College of American Pathologists
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  Lynch syndrome testing—when and how?




December 2007
Feature Story

William Check, PhD

Of the approximately 150,000 people who develop colorectal cancer each year, the vast majority are sporadic cases. But a few percent of colorectal cancer cases occur in people who have a genetic predisposition. Most of those with a genetic predisposition don't know they are carrying a genetic defect that greatly increases their risk of colorectal cancer and a few other selected cancers. "Until recently, there has not been a way, other than family history, to readily identify these individuals," says Stephen N. Thibodeau, PhD. "Now there is." Dr. Thibodeau, who is co-director of the clinical molecular genetics laboratory at the Mayo Clinic and professor of laboratory medicine and pathology at Mayo Medical School, gave a presentation on this procedure, called microsatellite instability, or MSI, testing, at the 2006 Association for Molecular Pathology meeting.

Hereditary nonpolyposis colon cancer, or HNPCC, is one of several hereditary forms of colorectal cancer. HNPCC is an autosomal dominantly inherited tendency to develop colorectal cancer and other tumors such as endometrial cancer, gastric cancer, and urothelial carcinoma of the upper urinary tract. Colorectal cancer and other tumors generally occur at a significantly younger age than is observed among patients with sporadic tumor of the same type. Historically, the diagnosis has been made clinically based on a pedigree that shows a strong family history of colorectal cancer or other HNPCC-associated tumor type. However, smaller families, incomplete histories, and incomplete penetrance often complicate a clinical diagnosis of HNPCC.

Accordingly, in 1991 an international consortium held in Amsterdam established the following "Amsterdam criteria" for HNPCC:

  • Historically verified colorectal cancer in three or more relatives, one of whom is a first-degree relative of the other two.
  • Presence of disease in two or more successive generations.
  • Age of colorectal cancer onset of less than 50 years in at least one person.
  • Exclude familial adenomatous polyposis.

The Amsterdam criteria were expanded in 1999 (Amsterdam criteria II) to recognize the importance of all HNPCC-associated tumors, and not just colon cancer, in the syndrome.

With the use of such highly selected families, that is, those meeting the Amsterdam criteria, the genes involved for a substantial fraction of clinically diagnosed HNPCC were eventually identified. These genes are those involved in one particular pathway: DNA mismatch repair. Thanks to these early findings and subsequent research, a clear story has emerged over the past decade that has important research and clinical implications.

Based on the presence or absence of functional DNA mismatch repair, or MMR, colon cancer is now divided into at least two broad categories. Tumors with defective MMR (dMMR) are characterized by the presence of a particular tumor phenotype, termed microsatellite instability, and by the absence of protein expression for any one of a number of genes involved in DNA mismatch repair, including hMLH1, hMSH2, hMSH6, or PMS2. Tumors with dMMR have been identified in approximately 15 percent to 20 percent of sporadic colon cancer and in a subset of patients with HNPCC (~two-thirds), now referred to as Lynch syndrome. Tumors with dMMR have a distinct MSI phenotype termed MSI-H (MSI at ?30 percent of loci examined).

In sporadic colon cancer, the MSI-H phenotype is associated with distinct clinicopathologic features, including proximal tumor site, high grade, early stage, and diploidy. This phenotype has consistently been associated with a more favorable outcome. Among sporadic colon cancer, the majority of MSI-H cases are due to inactivation of hMLH1 (~95 percent), with hMSH2 and hMSH6 accounting for a smaller percentage, approximately five percent and less than one percent respectively. Germline mutations in these same mismatch repair genes are also responsible for Lynch syndrome, with hMLH1 and hMSH2 accounting for the majority of cases (approximately 40 percent each) and hMSH6 and PMS2 again accounting for a smaller percentage, about 10 percent and five percent respectively. Among all cases involving hMSH2 and hMSH6 (sporadic or inherited), the presence of a germline mutation appears to be the most frequent mechanism of gene inactivation. For hMLH1, however, current data suggest that the most common mechanism of gene inactivation among unselected cases (~90 percent of cases) is promoter hypermethylation and, less frequently, by mutations in the gene itself. Thus, the molecular etiology of those tumors exhibiting dMMR is very heterogeneous, involving several different genes and numerous mechanisms of gene inactivation, including epigenetic (promoter hypermethylation), somatic, and germline alterations.

Identifying HNPCC patients is useful because colonoscopic and other organ screening measures of mutation carriers offer the chance to reduce cancer morbidity and mortality in such individuals. Identifying other family members with the gene defect is also important for their screening. The overall goal is to prevent death from metastatic disease, so preventive screening of HNPCC patients is essential.

