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Advances in Anatomic Pathology:
doi: 10.1097/PAP.0b013e3181bb6bdc
Review Articles

Colorectal Cancer Due to Deficiency in DNA Mismatch Repair Function: A Review

Bellizzi, Andrew M. MD; Frankel, Wendy L. MD

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Department of Pathology, The Ohio State University Medical Center, Columbus, OH

Reprints: Wendy L. Frankel, MD, Department of Pathology, The Ohio State University Medical Center, E-411 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210-1218 (e-mail: wendy.frankel@osumc.edu). All figures can be viewed online in color at http://www.anatomicpathology.com.

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Abstract

Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome attributable to deleterious germline mutations in mismatch repair (MMR) genes. The syndrome is typified by early-onset, frequently right-sided colorectal cancers (CRCs) with characteristic histologic features and tendency for multiplicity and an increased risk for extracolonic tumors at particular sites; it accounts for 1% to 5% of CRC. Deficient mismatch repair (dMMR) function manifests as immunohistochemically detectable absence of one or more MMR proteins and microsatellite instability (MSI). Approximately 15% of sporadic, noninherited CRC are characterized by high-level MSI, nearly always owing to transcriptional silencing of MLH1; these sporadic and LS cases exhibit considerable phenotypic overlap. Identification of CRC with dMMR is desirable to identify LS and because MSI status is prognostic and potentially predictive. This review will discuss the history of LS, the principles of MMR and MSI, the clinicopathologic features of LS-associated and sporadic high-level MSI CRC, the fundamentals of clinical testing for dMMR CRC, and the results of the Columbus-area Lynch syndrome study. We conclude with our approach to population-based LS screening based on institutional experience with nearly 2000 cases.

Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome owing to deleterious germline mutations in various DNA mismatch repair (MMR) genes (MSH2, MLH1, MSH6, and PMS2).1–3 The syndrome is typified by early-onset (mean age 45 in cancer-clinic-based series; about a decade older in population-based series), frequently right-sided colorectal cancer with characteristic histologic features and a propensity for multiplicity (synchronous and metachronous tumors).1–11 There is also an increased cancer risk at a defined subset of extracolonic sites including the endometrium, stomach, small intestine, ovary, renal pelvis, ureter, brain (glioblastoma multiforme in the Turcot variant) and skin (sebaceous tumors and keratoacanthomas in the Muir-Torre variant).12–17 Loss of the cognate wild type allele leads to deficient mismatch repair function (dMMR), which results in microsatellite instability (MSI). LS accounts for 1% to 5% of colorectal cancers (CRCs).5–7,18–21

Approximately 15% of “sporadic” CRC (ie, not due to deleterious germline mutation in a MMR gene) also demonstrate high-level microsatellite instability (MSI-H).5,18,20,22–26 In nearly all cases this is attributable to (biallelic) transcriptional silencing of the MMR gene MLH1 because of promoter hypermethylation.27–29 These cases demonstrate considerable phenotypic overlap with LS.

The clinical detection of CRC with dMMR function is desirable for 3 reasons: (1) the identification of probands with Lynch syndrome (with management implications for the patient and facilitating the identification of affected relatives who benefit from increased CRC screening); (2) MSI-H carcinoma is associated with a more favorable prognosis (compared with stage-matched microsatellite stable tumors); and (3) MSI status may be predictive of response to various chemotherapeutic agents including 5-fluorouracil and irinotecan.14,30–40 This review will discuss the history of Lynch syndrome, related terminology, MMR, and MSI, the histology of LS and sporadic MSI-H CRC, various strategies for identifying dMMR/MSI-H CRC, and results from the Columbus-area Lynch syndrome study. We conclude with our departmental approach and philosophy, based on collective experience with nearly 2000 CRCs.

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HISTORICAL PERSPECTIVE

Reportedly inspired by his fatalistic seamstress, Aldred Scott Warthin (former Professor and Chair of Pathology at the University of Michigan) made study of the hereditary nature of cancer through detailed pedigree analysis and pathologic documentation of familial cancers at his institution.41,42 In 1913 he reported her family's pedigree (“cancer family G”) along with other “cancerous fraternities”.43 This is the first known documentation of a kindred with Lynch syndrome. Of note, his seamstress later died of endometrial cancer (EC), and a germline MSH2 mutation was detected in a family member in 2000.44 Aside from occasional updating of “cancer family G” by Warthin and his colleagues, progress in the understanding of LS would wait over half a century.45,46

