EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome

The optimal study design for this purpose would be population based, enrolling a large group of individuals consecutively diagnosed with CRC. Initially, MMR gene mutation testing, accounting for as many major mutations as possible, would be performed on all of these cases. MSI testing would then be performed on samples from all cases with a mutation. Testing (both for MSI and MMR mutations) would utilize the technology currently in use. We restricted the search to articles published in 2003 and later, to help ensure that retrieved studies utilized current testing technologies. By that time, testing laboratories often would have (1) incorporated the basic National Cancer Institute (NCI) panel for MSI testing; (2) included additional mononucleotide markers to improve performance^,; (3) routinely tested for mutations in MSH6 and, possibly, PMS2; and (4) routinely tested for large deletions in MMR genes using multiplex ligation-dependent probe amplification. We searched PubMed from 2003 through June 2007, using the MeSH terms “(Colorectal Neoplasms or Hereditary Nonpolyposis) and (MSI or microsatellite instability),” restricted to humans and the English language. Overall, 212 articles were identified. Two of us (G.E.P. and S.M.) reviewed the 212 abstracts and agreed that 28 full articles should be reviewed for appropriateness. Of these 28 articles, 11 met the following inclusion criteria.^{,  ,  ,  ,  ,  ,  ,  ,  ,  ,}  (1) MMR gene mutations were identified without knowledge of MSI status (in at least an identifiable subset of the data); (2) MSI testing was attempted on all patients with Lynch syndrome; (3) the MSI testing methodology was described in sufficient detail to rate test quality; and (4) the MMR gene with the mutation was identifiable.

The analysis was restricted to individuals with CRC (a few excluded studies included only patients with endometrial or breast cancer). In some studies, a few individuals had multiple CRC tumors tested. We chose the earliest sample with complete test results (i.e., MSI and IHC), to best simulate what might happen as part of routine evaluation in the future. In some studies, there were a few instances of multiple family members being tested. We chose to use the family member with the youngest age of onset for a CRC who had complete test results. Assessment of MSI test quality was defined before reviewing the articles and consisted of four questions: (1) did the authors discuss whether microdissection was performed, and whether it was manual or via laser; (2) how many mononucleotide markers were included in the panel; (3) were both tumor and normal tissue used in determining MSI status; and (4) was a minimum proportion of tumor cells required (e.g., 30% or higher).

Of the 11 studies examined,^{,  ,  ,  ,  ,  ,  ,  ,  ,  ,}  only one was close to being population based, but it was restricted to CRC diagnosed under age 55. Because of the relatively low prevalence of Lynch syndrome (about 2–4%), comprehensive MMR gene testing among all CRC patients in the general population would be expensive. It is for this reason that the 11 studies used various definitions of high-risk populations, such as the Amsterdam or the Bethesda criteria, some other family history–based definition, or early age of onset for the proband (e.g., <55 years of age). If a study included some patients in whom MMR gene mutations were sought because of a positive MSI test result, that study was used only if an identifiable subset of the study population was identified with nonbiased criteria (e.g., family history). The studies were performed in North America, Europe,^{,  ,  ,,  ,  ,  ,} Asia, and Australia. The smallest included five individuals/families with Lynch syndrome; the largest included 26 patients (average 15); MSI results were available for a total of 150 Lynch syndrome patients.

Table 1 shows the estimated clinical sensitivities for MSI testing (positive defined as MSI-high, negative as MSI-low or MSI-stable) to identify MMR mutations. Eleven studies reported MSI results for 81 Lynch syndrome patients with mutations in MLH1. Combining the study results using a random effects model, the sensitivity of MSI testing was 85% (95% CI, 75–92%). The bolded rows indicate summary information for a particular MMR gene. Non-bolded clinical sensitivity estimates (last column) indicate results stratified by MSI test quality. The same studies identified 59 Lynch syndrome patients with a MSH2 mutation and found the sensitivity of MSI testing to be 85% (95% CI, 73–93%). Five studies identified 67 patients with MSH6 mutations and found the sensitivity of MSI testing to be 69% (95% CI, 46–85%). When assessing test quality, we found that all 11 studies reported using both tumor and normal tissue to assign MSI status. None of the studies explicitly reported laser microdissection, which has been reported to be the optimal method for sample preparation. In addition, none of the studies reported a minimum proportion of tumor cells. Roughly half of the studies, however, relied solely on the 1998 NCI recommended panel that includes only two mononucleotide markers (labeled as a 2 under the “MSI test quality” column), whereas the remaining studies utilized three or more mononucleotide markers (labeled as a 3+ under the MSI test quality column). When the results are stratified by this quality measure (last column in Table 1), the clinical sensitivities of studies using three or more mononucleotide markers are consistently higher (91% vs. 80% for MLH1, 87% vs. 84% for MSH2, and 77% vs. 55% for MSH6). This provides evidence supporting several methodological studies^,,, and a more recent NCI report suggested that additional mononucleotide markers (up to five) be included in an MSI panel for clinical testing. Only one study reported a Lynch syndrome patient with a PMS2 mutation and found an MSI-stable result, but only 4 of the 10 MSI test markers provided interpretable results.

A high proportion of Lynch syndrome patients with mutations in the MLH1 and MSH2 genes can be identified via MSI-high test results. The best estimate of sensitivity with the use of two mononucleotide markers in the NCI recommended panel is 80–84%, but with the use of at least three mononucleotide markers, the sensitivity can be increased to 87–91%. Clinical sensitivity seems to be lower for MSH6, with the corresponding estimates of 55% and 77% depending on the number of mononucleotide markers used. The greater discrepancy between these two estimates is likely due to a known reduction in the sensitivity of dinucleotide microsatellites to MSH6 mutations.

