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Screening for the Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer) Heather Hampel, M.S., Wendy L. Frankel, M.D., Edward Martin, M.D., Mark Arnold, M.D.,Karamjit Khanduja, M.D., Philip Kuebler, M.D., Ph.D., Hidewaki Nakagawa, M.D., Ph.D., Kaisa Sotamaa, M.D.,Thomas W. Prior, Ph.D., Judith Westman, M.D., Jenny Panescu, B.S., Dan Fix, B.S., Janet Lockman, B.S., Ilene Comeras, R.N., and Albert de la Chapelle, M.D., Ph.D.* N Engl J Med 2005;352:1851-60. Background < Amsterdam Criteria I> There should be at least 3 relatives with colon cancer and: 1.Large-scale for germ-line : increasingly possible One should screening be a first degree relative onmutations the other two; 2. At least two successive generations should be affected; 3. At least one should be diagnosed before age 50; The Lynch syndrome (hereditary nonpolyposis colorectal cancer) 4. Familial adenomatous polyposis (FAP) should be excluded; : caused by mutations DNA mismatch-repair genes 5. Tumors should be verified in by the pathological examination (MLH1, MSH2, MSH6, PMS2) : Amsterdam criteria , Bethesda guideline The sensitivity of the criteria : only 40-80 % among person at high risk of cancer -> The strategies for diagnosing Lynch syndrome need to be improved -> presence of germ line mutation in mismatch repair gene < Revised Bethesda Guidelines for testing colorectal tumors for MSI > 1. Amsterdam I criteria 2. Indivisuals with 2 HNPCC cancer (including synchronous/ metachronous colorectal cancer) 3. Indivisuals with colorectal cancer and a first degree relative with colorectal cancer or HNPCC extracolonic cancer or colorectal adenoma (<50yr) 4. Colorectal or endometrial cancer (<50yr) 5. Rt sided colorectal cancer with undifferentiated pattern on histology (<50yr) 6. Signet cell type colorectal cancer (<50yr) 7. colorectal adenoma (<40yr) Searching of mutation in four mismatch-repair genes : difficult and expensive -> molecular prescreening for microsatellite instability Microsatellite instability (MSI) : expansion or contraction of short repeated DNA sequences that are caused by the insertion or deletion of repeated unit : the presence of MSI -> defect in a DNA mismatch repair gene : hallmark of the Lynch syndrome (>90 % of tumors) -> prescreening for the syndrome : more favorable prognosis, do not benefit from chemotherapy with fluorouracil -> important because of prognosis, therapeutic implication Aims of this study : frequency of Lynch syndrome in pts with colorectal cancer : examine strategies for molecular screening to identify the Lynch syndrome : compare effectiveness two techniques to prescreen for mismatch repair deficiency genotyping for MSI <-> immunohistochemical analysis Methods <Patients> 1066 patients with new diagnosis of colorectal carcinoma at the major hospitals in metropolitan Columbus, Ohio Primary screening : genotyping of tumor for MSI Positive for MSI : search for germ-line mutations in the MLH1, MSH2, MSH6, PMS2 genes with the use of immunohistochemical staining for mismatch-repair proteins, genomic sequencing, deletion studies Family members of carriers of the mutation were counseled : those at risk -> mutation testing <Microsatellite instability> To determine microsatellite instability of the tumor : ascertain genotypes using polymorphic markers (BAT25, BAT26, D2S123,D5S346, and D18S69 or D17S250) High-frequency MSI : two or more marker Low-frequency MSI : only one marker Negative : none of the markers <Immunohistochemical staining for mismatch repair protein> Immunoperoxidase staining on formalin-fixed tissue Primary antibodies : MLH1, MSH2, MSH6 or GTBP, PMS2 <Detection of mutations> To search for germ-line mutations, DNA was directly sequenced with the use of primers <Methylation of the promoter region of MLH1> A region in the 5' part of the promoter region :was studied with the use of the methylation-specific PCR Another region closer to exon 1 :was studied with use of combined bisulfite restriction analysis Results Figure 1. Analytic Strategy of the Study and Numbers of Patients for Each Analysis. IHC denotes immunohistochemical, MSI microsatellite instability, and MLPA multiplex ligationdependent probe amplification. 3pts 15pts * Of the 119 relatives who received counseling and were offered testing, 2 chose not to undergo testing. † Of these 52 relatives, 14 had previously had a cancer related to the Lynch syndrome and 38 were unaffected at the time of testing. None of the 52 relatives had previously received a diagnosis of the Lynch syndrome. * Only 20% of pts need molecular testing Considerable savings of time, effort and cost over the method Figure 2. Proposed Strategy for Screening Patients with Colorectal Cancer for Lynch Syndrome Mutations and Mismatch-Repair Deficiency. Figure 3. Immunohistochemical Staining for Mismatch-Repair Proteins in Colorectal Adenocarcinoma. Panel Panel Panel Panel A shows positive staining for MLH1, Panel B negative staining for MLH1, C positive staining for MSH2, Panel D negative staining for MSH2, E positive staining for MSH6, Panel F negative staining for MSH6, G positive staining for PMS2, and Panel H negative staining for PMS2. Conclusions Routine molecular screening of patients with colorectal adenocarcinoma for the Lynch syndrome identified mutations in patients and their family members that otherwise would not have been detected. These data suggest that the effectiveness of screening with immunohistochemical analysis of the mismatch-repair proteins would be similar to that of the more complex strategy of genotyping for microsatellite instability.