 900 € [US $19 370] per detected mutation in combination with the revised Bethesda guidelines and 49 020 € [US $63 726] per detected mutation without previous selection of patients according to these clinical criteria) with respect to the corresponding individual strategies.Predictors of MSH2or MLH1Germline MutationsTo further refine clinical criteria for selecting patients who should be submitted to genetic testing, variables associated with MSH2or MLH1germline mutations were identified. Table 4shows parameters significantly associated in the univariate analysis. When only combined variables corresponding to the original set of recommendations were included in the logistic regression analysis, the revised Bethesda guidelines turned out to be the most discriminating set of parameters (odds ratio [OR], 33.3; 95% confidence interval [CI], 4.3-250; P = .001). In that sense, specific characteristics of the revised Bethesda guidelines, which allowed the identification of 2 additional gene carriers with respect to those identified by the original Bethesda guidelines, were criteria number 5 (colorectal cancer diagnosed in ≥2 first-degree or second-degree relatives with HNPCC-related tumors, regardless of age) in 2 cases and number 3 (colorectal cancer with the microsatellite instability–high histology diagnosed in a patient aged <60 years) in 1 case. Furthermore, when individual characteristics were added to the logistic regression model, family history of colorectal cancer diagnosed in at least 1 first-degree relative (OR, 3.6; 95% CI, 1.05-12.7; P = .04) was selected as an independent predictor of MSH2/MLH1germline mutations, along with the revised Bethesda guidelines (OR, 13.7; 95% CI, 1.47-125; P = .02).Table 4.Predictors of MSH2/MLH1Germline Mutations (Univariate Analysis)*CharacteristicNo. (%) of PatientsPValuePositive Predictive Value, %Germline Mutation (n = 11)No Germline Mutation (n = 1211)Colorectal cancer in ≥1 first-degree relative6 (54.5)151 (12.5).0013.8Colorectal or endometrial cancer in ≥1 first-degree relative6 (54.5)180 (14.8).0033.2Personal history of colorectal or endometrial cancer†4 (36.4)90 (7.4).0074.3Colorectal cancer diagnosed at age <50 y3 (27.3)55 (4.5).015.2Colorectal or endometrial cancer in ≥1 first-degree relative, personal history of colorectal or endometrial cancer, or age at diagnosis of proband colorectal cancer <50 y‡§8 (72.7)306 (25.3).0012.5Fulfillment of Amsterdam criteria4 (36.4)14 (1.1)<.00122.2Endometrial cancer in ≥1 family member4 (36.4)63 (5.2).0025.9Colorectal cancer diagnosed at age <50 y within a family, endometrial cancer in ≥1 family member, or fulfillment of Amsterdam criteria‡&par;7 (63.6)156 (12.9)<.0014.3Synchronous, metachronous colorectal, or other HNPCC-related cancer¶5 (45.5)98 (8.1).0014.9Colorectal or other HNPCC-related cancer¶ and/or a colorectal adenoma in ≥1 first-degree relative, 1 of the cancers diagnosed at age <50 y, and the adenoma diagnosed at age <40 y4 (36.4)61 (5.0).0026.2Colorectal or endometrial cancer† diagnosed at age <50 y3 (27.3)78 (6.4).033.7Right-sided colorectal cancer with an undifferentiated pattern (solid/cribriform) on histopathology diagnosed at age <50 y1 (9.1)5 (0.4).0516.7Signet-ring cell-type colorectal cancer diagnosed at age <50 y1 (9.1)14 (1.1).136.7Fulfillment of Bethesda guidelines‡8 (72.7)216 (17.8)<.0013.6Synchronous, metachronous colorectal, or other HNPCC-related tumors,# regardless of age5 (45.5)99 (8.2).0014.8Colorectal cancer with the MSI-high histology** diagnosed at age <60 y3 (27.3)52 (4.3).015.4Colorectal cancer diagnosed in ≥1 first-degree relatives with an HNPCC-related tumor, with ≥1 cancer diagnosed at age <50 y4 (36.4)57 (4.7).0016.6Colorectal cancer diagnosed in ≥2 first- or second-degree relatives with HNPCC-related tumors, regardless of age5 (45.5)83 (6.8).0015.7Fulfillment of revised Bethesda guidelines‡10 (90.9)277 (22.9)<.0013.5Abbreviations: HNPCC, hereditary nonpolyposis colorectal cancer; MSI, microsatellite instability.*When not specified, characteristics are referred to probands.†Patients with endometrial cancer also had colorectal cancer, which qualified inclusion into the study.‡Combinations of individual variables according to the original sets of recommendations.§Criteria suggested by Aaltonen et al.&par;Criteria suggested by Wijnen et al.¶Including endometrial, ovarian, gastric, hepatobiliary, small-bowel, or urinary tract cancer.#Including endometrial, ovarian, gastric, hepatobiliary, small-bowel, urinary tract, pancreatic, and brain cancer, sebaceous gland adenomas, and keratoacanthomas.