All of the studies would be ranked as quality Level 2 or 3; the lower ranking because the settings are all high-risk population (e.g., families who satisfy Amsterdam criteria) rather than population based. This may lead to an overestimate of sensitivity, if MSI test results were related to penetrance. However, this does not seem to be the case. When the population-based results for those younger than 55 years (80% for MLH1 and 82% for MSH2) are compared with the other four studies that were based on strong family histories regardless of age (82% for MLH1 and 88% for MSH2), the results are remarkably similar. The quality of evidence is considered adequate for estimating MSI sensitivity for MLH1, MSH2, and MSH6. Few data are available from individuals other than non-Hispanic whites. The one study in Asians included only five patients with Lynch syndrome, and it found an average MSI sensitivity of 80%. No estimate of clinical sensitivity for PMS2 was made due to the low number of reported results. In subsequent modeling, the sensitivity of MSI testing for PMS2 will be considered equivalent to that for MSH6—lower than that found for MLH1 and MSH2.

It is unlikely that clinical sensitivity would ever reach 100%, even if the laboratory test were to be improved and preanalytic/postanalytic errors eliminated. There is room for improvement, however, as it is likely that none of the studies under review used the most sophisticated MSI test now possible for identifying CRC patients with Lynch syndrome. Methodological studies have shown the importance of laser microdissection,^, the proportion of tumor tissue tested, and the number of cells tested.^, Other studies have provided ways to improve testing of poor quality samples.^, If these techniques had been included in studies under review, even higher clinical sensitivities might have been obtained. Several researchers have examined the possible reasons for an MSI-low or MSI-stable in a confirmed Lynch syndrome patient.^,,, In the majority of instances, a methodological reason for the initial false-negative result was identified. One study found that, for certain large deletions in MSH2, one of the mononucleotide markers (BAT26) appeared MSI-stable. It was inferred that this was due to the complete absence of the target BAT26 sequences in the tumor sample resulting in amplification of contaminated normal DNA.

External proficiency testing results from the United States and elsewhere indicated that current practice for MSI testing is not optimal in most laboratories. Based on this, it is likely that clinical sensitivity in routine practice for mutations in MLH1 and MSH2 will generally be toward the lower end of the range found earlier (about 84–90%). Corresponding clinical sensitivity for MSH6 will be even lower.

In this review, the false-positive rate (1 – specificity) is calculated initially, and then converted into the clinical specificity. The false-positive rate can be defined as the proportion of tested individuals in the population of interest (consecutive patients with CRC) without the disorder of interest (Lynch syndrome) that has MSI-high test results. By definition, Lynch syndrome is CRC caused by a MMR gene mutation. Most MSI-high test results that are not associated with a germline MMR gene mutation are explained by a nonfunctional MMR gene (MMR gene proteins are nonfunctional because of a somatic event). The most common cause is somatic methylation of the promoter region for MLH1, often referred to as sporadic MSI CRC. Lynch syndrome and sporadic MSI CRC have morphologic features in common (lymphocytic infiltration, mucin secretion, and poor differentiation) that clearly separate them from other sporadic CRC. Lynch syndrome, however, is more often associated with a positive family history and early age of onset, whereas MSI CRCs are more common in women and occur at later ages. Thus, the “false positive” MSI-high test results indicate that the cancer is likely caused by failure of the MMR gene, by a different mechanism (i.e., nonheritable MLH1 promoter methylation).

For the setting of population-based testing of newly diagnosed CRC cases, the optimal study design would be a consecutive series of individuals, all of whose tumors are tested for both MSI and MMR gene mutations (preferably for both point mutations and large deletions in MLH1, MSH2, MSH6, and PMS2). Among those individuals without an identified mutation (non-Lynch syndrome), the proportion with an MSI-high test result would be the false-positive rate from which the specificity can be computed. Studies that enroll only Amsterdam criteria–positive patients, or limit enrollees by age of onset (e.g., <40 years) would not be appropriate. However, studies that do not identify all cases of Lynch syndrome (e.g., only identify mutations among MSI-high patients) may also be acceptable, as the incidence of sporadic MSI CRC is high when compared to the incidence of Lynch syndrome.

Although we would have preferred to restrict studies included in this section to the time period of 2003 and later (so that MSI testing methodologies would be similar to those used to define clinical sensitivity), the limited number of available studies did not allow for this. Six studies provided sufficient information to compute the clinical specificity of MSI testing. The consensus estimate for the clinical specificity of MSI testing is 90.2% (95% CI, 87.7–92.7%), using a random effects model. When expressed as a false-positive rate, the estimates are 9.8% (95% CI, 7.3–13.0%). These results are heterogeneous, and this is only partially explained by study design or MSI test composition (numbers and types of microsatellites tested).

In all studies, we used only MSI-high results as being positive and included both MSI-low and MSI-stable as negative. Table 2 summarizes the results of MSI testing from studies that reported on consecutive newly diagnosed CRC from a general population. One of the studies restricted enrollment to patients under age 55. Another reported being conducted at a referral center, but enrolled consecutive newly diagnosed cases. Overall, the studies tested 3842 patients (after those with known mutations were removed); 356 MSI-high test results were identified in that group. Formal meta-analysis shows significant heterogeneity (Q = 40, P < 0.001), because one U.S. study reported a false-positive rate of 17.6%, whereas the remaining studies averaged about 9%.