Dr. Thibodeau says the initial purpose of the Amsterdam criteria was to help identify a homogeneous group of families with hereditary colon cancer syndrome for research purposes, such as linkage studies. "But these criteria are not sufficient to identify all cases of HNPCC, and as a result, these criteria have limitations for clinical purposes," he says. Many cases would be missed if these were the only criteria used, and "it is important to recognize," Dr. Thibodeau says, "that not all patients who fulfill the Amsterdam criteria have evidence of defective MMR." It has become increasingly important to distinguish families who meet the Amsterdam criteria for HNPCC and have defective MMR genes (Lynch syndrome) from those who meet the Amsterdam criteria but do not have defective MMR genes. The latter group has a lower incidence of colorectal cancer and perhaps other cancers as well, which has important implications for surveillance and counseling (Lindor NM, et al. JAMA. 2005; 293:1979-1985).

The Amsterdam criteria for a diagnosis of HNPCC were established before the discovery of MSI and the identification of the DNA MMR genes that are mutated in most patients with HNPCC. The greater understanding of HNPCC and its etiology led to a revised set of criteria—the Bethesda criteria—to help identify which patients are most likely to have Lynch syndrome and would benefit most from MSI testing. These criteria have since been revised (Umar A, et al. J Natl Cancer Inst. 2004;96:261-268) and include the following:

  • Colorectal cancer in patients under 50 years of age.
  • Synchronous or metachronous colorectal or other HNPCC-associated tumors, regardless of age.
  • Colorectal cancer with MSI-H [a high level of MSI] morphology in patients under 60 years of age.
  • Colorectal cancer with one or more first-degree relatives with HNPCC tumors, one diagnosed under age 50.
  • Colorectal cancer in two or more first- or second-degree relatives with HNPCC-related tumor, regardless of age.

With the discovery of a genetic cause for a substantial subset of HNPCC, "testing of tumors, especially colorectal tumors, for MSI by PCR can now be utilized as a useful phenotypic marker to help identify patients with this syndrome," Dr. Thibodeau says. Defective MMR can also be assessed with immunohistochemistry, or IHC. This generally involves evaluating tumors for expression of the protein products of the four DNA MMR genes—hMLH1, hMSH2, hMSH6, and PMS2. More than 95 percent of tumors with defective DNA MMR show loss of expression of one or more of these four genes. "The real value" of these two screening tests, Dr. Thibodeau says, "is to be able to better define the risk of a patient for having a hereditary cancer syndrome or Lynch syndrome."

Given a patient whose family history suggests hereditary nonpolyposis colorectal cancer syndrome or a patient who has other risk factors (such as young age of onset or multiple tumors), the method at Mayo Clinic is to screen the patient's tumor to identify whether it has evidence of defective DNA MMR (based on MSI) and which gene is most likely involved (based on IHC). "If there is no evidence of dMMR," Dr. Thibodeau says, "subsequent gene testing for the presence of a germline DNA MMR mutation is not likely to be very productive." If MSI or immunohistochemistry is abnormal, that is very strong evidence for the possibility of a genetic defect in one of the MMR genes, and the IHC results point to which gene is most likely affected. Sequencing of the MMR gene of interest would follow if the patient is interested in this testing after appropriate genetic counseling. In at-risk patients, Dr. Thibodeau cautions that a negative tumor test for defective MMR (MSI and IHC) does not rule out other hereditary causes of colorectal cancer. (Mayo Clinic's algorithm has been published: Baudhuin LM, et al. Fam Cancer. 2005;4:255-265).

Several issues are generally considered when testing for Lynch syndrome. The first is the relative value of MSI versus IHC and whether both should be performed or only one or the other. At Mayo, testing for defective MMR status by MSI and immunohistochemistry is generally performed simultaneously. "For most cases, we do both of these tests in our laboratory," says Kevin C. Halling, MD, PhD, assistant professor of laboratory medicine and pathology at Mayo Medical School and co-director of the clinical molecular genetics laboratory. "We find them complementary. Each provides important information. MSI testing provides for a more global view of whether or not the tumor exhibits defective DNA MMR, while IHC provides a very valuable bit of information—which mismatch repair gene is defective." And because the results of MSI and IHC testing are highly associated, Dr. Thibodeau says, they provide "valuable quality control information when performed together."

The second issue is when to perform the tumor testing. "Should one go directly to gene testing or should one utilize tumor screening first to identify those who are at highest risk? The approaches taken by various groups are complicated and differ based on a number of factors," Dr. Thibodeau says, "including the availability of laboratory testing, availability of tumor material, level of risk the patient presents with to begin with, etc. There clearly are pros and cons with both approaches."