**Including presence of tumor-infiltrating lymphocytes, Crohn-like lymphocytic reaction, mucinous/signet-ring differentiation, or medullary growth pattern.Table 5summarizes performance characteristics of strategies based on the combination of the 2 independent variables identified in the multivariate analysis. Genetic testing driven by this combination of clinical parameters was not substantially more accurate than when the revised Bethesda guidelines were only used (Table 3).Table 5.Performance Characteristics of Clinical Criteria for the Identification of MSH2/MLH1Gene Mutation Carriers (Multivariate Analysis)*StrategyPerformance Characteristic, %SensitivitySpecificityPositive Predictive ValueNegative Predictive ValueOverall AccuracyFulfillment of clinical criteria10070.32.910070.5Fulfillment of clinical criteria and presence of MSI90.997.928.699.997.9Fulfillment of clinical criteria and loss of protein expression81.898.128.199.897.9Fulfillment of clinical criteria and presence of MSI or loss of protein expression90.997.726.399.997.6Fulfillment of clinical criteria, presence of MSI, and loss of protein expression72.798.328.699.798.1Abbreviation: MSI, microsatellite instability.*Based on the combination of the revised Bethesda guidelinesand family history of colorectal cancer diagnosed in at least 1 first-degree relative.COMMENTOur study represents the first reported attempt to our knowledge to determine the effectiveness of different strategies for identifying MSH2/MLH1gene carriers in the context of the recently proposed revised Bethesda guidelines. The strength of our study relies on the fact that it was performed on a general population basis; it involved the largest number of patients evaluated so far; microsatellite instability analysis and protein immunostaining were performed in a parallel and blinded fashion; results were evaluated according to the presence of germline mutations; and finally, costs were also considered. Results of our study suggest that microsatellite instability testing and protein immunostaining are equivalent strategies in terms of cost-effectiveness, and that when either of these screening methods are performed in patients selected according to the revised Bethesda guidelines, they are highly accurate in identifying patients with MSH2/MLH1-associated HNPCC. However, the relatively low number of identified mutations, as well as the uncertain significance of the 3 missense mutations, may constitute a drawback of the study and, consequently, could have influenced the results. Nonetheless, recalculation of effectiveness and efficiency of the revised Bethesda guidelines, either alone or in combination with microsatellite instability testing or immunostaining, and considering only those unambiguous pathogenic mutations does not modify the conclusions of the study.Because molecular definition of HNPCC was established,identification of MSH2/MLH1gene carriers has become a critical and difficult issue.Currently, several approaches including the use of different sets of clinical criteria and methods for determining mismatch repair deficiency (ie, microsatellite instability testing and immunostaining) are being used and there is no unique and universally accepted strategy.Although it can be anticipated that epidemiological diversity among geographical areas and technical disparity among laboratoriesmay preclude the establishment of a definitive, flawless approach, our results suggest that the revised Bethesda guidelines seem to be the most accurate clinical criteria tested so far for the identification of patients at risk for such an inherited disorder.Indeed, its performance characteristics, either alone or in combination with microsatellite instability testing and/or immunostaining, a highly discriminating predictive value in the logistic regression model with respect to other sets of recommendations, and the lack of benefit of adding other independent predictors in terms of both effectiveness and efficiency warrants the use of these criteria in clinical practice. These results are further reinforced by those obtained in a parallel investigation of our group, in which original and revised Bethesda guidelines were compared directly. In that analysis, the latter provided a more accurate and cost-effective approach than the original criteria (unpublished data).Although current recommendations for HNPCC identification rely on microsatellite instability testing as screening strategy,immunostaining has been proposed as an alternative approach.Because protein immunostaining is often easier to perform than DNA analysis in a clinical setting, the use of this technique appears to offer a relatively convenient and rapid method for the prescreening of