As a way of assessing heterogeneity, we examined several possible biases. Two studies^, used only BAT26 (a mononucleotide marker) to define MSI status. Such a test is likely to have a higher specificity (lower false-positive rate) than a more complete panel. Consistent with this expectation, both of these studies have higher specificities (lower false-positive rates) than the consensus figure. There is no obvious reason why specificity was so low in one study (82.4%). Given that this was a referral center, some patients might have been enrolled after having already undergone MSI testing at an outlying institution. We kept this study in the analysis, as it offsets the studies biased toward high specificity and results in assigning a broad confidence interval.

Individual study quality for the six studies that estimated MSI specificity are all rated at quality Level 2 or 3. The quality of evidence is at least adequate because of the observed heterogeneity in the results. However, there are reasonable explanations (provided above) for most high and low estimates, and it might also be reasonable to assign a convincing quality of evidence.

The optimal study design for determining the clinical sensitivity of IHC testing is similar to that for MSI testing. Even though less obvious improvements may have occurred in IHC testing as compared with MSI testing, we restricted publications to 2003 and later for consistency and to avoid any temporal differences. The inclusion criteria were (1) MMR gene mutations were identified without knowledge of IHC status (in at least an identifiable subset of the data); (2) IHC testing was attempted on all patients with Lynch syndrome; and (3) the MMR gene with the mutation was identifiable. We searched the English language literature from January 2003 through June 2007, using the MeSH terms “(Colorectal Neoplasms or Hereditary Nonpolyposis) and (IHC or immunohistochemical),” restricted to humans. Overall, four articles were identified. Two of us (G.E.P. and S.M.) reviewed the abstracts and agreed that all four full articles should be reviewed. Although it was determined that none of these four satisfied the inclusion criteria, we identified nine additional articles that did. These were identified through reference lists from the four retrieved articles, through inclusion in the MSI review, or through inclusion in the original evidence report. In the few instances where articles reported on multiple cancers in the same individual or for multiple members of the same family, rules similar to those described for determining MSI sensitivity were used. All nine studies used various definitions of high-risk populations, such as the Amsterdam or the Bethesda criteria, some other family history–based definition, or early age of onset for the proband (e.g., <55 years). The studies were performed in North America, Europe,^{,  ,  ,,,,} Asia, and Australia. The smallest included three individuals/families with Lynch syndrome; the largest included 31 patients (average 16); IHC results were available for 149 Lynch syndrome patients.

Table 3 shows the estimated clinical sensitivities for IHC testing to identify MMR mutations (a correct test result is defined as a combination of IHC test results that indicate that sequencing is warranted). For example, the result is counted as correct when MLH1 protein is reported not to be present in the nucleus of tumor tissue (usually reported as negative or absent for protein in the tumor of a patient with a known MLH1 mutation). We performed the analysis of IHC sensitivity twice. In one analysis, we included samples in which failures occurred and considered these to be false negatives. In a second, less strict analysis, these failures were removed and one sample (reported to have reduced, but not negative expression of MLH1 protein) was reclassified as negative. Under the second set of columns labeled “Less Strict Interpretation,” Table 3 shows that seven studies reported IHC results for 58 Lynch syndrome patients with mutations in MLH1 and found the sensitivity of IHC testing to be 83% (95% CI, 65–93%). The same seven studies identified 40 Lynch syndrome patients with an MSH2 mutation and found the sensitivity of IHC testing to also be 83% (95% CI, 65–92%). Finally, five studies identified 33 patients with MSH6 mutations and found the sensitivity of IHC testing again to be 83% (95% CI, 66–93%). As expected, the “strict interpretation” results are consistently lower by three to nine percentage points. One study reported IHC test results for mutations in PMS2 (7 of 7 were absent protein staining). These results were not included in the table.

All of the studies providing IHC sensitivity estimates are of quality level 2 or 3 because of the high-risk population studied. The quality of evidence for sensitivity for MLH1, MSH2, and MSH6 mutations is adequate. Few data are available to estimate sensitivity for PMS2, but performance is likely to be similar to that found for the other MMR genes.

Table 4 shows the consensus estimate for the clinical specificity of IHC testing, based on three studies. The overall estimate of specificity is 88.8% (95% CI, 67.6–94.8%), using a random effects model. When expressed as a false-positive rate, the estimate is 11.2% (95% CI, 5.2–22.4%). Analysis shows the results to be heterogeneous (Q = 49, P < 0.001), only part of which might be explained by study design or IHC test composition. All three studies are graded Level 2 or 3. Given that the heterogeneity between studies cannot be fully explained, the level of evidence is considered adequate.

Few, if any, studies have performed comprehensive identification of all Lynch syndrome patients from a general population of newly diagnosed CRC patients. However, several of the studies included for the analysis of clinical specificity identified a high proportion of Lynch syndrome patients and an analysis of their data can be instructive, even though it may not be definitive. Table 5 shows the number of mutations identified in each of the four major MMR genes from the six studies previously examined (Tables 2 and 4). Only one study attempted to identify mutations in all four genes. Since that study was published, further PMS2 testing has identified three additional deleterious PMS2 mutations (Hampel, unpublished study). Another study sequenced three genes (omitting PMS2) and the remaining four studies sequenced only MLH1 and MSH2. The two studies from Finland^, report a very different ratio between MLH1 and MSH2 mutations identified (26:3), compared with the remaining studies (28:39). This is a result of a founder effect, which will not be representative of rates in other populations. The four analyzed studies are from Scotland, Spain, and the United States.^, We referenced the ratio of mutations in MLH1, MSH6, and PMS2 to the most common MMR gene to have mutations—MSH2. After weighting by the number of observations, the overall proportions were 32% MLH1, 39% MSH2, 14% MSH6, and 15% PMS2. Although based on small numbers from only one or two studies, it is interesting that slightly over one quarter of the MMR gene mutations identified occur on the MSH6 and PMS2 genes. In the future, it is likely that even more markers for detecting Lynch syndrome will be identified as more comprehensive DNA analyses become possible, and this missing information could be expressed by having the four MMR gene proportions add up to <100%. Because of the lack of information regarding which gene(s) might have future mutation/deletions/rearrangements identified, however, we have chosen to represent the distribution of MMR gene mutations among those currently identifiable (i.e., add up to 100%).

