Colorectal cancer (CRC) risk assessment involves evaluation of an individual's personal and family history for characteristics of an inherited susceptibility to develop CRC. These characteristics include early age at diagnosis, multiple primary cancers, polyposis, family history of syndrome-specific malignancies, and certain histologic features. In the absence of polyposis, the genetic testing algorithm for hereditary CRC focuses largely on identifying Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer. Previously, an understanding of the workup of individuals suspected of having LS could be left to those specializing in genetics. Recently, screening of all patients with newly diagnosed CRC for LS independent of family history has been endorsed by the CDC-sponsored Evaluation of Genomic Applications in Practice and Prevention Working Group (EGAPP) and others. However, although highlighting the EGAPP recommendations, the current NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Colorectal Cancer Screening continue to endorse mismatch repair (MMR) screening in individuals with a strong family history for colon cancer (to view the most recent version of these guidelines, visit the NCCN Web site at www.NCCN.org).1–3 Several centers, including Fox Chase Cancer Center (FCCC), are implementing universal screening programs. A need is emerging for clinicians to be able to converse with patients about the value of tests to evaluate hereditary CRC risk, and to facilitate the interpretation of the results.
Updates to the understanding of the pathogenesis of LS present an enormous challenge for clinicians attempting to remain knowledgeable about hereditary cancer syndromes and seeking to provide ongoing comprehensive genetic risk information to current and past patients. Although tumor testing and risk evaluation have become integral aspects of medical oncology care, the complete process of cancer genetic counseling, from the construction of a 3-generation pedigree to the interpretation of results, is a complex task that may require more time and resources than many physicians are able to provide. The cancer genetics specialist is expert at choosing the appropriate test, with the ability to integrate new information based on updates in testing technique, the identification of new genes, and increased understanding of the variable presentation of susceptibility conditions. Cancer genetics specialists can be found through the National Society of Genetic Counselors (www.nsgc.org) and the National Cancer Institute (www.cancer.gov or 1–800–4–CANCER).
Lynch Syndrome Etiology and Identification
LS is the most common cause of hereditary CRC, accounting for 2.7% of all CRC cases4,5 and 2.1% of all endometrial cancer cases.6,7 The prevalence of LS in the general population is estimated to be 1 in 300.3 LS is associated with increased risks for multiple malignancies in addition to CRC and endometrial cancer, including cancer of the stomach, ovary, renal pelvis, ureter, biliary tract, small bowel, brain, and sebaceous glands.8,9 LS is caused by mutations in the MMR genes MLH1, MSH2, MSH6, and PMS2,10–13 and by deletions in the EPCAM gene.14–16 The characteristic phenotype of LS tumors is microsatellite instability (MSI), a sign of defective MMR.17,18 Microsatellites are areas of the genome at which the DNA sequence is repetitive (e.g., (CA)n). During DNA replication, errors may occur that are usually corrected by the MMR protein complex, including the MLH1, MSH2, MSH6, and PMS2 proteins.19,20 Individuals with deficient MMR (dMMR), such as those with LS, will have difficulty repairing mismatches and their tumors will exhibit expansions and contractions in the microsatellite regions. These tumors will also show loss of immunohistochemical expression of one or more of the MMR proteins.21 MMR testing using MSI and immunohistochemical expression can be performed on formalin-fixed, paraffin-embedded tissue. An advantage of immunohistochemical testing is that it can be performed in the pathology department of most hospitals, whereas MSI testing requires molecular facilities. Based on a recent extensive evidence review by the EGAPP, the sensitivity of MSI testing is estimated to be 69% to 85% and the sensitivity of immunohistochemical testing is 83% in LS CRCs.22 Nonetheless, research has also highlighted several pitfalls of routine immunohistochemical testing for the LS proteins, including issues related to quality control and interobserver variability in interpretation.23,24 The dMMR phenotype is also present in LS-associated endometrial cancers at a high frequency.25
Sporadic Mismatch Repair Deficiency