Henry T. Lynch came upon the disease that would ultimately bear his name through consultation on a patient with a strong history of CRC in the absence of overt polyposis.41,42 Pedigree analysis of that patient's family formed the basis of Lynch's 1966 “Hereditary factors in cancer. Study of 2 large midwestern kindreds”, published in the Arch Intern Med.9 The paper caught the eye of A. James French, then Chair of Pathology at Michigan, who encouraged Lynch to take stewardship of Warthin's materials on “cancer family G”. Lynch and Krush's updating of the family, published in 1971, contains data on over 650 family members. Many of the syndrome's salient features were noted: (1) increased incidence of adenocarcinomas, mainly of the colon and endometrium; (2) increased risk for multiple tumors; (3) autosomal dominant inheritance; and (4) early onset of cancer.10 Despite Lynch and Krush's insight, their views met with considerable skepticism from the scientific community at large, as contemporary thinking was dominated by the notion that environmental factors were the principal determinant of cancer.41,42

Lynch's ideas slowly gained traction, particularly in the international community, culminating in the organization of the “International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer”. The group, composed of “30 leading experts from 8 different countries” met in Amsterdam in the summer of 1990. The major output from that meeting was the formulation of a series of clinical criteria (now known as the Amsterdam criteria and colloquially known as the “3-2-1” rule) to serve as a common starting point for future research: (1) at least 3 relatives with histologically confirmed colorectal cancer, 1 of whom is a first degree relative of the other 2; familial adenomatous polyposis should be excluded; (2) at least 2 successive generations involved; and (3) at least 1 of the cancers diagnosed before age 50.11 The Amsterdam criteria (AC) were updated at the 1998 group meeting, acknowledging the significance of a subset of extracolonic tumors (ie, endometrium, small bowel, ureter, and renal pelvis). These revised AC-II are presented in Table 1.

Table 1
Table 1
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The molecular genetic basis of Lynch syndrome was largely elucidated in a rapid succession of reports from 1993 to 1994. Peltomaki and colleagues linked the disease (in 2 large kindreds meeting the original AC) to a locus on chromosome 2p. Then, 3 groups independently reported a peculiar molecular phenotype in CRC characterized by widespread alterations in the sequence length of simple repetitive sequences, a phenomenon, which the groups variously termed “replicative errors (RERs)”, “MSI”, and “ubiquitous somatic mutations in simple repetitive sequences”. Aaltonen et al47 found the RER-positive phenotype in 11/14 (79%) “hereditary nonpolyposis colon cancer (HNPCC) tumors” and 6/46 (13%) sporadic CRC. The sporadic RER+ tumors shared a right-sided predominance and near diploid status with the HNPCC cases. Thibodeau et al48, found some degree of MSI in 25/90 (28%) CRC, linking this phenomenon to proximal location and to improved survival and demonstrating an inverse relationship with loss of heterozygosity. Ionov's group reported ubiquitous somatic mutations in simple repetitive sequences in 12% of CRC, which was relatively more common in women and was associated with right-sided location, poorly differentiated histology, fewer KRAS and p53 mutations, and lower tumor stage.49 By the end of 1993 the relevant gene on 2p, MSH2, had been cloned and germline mutations identified in Lynch families.50,51 A second disease locus was linked to 3p, and by early 1994 MLH1 germline mutations were noted in Lynch kindreds.52–54 The demonstration of disease causing mutations in PMS2 and MSH6 would follow.55–57

The next 15 years have seen the application of this knowledge to the better understanding of diseas incidence and phenotype and to the exploration of clinical issues of detection and management. Especially notable in this time period are the 2 National Cancer Institute-sponsored (NCI) workshops dedicated to the issuance of guidelines (“Bethesda guidelines”) to identify patients who would benefit from clinical testing for Lynch syndrome.1,3 The Revised Bethesda guidelines (BG) are presented in Table 2.

Table 2
Table 2
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TERMINOLOGY

Lynch referred to “cancer family syndrome” (CFS) in his early reports.9,10 He introduced the term “HPNCC” in 1985, emphasizing the heritable nature of a predisposition to CRC, in the absence of widespread polyposis; the term gained widespread acceptance.1,11,58 Boland and Troncale first made reference to “Lynch syndrome” in a 1984 case series.59 HNPCC and LS have largely been used synonymously.2,3

The term “HNPCC” is problematic for several reasons, and we encourage use of the term “Lynch syndrome”, a view shared by many others.13,60 First, HNPCC is an inexact, and potentially misleading, descriptor of the disease phenotype. It implies an absence of colorectal polyps, which is not the case. Lynch patients have been shown to harbor similar numbers of polyps as the general population.61 It also fails to acknowledge the wider spectrum of associated neoplasms including carcinomas of the endometrium, stomach, small bowel, ovary, pelviureter, and skin. More importantly, the term HNPCC has been variously applied to 2 overlapping groups of patients: (1) those meeting the AC; and (2) those evidencing the clinicopathologic and molecular features outlined in the introduction (preferably with demonstrable germline mutation in a MMR gene). This confusion is at least partially attributable to the association of HNPCC with the AC before the demonstration of the molecular genetic basis of the disease.