All four studies used in determining the distribution of MMR genes are of lower quality (Level 3 or 4). Only one of them identified PMS2 mutations, and the estimated proportion of mutations in this gene is provided with the least confidence. Overall, the quality of evidence is inadequate given the small numbers involved. The distribution of MMR genes would be more important if MSI were to be the preliminary test, as the sensitivity has been shown to be lower for MSH6 mutations. The distribution is less important if IHC is the preliminary test, as the sensitivity is constant over the range of MMR genes. Fewer data are available for the performance of either test to detect PMS2 mutations.

Somatic BRAF mutations have been detected in various cancers, including melanoma and CRC. About 90% of the mutations in the BRAF gene are accounted for by a transversion at nucleotide position 1799 (1799T>A) identified as V600E. The majority of CRCs with BRAF V600E mutations are associated with MSI positive results. Nearly always, a deleterious MMR gene mutation (i.e., Lynch syndrome) is not present when the BRAF V600E mutation is identified. There is a strong relationship between the V600E mutation and hypermethylation of the MLH1 MMR gene. MLH1 is by far the most common MMR gene to be associated with absent IHC staining, because of the high proportion of these tumors with associated hypermethylation. Researchers have hypothesized that performing BRAF testing on tumors with absent MLH1 staining might identify a group that is nearly entirely composed of sporadic CRC that would not benefit from MLH1 sequencing. This would result in important cost savings, as BRAF testing is relatively inexpensive in comparison to direct sequencing of MLH1.

Among the subpopulation of newly diagnosed CRC patients that has absent MLH1 staining, two mutually exclusive groups can be defined: (1) Lynch syndrome (those with a MMR gene mutation) and (2) sporadic cancer (those without a MMR gene mutation). In this subpopulation, the V600E BRAF mutation identifies sporadic cancer (not Lynch syndrome). Sensitivity, therefore, is defined as the proportion of sporadic cancers associated with the BRAF mutation. Specificity is defined as the proportion of Lynch syndrome cases without the BRAF mutation. In this scenario, specificity needs to be very high so that individuals with Lynch syndrome will be offered MLH1 sequencing and not be categorized as having sporadic cancers.

Four published studies provide some information on the sensitivity and specificity of BRAF testing for sporadic cancer among individuals with absent MLH1 staining. All are relatively small studies with important deficiencies. The results are summarized in Table 6.

Additional supporting information on the very high BRAF mutation testing specificity (proportion of Lynch syndrome not having the BRAF mutation) can be obtained from other published studies that did not perform IHC testing. For example, if a study reports that none of 40 Lynch syndrome patients were found to have the BRAF mutation, then the specificity (as defined earlier) must have been 100%, even though the actual number of tumors with absent MLH1 staining is not known. In seven studies, no BRAF mutations were found among 105 cases of Lynch syndrome with deleterious MLH1 mutations. Some of these studies, however, enrolled only subjects whose tumors were MSI-high (most of whom would have had absent stains) and/or had a positive family history. Regardless, this provides some additional support that the specificity approaches 100%. These studies also tended to enroll subjects at high risk of Lynch syndrome and, therefore, may not represent the findings in the general population.

Additional recruitment and molecular studies were performed after the publication of a population-based study of CRC and Lynch syndrome in the Columbus, OH catchment area (personal data provided by Dr. Albert de la Chapelle and Ms. Heather Hampel). Among a population-based cohort of 500 newly diagnosed CRC cases, 483 individuals had their tumor tested via IHC for absence of MLH1, MSH2, MSH6, and PMS2 protein. Of these 483 individuals, 71 (14.7%) had absent staining (abnormal result); 56 also were MSI-high. In 48 of the 71 tumors (58%, 95% CI, 44–64%), MLH1 staining was absent. Sequencing of exon 15 of the BRAF gene (which includes the V600E mutation) was successful for 39 of the 48 (81%) tumors. Two had insufficient tissue, and seven failed (causes for the failures were not reported). Three of the 39 individuals were identified as having Lynch syndrome associated with an MLH1 mutation. All three were negative for the BRAF mutation. As part of an earlier recruitment, five additional tumors from patients with germline MLH1 mutations were identified and tested for the BRAF mutation; no BRAF mutations were found. Thus, the specificity of BRAF mutation testing in this series, overall, was 8/8 (100%, 95% CI, 63–100%). Of the remaining 36 “sporadic” cancers, 25 had the BRAF mutation, yielding a sensitivity of 69% (95% CI, 52–84%). Identification of Lynch syndrome is close to complete in this series. Sequencing and multiplex ligation-dependent probe amplification testing were performed on all patients with an abnormal IHC result and/or an MSI-high result. All remaining individuals were tested for the two most common large MMR gene deletions, but none were identified. In this population-based cohort, the sensitivity of 69% and specificity of 100% are nearly identical to the summary estimates of the literature from high-risk populations (68% and 100%, respectively, from Table 6).