Approximately 15% of all CRCs will exhibit the dMMR17,18,26 phenotype, yet only a portion (approximately 3%) are from LS.4,5,27 The remaining 12% will exhibit MSI and show loss of MLH1 and PMS2 on immunohistochemical analysis. In these cases, dMMR is caused by acquired hypermethylation of the MLH1 gene promoter.28,29 This feature is a somatic change associated with advanced age, female sex, and location (with the proximal colon affected more often than the distal).30,31 The addition of methyl groups in the MLH1 promoter blocks transcription factor binding and effectively silences the MLH1 gene in colonic epithelial cells.29,32 Because the MLH1 and PMS2 proteins are heterodimers, PMS2 will generally also degrade if the MLH1 gene is not transcribed, resulting in loss of both MLH1 and PMS2 on clinical immunohistochemical staining.20
Tumor-based molecular testing for the common BRAF V600E mutation has been shown to be useful in distinguishing sporadic from LS-associated dMMR. BRAF encodes a kinase involved in response to growth signals. BRAF mutation leads to constitutive activity of the kinase.33 BRAF V600E accounts for approximately 90% of all activating BRAF mutations.33 Clinically, most CRCs with dMMR from acquired hypermethylation of MLH1 (40%–83%) are characterized by the BRAF V600E mutation, whereas this mutation has not been seen in tumors from germline MLH1 loss,34–36 although at least one case has been reported.35 Thus, the identification of BRAF V600E in a CRC tumor exhibiting MSI supports that further evaluation for germline MLH1 mutations is unwarranted.
Clinical Criteria for LS
The identification of individuals with LS has been a consistent challenge, owing largely to its variable presentation and the difficulty of obtaining an accurate and comprehensive family history. Many clinical definitions and testing algorithms for LS have been proposed over time. Before the elucidation of the molecular basis of LS, the Amsterdam criteria I were developed to provide criteria for recruiting patients into collaborative studies. These criteria describe families 1) that have had 3 cases of CRC, with 2 of the affected individuals being first-degree relatives of the third; 2) with CRCs occurring in at least 2 generations; 3) with 1 CRC diagnosed before the age of 50 years; and 4) in which familial adenomatous polyposis is excluded. The more recent Amsterdam criteria II acknowledged the importance of extracolonic cancers in LS.37 However, research has shown that the Amsterdam criteria are too restrictive, identifying only 45% to 56% of individuals with LS gene mutations.38,39
The Bethesda guidelines were originally developed to guide tumor-based MSI screening for LS in patients with CRC and were updated to their current form (the revised Bethesda guidelines) in 2003.40,41 The Bethesda guidelines consider personal and family history in addition to histologic features more common in high-level MSI (MSI-H) tumors, such as Crohn's disease–like lymphocytic reaction, the presence of tumor-infiltrating lymphocytes, and mucinous and signet ring appearance.40 These modifications make the Bethesda guidelines more sensitive than the Amsterdam criteria (94% vs. 61%) for detecting individuals with germline LS mutation, but at the cost of loss of specificity (25% vs. 67%).42
Risk Assessment for Lynch Syndrome
At FCCC, the approach to the evaluation of possible LS in a family has been to begin with MSI and immunohistochemical testing on the tumor of the family member with the youngest age at diagnosis of CRC or endometrial cancer. The advantage of beginning with MSI and immunohistochemical testing in tandem is the sensitivity of tumor-based tests for detecting the MMR phenotype,5,27,43 which helps to establish a clinical diagnosis of LS in families with suggestive histories.
A proficient MMR (pMMR) phenotype from tumor-based testing in an appropriate family member is extremely reassuring (i.e., highly sensitive) that LS is not the underlying cause of that tumor, although phenocopy cancers (i.e., a sporadic case of cancer within a putative LS family) must always be considered. Molecular genetic testing will miss approximately 15% of individuals with clinical LS. Recent updates in testing technique and the discovery of new genes associated with LS have exposed the weakness of molecular LS testing for detecting mutation carriers. Even novel mechanisms of LS not owing to mutations in MMR genes, such as EPCAM deletions and constitutional MLH1 epimutations, will result in the dMMR phenotype.15,16,44 In addition, immunohistochemical testing in particular can define MMR status and guide the genetic testing process, pinpointing the specific gene in which the mutation exists. For example, loss of MSH6 only on immunohistochemical testing indicates that genetic analysis of MSH6 should be pursued. This can save time and cost associated with pursuing testing in the 4 other LS genes. Table 1 summarizes the abnormal immunohistochemical testing results that are most frequently encountered in clinical practice and their interpretations. The NCCN Guidelines for Colorectal Cancer Screening1 provide a more detailed table that combines MSI, BRAF V600E, and methylation results (available online at www.NCCN.org [LS-A, page 2 of 2]).
