Incorporation of somatic <Emphasis Type="Italic">BRAF</Emphasis> mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.     

Thyroid cancer in a patient with a germline <Emphasis Type="Italic">MSH2</Emphasis> mutation. Case report and review of the Lynch syndrome expanding tumour spectrum

Lynch syndrome (HNPCC) is a dominantly inherited disorder characterized by germline defects in DNA mismatch repair (MMR) genes and the development of a variety of cancers, predominantly colorectal and endometrial. We present a 44-year-old woman who was shown to carry the truncating MSH2 gene mutation that had previously been identified in her family. Recently, she had been diagnosed with an undifferentiated carcinoma of the thyroid and an adenoma of her coecum. Although the thyroid carcinoma was not MSI-high (1 out of 5 microsatellites instable), it did show complete loss of immunohistochemical expression for the MSH2 protein, suggesting that this tumour was not coincidental. Although the risks for some tumour types, including breast cancer, soft tissue sarcoma and prostate cancer, are not significantly increased in Lynch syndrome, MMR deficiency in the presence of a corresponding germline defect has been demonstrated in incidental cases of a growing range of tumour types, which is reviewed in this paper. Interestingly, the MSH2-associated tumour spectrum appears to be wider than that of MLH1 and generally the risk for most extra-colonic cancers appears to be higher for MSH2 than for MLH1 mutation carriers. Together with a previously reported case, our findings show that anaplastic thyroid carcinoma can develop in the setting of Lynch syndrome. Uncommon Lynch syndrome-associated tumour types might be useful in the genetic analysis of a Lynch syndrome suspected family if samples from typical Lynch syndrome tumours are unavailable.     

Prediction of the outcome of genetic testing in HNPCC kindreds using the revised Amsterdam criteria and immunohistochemistry

Background and aims: Hereditary non-polyposis colorectal cancer (HNPCC) may be caused by mutations in the mismatch repair (MMR) genes MLH1, MSH2 or MSH6. Family history (Amsterdam criteria) has traditionally been used to select patients for mutation testing. It has been demonstrated that germline mutations in the MMR genes are associated with lack of the corresponding gene product as assessed with immunohistochemistry (IHC) in tumour specimens. The aim of the study was to assess the value of the Amsterdam criteria II and IHC in predicting germline mutations. Methods: Fifty-six families that were previously tested for MLH1, MSH2 and MSH6 mutations were selected for this study. All pedigrees were extended and verified and the families were scored according to the original (I) and the revised Amsterdam criteria (II). The probabilities for MLH1 and MSH2 mutations were calculated by logistic regression. In addition, all available tumour material from indexed family members was examined by IHC for the presence of the three gene products. Results: Three out of seven (39%) families where the mutation could be identified complied with the Amsterdam criteria I, while all seven (100%) met the Amsterdam criteria II. All families carrying a MLH1 or MSH2 mutation had > 15% calculated probability of finding a mutation. Tumours from all seven mutation carriers lacked the immunohistochemical expression of the corresponding MMR gene. Conclusion: The results indicate that the Amsterdam criteria II in combination with immunohistochemistry of the mismatch repair proteins in tumours may be a cost-effective approach to select families for mutation analysis.     

Lynch-like syndrome is as frequent as Lynch syndrome in early-onset nonfamilial nonpolyposis colorectal cancer

Early-onset (<50 years-old) nonpolyposis nonfamilial colorectal cancer (EO NP NF CRC) is a common clinical challenge. Although Lynch syndrome (LS) is associated with EO CRC, the frequency of this syndrome in the EO NF cases remains unknown. Besides, mismatch repair deficient (MMRd) CRCs with negative MMR gene testing have recently been described in up to 60% of cases and termed “Lynch-like syndrome” (LLS). Management and counseling decisions of these patients are complicated because of unconfirmed suspicions of hereditary cancer. To define the prevalence of MMR deficient CRCs, LS and LLS in patients with EO NP NF CRC, we recruited 102 patients with a first diagnosis of NP NF CRC ≤ 50 years old during 2003–2009 who underwent genetic counseling at our institution in Argentina. Tumor immunohistochemical (IHC) MMR for protein expression and microsatellite instability (MSI) status were evaluated, and in those with loss of MLH1 expression by IHC, somatic BRAF V600E mutation and both somatic and germline MLH1 methylation levels were studied. Tumors characterized as MMRd without somatic BRAF mutation nor MLH1 methylation were sent for germline analysis. Twenty one (20.6%) tumors were MMRd. Fourteen of 16 putative LS cases underwent germline testing: 6 pathogenic mutations were identified and 8 cases had no identifiable pathogenic mutations. The prevalence of LS and LLS in this cohort was 5.8% (6/102) and 7.8% (8/102), respectively. As a conclusion we found that 20% of patients with EO NP NF CRC have MMRd tumors, and at least half of these are likely to have LLS.     