All of the published studies are in high-risk populations and are assigned a quality rank of 2 or 3. However, the results are homogeneous. For this reason, we sought gray data to provide evidence that the published estimates would be applicable in the general population. Given the consistency between the published and gray data, the quality of evidence for sensitivity and specificity is adequate.

This supplemental evidence review did not involve a formal search or statistical summary concerning the literature on methylation testing. The literature suggests, however, that BRAF V600E mutation testing and methylation testing of the MLH1 promoter region among CRC cases with absent MLH1 protein might avoid similar numbers of sequencing tests with little loss in Lynch syndrome detection.^{,,  ,  ,  ,} 

Informed consent issues were not discussed in the original evidence report. The process of identifying the subset of individuals with Lynch syndrome from among those with CRC is most appropriately accomplished by a judicial, stepwise application of informed consent. Two possible options can be considered for informing CRC patients about testing for Lynch syndrome: (1) counsel everyone at the outset, or (2) perform MSI or IHC testing as part of routine practice without consent and reserve counseling for those whose test results indicate that mutation testing be considered. The first option assures that choices are possible for every aspect of the testing process. However, most (97% or 98%) of these patients would receive counseling about Lynch syndrome that is not relevant for their situation, and these same patients would worry about a medical condition that they would be found not to have. The second option does not allow choices about MSI or IHC testing, but instead limits the focus of attention to those with abnormal MSI or IHC results. This is because counseling is directed only at CRC patients with positive preliminary test results, including the 2–4% of patients whose cancers are due to a mutation in a MMR gene. In addition, those with MSI or IHC test results that indicate consideration of mutation testing retain the ability to decide about the most critical choices—definitive mutation testing and transmitting information to other family members.

There are some who feel that IHC testing is analogous to genetic testing, because individuals with results indicating the absence of MSH2 and/or MSH6, or PMS2 staining in their tumor are highly likely to have a germline mutation in these genes. However, patients with abnormal IHC results are not obliged to pursue genetic testing unless they choose to do so, and there are patients with absence of these proteins in their tumors who do not have an identifiable mutation in the corresponding gene. This could be compared to performing estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu (a gene that plays a key role in the regulation of normal oncogene cell growth) testing on breast cancer patients. It is known that patients with triple negative (ER−, PR−, and her-2/neu−) breast cancers have a high likelihood of carrying a BRCA1 gene mutation; however, informed consent is not needed for these tests, as they affect prognosis and management. There are clear data indicating that MSI status affects the prognosis for CRC patients, but not management.^, It has been proposed that a waiver of consent to perform MSI and IHC screening be considered for individuals with CRC. Although it is beyond the scope of this review to make recommendations regarding questions of informed consent for MSI and IHC testing, it is hoped that this presentation of evidence from both perspectives may help to inform the decision making of those in health policy domains, where these issues must ultimately be weighed.

Testing for Lynch syndrome is becoming more commonplace, and some institutions have begun screening using IHC or MSI on biopsy or surgical specimens. Also, rectal cancer patients often receive neoadjuvant chemotherapy and radiation therapy, allowing time for counseling and genetic testing before surgery and/or chemotherapy. As a result, surgical and oncological care of newly diagnosed CRC patients may be influenced by knowledge that a patient has Lynch syndrome. Subtotal colectomy with ileorectal anastomosis is recommended as a reasonable choice to be presented to patients with Lynch syndrome as the preferred surgery at the time of diagnosis with CRC. Although favored, it has not been proven superior to segmental resection with follow-up colonoscopic surveillance (category of evidence III, Grade C), based on a nonexperimental, descriptive study, insufficient evidence, and a 2006 multisociety evidence review of risk-reducing surgeries in inherited cancers. Patients with rectal cancers should be offered proctocolectomy with ileal pouch anal anastomosis or anterior proctosigmoidectomy with primary reconstruction.

Persons with CRC and Lynch syndrome have a 16% risk for developing a second primary CRC within 10 years of their first diagnosis. This influences the above recommendation for subtotal colectomy, instead of segmental resection with follow-up colonoscopy. A decision analysis study found that subtotal colectomy, in Lynch syndrome patients younger than 47 years, who have been diagnosed with CRC, leads to a 1–2.3 year increase in life expectancy. This was not adjusted based on quality of life, because there were no data available on quality of life differences after these surgeries among individuals with Lynch syndrome. Among patients with sporadic CRC, segmental resection has been shown to result in better quality of life than subtotal colectomy, but this may be offset by the need for frequent, lifelong colonoscopy and fear of CRC among persons with Lynch syndrome. Surgical risks of subtotal colectomy with ileorectal anastomosis are discussed later.

No alteration in chemotherapeutic management is currently recommended in CRC patients with Lynch syndrome. Both laboratory and clinical studies, though few in number, find that MSI-high tumors are resistant to fluorouracil (5-FU)-based chemotherapy and are more sensitive to CPT11 (irinotecan).^, Prospective clinical trials are needed, however, before altering the care of CRC patients with Lynch syndrome. If this finding is confirmed, MSI or IHC testing (as a surrogate for MSI) will likely become standard in the pathologic evaluation of all CRCs.

The reviews discussed above^{,  ,}  were not available at the time the original evidence report was performed. However, no prospective trial results for clinical management options for Lynch syndrome patients have been published since that report's completion.

