Overview of testing for Lynch syndrome/hnpcc

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1 Overview of testing for Lynch syndrome/hnpcc This overview provides detailed information about interpreting MSI/IHC testing and genetic testing for Lynch syndrome/hnpcc. It is intended to be a reference for providers. Ordering and interpreting genetic testing may be complex and is best done by experienced specialists. See the MSI & IHC testing tool for more information about MSI/IHC testing technology, and the Risk assessment tool for more information about when to order testing for Lynch syndrome. When to order microsatellite instability (MSI) and/or immunohistochemistry for the four mismatch repair proteins (IHC) screening tests for Lynch syndrome (LS) There are different approaches to evaluation of LS using MSI, IHC and genetic testing. Each approach has advantages and disadvantages and may depend on provider preferences or institutional resources. This overview describes an approach with the greatest cost-benefit ratio based on available information for the evaluation of LS. Screening for all CRC cases When screening all cases of colorectal cancer (CRC) for LS, select either the MSI or the IHC test on tumor tissue.* Either the IHC or MSI test is acceptable, but IHC is more cost-effective when screening all CRC cases for LS. Each test will miss about 5 15 percent of all LS cases. One out of every five to eight colorectal cancer patients will be MSI-high or have abnormal IHC results. The ability to perform the test may be subject to the amount and type of tissue available. Note that some institutions are testing all uterine cancers for LS through MSI/IHC analysis. Screening for high-risk families When screening a high-risk family for LS, consider ordering both MSI and IHC testing. See the Risk assessment tool and MSI & IHC testing tool for more information about assessing risk and identifying candidates for LS evaluation. Priority should be given to performing MSI and IHC on a colorectal or endometrial adenocarcinoma from an affected family member. If there are no tumors available, testing can begin on an adenomatous colon polyp however, normal MSI and IHC results in a polyp cannot rule out a diagnosis of LS. Approximately one out of every five patients whose colorectal cancer is MSI-high or has abnormal IHC is found to have LS (the odds are higher for patients whose abnormal IHC includes loss of MSH2, MSH6 or PMS2 alone). Colorectal Cancer Fact Sheets: Testing for Lynch syndrome 1

2 *Note: In 2009 the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) working group reviewed existing evidence and proposed that MSI/IHC testing or genetic testing of the MMR genes be offered to all individuals diagnosed with CRC. The motivations for the recommendation are that Lynch syndrome accounts for approximately 2 4 percent of new diagnoses of CRC; testing and screening can reduce morbidity and mortality in relatives; and that, Family history is an important risk factor for CRC in the general population. Among individuals with newly diagnosed CRC, however, family history is less useful as the first step in identifying Lynch syndrome than strategies involving the analysis of tumor samples (e.g., MSI, IHC). The application of Amsterdam and Bethesda criteria has resulted in variable and generally poor performance in identifying Lynch syndrome. Therefore, the EGAPP working group does not recommend the use of family history to exclude individuals with newly diagnosed cancer from the offer of genetic testing. (EGAPP working group, 2009) These recommendations have not yet been adopted by the majority of medical centers; although, consideration of the benefits and limitations of adoption is underway. How to interpret MSI results If the tumor is MSI high (meaning that a high number of microsatellite repeats were found), order IHC to help direct genetic testing; then, proceed as described below. An alternative is to proceed directly to genetic testing using a blood sample from the patient. In this case, you start with MLH1 and MSH2 gene testing because they are the most common causes of LS. If negative, proceed to MSH6 testing followed by PMS2 testing. Some providers would elect this approach of genetic testing as the next test in cases of a strong family history of LS. However, it is not the most cost-effective approach because if genetic testing is negative, you would proceed to IHC to determine if a mutation was missed by genetic testing. If the tumor is MSI negative, the patient is unlikely to have LS. If there is a high index of suspicion, consider IHC testing because MSI will miss about 5 15 percent of LS cases. If IHC is also normal, no further genetic testing for LS is indicated. Consider the possibility of familial colorectal cancer and other rare genetic syndromes. If you still suspect LS or another colorectal syndrome, refer to a genetic specialist for further evaluation. How to interpret IHC results If IHC is normal, no loss of expression If MLH1, MSH2, MSH6 and PMS2 are present, the patient is unlikely to have LS. If there is a high index of suspicion, consider MSI testing because IHC will miss about 5 15 percent of LS cases. If MSI is also negative, no further genetic testing for LS is indicated. Consider the possibility of familial colorectal cancer and other rare genetic syndromes. If you still suspect LS or another colorectal syndrome, refer to a genetic specialist for further evaluation. If MLH1 and PMS2 are absent If MLH1 and PMS2 are absent, the patient likely has acquired methylation of the MLH1 Colorectal Cancer Fact Sheets: Testing for Lynch syndrome 2