Lindor et al13 have demonstrated that up to 40% of patients meeting the AC-I lack evidence of hereditary deficiency in MMR. They studied 161 AC-I pedigrees, which they sorted into 2 groups based on tumor MSI status (in this study MSI-H status in the setting of strong family history served as a surrogate for germline MMR gene mutation). The standardized incidence ratio for CRC in the first-degree and second-degree relatives of the patients with MSI-H tumors was 6.1; these family members were also at a statistically significantly increased risk for the typical array of Lynch-associated extracolonic tumors. The CRC risk in families with low-level microsatellite unstable (MSI-L) or microsatellite stable (MSS) tumors was significantly lower (standardized incidence ratio 2.3), and they failed to demonstrate an increased risk for extracolonic tumors. On the basis of these results they concluded that the population meeting the AC is composed of at least 2 groups: (1) those families with evidence of hereditary dMMR exhibiting the clinical features of the cancer predisposition syndrome described by Lynch; and (2) those families without evidence of hereditary dMMR. They attributed the increased CRC risk in this second group to a combination of chance familial clustering, shared environment, and some as yet undiscovered gene(s). For the first group, they encouraged use of the diagnostic term “Lynch syndrome”, and for the second, they proposed the descriptor “familial colorectal cancer type X”. These findings have been externally validated.62

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MISMATCH REPAIR

DNA replication is associated with a finite error rate, including the incorporation of mispaired bases (eg, G with A) and slippage of the DNA strands during replication (resulting in the formation of insertion/deletion loops). Failure to repair these errors results in point and frameshift mutations, respectively. The DNA MMR apparatus recognizes errors that elude the proofreading function of DNA polymerase. The system is highly conserved from bacteria to humans, and as the system had been previously characterized in single-celled organisms, the link between MSI and dMMR in human cancer was first recognized by microbial geneticists.63,64 The human MMR genes are named after their prokaryotic counterparts. For example, MSH2 is “MutS homologue 2”. (The “mut” refers to the generalized hypermutability noted in bacterial strains with loss of MutS function).

The MMR proteins function as heterodimers. The MSH2-MSH6 complex recognizes mispaired bases and insertion/deletion loops. It recruits MLH1-PMS2, which subsequently directs the remainder of the MMR machinery.64 MSH2 and MLH1 are the dominant (obligate) constituents of their respective pairs. In the absence of MSH6, MSH2 can pair with MSH3, and in the absence of PMS2, MLH1 can pair with PMS1. This may partially explain the somewhat attenuated Lynch phenotype attributed to MSH6 and PMS2 mutation, (reduced penetrance and older age of onset relative to MSH2 and MLH1 mutants—with the caveat that MSH6 mutants may have a relatively increased risk of EC) although this attribution is admittedly speculative.65–70

The MSH6 and PMS2 proteins are unstable in the absence of their respective dominant partner. This was demonstrated by quantitative reverse transcription-polymerase chain reaction and Western blot in a series of dMMR cell lines.71 Also, the majority of Lynch mutations are nonsense or frameshift mutations, leading to truncated (unstable) proteins; missense mutations may destabilize the resulting mRNA or protein or interfere with protein-protein interaction.72 These features of the MMR proteins have clinical diagnostic consequences as relates to the interpretation of MMR immunohistochemistry (IHC) (“Clinical Testing”).

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MICROSATELLITE INSTABILITY

Microsatellites are simple repetitive DNA sequences scattered throughout the genome, composed of 1 to 6 base pair units that may repeat up to 100 times. They are inherently hypermutable because of their propensity for strand slippage during DNA replication. As discussed above, the correction of the resulting insertion/deletion loops requires the intact function of the DNA MMR system. With loss of MMR function insertion/deletion loops are not repaired, resulting in the variable expansion or contraction of microsatellites. This phenomenon is referred to as MSI (demonstrated on MSI testing as a typical pattern of bands on a gel or peaks on a sequence analyzer). Genes that contain simple repetitive sequences in critical regions are susceptible to this same phenomenon. The resulting frameshift mutations in MSI-H tumors lead to loss of function in a well-described group of tumor suppressors including TGFβRII, BAX, IGFRII, and, interestingly MSH3 and MSH6.73–76 As such, MSI-H tumors have been described as exhibiting a “mutator phenotype”.