Although acquiring permissions and tumor specimens from relatives of a proband can be challenging, tumor blocks are retained for at least 10 years and are generally adequate for testing. Tumor-based MMR screening several years after a CRC or endometrial cancer diagnosis may be associated with out-of-pocket costs to families if the proband is deceased, but these costs are balanced by the value of the information the screening provides. Particularly because germline DNA-based testing for LS in unaffected individuals remains uncovered by some insurance carriers, this approach can also help reduce patient out-of-pocket costs through focused testing as opposed to complete analysis of the 5 LS-associated genes, which may cost up to $8000 or more.
Recently, FCCC began transitioning to a universal (reflex) LS screening strategy (Figure 1). Supported by the EGAPP recommendations, and partially modeled based on the experience reported by Ohio State University, the FCCC reflex MMR screening strategy will use immunohistochemical testing as the primary screen for MMR in all new patients with surgically resected or biopsied CRC and endometrial cancer cases, with MSI performed only when a failed or uninterpretable immunohistochemical test is encountered. Individuals for whom immunohistochemistry shows a loss of PMS2, MSH2, and/or MSH6 will be directed to genetic counseling and germline DNA testing. When loss of MLH1/PMS2 is seen on immunohistochemistry, individuals with endometrial cancer will be directed for genetic counseling for consideration of methylation or germline testing. Those with CRC will have BRAF
Interpreting Common Abnormal Results on Immunohistochemical Testing
Beyond its use in identifying LS, evaluation of MMR status may, in the near future, become an increasingly integral part of the prognostication and treatment planning for patients with newly diagnosed CRC. The superior prognosis of patients with dMMR tumors over those with pMMR has been established,46,47 and data strongly suggest that stage II colon tumors with dMMR are unlikely to benefit from 5-FU–based adjuvant chemotherapy.48,49
Difficulties in LS Evaluation: Clinical Cases
Risk assessment for LS is complex, and difficulties in clinical decision-making are common. In the following cases, the authors illuminate several difficulties they have encountered in their clinical practice. Table 2 provides a referenced summary that includes these scenarios and several others that are not discussed in detail.
Case 1
Personal History: A 56-year old man with recently diagnosed stage IIA CRC was referred for cancer risk assessment by his medical oncologist. The physician ordered MSI and immunohistochemical testing on the colon tumor specimen to inform use of adjuvant 5-FU–based chemotherapy. This testing showed MSI-H and loss of MLH1 and PMS2 expression on immunohistochemistry (Figure 2A).
Family History: The paternal history included a father with CRC (age, 60 years), an uncle with CRC (age, 64 years), and an aunt with gastric cancer.
Reflex mismatch repair screening at Fox Chase Cancer Center.
Abbreviations: CRC, colorectal cancer; dMMR, deficient mismatch repair; EC, endometrial cancer; IHC, immunohistochemistry; MMR, mismatch repair; MSI, microsatellite instability testing; MSI-H, high-level microsatellite instability; pMMR, proficient mismatch repair.
*MSI only if immunohistochemistry failure.
†BRAF V600E analysis only in colorectal cancer, because this mutation seems to be absent in most endometrial cancers.55,56
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
Reflex mismatch repair screening at Fox Chase Cancer Center.
Abbreviations: CRC, colorectal cancer; dMMR, deficient mismatch repair; EC, endometrial cancer; IHC, immunohistochemistry; MMR, mismatch repair; MSI, microsatellite instability testing; MSI-H, high-level microsatellite instability; pMMR, proficient mismatch repair.
*MSI only if immunohistochemistry failure.