Sporadic endometrial adenocarcinoma with MMR deficiency due to biallelic <Emphasis Type="Italic">MSH2</Emphasis> somatic mutations

The invalidation of the Mismatch Repair (MMR) system is responsible for a so-called “deficient MMR” phenotype (dMMR) characterized by microsatellite instability and abnormal pattern of expression of MMR proteins in tumor tissue. This phenotype occurs in at least 20% of sporadic endometrial adenocarcinomas by epigenetic silencing of MLH1 gene. It is also observed in virtually all tumors occurring in patients with Lynch syndrome by monoallelic germline mutation in one of the MMR genes. The determination of this phenotype (dMMR vs. proficient MMR—pMMR) has therefore a pivotal place in the diagnosis algorithm for Lynch syndrome by monoallelic germline mutation in one of the MMR genes. The determination of this phenotype (dMMR vs. proficient MMR—pMMR) has therefore a pivotal place in the diagnosis algorithm for Lynch syndrome. We report the case of a woman with an early-onset endometrial adenocarcinoma who was suspected to be affected with Lynch syndrome based on tumor dMMR phenotype (MSI associated with loss of expression of MSH2 and MSH6 proteins). After complete germline and somatic evaluations, this phenotype was eventually explained by two MSH2 somatic mutations and the diagnosis of Lynch-like syndrome due to an unidentified MSH2 germline mutation was ruled out. Somatic mosaicism at low mutation rate was unlikely as no mutation was detected by DNA analysis from various tissue samples. Nevertheless, the three patient’s children were tested for the two mutations and these tests were negative. Biallelic somatic mutations of one MMR gene is a mechanism of invalidation of the MMR system in sporadic cases. Clinicians have to be aware of this mechanism because of the great clinical implication for the patients and their relatives.     

Immunohistochemical null-phenotype for mismatch repair proteins in colonic carcinoma associated with concurrent <Emphasis Type="Italic">MLH1</Emphasis> hypermethylation and <Emphasis Type="Italic">MSH2</Emphasis> somatic mutations

Microsatellite instability, a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual “null” IHC staining pattern with complete loss of all four MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient’s age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.     

Selection of patients with germline <Emphasis Type="Italic">MLH1</Emphasis> mutated Lynch syndrome by determination of <Emphasis Type="Italic">MLH1</Emphasis> methylation and <Emphasis Type="Italic">BRAF</Emphasis> mutation

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.     

Constitutional MLH1 methylation presenting with colonic polyposis syndrome and not Lynch syndrome

At least one-third of patients meeting clinical criteria for Lynch syndrome will have no germline mutation and constitutional epimutations leading to promoter methylation of MLH1 have been identified in a subset of these patients. We report the first case of constitutional MLH1 promoter methylation associated with a colonic polyposis syndrome in a 39 year-old man with a family history of colorectal cancer (CRC) and a personal history of 21 polyps identified over 8 years as well as the development of two synchronous CRCs over 16 months who was evaluated for a hereditary cancer syndrome. Immunohistochemistry (IHC) of multiple tumors showed absent MLH1 and PMS2 expression, though germline testing with Sanger sequencing and multiplex ligation-dependent probe amplification of these mismatch repair genes (MMR) genes was negative. A next generation sequencing panel of 29 genes also failed to identify a pathogenic mutation. Hypermethylation was identified in MLH1 intron 1 in tumor specimens along with buccal cells and peripheral white blood cells, confirming the diagnosis of constitutional MLH1 promoter methylation. This case highlights that constitutional MLH1 methylation should be considered in the differential diagnosis for a polyposis syndrome if IHC staining shows absent MMR gene expression.     

