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© The Author 2007. Published by Oxford University Press.
EDITORIALS |
Toward a Consensus in Molecular Diagnosis of Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome)
Affiliations of authors: Department of Preventive Medicine, Creighton University School of Medicine, Omaha, NE (HTL, JFL); Department of Gastrointestinal Medicine and Nutrition, The University of Texas M. D. Anderson Cancer Center, Houston, TX (PML)
Correspondence to: Henry T. Lynch, MD, Department of Preventive Medicine, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178 (e-mail: htlynch{at}creighton.edu).
Before molecular genetic diagnostics came of age in the 1990s, a comprehensive family history was the only basis on which familial risk of colorectal cancer could be estimated. In the case of hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome, the historic perspective offered by Warthin in 1895 has not changed appreciably (1). Warthin's seamstress confided to him that one day she would die of cancer of the colon or of the female organs, "because everyone in my family died of these diseases." Her prophecy proved accurate because she died of endometrial cancer at a relatively young age. In 2007, many patients still base their expectations and plan their behaviors around a similar sense of fatalism, and little else.
The seamstress's family, now known as family G, was subsequently updated (2) following the report of two strikingly similar families, which, for the lack of a better term, were called "cancer families" (3). Noteworthy in these families was the predilection to an excess of proximal colonic cancer (4). This clinical feature, mandating colonoscopy, was followed by the identification of so-called "cardinal clinical features" of the syndrome (5), which included an excess of synchronous and metachronous colorectal cancers; early age of onset (average 45 years); accelerated carcinogenesis; a litany of integral extracolonic cancers with carcinoma of the endometrium as the most common, carcinomas of the ovary, stomach (particularly in HNPCC families indigenous to Japan and Korea), small bowel, pancreas, and upper uroepithelial tract; brain tumors in the Turcot syndrome variant of HNPCC (6); and cutaneous stigmata (sebaceous adenomas, sebaceous carcinomas, and multiple keratoacanthomas) in the Muir–Torre syndrome variant of HNPCC (7–9).
The clinical features of HNPCC have been expanded and clarified (5). Molecular diagnostics have changed the landscape considerably in the past 15 years (10–15). In a given family suspected of having HNPCC, it is now quite common to identify a germline mutation in one of the mismatch repair (MMR) genes. This not only confirms the diagnosis of HNPCC but also identifies those family members who are mutation carriers and require aggressive management. Because offspring of affected parents in these families would otherwise be considered to be at risk even without mutational testing, the real benefit of targeted mutational screening often lies in its ability to characterize and thus exclude noncarriers (16). But these are often the easy cases—a family meets the rather compelling Amsterdam criteria for HNPCC, and an affected member undergoes informative mutational testing, with or without prior screening of tumor tissue for evidence of microsatellite instability (MSI) or abnormalities detected by immunohistochemistry (IHC) that points to an underlying MMR mutation.
We now know that many, if not most, diagnoses of mutations in MMR genes do not proceed in such an orderly fashion. In some large series, only a minority of patients actually meets stringent or modified Amsterdam criteria (17,18). For these reasons, and in recognition of the diagnostic potential of microsatellite testing, much broader criteria for selecting patients for MSI analysis have been provided (19,20). Although MSI and IHC are quite sensitive for HNPCC, they are not specific, because 15% or more of nonfamilial or sporadic colorectal cancers exhibit MSI, due to hypermethylation of the MLH1 promoter (a phenomenon that exposes a corresponding limitation in the use of IHC because MLH1 protein expression is lost in such tumors as well) (21). In addition, whether or not the clinical picture is compelling or MSI/IHC informative, mutational testing is often disappointing, with tests that are either negative for a deleterious mutation or positive, maddeningly so, for a mutation of "uncertain significance." For all these reasons, a tremendous amount of effort has recently been devoted to constructing strategies for optimizing the selection of subjects for, and interpreting results of, MMR mutational tests.
In this issue of the Journal, Lagerstedt Robinson et al. (22) characterized the molecular genetic status of a large, Swedish HNPCC registry. Using current diagnostic strategies for efficiently detecting MMR mutations, the range of challenges to such detection were encountered.
Reproducing the conventional clinical setting, 285 families had been accumulated on the basis of referral for family history or early-onset colorectal cancer. Careful collection and verification of reported family history of cancer ensued, and families were classified as having Amsterdam criteria, specified lesser strength of family history, or onset before age 50 years in singleton patients. Tumor tissue from the youngest available family member was subjected to MSI analysis according to conventional techniques. IHC was also performed, but only on MSI-positive tumors. Mutational testing of the four commonly mutated MMR genes (MSH2, MLH1, MSH6, and PMS2), including rearrangement studies for detection of large deletions, was conducted on the youngest member of families meeting Amsterdam criteria (extended to include non-Amsterdam families with a patient younger than age 50 years) and on all other patients showing MSI. By means of the fairly stringent selection and testing schemes, pathologic mutations were found in 88% of Amsterdam families, 59% of non-Amsterdam families, and 80% of the early-onset MSI-positive singleton patients. In families meeting Amsterdam features but without demonstrated MSI, 29% were found to have pathologic germline mutations. In a few instances, loss of staining with IHC was found in tumors that were MSI negative. MMR mutations were found in many tumors, with some but not all interpreted as likely being pathologic according to accepted criteria, mainly cosegregation with disease in multiple similarly affected families. Careful parsing of the data led the authors to conclude that MSI alone could not be used as a basis for selecting patients for mutational testing, given the modest but real fraction of patients with MSI-negative tumors in which mutations were found. In addition, because IHC predicted mutations in a few MSI-negative tumors, but missed other MSI-positive tumors, neither MSI nor IHC was felt to serve as a stand-alone screen.