tumors with mismatch repair defects.Equivalence of both strategies has been demonstrated in high-risk colorectal cancer populations, in which detection of microsatellite instability or loss of MSH2/MLH1 expression were both useful criteria for selecting patients who should be submitted to genetic testing.In addition, immunostaining is the method of choice to direct the search for germline mutations,and it may help to solve the status of MSH2germline variants of uncertain significance because somatic inactivation of MSH2is a rare event in sporadic microsatellite instability tumors.Finally, results of recent investigations suggest that systematic evaluation of other mismatch repair proteins, such as MHS6and PMS2,may contribute to increase the effectiveness of immunostaining.Despite the high correlation between MSH2or MLH1germline mutations and the absence of protein expression, some conflicting results were observed in our study. Indeed, 2 patients with missense mutations in either MSH2or MLH1exhibited loss of expression of the opposite protein. These contradictory results bring about the issue of determining the pathogenic significance of missense mismatch repair gene mutations. With respect to the MLH1:Lys618Ala variant, although it has been shown to segregate with the HNPCC phenotype,it has also been reported in healthy controls.Regarding the MSH2:Ile145Met variant, it has been reported in other HNPCC families (http://www.insight-group.org), but functional results raised some concerns with respect to its pathogenicity.To overcome this situation, calculations were repeated considering these 3 missense mutations as not proven pathogenic and, although minor variations occurred, the results did not change, thus reinforcing the conclusions of the study.In our study, microsatellite instability testing was systematically performed by analyzing the BAT-26marker alone instead of using the 5-marker panel proposed by the National Cancer Institute.The use of this single marker, in a similar manner as it has been performed in previous investigations,was justified by its high sensitivity, which ranged between 93% and 97%.Indeed, in the great majority of tumors, analysis of mononucleotide repeats BAT-25and BAT-26is sufficient to establish the microsatellite instability status without reference to the germline DNA, because these markers are quasi-monomorphic in white populations.In addition, Loukola et aldemonstrated that the use of BAT-26alone was feasible in screening for individuals with HNPCC because this marker was able to identify all gene mutation carriers. Nevertheless, there is still some controversy with respect to this issue because some studies have suggested that BAT-26can miss some cases with mismatch repair defects (ie, those related to the MSH2gene).With this limitation, we designed our investigation with the hypothesis that simultaneous protein immunostaining would contribute to identify germline mutations in patients with BAT-26stable tumors. However, our results demonstrated that combination of both microsatellite instability testing and protein immunostaining did not provide any advantage with respect to each of these approaches individually. More important, performance of the 4 remaining markers of the Bethesda panel did not contribute to identify any additional gene mutation carrier because all tumors stable for the BAT-26marker and showing loss of protein expression were also stable for the whole panel. Finally, the frequency of MSH2/MLH1germline mutations observed in our study (0.9%) is very similar to that reported in other investigations using different screening strategies; therefore, arguing against the possibility of underestimating the incidence of HNPCC as a result of the screening method used. However, some geographical differences may exist, as noted by the relatively low frequency of MSH2/MLH1gene carriers among patients fulfilling the Amsterdam criteria found in our study, similar to the previously reported studies in Spain.Taking into account all these considerations, evaluation of the BAT-26marker alone seems to be a simple, rapid, and reliable method to screen for microsatellite instability when the final goal is to select patients who should be tested for MSH2/MLH1germline defects.In the last few years, it has been demonstrated that colorectal cancer surveillance of HNPCC gene carriers is effective and considerably less costly than no colorectal cancer surveillance.In addition, screening patients with newly diagnosed colorectal cancer using the original Bethesda guidelines and microsatellite instability testing to drive to subsequent genetic testing for HNPCC is