Epidemiological,clinical and molecular characterization of Lynch-like syndrome: A population-based study

Colorectal carcinomas that are mismatch repair (MMR)-deficient in the absence of MLH1 promoter methylation or germline mutations represent Lynch-like syndrome (LLS). Double somatic events inactivating MMR genes are involved in the etiology of LLS tumors. Our purpose was to define the clinical and broader molecular hallmarks of LLS tumors and the population incidence of LLS, which remain poorly characterized. We investigated 762 consecutive colorectal carcinomas operated in Central Finland in 2000–2010. LLS cases were identified by a stepwise protocol based on MMR protein expression, MLH1 methylation and MMR gene mutation status. LLS tumors were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations in 578 cancer-relevant genes. Among 107 MMR-deficient tumors, 81 (76%) were attributable to MLH1 promoter methylation and 9 (8%) to germline mutations (Lynch syndrome, LS), leaving 14 LLS cases (13%) (3 remained unclassified). LLS carcinomas were diagnosed at a mean age of 65 years (vs. 44 years in LS, p < 0.001), had a proximal to distal ratio of 1:1, and all were BRAF V600E-negative. Two somatic events in MMR genes were identifiable in 11 tumors (79%). As novel findings, the tumors contained an average of 31 nonsynonymous somatic mutations/Mb and 13/14 were CIMP-positive. In conclusion, we establish the epidemiological, clinical and molecular characteristics of LLS in a population-based study design. Significantly more frequent CIMP-positivity and lower rates of somatic mutations make a distinction to LS. The absence of BRAF V600E mutation separates LLS colorectal carcinomas from MLH1-methylated colorectal carcinomas with CIMP-positive phenotype.     

Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency

BackgroundPatients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs.Patients and methodsMSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation.ResultsAmong 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44).ConclusionsLS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.     

Muir–Torre syndrome or phenocopy? The value of the immunohistochemical expression of mismatch repair proteins in sebaceous tumors of immunocompromised patients

Primary and secondary immunodepressive conditions are associated with an increased incidence of sebaceous tumors. Microsatellite instability (MSI) and lack of expression of mismatch repair (MMR) proteins, typical markers of Muir–Torre/Lynch heredo-familial settings, can be recognized also in immunocompromised patients. We aimed to carry on a systematic examination of clinical, immunohistochemical, biomolecular features of sebaceous tumors arising in immunocompromised and immunocompetent patients between 1986 and 2012. Microsatellite screening, immunohistochemical analysis and genetic testing were performed for hMLH1, hMSH2 and hMSH6. Methylation status of MMR genes was checked in cases with immunohistochemistry (IHC) loss of MMR proteins expression and no germline mutations. Fifteen patients had a personal history of visceral carcinomas fulfilling diagnostic criteria for Muir–Torre syndrome. In this cohort, IHC analysis, MSI status and genetic testing were in agreement, showing eight MSH2 and two MLH1 germline mutations. Five patients were immunosuppressed and their sebaceous tumors showed a lack of MSH2/MSH6 expression, although just one case with positive family history for visceral cancer harbored a germline mutation. In immunosuppressed patients, loss of IHC for MMR proteins is not necessarily secondary to MMR germline mutations. IHC false positives are probably due to epigenetic alterations. MSI and lack of expression of MMR proteins can be recognized also in immunocompromised patients without MMR germline mutations.     

Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n = 614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.     

Prognosis in DNA Mismatch Repair Deficient Colorectal Cancer: are all MSI Tumours Equivalent?

Microsatellite instability (MSI) in colorectal tumours is the hallmark of defective DNA mismatch repair (MMR) and high level MSI can be detected in up to 15% of incident colorectal cancers. MSI in sporadic colorectal tumours is primarily due to epigenetic silencing of MLH1 while MSI is almost universal in tumours from HNPCC family members due to germline MMR gene mutation with loss or mutational inactivation of the second copy as a somatic event. There is evidence that tumour MSI is associated with a better outcome than the generality of large bowel malignancy. However, although MSI occurs in both sporadic colorectal cancer and in tumours arising in patients with germline MMR gene mutations, cancer survival should not be considered to be equivalent for these two groups with MSI tumours simply because both exhibit similarities in molecular phenotype. Here, we review the evidence on prognosis in patients with sporadic MSI tumours compared to those who have inherited a germline DNA MMR repair gene defect. In addition, we explore whether there are variables that afford opportunity to distinguish three groups on the basis of MSI status, namely: sporadic MSI tumours; MSI tumours in carriers of germline MMR gene defects; microsatellite stable (MSS) tumours. Differences in prognosis between these three groups is important because it underpins the rationale for surveillance and early identification of tumours in MMR gene carriers, as well as refining understanding of the influence of MSI on cancer progression. Furthermore, we discuss the effect of MSI on the effectiveness of chemotherapy regimens.     

Mismatch repair gene mutation analysis and colonoscopy surveillance in Chinese lynch syndrome families

Background

Lynch syndrome (or HNPCC) is a colorectal cancer syndrome caused by germline mutations in either one of the DNA mismatch repair (MMR) genes hMLH1, hMSH2, hMSH6 or hPMS2. Mutations in hMLH1 and hMSH2 are most prevalent. Here we aimed to determine the cancer risk of MMR gene mutation carriers and, in addition, the efficacy of colonoscopy surveillance in Chinese Lynch syndrome family members with and without MMR gene mutations.