A 1997 NCI workshop established a reference panel of microsatellites for clinical and research testing and defined diagnostic criteria for the MSI-H, MSI-L, and MSS phenotypes. The core panel consists of 2 mononucleotide repeats (BAT25, BAT26) and 3 dinucleotide repeats (D5S346, D2S123, D17S250). Nineteen “alternative loci” are also provided. When analyzing 5 loci, MSI-H is defined as instability at ≥2 loci and MSI-L as instability at 1 locus. Given uncertainty over the ability of a finite panel to definitively distinguish MSI-L from MSS, tumors exhibiting instability at 0 loci are defined as “MSS or MSI-L.” When greater than 5 loci are studied, MSI-H is defined as instability at ≥30% to 40% of loci tested and MSI-L as instability at <30% to 40% of loci tested.77 A 2002 NCI workshop addended these guidelines, recommending the testing of additional mononucleotide markers in tumors with instability at only dinucleotide loci, as mononucleotide markers are more reliable in the identification of MSI-H tumors.3

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HISTOLOGY

Mecklin et al61 reported the first systematic histologic evaluation of CRC and adenomas in “CFS” patients in 1986, predating the recognition of MSI and the genetic basis of Lynch syndrome by 7 years. They noted an increased incidence of mucinous carcinomas in cases versus sporadic controls (39% vs. 20%). Poorly differentiated tumors were also more common (24% vs. 12%), but that result failed to reach statistical significance. Although the number of adenomas was similar in the 2 groups, the adenomas of CFS patients tended to contain higher-grade dysplasia and a more substantial villous component, suggesting they were more “advanced”.

Since that time an array of tumor types and histologic features have come to be associated with Lynch syndrome and MSI-H CRC: mucinous, signet-ring cell, and medullary carcinoma, tumor infiltrating and peritumoral lymphocytes, a “Crohn-like” inflammatory response, poor differentiation, tumor heterogeneity, and a “pushing” tumor border.22,25,26,61,78–84 Greenson et al26,83 have further emphasized the presence of any mucinous component, a lack of dirty necrosis, and well-differentiated tumors as correlating with MSI-H status.

By convention, mucinous adenocarcinoma is defined as a tumor composed of >50% mucin and signet-ring cell adenocarcinoma as a tumor containing >50% signet-ring cells. The mucin in mucinous adenocarcinomas is predominantly extracellular, whereas signet-ring cells contain prominent intracytoplasmic mucin, typically displacing the nucleus to one side of the cell.85 Medullary carcinomas are characterized by a constellation of cytoarchitectural features including large cell size, vesicular chromatin and prominent nucleoli, sheet-like to occasionally trabecular growth, and overall circumscription. Tumor infiltrating lymphocytes (TILs) are especially prominent in this tumor type.85

TILs refer to the lymphoid component intimately admixed with the tumor; they have been shown to consist largely of CD3/CD8 coexpressing cytotoxic T-cells [their prominence is speculated to represent: (1) a response to abundant tumor neoantigen formation owing to the “mutator phenotype”; and (2) a possible basis for the improved prognosis in MSI-H tumors].80 Various methods (and thresholds) for counting TILs have been reported, including evaluation of hematoxylin and eosin or CD3-immunostained slides. One practical method involves scanning the slide for a region with TIL, counting 5 consecutive 40× fields, and calculating the mean number of TIL/high-power field (HPF); studies using this method defined a positive result as >2 TIL/HPF.26,83 Peritumoral lymphocytes refer to the lymphoid cuff at the leading edge of the tumor, whereas the “Crohn-like” reaction is composed of “prominent” nodular lymphoid aggregates at the infiltrating edge of the tumor, typically identified at the junction of the muscularis propria and pericolonic adipose tissue. Thresholds for a positive “Crohn-like” reaction have included “2 or more large lymphoid aggregates in a section”, “a single 4× field of at least 3 nodular aggregates of lymphocytes”, “a minimum of 3 lymphoid aggregates per section”, and “at least 4 nodular aggregates in a low power field (4×)”.25,82,83,86

Tumors are generally graded as well, moderately, or poorly differentiated. World Health Organization criteria state that tumors with heterogeneous patterns of differentiation should be categorized based on the highest-grade component, with the important caveat that foci of poor differentiation at the leading edge of a tumor (ie, tumor budding, epithelial-mesenchymal transition) are insufficient to classify a tumor as poorly differentiated.85 Tumor heterogeneity refers to the presence of 2 or more distinct growth patterns within a tumor; Alexander and colleagues provide the example of a tumor with mixed mucinous and medullary features.86 A “pushing” or “expansile” tumor margin is distinguished from an “infiltrative” one; this assessment is best made at low power. In their investigations Greenson et al26,83 contrast typical “dirty or garland necrosis” from large areas of infarct-like “geographic necrosis”; again, lack of the former correlates with MSI-H status. Examples of various MSI-H-associated tumor types and histologic features are presented in Figure 1.