Table 7 summarizes studies that report the number of relatives at risk for Lynch syndrome, the number receiving genetic counseling, and the number undergoing genetic testing. One thousand eight hundred eighty-six relatives of 234 individuals with Lynch syndrome (eight per proband) were identified. This is likely to be an overestimate of the numbers of relatives actually approached as part of a screening program, as some studies targeted large families for study. If the two studies that targeted large families are removed, the remaining four studies provide estimates ranging from 2.1 to 12 first-degree relatives approached. The wide discrepancy is likely due to the amount of effort and resources, and the technique applied to identifying, contacting, and approaching relatives. Genetic counseling, for example, may only be offered at a central site requiring patient travel, or counselors might travel to family events such as reunions to maximize availability. Although not reported, there would likely be at least as many second-degree relatives.

Among the total number of 1886 relatives identified, 873 or 52% (95% CI, 37–66%) were counseled. The resulting analysis showed heterogeneity (Q = 130, P < 0.001), mainly due to the later study by Aktan-Collan et al. With this study removed, the consensus estimate is 46% (95% CI, 41–50%). Among those receiving counseling, 732 or 95% (95% CI, 93–97%) underwent genetic testing. These results were homogeneous (Q = 12, P = 0.06). Gene testing offers possible benefits and harms to blood relatives of individuals with Lynch syndrome, whether the results are positive or negative. Those relatives found to carry a MMR gene mutation are encouraged to comply with increased surveillance (e.g., colonoscopy). Those who do not carry a MMR gene mutation may derive psychological benefits (see next section) and can follow surveillance recommendations for the general population.

Although the original evidence report summarized factors that might affect the acceptance of genetic testing, it did not estimate the number of family members of Lynch syndrome patients that could be identified, nor did it estimate the uptake of genetic counseling and testing by family members.

Few publications report data on worry about insurance and/or employment due to MMR gene mutation testing results. In one study, 90 women were referred for predictive genetic testing (MMR or BRCA1/2). One woman (1%, 95% CI, 1–6%) chose not to pursue genetic testing, because of concerns regarding future insurance and employment implications. Another study invited patients who met either the Amsterdam or the Bethesda criteria, and their families, to participate in genetic counseling and testing after MMR gene testing became available. Of the 23 patients and family members who chose to participate, 18% were concerned about problems with insurance and/or employers, compared with 33% of the 48 nonparticipants who returned a questionnaire.

This question was not directly addressed by the original evidence report. Both studies summarized above, however, were included. The insurance concern cited in the study by Arver et al. was part of the report (original evidence report– Table 26). The study by Keller et al. was summarized in Extra Table 1 in the original evidence report, however, no mention was made regarding insurance concerns. In May 2008, the Genetic Information Nondiscrimination Act was signed into law. This bill is designed to protect Americans against discrimination based on their genetic information when it comes to health insurance and employment.

Relevant findings from studies mentioned in this section are summarized in Table 8. Recent data suggest a colon cancer risk ranging between 22% and 58% by age 70 for individuals with a MMR mutation.^, Based on all available data, reasonable estimates for penetrance by age 70 might be 45% for men, and 35% for women. These penetrance estimates are lower than previously thought. One likely explanation is that more recent studies tend to be population based. Earlier studies focused on families with many affected members, where risk might be intensified by other genetic and environmental influences. A review in 2002 exemplifies the earlier thinking about penetrance. It documented a consistent finding among seven studies that risk for colon cancer by age 70 years is higher among male MLH1 and MSH2 mutation carriers (80%) than among females (40%). By contrast, a cohort study of colon cancer cases diagnosed before age 45 years, reported in 2006, cites a lower risk for colon cancer among men (45%), but still finds a reduced risk among women (38%). This study included MLH1, MSH2, MSH6, and PMS2 mutation carriers and suggested that CRC risk may be 10% lower for MSH6 and PMS2 mutation carriers than for MLH1 and MSH2. This finding is supported by two studies that show the risk for colon cancer in carriers of MSH6 mutations to be 58% and 32% by age 80, respectively.^, Reanalysis of data that adjusted for the ascertainment of cases collected from a previous study showed that the cumulative risk for CRC among MLH1 or MSH2 mutation carriers is 27% for men and 22% for women, by age 70 years. There is a great deal of inconsistency among data pertaining to this question. Because of the heterogeneity of population sampling, MMR genes tested and other variables, it is not possible to create a single summary estimate. There are, however, two consistent findings. First, in all studies, females have a lower risk of developing CRC than their male counterparts, usually by 20–40%. Second, there is a clear trend toward lower penetrance estimates when studies are population based. Using these two findings, reasonable estimates for penetrance by age 70 might be 45% for men, and 35% for women. It should be noted that our analyses have been focused primarily upon cohort, and generally on more recent studies, that attempt to avoid the “high-risk family” biases.

The risk for other Lynch syndrome–related cancers (e.g., endometrial, stomach, and brain) has been estimated to be 22% for men and 34% for women, up to age 70 years. The gender difference is mainly due to the risk of endometrial cancer in women, and it may be possible to impact the incidence by screening and/or risk-reducing activities (discussed later). The proportion of female MLH1/MSH2 mutation carriers that develops endometrial cancer by age 70 has been reported in five studies summarized in Table 8.^{,,  ,  ,}  The estimates range from around 30% to 60%. The proportion of female MSH6 mutation carriers that develops endometrial cancer by age 80 seems to be similar, at 52% and 64%.^, These estimates seem high compared with the overall rates of Lynch syndrome–related cancers. A recent methodological study suggests that the studies on the risk of endometrial cancer among relatives may also suffer from the same bias of strong family history discussed for CRC. Overall, the penetrance for endometrial cancer by age 70 in the general Lynch syndrome population may be as low as 20–25%.