Methods

A Lynch syndrome family registry encompassing 106 families in Northern China was recently established. Detailed pedigree data for each family were collected and hMLH1 and hMSH2 gene mutation analyses were performed. Germ-line mutations were identified in probands from 42 of these families, and additional genetic analyses were performed in each member of these 42 families to identify mutation and non-mutation carriers. Among the family members included, 180 received colonoscopy and the remaining cases were followed without colonoscopy.

Results

Overall 54.8 % of the Lynch syndrome family members carried MMR gene mutations, and these mutation carriers exhibited significantly higher colorectal cancer and other Lynch syndrome-associated cancer risks as compared to non-mutation carriers. The cumulative risk for all Lynch syndrome-related cancers at age 70 was 93.8 % for both hMLH1 and hMSH2 mutation carriers, and 81.7 % and 93.1 % for colorectal cancer at this age, respectively. Whereas 43 of 102 (42.2 %) mutation carriers exhibited significant colonoscopy findings, including 10 colorectal cancers, none of 78 non-mutation carriers exhibited significant findings, and no cancers were detected. In addition, in the mutation carriers, colonoscopy surveillance led to the detection of more early stage cancers than in the non-surveillance group (70.0 % versus 36.5 %, p?<?0.01).

Conclusion

In Lynch syndrome family members, we recommend pre-symptomatic MMR gene mutation analysis in order to identify high risk individuals for colonoscopy surveillance.     

Molecular biology from bench-to-bedside – Which colorectal cancer patients should be referred for genetic counselling and risk assessment

Lynch syndrome is associated with deficiency of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. However, most MLH1 deficient tumours are sporadic in origin, and they can be identified if harbouring a BRAF V600E mutation or hypermethylation of the MLH1 gene promoter.The aim of this study was to validate our previously suggested clinically applicable strategy based on molecular characteristics for identifying which patients to refer for genetic counselling.The strategy was validated in an unselected cohort of 287 colorectal cancer patients. All tumours were tested for MLH1, PMS2, MSH2 and MSH6 protein expression with immunohistochemistry. DNA from MLH1 negative tumours was sequenced for BRAF mutations. If BRAF was wild-type, MLH1 promoter was subsequently analyzed for promoter hypermethylation.Most tumours, 251 (88%), stained positive for all four proteins. Six (2%) had negative MSH2 and one (<1%) isolated loss of MSH6. MLH1 and PMS2 were negative in 29 cases (10%). DNA quality allowed BRAF analysis in 27 of these with 14 mutations and 13 wild-type. DNA quality allowed methylation analysis in 11 of the 13 BRAF wild-type, and all but one were methylated. Subsequently, Lynch syndrome could not be ruled out in 12 patients. A follow-up at 8–10 years revealed four definite cases of Lynch syndrome and three highly suspicious.An easy and clinically applicable step-wise approach with immunohistochemistry (100%), BRAF sequencing (10%) and methylation analysis (5%) identified several patients with hereditary cancer. It is feasible to perform a molecular screening to select patients for genetic counselling.     

Large genomic rearrangements and germline epimutations in Lynch syndrome

In one‐third of families fulfilling the Amsterdam criteria for hereditary nonpolyposis colorectal cancer/Lynch syndrome, and a majority of those not fulfilling these criteria point mutations in DNA mismatch repair (MMR) genes are not found. The role of large genomic rearrangements and germline epimutations in MLH1, MSH2 and MSH6 was evaluated in 2 such cohorts. All 45 index patients were mutation‐negative by genomic sequencing and testing for a prevalent population‐specific founder mutation, and selectively lacked MMR protein expression in tumor tissue. Eleven patients (“research cohort”) represented 11 mutation‐negative families among 81 verified or putative Lynch syndrome families from the nation‐wide Hereditary Colorectal Cancer Registry of Finland. Thirty‐four patients from 33 families (“clinic‐based cohort”) represented suspected Lynch syndrome patients tested for MMR gene mutations in a diagnostic laboratory during 2004–2007. Multiplex ligation‐dependent probe amplification (MLPA) and methylation‐specific (MS)‐MLPA were used to detect rearrangements and epimutations, respectively. Large genomic deletions occurred in 12/45 patients (27%), being present in 3/25 (12%), 9/16 (56%) and 0/4 (0%) among index patients lacking MLH1, MSH2 or MSH6 expression, respectively. Germline epimutations of MLH1, one of which coexisted with a genomic deletion, occurred in 2 patients (4%) and were accompanied by monoallelic expression in mRNA. Large genomic deletions (mainly MSH2) and germline epimutations (MLH1) together explain a significant fraction of point mutation‐negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of such families. © 2008 Wiley‐Liss, Inc.     