Figure 1
Figure 1
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Test characteristics (ie, sensitivity, specificity, positive, and negative predictive value) have been calculated for these histologic features, either taken alone or in various combinations. In several studies, the presence of TIL has represented the most sensitive histologic component.25,26,83 By increasing the threshold for positivity, TIL can be highly specific as well.83 Medullary histology may be the most specific histologic feature of MSI-H carcinoma.86 In 1 report, the combination of >2 TIL/HPF, and/or any mucinous differentiation, and/or lack of dirty necrosis was 100% sensitive in identifying MSI-H tumors.83 Two recent studies have reported prediction models to identify likely MSI-H tumors for further testing. Each model was generated based on thorough morphologic evaluation and MSI analysis followed by logistic regression of over 1000 CRCs. The “MsPath” (MSI by pathology) model combines age, tumor site, tumor type, grade, Crohn-like reaction, and TIL; the authors report the sensitivity of an MsPath score ≥1.0 as 93% sensitive and 55% specific for the identification of MSI-H CRC; the manuscript includes a nomogram.25 The “MSI probability score” combines a similar set of clinicopathologic features, and the authors report a sensitivity of 92% and specificity of 46% for a score of 1; a nomogram and a link to a website are provided.26 It should be emphasized that given the low pretest probability of disease (ie, 15% to 20% for MSI-H carcinoma, 1% to 5% for Lynch syndrome) the positive predictive value of histology is modest; thus, a large number of CRCs identified as “possibly microsatellite unstable” will prove to be stable.

Jeremy Jass has argued that the constellation of histologic features classically associated with Lynch-associated and sporadic MSI-H CRC largely represent those of the later. Most histologic studies have focused on sporadic tumors or have analyzed MSI-H tumors in general (which will consist predominantly of sporadic tumors). His group performed a large comparison of HNPCC-associated (n=112) and sporadic MSI-H tumors (n=57); patients in the first group met AC, and/or BG, and/or exhibited germline MMR mutations, whereas patients in the second group had no significant family history and MLH1 mutations were excluded by a screening assay. The following features were significantly less common in HNPCC tumors (all P<0.05): proximal location (68% vs. 84%), poor differentiation (34% vs. 57%), mucinous histology (22% vs. 43%), and tumor heterogeneity (26% vs. 78%). In contrast, TIL (73% vs. 56%) and peritumoral lymphocytes (34% vs. 17%) were more frequent in HNPCC-tumors, although these results did not reach statistical significance. A contiguous serrated adenoma was noted in 29% of sporadic MSI-H CRCs versus <1% of HNPCC-CRCs (P<0.001), with foci of serration frequently retained in the invasive component.82 In another study, tumor budding was more frequent in “HNPCC-associated” tumors than sporadic MSI-H cases (20% vs. 0%) (The relative paucity of tumor budding in both these groups is also speculated to relate to improved prognosis).87

Frequent mucinous histology and serration in sporadic MSI-H tumors is attributed to their origin from (hypermucinous) serrated precursors. Other features of the “serrated pathway of neoplasia” retained in sporadic MSI-H CRC include high-level promoter methylation and activating BRAF mutation.29,82,88–93 Each of these features can be exploited diagnostically in the separation of Lynch-associated and sporadic MSI-H CRC. A comparison of the clinical, histologic, immunophenotypic, and molecular features of Lynch-associated and sporadic MSI-H tumors is presented in Table 3.

Table 3
Table 3
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CLINICAL TESTING

Clinical testing for dMMR function in CRC has 2 potential endpoints: (1) to identify the Lynch syndrome; or (2) to identify all MSI-H CRC and is associated with 2 fundamental screening strategies: (1) select patients for testing based on clinical and/or pathologic criteria; or (2) test all CRC. In this light, the AC and BG have been evaluated in population-based cohorts for their ability to identify patients with LS. In general, the AC-II achieve sensitivities on the order from 40% to 50%, whereas the revised BG perform at about 90%.4–7,18–21,94 It should be emphasized that these test characteristics are achieved in an idealized setting, with Finnish investigators having access to national cancer registries and other researchers describing the use of “hospital clinical geneticists” and “detailed questionnaires”. Practical barriers to the use of the BG include their complexity and the vagaries in documenting a reliable family cancer history.95 Other limitations of current clinical guidelines include their reduced sensitivity in smaller families and in populations in whom routine screening colonoscopy alters the natural history of the disease. And obviously, these strategies do not target the larger population of MSI-H CRC. The fulfillment of clinical criteria for LS triggers further testing.