A recent literature review evaluated how predictive testing affects surveillance/screening behaviors. Uptake of colonoscopy for MMR mutation carriers was high in six^{,  ,  ,  ,  ,  ,}  of seven studies, ranging from 53% to 100% (top half of Table 9). Five studies reported the uptake of colonoscopy within 1–2 years after receiving genetic test results,^{,  ,  ,,} the other two studies reported the uptake of colonoscopy since receiving their genetic test results.^, Overall, the average uptake rate was 79% (95% CI, 67–87%) with some evidence of heterogeneity (Q = 14, P = 0.03). This is due to the study of Hadley et al. (53%). If that study were to be removed, the uptake rate would be 81% (95% CI, 72–87%) with no evidence of heterogeneity (Q = 8, P = 0.2). Two recent reviews regarding clinical management of individuals with Lynch syndrome^, reached similar conclusions regarding colonoscopy surveillance for CRC and endometrial cancer. Colonoscopy is recommended every 1–2 years, beginning at ages 20–25.^,,, The 1–2 year frequency is graded level of evidence IIIC. Individuals with MSH6 mutations are recommended to begin colonoscopy at age 30, given the lower colon cancer risks associated with mutations in this gene.

Colonoscopy risks include adverse events related to the bowel preparation. The most common events are nausea, abdominal pain, and dizziness, which seem to be comparable between the two most common bowel cleansing agents, sodium phosphate (NaP) and polyethylene glycol. Biochemical changes associated with NaP are largely asymptomatic, but require caution in patients with cardiovascular or renal impairment. Significantly more patients are able to complete the NaP preparation than that for polyethylene glycol. There have been rare serious or fatal events from the bowel preparation.

The most common serious, adverse events associated with the colonoscopy procedure (Table 10) are bleeding (1.1/1000, 95% CI, 0.8–1.4), perforation (3.3/1000, 95% CI, 2.3–4.6), and death (0.08/1000, 95% CI, 0.05–0.14).^{,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,}  Factors associated with increased risk for adverse events are female gender, increasing age, and history of surgery, or diverticular disease.

Although there are no randomized trials to document whether systematic surveillance is effective in reducing Lynch syndrome–related morbidity and mortality, one long-term study from Finland (begun in 1982) reports follow-up on 252 family members (age 20–66 years) of 22 index cases. Initially, the families were selected on the basis of family history, and 133 chose colonic examination (initially at a 5-year interval, and then at 3-year intervals); 78 chose to forego colon cancer screening and 41 were not initially contacted. After 14.5 years, 8 of 133 of those who chose screening (6%) and 19 of 119 (16%) of those who chose not to be screened had developed CRC. Mutation testing became available in 1996–1998, and all of the colon cancers were found to have developed in family members who carried a mutation—8 of 44 (18%) and 19 of 46 (41%), respectively. There were no CRC deaths among those who chose colon cancer screening, and nine among those who chose not to be screened. Overall deaths were 10 (8%) in those choosing screening, and 26 (22%) in those choosing not to be screened. This study reports a 62% reduction in risk for CRC and a significant reduction in CRC-associated mortality among family members of index Lynch syndrome cases who were found to carry a mutation. All results are computed using an “intention to treat” analysis. Crossover clearly occurred, with a proportion of individuals initially choosing to forgo surveillance later agreeing. This would likely result in an underestimation of the actual reduction in CRC risk among the surveillance group.

Further evidence for the efficacy of surveillance was reported recently. In this cohort study, 2788 members from 146 Lynch syndrome families in the Netherlands were followed to assess mortality caused by CRC. The standardized mortality ratio for CRC showed a 70% decrease over three time periods: 1960–1974, 1975–1989, and 1990–2004 (P < 0.001). When comparing the subjects who did (n = 897) or did not (n = 1073) have surveillance colonoscopies, a significant difference in standardized mortality ratio was observed (6.5 vs. 23.9, respectively; P < 0.001). Evidence for efficacy of periodic colonoscopy is graded as level IIb.

Risk-reducing colorectal resection is generally not recommended for MMR gene mutation carriers who do not have CRC (or a Lynch syndrome–related cancer).^, Subtotal colectomy with ileorectal anastomosis has an overall 30-day mortality rate of 0.9% and an overall 30-day morbidity rate of 26.0%. Morbidity is explained by the following complications: anastomotic leak (6.5%), small bowel obstruction (14.4%), fistula (2.8%), and anastomotic stricture (1.4%). Fistula and anastomotic stricture are significantly more common in patients having surgery for Crohn disease, so these risks would be lower in patients with Lynch syndrome. Median stool frequency is 3/day 1 year after surgery and does not change with longer follow-up. Among patients with familial adenomatous polyposis, the quality of life is significantly poorer than that of the general population after total colectomy with ileorectal anastomosis (P < 0.001).

The difficulty of this surgery and concerns about quality of life after surgery, combined with the efficacy of colonoscopic surveillance, leads patients to rarely choose this option. Only one study was found that assessed the uptake of risk-reducing colectomy. Of 32 MMR mutation carriers, none had undergone this risk-reducing surgery 12 months after receiving their mutation status. One mutation carrier indicated an intention to do so. It could be discussed as an alternative to regular colonoscopy, given the high cancer risk, concern about the safety of (or compliance with) repeated colonoscopy, and in the setting of high patient anxiety.