cost-effective, especially if the benefits to the patients’ immediate relatives are considered.However, there is very little information with respect to other strategies.In that sense, no study has evaluated the efficiency of the revised Bethesda guidelines or compared microsatellite instability testing and protein immunostaining in terms of cost-effectiveness for the identification of MSH2/MLH1germline mutations. Our results suggest that clinical selection of patients according to the revised Bethesda guidelines along with either microsatellite instability testing or immunostaining was more efficient than any of these approaches separately. However, because of the noteworthy variations in costs of medical procedures among countries and health systems as well as the minimal difference observed, the superiority of immunostaining vs microsatellite instability testing cannot be clearly established.In conclusion, our results demonstrate that the revised Bethesda guidelines constitute a very useful approach to select patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and protein immunostaining are equivalent and highly cost-effective strategies to further select those patients who should be tested for MSH2/MLH1germline mutations. Considering this equivalence and that immunostaining is more available than DNA analysis in a clinical setting, the use of immunohistochemistry may help identify a larger proportion of patients with HNPCC.Corresponding Author:Antoni Castells, MD, Department of Gastroenterology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain (castells@clinic.ub.es).Author Contributions:Dr Castells had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Piñol, Castells, Andreu, Llor, Payá.Acquisition of data: Piñol, Castells, Andreu, Llor, Xicola, Rodriguez-Moranta, Jover, Bessa.Analysis and interpretation of data: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Rodríguez-Moranta, Jover, Bessa.Drafting of the manuscript: Piñol, Castells, Andreu.Critical revision of the manuscript for important intellectual content: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Llor, Xicola, Rodríguez-Moranta, Payá, Jover, Bessa.Statistical analysis: Castells.Obtained funding: Castells, Llor.Administrative, technical, or material support: Andreu, Llor, Bessa.Study supervision: Piñol, Castells, Andreu, Castellví-Bel, Alenda, Llor, Xicola, Rodríguez-Moranta, Jover, Bessa.Histopathological advice: Alenda, Payá.Financial Disclosures:None reported.Gastrointestinal Oncology Group of the Spanish Gastroenterological Association Investigators and Study OrganizationWriting Committee and Working Group:Antoni Castells (co-chair), Montserrat Andreu (co-chair), and Virgínia Piñol.Participants:Hospital 12 de Octubre, Madrid, Spain: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, Alberto Ibáñez; Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francisco Rodríguez-Moranta, Francesc Balaguer, Antonio Soriano, Rosa Cuadrado, Maria Pellisé, Rosa Miquel, J. Ignasi Elizalde, Josep M. Piqué; Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, Javier Ortego; Hospital Cristal-Piñor, Complexo Hospitalario de Ourense, Ourense: Joaquin Cubiella (local coordinator), Mª Soledad Díez, Mercedes Salgado, Eloy Sánchez, Mariano Vega; Hospital del Mar, Barcelona: Montserrat Andreu (local coordinator), Xavier Bessa, Agustín Panadés, Asumpta Munné, Felipe Bory, Miguel Nieto, Agustín Seoane; Hospital Donosti, San Sebastián: Luis Bujanda (local coordinator), Juan Ignacio Arenas, Isabel Montalvo, Julio Torrado, Ángel Cosme; Hospital General Universitario de Alicante, Alicante: Artemio Payá (local coordinator), Rodrigo Jover, Juan Carlos Penalva, Cristina Alenda; Hospital General de Granollers, Granollers: Joaquim Rigau (local coordinator), Ángel Serrano, Anna Giménez; Hospital General de Vic, Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, Ramon Barniol; Hospital General Universitario de Guadalajara, Guadalajara: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, Jaime Sánchez; Hospital General Universitario de Valencia, Valencia: Enrique Medina (local coordinator), Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, Elisa Orti; Hospital do Meixoeiro, Vigo: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, Eugenia Sanchez; Hospital San Eloy, Baracaldo: Luis Bujanda (local coordinator), Carmen Muñoz, María del Mar Ramírez, Araceli Sánchez; Hospital Universitari Germans Trias i Pujol, Badalona: Xavier Llor (local coordinator), Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, Elisenda Pons, José M. Hernández, Miquel A. Gassull; Hospital Universitari Mútua de Terrassa, Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, Maria