†BRAF V600E analysis only in colorectal cancer, because this mutation seems to be absent in most endometrial cancers.55,56
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
Reflex mismatch repair screening at Fox Chase Cancer Center.
Abbreviations: CRC, colorectal cancer; dMMR, deficient mismatch repair; EC, endometrial cancer; IHC, immunohistochemistry; MMR, mismatch repair; MSI, microsatellite instability testing; MSI-H, high-level microsatellite instability; pMMR, proficient mismatch repair.
*MSI only if immunohistochemistry failure.
†BRAF V600E analysis only in colorectal cancer, because this mutation seems to be absent in most endometrial cancers.55,56
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
Testing Strategy: Germline DNA testing of the MLH1 gene showed no mutation. Tumor analysis for the BRAF V600E mutation was negative. However, because this test is not completely sensitive for hypermethylation of the MLH1 gene promoter, MLH1 methylation–specific polymerase chain reaction was
Challenges for Lynch Syndrome Risk Assessment
Discussion: The dMMR phenotype is not exclusive to LS, and in fact is more frequently from a non–hereditary cause. The 12% of cancers that have somatic methylation of the MLH1 promoter will have
Pedigrees for case 1 (A), case 2 (B), and case 3 (C).
Abbreviations: CRC, colorectal cancer; DCIS, ductal carcinoma in situ.
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
Pedigrees for case 1 (A), case 2 (B), and case 3 (C).
Abbreviations: CRC, colorectal cancer; DCIS, ductal carcinoma in situ.
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
Pedigrees for case 1 (A), case 2 (B), and case 3 (C).
Abbreviations: CRC, colorectal cancer; DCIS, ductal carcinoma in situ.
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 9, 11; 10.6004/jnccn.2011.0106
In planning a testing strategy for this patient, the authors had 2 main testing options: assess for the presence of LS (MLH1 gene analysis), or test for evidence of a sporadic cause (tumor hypermethylation and/or BRAF V600E gene analysis). If the personal and family histories are consistent with LS, MLH1 analysis is a reasonable place to start, whereas if the patient has no family history or was not diagnosed at a young age, beginning with testing for hypermethylation and/or the BRAF V600E mutation may be less time-consuming and more cost-effective. Because of the number of family members affected with LS cancers, the authors chose to pursue MLH1 gene analysis first. Although no MLH1 mutation was identified, they could not assume LS was absent. To guard against the possibility of mutation undetected using standard testing techniques, they pursued evidence of an epigenetic cause for the dMMR results. Although the BRAF V600E mutation will not be found in all tumors with epigenetic silencing of MLH1, promoter methylation testing is technically complex and offered by few clinical laboratories. Therefore, the authors first pursued BRAF testing, which is available in their molecular laboratory, followed by methylation when the BRAF V600E mutation was not found. The patient's positive hypermethylation results provide an answer for his loss of MLH1/PMS2 tumor results determined through MSI-H/immunohistochemical testing. The results also provide evidence that the underlying cause of his cancer was unlikely LS. Although coexistence of MLH1 hypermethylation and germline MLH1 gene mutations has been reported,35,50 this seems to be a rare occurrence and particularly unlikely in this patient given that MLH1 genetic testing showed no mutation.
Clinical Implications: Given his family history of CRC, the possibility of a different gene or combination of genes affecting risk in the family must be considered. The patient's risk of a second primary CRC is difficult to quantify, but it would be reasonable for him and close relatives to be followed up closely with colonoscopy. The patient was advised to pursue a colonoscopy interval of 2 to 3 years, and his children were advised to begin colonoscopy in their 40s (10 years earlier than the patient), to be repeated every 3 to 5 years. He was advised to forego screening for the extracolonic cancers associated with LS, and was encouraged to inform the authors if he or family members developed other cancers or had more than 10 cumulative adenomas on future screenings, because further testing (e.g., for an APC gene–associated polyposis) may then be justified.
Case 2
Personal History: A 61-year-old woman was referred for risk assessment because of a personal and family history of cancer. She was diagnosed with stage I endometrial cancer at 52 years of age (Figure 2B).