The “MsPath” and “MSI probability score” described above hold promise as tools for identifying MSI-H CRC, again selecting cases for further clinical testing. It should be noted, though, that these will only offer an estimate of the likelihood of MSI-H status (not of LS), and follow-up testing may still be warranted in at least half of total cases.25,26 If the predictive significance of MSI-H status (ie, therapeutic relevance) can be definitively proven in prospective, randomized controlled clinical trials, then the “test all CRC” strategy for an endpoint of MSI-H should become the standard of care. MSI testing is considered the “gold standard” for the demonstration of MSI-H status.

As nondirected germline mutation testing for Lynch syndrome is prohibitively expensive (approximately $1000/gene), MSI testing, and MMR IHC have been evaluated as screening tests.96 Each of these tests has associated advantages and disadvantages. These are outlined in Table 4. MSI testing involves the PCR-based interrogation of a panel of microsatellite markers in matched tumor and normal tissue (generally adjacent non-neoplastic colon or peripheral blood). It requires microdissection and the services of a molecular diagnostics laboratory. Its inability to suggest a particular gene for mutation analysis is a clear disadvantage, and if further testing is restricted to MSI-H cases, it may also be slightly less sensitive than IHC in the identification of LS due to MSH6 mutation (which may present as MSI-L CRC).97

Table 4
Table 4
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IHC is readily available in the majority of diagnostic anatomic pathology laboratories. Antibodies to the 4 proteins implicated in LS (through germline mutation of the associated gene) are commercially available. An abnormal result is the complete absence (loss) of nuclear immunoreactivity for one or more of the proteins in the tumor. All 4 proteins are normally expressed in non-neoplastic tissue, and thus stroma, lymphocytes, and non-neoplastic crypts serve as critical internal controls. An antigen retrieval step has been shown to be important; in one study inadequate antigen retrieval was associated with weak staining and poor specificity for LS (whereas high background staining was associated with poor sensitivity).98 Weak or heterogeneous staining patterns are occasionally encountered and may be attributable to variation in tissue fixation or other technical factors. Repeating the IHC may be helpful in these cases. A key advantage to the use of IHC in Lynch screening is its ability to direct gene testing. Loss of MSH2 function (because of deleterious mutation) manifests as absent immunohistochemical (IHC) expression of MSH2 and MSH6, whereas loss of MLH1 function (because of deleterious mutation or promoter hypermethylation) is detectable as absent immunohistochemical expression of MLH1 and PMS2. Isolated absence of MSH6 or PMS2 protein suggests mutation in the respective gene. Representative immunohistochemical staining patterns are presented in Figures 2 and 3.

Figure 2
Figure 2
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Figure 3
Figure 3
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The results of MMR IHC and MSI testing have been shown to be largely concordant.6,7,20,21,68,69,99–107 In 1 large study, combined use of MLH1 and MSH2 IHC achieved a sensitivity of 92.3% and a specificity of 100% for the identification of MSI-H tumors (1144 cases tested, 302 of which were MSI-H).100 The addition of MSH6 and PMS2 to the panel should lead to further increased sensitivity, as MSH6 and PMS2 mutation are increasingly recognized as etiologic in a significant minority of Lynch cases.65,66,68,69,102 A potential drawback to IHC is its inability to detect missense mutations that fail to destabilize the resulting mRNA or protein. Owing to this, Burgart has estimated the maximum sensitivity of MMR IHC for detecting LS at 95%.106 In our experience, IHC analysis identifies LS and MSI-H CRC in >90% of cases; MSI testing is similarly sensitive for identifying LS.6,7,19–21,105

In tumors exhibiting absence (loss) of MLH1 protein expression, an additional layer of testing may be used before proceeding to MLH1 mutation analysis: MLH1 promoter methylation testing and/or BRAF mutation analysis. Each of these takes advantage of the unique developmental history of sporadic MSI-H CRC, as discussed previously. In the first instance, methylation-specific PCR is used to determine the methylation status of the MLH1 promoter. Methylated cases are likely sporadic in nature, and thus, MLH1 germline mutation analysis may not be necessary. There is one important caveat. Although the “second-hit” in the majority of LS is either loss of heterozygosity or somatic mutation, in rare cases MLH1 promoter hypermethylation has been described.28,29,90,108,109 It is also more expensive than BRAF analysis, as well as technically challenging.