More than 75% of women with a MMR gene mutation who develop endometrial cancer will be diagnosed at Stage I. However, a proportion of these women with Stage I disease will still develop metastatic disease at a later time. This is based on documentation that 10–15% of women in the general population with sporadic early stage tumors will die from metastatic disease.^, Transvaginal ultrasound is not highly effective at identifying endometrial cancers in women with Lynch syndrome.^{,  ,}  Endometrial biopsy, however, is effective at identifying both premalignant and malignant lesions. Two reviews dealing with this aspect of Lynch syndrome management reached similar conclusions. Endometrial cancer surveillance, including transvaginal ultrasound in combination with endometrial aspiration biopsy, should be performed every 1–2 years, beginning at age 30–35.^, The level of evidence for this surveillance guideline is IIIC. Transvaginal ultrasound is relatively noninvasive and inexpensive. In a large study using endometrial biopsy with an aspiration method, 96% of patients found the pain acceptable. A recent study suggests hysteroscopy with biopsy may be a feasible method to screen for endometrial cancer among women with Lynch syndrome (n = 11) and women who meet Amsterdam II criteria (n = 46). Of 91 attempted hysteroscopies (10 failed), 2 endometrial carcinomas were detected. Two studies performed outside the United States assessed uptake for endometrial cancer surveillance among women. In both studies, increased adherence to surveillance was noted after genetic testing (bottom of Table 9).^, Overall, 63% of women complied with endometrial cancer surveillance (95% CI, 46–75%).

In a recent retrospective study, 61 of 315 women with MMR gene mutations chose risk-reducing surgery. The entire cohort was followed for approximately 10 years. No cases of endometrial or ovarian cancer developed in women who had risk-reducing surgery, whereas 33% of women who did not have surgery developed endometrial cancer and 5.5% developed ovarian cancer. The two recent reviews discussed in previous sections also addressed this aspect of Lynch syndrome management and were in agreement that risk-reducing hysterectomy and bilateral salpingo-oophorectomy are not recommended,^, but should be presented as an option.

A second study examined preventive behaviors 1 year after genetic testing for MMR mutations. There were 21 female mutation carriers and 48 females with no mutations. When the study began, five of the women reported having had a hysterectomy, and two reported having had an oophorectomy. In the 12 months after receipt of the genetic test results, none of the women had chosen to have a hysterectomy. Two of the women who reported having had a hysterectomy at baseline chose to have a bilateral oophorectomy. No information is given on mutation status of the women who chose risk-reducing surgery. A larger series in a surveillance study from Finland found that 34% (59 of 175) of women with Lynch syndrome underwent a hysterectomy. Of these, 43 (72%) elected surgery, because of the finding of a premalignant lesion on screening, simultaneous laparotomy for another reason, or for risk reduction.

Risks from hysterectomy (Table 11) vary depending on the surgical technique (abdominal, laparoscopic, and vaginal) but include infection (3–33%), bleeding (3.4–9.1%), organ injury (0.6–3.2%), and rarely death (none observed to 0.04%).^{,  ,  ,}  Because of the significant heterogeneity, summary estimates for complication rates were not computed. A meta-analysis of 27 trials with 3643 participants found that “return to normal activities” was quicker for vaginal compared with abdominal hysterectomy with weighted mean differences of 9.5 days (95% CI, 6.4–12.6), and for laparoscopic compared with abdominal hysterectomy with weighted mean differences of 13.6 days (95% CI, 11.8–15.4). There were no significant differences in return to normal activities between laparoscopic versus vaginal hysterectomy. There were more urinary tract injuries with laparoscopic than with abdominal hysterectomy (odds ratio 2.61, 95% CI, 1.22–5.6), but no other intraoperative visceral injuries showed a significant difference between surgical approaches. One study measured subjective outcomes by questionnaire survey at 4–6 weeks and 1 year after hysterectomy. Although subjective complaints (including abdominal pain, urinary incontinence, menopausal symptoms, and genital prolapse) increased significantly at the 1-year questionnaire (P < 0.001), 95% of patients reported being satisfied with the procedure.

Risks from bilateral salpingo-oophoectomy are numerous and it is beyond the scope of this article to summarize all of them. Two recent reviews on this topic have been published.^, Risk-reducing oophorectomy in premenopausal women induces the sudden onset of menopause. Effects may include a higher percentage of adipose tissue and lower muscle mass, adverse changes in multiple cardiovascular risk factors, higher risk of myocardial infarction, higher rates of atherosclerosis, increased risk of mortality, short-term memory declines, dementia, macular degeneration, increased risk of bone fractures and osteoporosis, sexual dysfunction and loss of desire, skin changes, and urogenital atrophy.

There are no studies addressing surveillance for other less common Lynch syndrome-associated cancers, so the recommendation below, extracted from the two recent reviews, is based on expert opinion. Persons with Lynch syndrome should stay in contact with their physicians and report signs or symptoms immediately (one review recommends annual visits beginning at age 21). There are no data regarding the efficacy or compliance with this screening recommendation.

Data pertaining to psychosocial outcomes of genetic counseling and testing have been looked at by comparing outcomes between carriers and noncarriers of MMR gene mutations, and changes in outcomes over time. Changes in distress among mutation carriers seem to be short term, and there is no indication of adverse effects of genetic testing.^{,,,  ,  ,}  Unaffected noncarriers derive psychological benefits, such as short- and long-term decreases in colon cancer worry, general anxiety, and depression, other benefits include removal of uncertainty, assurance that offspring are not at high risk, avoidance of intensive surveillance, and removal of the threat of discrimination.Table 12 summarizes differences in outcomes at selected time intervals after genetic testing between mutation carriers and noncarriers.

The original evidence report noted the changes in psychosocial outcomes over time, and the differences in outcomes between mutation carriers and noncarriers. The study with the longest follow-up (3 years) was not available for this report. The evidence report also discussed the efficacy of pretest genetic counseling for informing family members of potential risks and benefits of testing. Psychological benefits of genetic testing were not reviewed.