Esteve; Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, Joan Viñas; Hospital Universitario de Canarias, Tenerife: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, Antonio Martín; Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, Teresa Sala; Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator), María del Valle García, Patricia López, Fernando López, Rosa Ortega, Javier Briceño, Javier Padillo; Fundació Hospital Son Llatzer, Palma de Mallorca: Àngels Vilella (local coordinator), Carlos Dolz, Hernan Andreu.Funding/Support:This study was supported by grants FIS 01/0104-01, 01/0104-02, and 01/0104-03 from the Fondo de Investigación Sanitaria; RC03/02 and RC03/10 from the Instituto de Salud Carlos III; SAF 04-07190 from the Ministerio de Ciencia y Tecnología; and from Merck, Sharp and Dhome, Spain. Dr Piñol received a research grant from the Institut d’Investigacions Biomèdiques August Pi i Sunyer; Dr Llor a contract from Programa Ramon y Cajal (Ministerio de Ciencia y Tecnología); and Dr Rodríguez-Moranta from the Hospital Clínic and Instituto de Salud Carlos III.Role of the Sponsor:The funding sources had no involvement in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.Acknowledgment:We thank Josep M. Piqué, MD, for his critical review and helpful comments on the manuscript, as well as his enthusiastic and continuous support to the EPICOLON project.REFERENCESAKRustgiHereditary gastrointestinal polyposis and nonpolyposis syndromes.N Engl J Med1994331169417027969362JPTerdimanJRGumJrPGConradEfficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.Gastroenterology2001120213011208710FMGiardielloJDBrensingerGMPetersenAGA technical review on hereditary colorectal cancer and genetic testing.Gastroenterology200112119821311438509AUmarJIRisingerETHawkJCBarrettTesting guidelines for hereditary non-polyposis colorectal cancer.Nat Rev Cancer2004415315814964310HFVasenJPMecklinPMKhanHTLynchThe International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC).Dis Colon Rectum1991344244252022152HFVasenPWatsonJPMecklinHTLynchNew clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC.Gastroenterology19991161453145610348829MARodriguez-BigasCRBolandSRHamiltonA National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst199789175817629392616AUmarCRBolandJPTerdimanRevised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst20049626126814970275CRBolandSNThibodeauSRHamiltonA National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res199858524852579823339LAAaltonenRSalovaaraPKristoIncidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease.N Engl J Med1998338148114879593786RSalovaaraALoukolaPKristoPopulation-based molecular detection of hereditary nonpolyposis colorectal cancer.J Clin Oncol2000182193220010829038CLambertiRKruseCRuelfsMicrosatellite instability—a useful diagnostic tool to select patients at high risk for hereditary non-polyposis colorectal cancer: a study in different groups of patients with colorectal cancer.Gut19994483984310323887WSSamowitzKCurtinHHLinThe colon cancer burden of genetically defined hereditary nonpolyposis colon cancer.Gastroenterology200112183083811606497JTWijnenHFVasenPMKhanClinical findings with implications for genetic testing in families with clustering of colorectal cancer.N Engl J Med19983395115189709044SNThibodeauAJFrenchJMCunninghamMicrosatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.Cancer Res199858171317189563488SNThibodeauAJFrenchPCRocheAltered expression of hMSH2and hMLH1in tumors with microsatellite instability and genetic alterations in mismatch repair genes.Cancer Res199656483648408895729JMCunninghamCYKimERChristensenThe frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas.Am J Hum Genet20016978079011524701WDietmaierSWallingerTBockerFKullmannRFishelJRuschoffDiagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res199757474947569354436NMLindorLJBurgartOLeontovichImmunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors.J Clin Oncol2002201043104811844828MChristensenNKatballeFWikmanAntibody-based screening for hereditary nonpolyposis colorectal carcinoma compared with microsatellite analysis and sequencing.Cancer2002952422243012436451Ade la ChapelleMicrosatellite instability phenotype of tumors: genotyping or immunohistochemistry? the jury is still out.J Clin Oncol20022089789911844809SSWahlbergJSchmeitsGThomasEvaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2and MLH1mutations in hereditary nonpolyposis colon cancer families.Cancer Res2002623485349212067992MScartozziFBianchiSRosatiMutations of hMLH1and hMSH2in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.J Clin