Somatic activating mutations in BRAF, a component of the Ras/Raf/MAP kinase pathway, occur in a wide variety of human tumors, including 15% of CRC.110 They are frequently encountered in sessile serrated adenomas and sporadic MSI-H CRC (around 75%), and are taken as evidence of their evolutionary link.29,88–91,93 We are aware of only a single description of BRAF V600E in an MLH1 germline mutant (a missense change in a patient also demonstrating MLH1 promoter methylation; thus not necessarily pathogenic).88 The majority of mutations are accounted for by a point mutation resulting in the substitution of glutamic acid for valine at codon 600 (V600E); in one study of CRC, 60/63 (95%) mutations were V600E.88 Thus, the presence of BRAF V600E in a CRC supports the interpretation that the case is sporadic, and MLH1 germline mutation analysis generally is not pursued. Testing for a single point mutation (rather than sequencing the entire gene) makes BRAF mutation analysis economically attractive ($100).96

We are occasionally asked to evaluate adenomas in patients with “possible Lynch syndrome”, in the setting of a strong family history in which tumor tissue is not available for analysis. MMR IHC is reasonably sensitive and highly specific in this setting.23,111,112 Halvarsson et al111 reported loss of MMR protein expression in 23/35 (66%) of adenomas from 26 “HNPCC” patients (88% of whom had proven germline MMR mutations). Absence of staining was particularly frequent in adenomas >5 mm in size (88%). Nineteen of the adenomas demonstrating MMR protein loss contained only typical, low-grade dysplasia. The pattern of absent staining predicted the involved gene in each case. In signing out these cases we comment that “whereas absent staining may be seen in Lynch syndrome and the pattern of loss useful in directing gene testing, intact expression does not exclude the diagnosis.”

Absent staining in this clinical setting must be distinguished from that seen in sessile serrated adenomas with superimposed cytologic dysplasia (SSAD) (the generally accepted precursor of sporadic MSI-H CRC).113 The architecture of the lesion and the pattern of loss are important considerations. SSADs are serrated (adenomas in LS are nearly always not), may demonstrate combined loss of MLH1 and PMS2 (not MSH2 and/or MSH6), and, in our experience, loss of expression is generally a late event, frequently corresponding with invasion (whereas loss of expression is often seen in LS adenomas with only low-grade dysplasia).

The demonstration of a deleterious germline mutation is the “gold standard” for the diagnosis of Lynch syndrome. It allows for relatively inexpensive, directed germline analysis in family members. Although the specifics of germline mutation analysis are beyond the scope of this review, we offer several points of note. In general, mutation analysis is performed from a peripheral blood sample, including sequence analysis of exons and intron-exon boundaries of the implicated gene. Most reported pathogenic mutations are nonsense or frameshift, resulting in a truncated protein; missense mutations are more difficult to classify.72 Large deletions may account for up to 20% of pathogenic mutations, and these are not detected with routine sequencing. Multiplex ligation-dependent probe amplification (MLPA) provides a quantitative measure of exon dosage and is considered the method of choice for the detection of large deletions.114–117 With hundreds of described mutations in LS, mutation analysis is complex, and the sensitivity of testing, although continuing to improve, is less than 100%. In the setting of a compelling family history, the inability to document an unequivocally pathogenic germline mutation should not exclude a patient from LS-appropriate surveillance. A Lynch syndrome mutational database is maintained by the International Society for Gastrointestinal Hereditary Tumors (formerly the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer).118

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COLUMBUS-AREA LYNCH SYNDROME STUDY

The Columbus-area Lynch syndrome study, performed under the direction of Albert de la Chapelle, was a population-based effort to determine the incidence of LS among newly diagnosed CRC and the feasibility of large-scale screening. From January 1999 to August 2004, 1566 patients were enrolled. The results from the first 1066 and final 500 patients were published separately.6,7 Each of the 1566 patients underwent MSI analysis using a modified “Bethesda panel” of 5 or 6 microsatellites. For the first 1066 patients, four protein MMR IHC was performed on all MSI-H and MSI-L CRC and on an additional set of 109 MSS cases with “high-risk” clinical features. For the final 500 patients, MMR IHC was performed on all cases with available tumor. Abnormal results were followed by mutation analysis, including MLPA testing as appropriate, and MLH1 promoter methylation testing.