In Fig. 1 (22), a clinical algorithm for testing is presented. Beginning with a positive family history or early cancer onset, MSI testing is performed, and positive tumors are then subjected to IHC followed by mutational testing. If no mutation is found, tumors are then tested for the presence of a BRAF mutation (a good surrogate for methylation testing to identify non-HNPCC epigenetic silencing of MLH1). Finally, patients who are mutation negative receive clinically oriented recommendations for empiric screening, which is only slightly less aggressive than that for mutation-positive patients (5,8,9).
So what can we conclude from these Swedish registry data and the diagnostic work-up strategy posed by the authors? First, the findings as to mutation frequency in the categories of subjects selected are consistent with those of previous large series and registry-based programs. Second, the roles for MSI and IHC in selecting subjects with greatest likelihood of mutation carriage have been described, as has been the low, but not negligible yield of conducting mutational testing notwithstanding negative MSI and IHC. Third, the difficulties in characterizing mutations of uncertain pathogenicity have been described.
The main contribution of the effort by Lagerstedt Robinson et al. is the further demonstration of a central role for MSI combined with IHC in selecting subjects for MMR mutation testing. Others (17) have found an even tighter correlation between MSI and IHC, often accounting for discrepant findings on the basis of technical limitations in the handling of tissues. Lagerstedt Robinson et al. have also reminded us that, at least in sufficiently compelling clinical settings (Amsterdam criteria), mutational testing may be warranted even when neither MSI nor IHC is informative. They were not able to provide data on mutation yield in MSI-negative/IHC-negative patients whose clinical picture was less striking because these patients were not tested for MMR mutations. That these patients were not tested was likely due to an (unstated) assumption that the yield would have been vanishingly low and therefore not a cost-effective undertaking. If the authors are to be criticized for failing to do such testing, then it is a failure that is shared by every other group of investigators that has conducted such studies (14,17,18).
The flow diagram or decision tree presented as Fig. 1 (22) is very similar to that provided by Hampel et al. (17) in a study of a large series of patients in Ohio. It is also more or less in keeping with our own clinical strategy. If one takes the decision tree presented by Lagerstedt Robinson et al. as a frame of reference for dealing with suspected HNPCC, what are the key issues at nodal points? Selection of patients for further consideration will determine how exhaustively the mutation search should be. The genetic counseling process will help frame the issues for the patient (5). An Amsterdam criteria–positive patient may have such a high probability of a mutation that MSI and IHC can be dispensed with altogether and models purporting to provide a priori probabilities without and with these screens can be used, much as they are in BRCA-testing strategies (23–25) [these articles were critiqued by Ford and Whittemore (26)]. As the clinical presentation of HNPCC becomes less obvious—lesser family history or early colorectal cancer onset alone—the greater is the role for MSI and IHC. MSI-negative/IHC-negative tumors perhaps warrant no further molecular genetic evaluation. In clinically marginal cases, if MSI is present and accompanied by loss of MLH1 protein, an argument may be made for proceeding next with BRAF mutation testing or methylation assay, rather than expensive MLH1 mutation testing, because the presence of a BRAF mutation pretty conclusively rules out HNPCC (27,28).
If a decision is made to perform MSI, some routinely do IHC staining at the same time and proceed with mutational testing if either test is informative. Performing both tests serves a quality assurance role. When discrepancies between MSI and IHC occur, as they do in up to 10% of cases, further assessment of technical issues accounting for the discrepancy can lead to performance improvement (15,29). Other centers, including this Swedish group, pursue a stepwise approach. If the tumor is MSI positive, IHC is then done to direct mutational testing to a specific MMR gene, which MSI alone cannot do (30). If the tumor is MSI negative, the clinician must weigh the low probability of an informative IHC test and the cost of performing it.
If MSI and/or IHC are informative and mutational testing is conducted, a substantial proportion of tumors will be found to have mutations of uncertain biologic relevance, as in the Lagerstedt Robinson et al. study in which 11% of evaluated tumors had mutations in this category. As their discussion emphasized, each tumor has to be carefully and conservatively evaluated, with only those mutations cosegregating with disease regarded as pathologic.
To help familiarize practitioners with testing and surveillance measures in HNPCC, a number of practice guidelines have been developed by various professional organizations and agencies (31–34). The findings and conclusions arrived at by Lagerstedt Robinson et al. are fairly consistent with these guidelines. Importantly, as practice guidelines strive to be as evidence-based as possible, consistent data from around the world provide support for their validity.
NOTES
The work of H. T. Lynch is partially supported by the Charles F. and Mary C. Heider Endowed Chair in Cancer Research, which he holds at Creighton University.
This article was supported by the National Institutes of Health grant #2U01 CA086389-06 and by revenue from Nebraska cigarette taxes awarded to Creighton University by the Nebraska Department of Health and Human Services. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Nebraska or the Nebraska Department of Health and Human Services.
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J Natl Cancer Inst 2007 99: 291-299.
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