Family History: Paternal history included a father with transitional cell carcinoma of the renal pelvis (age, 69 years), an uncle with CRC (age, 65 years), an aunt with CRC (age, 57 years), and an aunt with gastric cancer (age, 60 years). Her paternal grandmother had endometrial cancer (age, 35 years).
Testing Strategy: The patient was counseled that her family history included a clustering of cancers typical for LS families, and further assessment for LS was recommended. MSI testing on the patient's endometrial cancer showed a tumor that was MSI-H at 7 of 8 markers and showed loss of MSH2 and MSH6 on immunohistochemistry. This pattern of expression is consistent with LS, most frequently from a mutation in the MSH2 gene. Germline molecular analysis of MSH2 with reflex to MSH6 showed no mutation. When the patient was initially seen for risk assessment (in 2005), no further genetic testing was available. Because of her pattern of MSI and immunohistochemistry results in combination with her family history, she was given a putative diagnosis of LS and advised to follow associated screening guidelines. When clinical EPCAM testing became available, the patient was informed at her yearly clinic visit. She chose to pursue this testing, the results of which were negative (i.e., failed to detect a deletion).
Discussion: In contrast to Case 1, the underlying cause of dMMR is less uncertain in this patient. Even in the absence of a detectable LS mutation, the patient's dMMR (MSI-H and loss of MSH2 and MSH6 on immunohistochemistry) indicates she has LS, but LS that is from a currently undetectable molecular cause. Unlike the dual loss of MLH1 and PMS2 on immunohistochemistry, loss of MSH2 and MSH6 expression does not commonly occur sporadically, nor does loss of MSH6 or PMS2 alone (see Table 2). Germline MSH2 promoter hypermethylation has been documented in the literature, but this does not seem to be common.14,51 This case shows the value of MMR screening with MSI or immunohistochemistry testing before germline DNA testing, and its ability to detect LS even when a gene mutation is not identified. If molecular genetic testing had been pursued first, the authors may have been tempted to consider the patient a phenocopy. Aside from family history, no evidence would have justified LS screening.
This case highlights recent updates in the understanding of LS. Since the patient's initial evaluation in 2005, clinical LS genetic testing has expanded to include 2 additional genes (MSH6 and PMS2), and an entirely novel causative mechanism for the disease has been described (EPCAM).
Clinical Implications: Because the authors strongly suspect that this patient has hereditary dMMR because of clinical LS, appropriate medical management recommendations can be made for LS (for current recommendations, see the NCCN Guidelines for Colorectal Screening, available on the NCCN Web site at www.NCCN.org). She was advised that her first-degree relatives should consider themselves at risk for the currently undetected familial LS gene mutation.1,22
Case 3
Personal History: A 49-year-old man was referred for risk assessment by his gastroenterologist because of a personal history of colon polyps and a family history of CRC. He had 3 polyps (nonadenomatous) removed when he was in his 40s (Figure 2C).
Family History: A sister died of CRC (diagnosed at 40 years of age), and another had 2 adenomas at 47 years of age. Paternal history included a father with CRC (age, 61 years), an aunt with CRC (age, 75 years), an uncle with CRC (age, 42 years), and a grandmother with CRC (age, 76 years).
Testing Strategy: The patient was counseled that his family represented a classic hereditary CRC kindred. The authors recommended he pursue MSI and immunohistochemical testing on his sister's CRC to best define the family risk, but he expressed reluctance to involve his grieving brother-in-law, whose permission would be necessary to obtain the tumor specimen. The authors pursued germline genetic testing of MLH1, MSH2, and MSH6 (PMS2 testing was not yet available and EPCAM deletions had not been described) on the patient and this showed no mutation. The authors discussed with the patient that this was an uninformative result and reiterated the value of MSI and immunohistochemical testing on his sister's tumor.
The authors drafted a letter for the patient to share with family explaining MSI and immunohistochemical testing and the potential implications. After 1 year, his brother-in-law contacted the authors about pursuing MSI and immunohistochemical testing on his sister's tumor. Immunohistochemical testing showed an MSS tumor with intact expression of MLH1, MSH2, MSH6, and PMS2.