High-level MSI was detected in 12.7% of tumors. MMR IHC was 92% sensitive for the detection of MSI-H status. Forty-four (2.7%) unequivocally pathogenic germline mutations were identified (23 MSH2, 9 MLH1, 6 MSH6, and 6 PMS2). The 44 probands were diagnosed at a mean age of 51.4 years; 22/44 (50%) were over age 50 and 11/44 (25%) failed to meet AC or BG. The sensitivity of MSI-H status and abnormal MMR IHC for detecting LS are estimated at 95% (42/44) and 93% (41/44), respectively. Testing of the probands' family members resulted in the identification of an additional 109 mutations (mean of 3.3 additional mutations detected per proband). Speaking to the practical applicability of current clinical guidelines, of the 143 patients with germline mutations identified in the course of this investigation, only 1 (0.7%) carried a previous diagnosis of Lynch syndrome. We concluded that population-based screening for LS was feasible and that whereas MSI testing and MMR IHC performed similarly in this setting, IHC had the advantage of directing gene testing.

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THE OHIO STATE UNIVERSITY APPROACH

On the basis of these results, in March 2006 we implemented screening for Lynch syndrome in all newly diagnosed CRC. (In April of 2007 we implemented screening in all newly diagnosed ECs; we had conducted a similar population-based study in EC).119 Successful implementation took place in the context of broad-based support from the Departments of Medicine, Surgery, Gynecology, Pathology, and Clinical Cancer Genetics (CCG). Given its high-sensitivity and what we considered to be several practical advantages (Table 4) we chose MMR-IHC-based over MSI-based screening. At present, the identification of LS is our primary goal; the identification of tumors likely to be microsatellite unstable is a secondary benefit. As evidence supporting the predictive value of MSI status accumulates, our testing strategy may change. Our current diagnostic algorithm is presented in Figure 4.

Figure 4
Figure 4
Image Tools

MMR IHC (MLH1, MSH2, MSH6, PMS2) is preformed on all newly diagnosed CRC. The intact expression of all 4 proteins is the most likely result. In the absence of a compelling clinical history, (eg, CRC diagnosed <45 y, first-degree relative with CRC, multiple primary tumors) testing stops here. The second most likely result is absent MLH1 expression (nearly always accompanied by absent PMS2 expression). These cases are reflexed to BRAF mutation analysis. BRAF V600E mutants are generally excluded from further testing. In the absence of BRAF mutation, patients are counseled and directed for MLH1 mutation analysis, including MLPA testing as appropriate. In the absence of demonstrable mutation, testing may proceed to PMS2 mutation analysis and/or MLH1 methylation analysis. Patients with loss of MSH2 and/or MSH6 or isolated loss of PMS2 expression are counseled and directed for sequence analysis of the implicated gene, again, supplemented by MLPA as appropriate. The demonstration of a deleterious germline mutation in a MMR gene is the gold standard for the diagnosis of Lynch syndrome. In the setting of abnormal IHC, given the technical challenges inherent in mutation analysis, failure to demonstrate a germline mutation does not categorically exclude the diagnosis. Patients are counseled accordingly. Supplemental MSI testing may be helpful in these cases. Also, patients referred to CCG with especially compelling clinical histories may be screened with both modalities (ie, IHC and MSI testing) “up front”.

We acknowledge the controversies surrounding “genetic testing.” We do not consider MSI or MMR IHC testing to be “genetic testing”, but rather tests of tumor phenotype, and as such, we do not require “informed consent” prior to initiating MMR IHC testing.120 Some individuals may be more at ease with MSI testing, as the results do not implicate a particular gene. Abnormal results are forwarded to CCG, who, functioning as part of the implied clinical care team, communicate the results to the patient. Our clinicians had requested that CCG assume this function. Patients may or may not harbor a germline mutation, and they are free to choose or decline further counseling or clinical testing.

Given the proven impact of intensive screening in Lynch syndrome and the relative ease of screening for the disease, we consider the implementation of Lynch screening to be highly desirable. Several strategies exist, including those based on clinical characteristics, histology, MSI testing, MMR IHC, and combinations thereof. The individual strategy chosen should make sense in the context of the patient population served and the resources at hand. Given skills in morphology and laboratory medicine, pathologists are uniquely positioned to play a critical role in this endeavor.

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ACKNOWLEDGMENTS

The authors thank the advice and editorial assistance from Heather Hampel, MS and the secretarial assistance from Jacqui Lankford.

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A novel germline MLH1 mutation causing Lynch Syndrome in patients from the Republic of Macedonia
Hiljadnikova-Bajro, M; Josifovski, T; Panovski, M; Dimovski, AJ
Croatian Medical Journal, 53(5): 496-501.
10.3325/cmj.2012.53.496
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Keywords:

Lynch syndrome; hereditary non-polyposis colorectal cancer; mismatch repair; microsatellite instability; promoter methylation

© 2009 Lippincott Williams & Wilkins, Inc.

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