Molecular assessment of colorectal cancer through Lynch syndrome screening

Since 2017, the National Institute for Health and Care Excellence (NICE) has recommended molecular testing of all patients with newly diagnosed colorectal cancer (CRC) to identify those with suspected Lynch syndrome who should be referred to clinical genetics for germline testing. The pathway involves firstly determining the mismatch repair (MMR) expression status by immunohistochemistry (IHC) or performing microsatellite instability testing. This may be followed by BRAF V600E mutation testing and then MLH1 promoter hypermethylation analysis depending on the result. This approach identifies patients that are most likely to have underlying germline mutations in the MMR genes as opposed to somatic causes of deficient MMR. Here we demonstrate a case with loss of MLH1 protein expression and discuss the subsequent testing strategy according to NICE guidance.     

BRAF mutation in sporadic colorectal cancer and Lynch syndrome

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n?=?137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1 %) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100 % sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8 %; 14/18) and less frequently in the microsatellite-stable group (7.6 %; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.     

Molecular pathology of lung cancer: key to personalized medicine

The majority of lung adenocarcinoma patients with epidermal growth factor receptor- (EGFR) mutated or EML4-ALK rearrangement-positive tumors are sensitive to tyrosine kinase inhibitors. Both primary and acquired resistance in a significant number of those patients to these therapies remains a major clinical problem. The specific molecular mechanisms associated with tyrosine kinase inhibitor resistance are not fully understood. Clinicopathological observations suggest that molecular alterations involving so-called 'driver mutations' could be used as markers that aid in the selection of patients most likely to benefit from targeted therapies. In this review, we summarize recent developments involving the specific molecular mechanisms and markers that have been associated with primary and acquired resistance to EGFR-targeted therapy in lung adenocarcinomas. Understanding these mechanisms may provide new treatment avenues and improve current treatment algorithms.     

DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome

Poulogiannis G, Frayling I M & Arends M J
(2010) Histopathology 56, 167–179 DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC), and is characterized by the loss of function of the MMR pathway. Failure to repair replication‐associated errors due to a defective MMR system allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, giving rise to the phenomenon of microsatellite instability (MSI). A high frequency of instability at microsatellites (MSI‐H) is the hallmark of the most common form of hereditary susceptibility to CRC, known as Lynch syndrome (LS) (previously known as hereditary non‐polyposis colorectal cancer syndrome), but is also observed in ～15–20% of sporadic colonic cancers (and rarely in rectal cancers). Tumour analysis by both MMR protein immunohistochemistry and DNA testing for MSI is necessary to provide a comprehensive picture of molecular abnormality, for use in conjunction with family history data and other clinicopathological features, in order to distinguish LS from sporadic MMR‐deficient CRC. Identification of the gene targets that become mutated in MMR‐deficient tumours may explain, at least in part, some of the clinical, pathological and biological features of MSI‐H CRCs and holds promise for developing novel therapeutics.     

The cost-effectiveness of genetic testing strategies for Lynch syndrome among newly diagnosed patients with colorectal cancer

PurposeTo estimate the cost-effectiveness of genetic testing strategies to identify Lynch syndrome among newly diagnosed patients with colorectal cancer and to offer targeted testing to relatives of patients with Lynch syndrome.MethodsWe calculated incremental costs per life-year saved for universal testing relative to no testing and age-targeted testing for strategies that use preliminary genetic tests (immunohistochemistry or microsatellite instability) of tumors followed by sequencing of mismatch repair genes. We also calculated incremental cost-effectiveness ratios for pairs of testing strategies.ResultsStrategies to test for Lynch syndrome in newly diagnosed colorectal tumors using preliminary tests before gene sequencing have incremental cost-effectiveness ratios of ≤$45,000 per life-year saved compared with no testing and ≤$75,000 per life-year saved compared with testing restricted to patients younger than 50 years. The lowest cost testing strategies, using immunohistochemistry as a preliminary test, cost ≤$25,000 per life-year saved relative to no testing and ≤$40,000 per life-year saved relative to testing only patients younger than 50 years. Other testing strategies have incremental cost-effectiveness ratios ≥$700,000 per life-year saved relative to the lowest cost strategies. Increasing the number of relatives tested would improve cost-effectiveness.ConclusionLaboratory-based strategies using preliminary tests seem cost-effective from the US health care system perspective. Universal testing detects nearly twice as many cases of Lynch syndrome as targeting younger patients and has an incremental cost-effectiveness ratio comparable with other preventive services. This finding provides support for a recent US recommendation to offer testing for Lynch syndrome to all newly diagnosed patients with colorectal cancer.     