Discussion: This case exemplifies an issue commonly encountered in cancer genetics: who is the best person to test in a family? Although testing the patient was convenient because he was unaffected, the authors' only option was to pursue costly germline genetic testing for LS, which was likely to yield an uninformative result. His sister with adenomas was a slightly better candidate because, having manifested a clinical feature of LS, she was more likely to receive an informative result. Still, this would require involving an additional individual who may not be interested in testing or may face barriers to obtaining genetic services. Moreover, like the proband, even if no mutation was detected in her, this could not rule out LS, because she could be producing adenomas sporadically within an LS family. The ideal candidate for testing was his sister who was affected with CRC at 40 years of age. Identifying pMMR in an individual expressing the risk phenotype, (i.e., affected with CRC at a young age) is an excellent way to reduce suspicion of LS.
MSI and immunohistochemical testing on the tumor of a deceased individual is often overlooked as an option to define hereditary risk in a family. In this patient, the MSI and immunohistochemical test results were extremely valuable in elucidating cancer risks. Families meeting Amsterdam criteria I with MSS tumors have an overall lower CRC risk and later average age at diagnosis than individuals with LS52,53; they also have no significantly elevated extracolonic cancer risks52 and do not demonstrate the rapid progression to CRC seen in MMR mutation carriers.53,54 This phenotype has been named familial colorectal cancer type X because the molecular origin has not yet been identified.52 The underlying genetic cause is likely heterogeneous, encompassing mutations in single genes with dominant inheritance, accumulation of risk from multiple low penetrance alleles, and shared environmental and familial influences in the family. Unfortunately, the authors cannot determine whether the original proband or his deceased sister's daughters have inherited the predisposition to CRC, but more appropriate medical management recommendations can be made for the family.
Clinical Implications: A pMMR result on the proband's sister's tumor indicated that LS was an unlikely cause of the CRC phenotype in the family (although it remains possible that the sister was a phenocopy), and that extracolonic screening associated with LS was not warranted. Young individuals were advised to begin colonoscopy at 30 years of age (10 years before the earliest family diagnosis), and that screening frequency should reflect the hereditary risk in the family (the recommendation was for colonoscopy every 2 to 3 years, although a very conservative 1-year interval could also be considered despite an unknown molecular risk). Depending on the findings of future colonoscopies, the proband or other family members may be candidates for additional genetic testing.
Conclusions
Identifying LS remains a public health imperative. Recommendations and clinical strategies for more-expansive universal LS screening and an everexpanding repertoire of molecular testing options are emerging and have the potential to improve LS detection. General and specialty providers need a better understanding of this common cancer risk syndrome; how to best identify clinically affected individuals, mutation carriers, and family members; and the medical implications, including recommended preventive screening and surgical prophylaxis.
References
- 1↑
Burt RW, Barthel JS, Cannon J et al.. NCCN Clinical Practice Guidelines in Oncology: Colorectal Cancer Screening. Version 2, 2011. Available at: http://www.nccn.org/professionals/physician_gls/pdf/colorectal_screening.pdf. Accessed September 22, 2011.
- 2
Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med 2009;11:35–41.
- 3↑
Boland CR, Shike M. Report from the Jerusalem workshop on Lynch syndrome-hereditary nonpolyposis colorectal cancer. Gastroenterology 2010;138:2197.e1–7.
- 4↑
Salovaara R, Loukola A, Kristo P et al.. Population-based molecular detection of hereditary nonpolyposis colorectal cancer. J Clin Oncol 2000;18:2193–2200. Erratum in J Clin Oncol 2000;18:3456.
- 5↑
Hampel H, Frankel WL, Martin E et al.. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med 2005;352:1851–1860.
- 6↑
Ollikainen M, Abdel-Rahman WM, Moisio AL et al.. Molecular analysis of familial endometrial carcinoma: a manifestation of hereditary nonpolyposis colorectal cancer or a separate syndrome? J Clin Oncol 2005;23:4609–4616.
- 7↑
Hampel H, Frankel W, Panescu J et al.. Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients. Cancer Res 2006;66:7810–7817.