Oncol2002201203120811870161SSalahshorKKoelbleCRubioALindblomMicrosatellite instability and hMLH1and hMSH2expression analysis in familial and sporadic colorectal cancer.Lab Invest20018153554111304573RWardCMeldrumRWilliamsImpact of microsatellite testing and mismatch repair protein expression on the clinical interpretation of genetic testing in hereditary non-polyposis colorectal cancer.J Cancer Res Clin Oncol200212840341112200596AEDe JongMVan PuijenbroekYHendriksMicrosatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer.Clin Cancer Res20041097298014871975TFurukawaFKonishiKShitohMKojimaHNagaiTTsukamotoEvaluation of screening strategy for detecting hereditary nonpolyposis colorectal carcinoma.Cancer20029491192011920458VPiñolMAndreuACastellsAPayáXBessaRJoverFrequency of hereditary non-polyposis colorectal cancer and other colorectal cancer familial forms in Spain: a multicenter, prospective, nation-wide study.Eur J Gastroenterol Hepatol200416394515095851American Joint Committee on CancerManual for Staging of Cancer.5th ed. Philadelphia, Pa: JB Lippincott; 1997JMHoangPHCottuBThuilleRJSalmonGThomasRHamelinBAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res1997573003039000572ALoukolaKEklinPLaihoMicrosatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC).Cancer Res2001614545454911389088IGonzalez-GarciaVMorenoMNavarroStandardized approach for microsatellite instability detection in colorectal carcinomas.J Natl Cancer Inst20009254454910749909XPZhouJMHoangYJLiDetermination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites.Genes Chromosomes Cancer1998211011079491320JJGilleFBHogervorstGPalsGenomic deletions of MSH2and MLH1in colorectal cancer families detected by a novel mutation detection approach.Br J Cancer20028789289712373605YIonovMAPeinadoSMalkhosyanDShibataMPeruchoUbiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature19933635585618505985AHemminkiPPeltomakiJPMecklinLoss of the wild type MLH1gene is a feature of hereditary nonpolyposis colorectal cancer.Nat Genet199484054107894494NPapadopoulosNCNicolaidesYFWeiMutation of a mutl homolog in hereditary colon cancer.Science1994263162516298128251BLiuRParsonsNPapadopoulosAnalysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.Nat Med199621691748574961WMullerLJBurgartRKrause-PaulusThe reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer: results of an international collaborative study.Fam Cancer20011879214574003SSyngalEAFoxCEngRDKolodnerJEGarberSensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2and MLH1.J Med Genet20003764164510978352American Gastroenterological AssociationAmerican Gastroenterological Association medical position statement: hereditary colorectal cancer and genetic testing.Gastroenterology200112119519711438508PPeltomakiHFVasenMutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study.Gastroenterology1997113114611589322509TLiuPTannergardPHackmanMissense mutations in hMLH1associated with colorectal cancer.Hum Genet199910543744110598809JWijnenPMKhanHVasenHereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.Am J Hum Genet1997613293359311737RKariolaROtwayKELonnqvistTwo mismatch repair gene mutations found in a colon cancer patient: which one is pathogenic?Hum Genet200311210510912522549MRavnik-GlavacUPotocnikDGlavacIncidence of germline hMLH1and hMSH2mutations (HNPCC patients) among newly diagnosed colorectal cancers in a Slovenian population.J Med Genet20003753353610970186APercesepeFBorghiMMenigattiMolecular screening for hereditary nonpolyposis colorectal cancer: a prospective, population-based study.J Clin Oncol2001193944395011579115TCaldesJGodinoMde la HoyaPrevalence of germline mutations of MLH1and MSH2in hereditary nonpolyposis colorectal cancer families from Spain.Int J Cancer20029877477911920650MPalicioJBalmanaSGonzalezMismatch repair gene analysis in Catalonian families with colorectal cancer [letter].J Med Genet200239E2912070261HFVasenMvan BallegooijenEBuskensA cost-effectiveness analysis of colorectal screening of hereditary nonpolyposis colorectal carcinoma gene carriers.Cancer199882163216379576281SDRamseyLClarkeREtzioniMHigashiKBerryNUrbanCost-effectiveness of microsatellite instability screening as a method for detecting hereditary nonpolyposis colorectal cancer.Ann Intern Med200113557758811601929CMReyesBAAllenJPTerdimanLSWilsonComparison of selection strategies for genetic testing of patients with hereditary nonpolyposis colorectal carcinoma: effectiveness and cost-effectiveness.Cancer2002951848185612404277

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JAMA – American Medical Association

Published: Apr 27, 2005

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