James Eshleman, MD, PhD, associate professor of pathology and oncology and associate director of the molecular diagnostics laboratory at Johns Hopkins Medical Institutions, agrees this is a complicated area. "I don't think that there is a simple algorithm that fits every patient scenario," he says. Dr. Eshleman sees the approach differing with the patient's pre-test probability of having Lynch syndrome. "If someone comes in with an extraordinarily strong family history and colon cancer at a young age, it may make sense to go straight to gene sequencing," he says. "With a less strong family history, most people would do one of the functional tests first."

Whether the first functional test should be MSI or IHC depends to some degree on local expertise, in Dr. Eshleman's view. "Some places are very comfortable doing immunohistochemistry," he says, "while their molecular pathology laboratory may not have taken on MSI testing. In other places perhaps MSI is easy and routine to do." Dr. Eshleman cites one advantage to MSI testing: It has a good correlation with prognosis and possibly 5-fluorouracil responsiveness and is "essentially a nearly perfect functional assay for MMR defects." On the other hand, he adds, "Immunohistochemistry is widely available. It also provides some guidance as to which gene is likely defective. Almost every pathology lab does it. It takes some experience to validate the antibodies, but in general labs have the expertise to do it. I would argue that it is still in flux what the best approach would be."

At ACL Laboratory in Rosemont, Ill., MSI testing is done but not immunohistochemistry. "It would be useful to have IHC assays," says Jan A. Nowak, MD, PhD, a pathologist at Advocate Lutheran General Hospital in Park Ridge, Ill., and medical director of the Rosemont ACL Laboratory. "If one decides to go that route, you would need to test for all four MMR proteins and have pretty good validation that you are doing it well." Dr. Nowak wonders about the cost of doing IHC in addition to MSI testing. "It's also beginning to sound like HER2," he says, "leaving me with a sense of déjà vu. Will someone soon be telling us what to do?" Whatever happens, Dr. Nowak believes that the "CAP and pathologists should be ahead of the game."

Testing for MSI has increased in the past few years, Dr. Nowak says. The number of laboratories subscribing to MSI proficiency testing went from 35 three years ago to 72 in the CAP's 2007 MSI-A Survey. "Performance on that Survey has been good," Dr. Nowak says. About half of the participants also do immunohistochemistry.

Dr. Halling, vice chair of the CAP Molecular Oncology Committee (and chair as of Jan. 1), foresees subscriptions to this Survey approaching 100. The chief reason for the ongoing increase in MSI testing, he says, may be the greater understanding that pathologists and other physicians have of the existence of HNPCC and the value of doing the test to identify which patients may have it. "It takes a while for word to filter out to everybody," Dr. Halling says. "Increasingly clinicians understand that they should order this test in a colon cancer patient under age 50 or if the patient has a family history of colon cancer." No Survey exists yet for MMR protein immunostaining. "That is something we want to do. We're hoping the [CAP] committee that covers that area will come up with something and we may make a joint Survey," he says.

As with much testing, volume is one of the drivers that influences whether MSI testing is adopted in a particular diagnostic laboratory. "The complexity of MSI testing is well within the realm of what most molecular diagnostics labs can do," Dr. Thibodeau says. "And, as with all tests, proficiency in performing and interpreting the test results is critical."

While many laboratories now perform MSI testing, immunohistochemistry, or both, "relatively few perform gene sequencing," Dr. Halling says. "Many groups will send to a larger laboratory like Mayo or Myriad Genetics for that." With only about 10 laboratories in the U.S. now doing sequencing for defective MMR genes, there would not be much demand for proficiency testing. "But it will come," Dr. Halling predicts.

The possible need for guidelines for MSI testing is an "active topic for discussion" in the Clinical Practice Committee of the Association for Molecular Pathology, says Dr. Nowak, who is past chair of the committee. A start to establishing such guidelines was made under Dr. Nowak's chairmanship. Now, he says, "there may be some utility in publishing those guidelines."

Microsatellites are repetitive DNA elements with unit lengths from one to six nucleotides and repeat numbers of 10 to 60 that are found abundantly throughout the genome. The majority of microsatellite repeats are found in noncoding regions of the genome; they occur occasionally within coding regions of genes. (Interestingly, instability of microsatellite repeats within coding regions of some tumor suppressor genes such as TGF-beta have been implicated in colorectal tumorigenesis.) "Microsatellite loci are incredibly prone to a process called slippage," Dr. Eshleman says. "Because of their repetitive nature, during replication when DNA is somewhat fluid you get realignment. A CA pair on one strand may bind to a GT upstream or downstream on the other strand and form a small loop." The result is a single cell that has a mismatched pair. "In an MMR functional cell," Dr. Eshleman continues, "repair enzymes fix that. If MMR proteins are lacking or dysfunctional, slipped intermediates are maintained and get transmitted to daughter cells." Usually the altered microsatellite is shorter. This mechanism explains why MSI and defective MMR genes are so closely correlated.