- 8↑
Watson P, Lynch HT. Extracolonic cancer in hereditary nonpolyposis colorectal cancer. Cancer 1993;71:677–685.
- 9↑
Vasen HF, Wijnen JT, Menko FH et al.. Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis. Gastroenterology 1996;110:1020–1027. Erratum in Gastroenterology 1996;111:1402.
- 10↑
Leach FS, Nicolaides NC, Papadopoulos N et al.. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 1993;75:1215–1225.
- 11
Papadopoulos N, Nicolaides NC, Wei YF et al.. Mutation of a mutL homolog in hereditary colon cancer. Science 1994;263:1625–1629.
- 12
Nicolaides NC, Papadopoulos N, Liu B et al.. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 1994;371:75–80.
- 13↑
Miyaki M, Konishi M, Tanaka K et al.. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nat Genet 1997;17:271–272.
- 14↑
Niessen RC, Hofstra RM, Westers H et al.. Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer 2009;48:737–744.
- 15↑
Kovacs ME, Papp J, Szentirmay Z et al.. Deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome. Hum Mutat 2009;30:197–203.
- 16↑
Ligtenberg MJL, Kuiper RP, Chan TL et al.. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3’ exons of TACSTD1. Nat Genet 2009;41:112–117.
- 17↑
Ionov Y, Peinado MA, Malkhosyan S et al.. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558–561.
- 18↑
Aaltonen LA, Peltomaki P, Mecklin JP et al.. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994;54:1645–1648.
- 20↑
Iyer RR, Pluciennik A, Burdett V, Modrich PL. DNA mismatch repair: functions and mechanisms. Chem Rev 2006;106:302–323.
- 21↑
Engel C, Forberg J, Holinski-Feder E et al.. Novel strategy for optimal sequential application of clinical criteria, immunohistochemistry and microsatellite analysis in the diagnosis of hereditary nonpolyposis colorectal cancer. Int J Cancer 2006;118:115–122.
- 22↑
Palomaki GE, McClain MR, Melillo S et al.. EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome. Genet Med 2009;11:42–65.
- 23↑
Klarskov L, Ladelund S, Holck S et al.. Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 2010;41:1387–1396.
- 24↑
Overbeek LI, Ligtenberg MJ, Willems RW et al.. Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting. Am J Surg Pathol 2008;32:1246–1251.
- 25↑
Lu KH, Schorge JO, Rodabaugh KJ et al.. Prospective determination of prevalence of lynch syndrome in young women with endometrial cancer. J Clin Oncol 2007;25:5158–5164.
- 26↑
Jensen LH, Lindebjerg J, Byriel L et al.. Strategy in clinical practice for classification of unselected colorectal tumours based on mismatch repair deficiency. Colorectal Dis 2008;10:490–497.
- 27↑
Hampel H, Frankel WL, Martin E et al.. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008;26:5783–5788.
- 28↑
Herman JG, Umar A, Polyak K et al.. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 1998;95:6870–6875.
- 29↑
Deng G, Chen A, Hong J et al.. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 1999;59:2029–2033.
- 30↑
Malkhosyan SR, Yamamoto H, Piao Z, Perucho M. Late onset and high incidence of colon cancer of the mutator phenotype with hypermethylated hMLH1 gene in women. Gastroenterology 2000;119:598.
- 31↑
Kakar S, Burgart LJ, Thibodeau SN et al.. Frequency of loss of hMLH1 expression in colorectal carcinoma increases with advancing age. Cancer 2003;97:1421–1427.
- 32↑
Tate PH, Bird AP. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev 1993;3:226–231.
- 33↑
Wan PT, Garnett MJ, Roe SM et al.. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–867.
- 34↑
Kambara T, Simms LA, Whitehall VL et al.. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137–1144.
- 35↑
Wang L, Cunningham JM, Winters JL et al.. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 2003;63:5209–5212.
- 36↑
Domingo E, Laiho P, Ollikainen M et al.. BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41:664–668.
- 37↑
Vasen HF, Mecklin JP, Khan PM, Lynch HT. The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum 1991;34:424–425.