A state-wide population-based program for detection of lynch syndrome based upon immunohistochemical and molecular testing of colorectal tumours

We have previously established in a large retrospective study that testing for microsatellite instability (MSI) in colorectal cancer (CRC) from patients aged <60 years was an effective first screen to identify individuals with Lynch syndrome (LS). From these findings, MSI and/or immunohistochemical (IHC) screening was recommended for all newly diagnosed CRC patients aged <60 years in Western Australia, regardless of family history of cancer. In the current study we evaluated the utility of routine MSI/IHC screening by diagnostic pathology laboratories for the detection of previously undiagnosed individuals and families with LS. From January 2009 to December 2010, 270 tumours were tested for MSI and for expression of MLH1, PMS2, MSH2 and MSH6 using IHC. Cases showing MSI and/or loss of expression were also tested for the BRAF V600E hotspot mutation. Seventy cases were found to have MSI, of which 25 were excluded from further investigation as possible LS cases due to presence of the BRAF V600E mutation. The remaining 45 “red flag” cases were eligible for germline testing based on their MSI, IHC and BRAF status. From 31 cases tested to date, 15 germline mutations have been found. Thirteen were from individuals not previously recognized as LS and two were untested members from known LS families. Extrapolation of the mutation incidence (15/31, 48%) to all red flag cases (n = 45) suggests that approximately 22 mutation carriers exist in this cohort. This value approximates the number of CRC cases due to LS that could be expected to arise in the Western Australian population over a two-year period (n = 24), assuming that 1% of all CRCs are due to LS. Although further improvements in workflow can be made, our preliminary findings following the implementation of state-wide routine MSI and IHC testing in Western Australia indicate that the majority of LS cases are being identified.     

Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome

Lynch syndrome, the most common inherited colorectal cancer syndrome in adults, is an autosomal dominant condition caused by heterozygous germ-line mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Inheriting biallelic (homozygous) mutations in any of the MMR genes results in a different clinical syndrome termed biallelic mismatch repair deficiency (BMMR-D) that is characterised by gastrointestinal tumours, skin lesions, brain tumours and haematologic malignancies. This recently described and under-recognised syndrome can present with adenomatous polyps leading to early-onset small bowel and colorectal adenocarcinoma. An important clue in the family history that suggests underling BMMR-D is consanguinity. Interestingly, pedigrees of BMMR-D patients typically show a paucity of Lynch syndrome cancers and most parents are unaffected. Therefore, a family history of cancers is often non-contributory. Detection of BMMR-D can lead to more appropriate genetic counselling and the implementation of targeted surveillance protocols to achieve earlier tumour detection that will allow surgical resection. This review describes an approach for diagnosis and management of these patients and their families.     

Population‐based detection of Lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test

Approximately 1–2% of colorectal cancers (CRC) arise because of germline mutations in DNA mismatch repair genes, referred to as Lynch syndrome. These tumours show microsatellite instability (MSI) and loss of expression of mismatch repair proteins. Pre‐symptomatic identification of mutation carriers has been demonstrated to improve survival; however, there is concern that many are not being identified using current practices. We evaluated population‐based MSI screening of CRC in young patients as a means of ascertaining mutation carriers. CRC diagnosed in patients aged <60 years were identified from pathology records. No prior information was available on family history of cancer. PCR techniques were used to determine MSI in the BAT‐26 mononucleotide repeat and mutation in the BRAF oncogene. Loss of MLH1, MSH2, MSH6 and PMS2 protein expression was evaluated in MSI+ tumours by immunohistochemistry. MSI+ tumours were found in 105/1,344 (7.8%) patients, of which 7 were excluded as possible Lynch syndrome because of BRAF mutation. Of the 98 “red flag” cases that were followed up, 25 were already known as mutation carriers or members of mutation carrier families. Germline test results were obtained for 35 patients and revealed that 22 showed no apparent mutation, 11 showed likely pathogenic mutations and 2 had unclassified variants. The proportion of MSI+ cases in different age groups that were estimated to be mutation carriers was 89% (<30 years), 83% (30–39), 68% (40–49) and 17% (50–59). We recommend MSI as the initial test for population‐based screening of Lynch syndrome in younger CRC patients, regardless of family history. © 2008 Wiley‐Liss, Inc.