In clinical practice, testing for suspected Lynch syndrome is done on paraffin-embedded tissue using one or more 5-µm thick sections. In addition to testing tumor tissue, it is important to test normal tissue (usually adjacent tissue from the same organ, but any normal tissue such as peripheral blood will suffice). Tissue from colon cancer is preferred since most work has been done on that. "Although adenomas can be useful in some circumstances, colon adenocarcinomas are the tissue of choice and most often used," Dr. Thibodeau says.

For MSI testing, marker selection is important. Most but not all labs assess for MSI by analyzing five (Umar A, et al. J Natl Cancer Inst. 2004;96:261-268) to 10 microsatellites weighted toward mononucleotide markers, since these are more sensitive and specific for the type of MSI found in DNA MMR defective tumors. Mononucleotide repeats are especially important for identifying patients with germline hMSH6 mutations since tumors in these patients frequently show lower levels of instability and a predilection to show instability in mononucleotide compared with dinucleotide markers. In a validation study done in Dr. Thibodeau's laboratory, 10 specific microsatellite loci markers were used: BAT 25, BAT 26, BAT 34c4, BAT40, D5S346, mycL, ACTC, D10S197, D18S55, and D17S250. Microsatellite instability is defined as a variation in the size of PCR amplicons for the specific microsatellite loci as seen in tumor compared with normal tissue. Three MSI phenotypes have been defined: microsatellite stable (MSS, no instability), low-frequency instability (MSI-L, <30 percent of markers affected), and high-frequency instability (MSI-H, >30 percent of markers) (J Natl Cancer Inst. 2004;96:261-268).

Dr. Thibodeau and his colleagues have investigated the reproducibility between laboratories of MSI testing for Lynch syndrome. They recently reported the experience of a six-center multinational consortium with MSI quality control activities, as laboratories developed competency with MSI testing and interpretation (Lindor NM, et al. Cancer Biomark. 2006;2:5-9). "Each group used the same set of markers while trying to set up the assay," Dr. Thibodeau says. "It was proficiency-type testing with experienced and inexperienced labs in a consortium. In the first round, experienced labs did well and inexperienced labs did generally less well," he says. "Then we had a training session and after that repeated the evaluation." In the second there was substantial improvement. "The point of the paper is that there is a bit of a learning curve," Dr. Thibodeau says. Laboratorians have to learn how to score, what to look for, and how to optimize the assays. The good news: "Under the best conditions, this is a very robust assay," he says.

For IHC scoring, protein staining must be nuclear, and non-tumor cells (for example, normal colonic mucosa or lymphocytes) must be present and demonstrate normal staining. Among cases that show a loss of staining for one or more of the DNA MMR proteins (and thus evidence of defective DNA MMR), typical IHC patterns are loss of both hMLH1 and PMS2 with normal hMSH2 and hMSH6 staining, loss of hMSH2 and hMSH6 with normal hMLH1 and PMS2 staining, or loss of either hMSH6 or PMS2 alone. Mismatch repair proteins exist as heterodimers, Dr. Eshleman points out, which explains the IHC patterns. For instance, hMSH2 and hMSH6 are partners, with hMSH2 being dominant; hMLH1 and PMS2 are partners, with hMLH1 dominant. "To be stable in the cell, both partners have to be present," he says. "When you lose hMSH2 expression, you should also lose hMSH6 expression. But if you lose hMSH6 expression, you shouldn't lose hMSH2 expression. So by doing IHC tests for both partners, you have a kind of built-in-control."

Now let's say you are evaluating a patient with colon cancer and a suggestive family history. The patient's tumor tests abnormal by MSI and IHC. Can you now say that this patient has Lynch syndrome? No, because MSI and concomitant loss of DNA MMR protein expression are found in many colorectal cancer cases not due to Lynch syndrome. "Defective DNA MMR occurs in both sporadic and inherited colon cases," Dr. Thibodeau cautions. Defective MMR is observed in 15 percent to 20 percent of sporadic cases. In these non-hereditary cases, defective MMR is almost always due to somatic (nonheritable) changes, rather than heritable germline mutations. When hMSH2/ hMSH6 expression is lost by IHC, germline hMSH2 mutations are found in a very high proportion of cases. However, if hMLH1/PMS2 expression is lost, Dr. Thibodeau says, "it is a different story." In this case, there is a fairly high frequency of somatic hypermethylation of the hMLH1 gene promoter, which indicates that the colorectal tumor is likely a sporadic tumor and not inherited.