- 38↑
Wijnen JT, Vasen HF, Khan PM et al.. Clinical findings with implications for genetic testing in families with clustering of colorectal cancer. N Engl J Med 1998;339:511–518.
- 39↑
Mangold E, Pagenstecher C, Friedl W et al.. Spectrum and frequencies of mutations in MSH2 and MLH1 identified in 1,721 German families suspected of hereditary nonpolyposis colorectal cancer. Int J Cancer 2005;116:692–702.
- 40↑
Rodriguez-Bigas MA, Boland CR, Hamilton SR et al.. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst 1997;89:1758–1762.
- 41↑
Umar A, Boland CR, Terdiman JP et al.. Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 2004;96:261–268.
- 42↑
Syngal S, Fox EA, Eng C et al.. Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2 and MLH1. J Med Genet 2000;37:641–645.
- 43↑
Wahlberg SS, Schmeits J, Thomas G et al.. Evaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families. Cancer Res 2002;62:3485–3492.
- 44↑
Gazzoli I, Loda M, Garber J et al.. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor. Cancer Res 2002;62:3925–3928.
- 45↑
Pinol V, Castells A, Andreu M et al.. Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer. JAMA 2005;293:1986–1994.
- 46↑
Gryfe R, Kim H, Hsieh ET et al.. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000;342:69–77.
- 47↑
Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 2005;23:609–618.
- 48↑
Sargent DJ, Marsoni S, Monges G et al.. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 2010;28:3219–3226.
- 49↑
Ribic CM, Sargent DJ, Moore MJ et al.. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003;349:247–257.
- 50↑
Rahner N, Friedrichs N, Steinke V et al.. Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome. J Pathol 2008;214:10–16.
- 51↑
Rumilla K, Schowalter KV, Lindor NM et al.. Frequency of deletions of EPCAM (TACSTD1) in MSH2-associated lynch syndrome cases. J Mol Diagn 2011;13:93–99.
- 52↑
Lindor NM, Rabe K, Petersen GM et al.. Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: familial colorectal cancer type X. JAMA 2005;293:1979–1985.
- 53↑
Mueller-Koch Y, Vogelsang H, Kopp R et al.. Hereditary nonpolyposis colorectal cancer: clinical and molecular evidence for a new entity of hereditary colorectal cancer. Gut 2005;54:1733–1740.
- 54↑
Dove-Edwin I, de Jong AE, Adams J et al.. Prospective results of surveillance colonoscopy in dominant familial colorectal cancer with and without Lynch syndrome. Gastroenterology 2006;130:1995–2000.
- 55↑
Kawaguchi M, Yanokura M, Banno K et al.. Analysis of a correlation between the BRAF V600E mutation and abnormal DNA mismatch repair in patients with sporadic endometrial cancer. Int J Oncol 2009;34:1541–1547.
- 56↑
Pappa KI, Choleza M, Markaki S et al.. Consistent absence of BRAF mutations in cervical and endometrial cancer despite KRAS mutation status. Gynecol Oncol 2006;100:596–600.
- 57
Gylling AH, Nieminen TT, Abdel-Rahman WM et al.. Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors. Carcinogenesis 2008;29:1351–1359.
- 58
Choi MY, Lauwers GY, Hur C et al.. Microsatellite instability is frequently observed in rectal cancer and influenced by neoadjuvant chemoradiation. Int J Radiat Oncol Biol Phys 2007;68:1584.
- 59
Bao F, Panarelli NC, Rennert H et al.. Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma. Am J Surg Pathol 2010;34:1798–1804.
- 60
De Jong AE, Morreau H, Van Puijenbroek M et al.. The role of mismatch repair gene defects in the development of adenomas in patients with HNPCC. Gastroenterology 2004;126:42–48.
- 61
Pino MS, Mino-Kenudson M, Wildemore BM et al.. Deficient DNA mismatch repair is common in Lynch syndrome-associated colorectal adenomas. J Mol Diagn 2009;11:238–247.
- 62
Modica I, Soslow RA, Black D et al.. Utility of immunohistochemistry in predicting microsatellite instability in endometrial carcinoma. Am J Surg Pathol 2007;31:744–751.