3 Xxxxxx gene promoter, a situation that causes lack of MLH1 protein expression (but does not cause LS). However, LS is still possible. If the patient was diagnosed before age 50 or has at least one first-degree relative with colorectal or endometrial cancer, proceed to genetic testing of the MLH1 gene. In all other cases, consider MLH1 promoter methylation testing and/or BRAF mutation testing on the patient s colorectal tumor (this is not informative for endometrial tumors). If MSH2 and MSH6 are absent If MSH2 and MSH6 are absent, the patient likely has LS. Order MSH2 genetic testing using a blood sample from the patient as the first step. If no mutation is found, consider EPCAM genetic testing and consultation with a genetics specialist. EPCAM is not a mismatch repair protein, but it can affect MSH2 expression. Recent reports have identified EPCAM deletions in patients with Lynch syndrome. As the data about EPCAM and LS is rapidly changing, consider consulting with a genetics specialist. If no mutation is found, follow up with MSH6 genetic testing. If only MSH6 is absent If only MSH6 is absent, the patient may have LS. Order MSH6 genetic testing using a blood sample from the patient. If only PMS2 is absent If only PMS2 is absent, the patient may have LS. Order PMS2 genetic testing using a blood sample from the patient. Any other combination of IHC results Any other combination of abnormal IHC results is rare. If the result includes the absence of MLH1, consider follow up using the guidelines for patients with absence of MLH1 and PMS2. How to interpret genetic testing results Deleterious mutations Deleterious mutations in any of the four mismatch repair genes (MLH1, MSH2, MSH6, PMS2) or EPCAM are diagnostic for LS. If there is a known mutation in the family, at-risk family members can undergo singlemutation testing, which is very reliable and less costly than sequencing. Any individuals who have the mutation and individuals who defer genetic testing should follow the LS surveillance guidelines. Any individual who does not have a mutation that has previously been identified in his or her family should follow guidelines for cancer screening in the general population in the absence of other cancer risk factors. Colorectal Cancer Fact Sheets: Testing for Lynch syndrome 3

4 Xxxxxx Negative results (no mutation identified) Negative results (no mutation identified) can be complicated. First, be certain that the lab completed both sequencing and large-rearrangement testing on the gene (unless you ordered a single-mutation test for a known mutation in the family). Order large-rearrangement testing if it was not performed because these types of mutations are common in the LS gene. If there is a known LS gene mutation in the family, the result is considered a true negative, and the individual can follow guidelines for cancer screening in the general population as long as he or she does not have any other cancer risk factors. If MLH1 and PMS2 were absent on IHC or if IHC was not performed and genetic testing is negative, it is likely that the patient has acquired MLH1 promoter methylation, a condition that is not causative of LS. This can be: Confirmed through additional testing (MLH1 methylation testing and BRAF somatic mutation testing on the patient s tumor), or Assumed unless the family history is particularly striking, in which case more work-up may be indicated. If family history is striking, provide screening guidelines for at-risk relatives based on types of cancers and ages at diagnosis in the family (generally begin colonoscopy 10 years earlier than the youngest diagnosis in the family and repeat every 1 5 years). If MSH2 and MSH6 were absent on IHC and genetic testing is negative, have patient and at-risk relatives follow cancer screening recommendations for LS. If MSH6 or PMS2 were absent on IHC and genetic testing is negative, have patient and at-risk relatives follow cancer screening recommendations for LS. Variants of uncertain significance Variants of uncertain significance are found fairly frequently in the mismatch repair genes and result in inconclusive results. Contact the laboratory for more information about the variant, or refer the family to a genetic counselor who can help interpret this result. Do not offer predictive testing to at-risk relatives. Follow LS surveillance guidelines until the mutation is reclassified for: Patients with an MSI-high tumor or abnormal IHC result and at-risk relatives, and Families that meet Bethesda or Amsterdam criteria. How to interpret MLH1 promoter methylation/braf tumor testing results If V600E BRAF mutation is present, whether MLH1 promoter methylation is present or absent: the patient most likely does not have Lynch syndrome. Provide screening guidelines for the at-risk family members based on types of cancers and ages at diagnosis in the family (generally begin colonoscopy 10 years earlier than the youngest diagnosis in the family and repeat every 1 5 years with frequency based on findings of exams). There are reports of individuals with LS carrying BRAF mutations. While the presence of the V600E BRAF mutation significantly reduces the chance of LS, it is still a possibility. Consider the personal and family history and refer to a genetic specialist if there is still a suspicion of LS. If MLH1 promoter methylation is present but the V600E BRAF mutation is not present: there is a small possibility that the patient may have Lynch syndrome. He or she could have an MLH1 genetic mutation and have acquired MLH1 promoter methylation as the second hit in the tumor. In addition, there are rare cases of inherited MLH1 promoter methylation. Consider Colorectal Cancer Fact Sheets: Tesing for Lynch syndrome 4

5 ordering MLH1 gene testing, including multiplex ligation-dependent probe amplification (MLPA). If negative, consider ordering MLH1 promoter methylation testing in normal tissue (such as blood) from the patient. References: Ladabaum U et al. Strategies to identify the Lynch syndrome among patients with colorectal cancer: A costeffectiveness analysis. Ann Intern Med 2011 Jul 19; 155:69. Weissman SM, Burt R, Church J, Erdman S, Hampel H, Holter S, Jasperson K, Kalady MF, Haidle JL, Lynch HT, Palaniappan S, Wise PE, Senter L. Identification of Individuals at Risk for Lynch Syndrome Using Targeted Evaluations and Genetic Testing: National Society of Genetic Counselors and the Collaborative Group of the Americas on Inherited Colorectal Cancer Joint Practice Guideline. J Genet Couns Dec 14. [Epub ahead of print] Senter L, Clendenning M, Sotamaa K, Hampel H, Green J, Potter JD, Lindblom A, Lagerstedt K, Thibodeau SN, Lindor NM, Young J, Winship I, Dowty JG, White DM, Hopper JL, Baglietto L, Jenkins MA, de la Chapelle A. The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations. Gastroenterology Aug;135(2): Epub 2008 May 2. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (2009). Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome relatives, Genetics in Medicine, 11(1), Myundura M, Grosse SD, Hample H, Palomaki GE. The cost-effectiveness of genetic testing strategies for Lynch syndrome among newly diagnosed patients with colorectal cancer. Genet Med 2010 Feb; 12(2): :2/12:jt: Updated Feb 2012 Colorectal Cancer Fact Sheets: Tesing for Lynch syndrome 5