The probability of a patient having Lynch syndrome is strongly influenced by his or her family history, other risk factors, or both. It is here that the Amsterdam or Bethesda criteria provide valuable information, as demonstrated by results from two sets of patients—one unselected, the other selected to be moderate or high risk for HNPCC. Among the 788 unselected patients, Dr. Thibodeau showed, 14 percent were MSI-H and 99 percent of them had abnormal IHC findings. However, in 84 percent of these patients with defective MMR, loss of hMLH1/PMS2 was found. Based on current literature, the vast majority of these (approximately 90 to 95 percent) will be due to promoter hypermethylation, a somatic and epigenetic mutation. Only about 10 percent of the overall cases demonstrated loss of hMSH2/hMSH6.

In contrast, the results in 675 cases selected by multiple criteria, including the Amsterdam and Bethesda criteria, to be of moderate or high risk, showed that twice as many, 27 percent, were MSI-H, of which 94 percent were IHC positive. In these patients, 32 percent were found to have loss of hMSH2/ hMSH6 and another 10 percent had loss for hMSH6 alone by IHC. As indicated earlier, the loss of hMSH2 or hMSH6, or both, is highly associated with the presence of a germline mutation (Lynch syndrome). Thus, by using some selection criteria, there was a substantial enrichment for those cases most likely to have Lynch syndrome.

Dr. Thibodeau summarizes these findings: "As you increase the level of risk that a patient will have Lynch syndrome by selection, the proportion of colorectal cancer cases with defective DNA MMR due to hMLH1 hypermethylation decreases and the proportion due to germline mutation increases." It is important to distinguish these two types of colorectal cancer because they have different implications for the patient and the patient's family (one is heritable in an autosomal dominant fashion, one is not).

When hMLH1 is implicated in a case of apparently hereditary colorectal cancer, both promoter hypermethylation and germline mutation must be considered, especially if a germline mutation is not found in the hMLH1 gene. "You have to ask," Dr. Thibodeau says, "is there germline hMLH1 mutation and I didn't find it? Or is the test negative because the loss of hMLH1 expression is due to hypermethylation of the hMLH1 promoter?"

One way to help with this problem is to test directly for the presence of hMLH1 promoter methylation. For this, a number of quantitative and non-quantitative methods are available. For example, a study from the Institute of Pathology and Molecular Diagnostics at the University of Regensburg in Germany showed that, in MSI-H colorectal cancer, "[Q]uantitative MLH1 methylation analysis is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC" (Bettstetter M, et al. Clin Cancer Res. 2007;13:3221-3228).

Dr. Thibodeau says another way to discriminate such cases is to assess the colorectal tumor for the presence of a V600E mutation of the BRAF oncogene. To date, BRAF V600E mutations have been found frequently in cases with hMLH1 promoter hypermethylation but not in germline cases. Thus, the finding of a BRAF mutation in the tumor strongly suggests that the patient does not have a germline mutation. "Our data suggest that using both tests—hMLH1 promoter methylation and the BRAF V600E mutation-provides the best discrimination," Dr. Thibodeau says.

In typical clinical practice, patients are selected for MSI testing by gastrointestinal, oncologic, or medical genetics practitioners as being at high risk. Focusing on colon cancer patients with a family history will detect many, but not all, cases of heritable cancer. For this reason, many institutions are instituting limited screening in selected patients with a variety of protocols. At Mayo Clinic, reflex testing of selected surgical patients by pathology is practiced. "Reflex testing is a bit different," Dr. Thibodeau says. "It is done as part of surgical pathology practice when a patient has a newly diagnosed colon cancer." Indications of higher risk for MMR, such as age of onset under 50 years, are used. When a patient has surgery, the pathologist looks at the tumor. "If it is a young onset patient, we automatically reflex to MSI testing," Dr. Thibodeau says. "That allows us at the time of surgery to see whether the patient is at risk for having Lynch syndrome." Tumor is tested for MSI only and results are reported to the surgeon and colorectal neoplasia clinic. If the test result is abnormal, the patient is approached to see if additional testing is desired. "We don't do IHC until the patient consents," Dr. Thibodeau says. This is related to the fact that IHC reveals more information with respect to the likelihood of having a germline mutation.

William Check is a medical writer in Wilmette, Ill.