Worldwide prevalence of Lynch syndrome in patients with colorectal cancer: Systematic review and meta-analysis

PurposeLynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, with an estimated prevalence of 2% to 3% of CRC. A prevalence study is needed to provide accurate estimates of the true prevalence of LS.MethodsMEDLINE (Ovid), Embase, and Web of Science were searched. Prevalence was calculated by random effects meta-analysis models. I² score was used to assess heterogeneity across studies. Meta-regression was performed for between-study variance.ResultsA total of 51 studies were included in this review. The overall pooled yield of LS screening was 2.2% based on all methods of detection. Studies performing germline tests on all participants with CRC reported higher prevalence (5.1%) as opposed to studies only performing germline tests on participants with tumors with mismatch repair deficiency (1.6%) or microsatellite instability (1.1%). Selected cohorts of CRC had a higher prevalence of germline LS diagnoses.ConclusionLS prevalence across multiple ethnic, geographic, and clinical populations is remarkably similar. Universal germline testing of patients presenting with cancer identifies that most CRCs are attributed to LS. Young patients presenting with CRC and those who fulfill criteria for a familial risk provide the highest returns for LS identification. Our study supports the universal germline CRC screening for LS.     

Molecular pathology of Lynch syndrome

Lynch syndrome (LS) is characterised by predisposition to colorectal, endometrial, and other cancers and is caused by inherited pathogenic variants affecting the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. It is probably the most common predisposition to cancer, having an estimated prevalence of between 1/100 and 1/180. Resources such as the International Society for Gastrointestinal Hereditary Cancer's MMR gene variant database, the Prospective Lynch Syndrome Database (PLSD), and the Colon Cancer Family Register (CCFR), as well as pathological and immunological studies, are enabling advances in the understanding of LS. These include defined criteria by which to interpret gene variants, the function of MMR in the normal control of apoptosis, definition of the risks of the various cancers, and the mechanisms and pathways by which the colorectal and endometrial tumours develop, including the critical role of the immune system. Colorectal cancers in LS can develop along three pathways, including flat intramucosal lesions, which depend on the underlying affected MMR gene. This gives insights into the limitations of colonoscopic surveillance and highlights the need for other forms of anti-cancer prophylaxis in LS. Finally, it shows that the processes of autoimmunisation and immunoediting fundamentally constrain the development of tumours in LS and explain the efficacy of immune checkpoint blockade therapy in MMR-deficient tumours. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molecular typing of colorectal cancer: applications in diagnosis and treatment

Molecular typing of colorectal cancer (CRC) is at an early stage of development with analysis of KRAS mutation status and DNA mismatch repair (MMR) deficiency representing the two major approaches currently in use for diagnosis and treatment-related purposes. RAS proteins act as molecular switches that regulate cellular processes including cell growth and survival. Activating KRAS mutations are found in 40–50% of colorectal adenomas and cancers. Detection of KRAS mutations guide the decision to use anti-EGFR antibody therapy, which is not effective in KRAS mutant cancers. MMR deficiency results in failure to repair replication-associated DNA errors, allowing persistence of mismatch mutations all over the genome, especially in regions of repetitive DNA known as microsatellites, giving rise to microsatellite instability (MSI). A high frequency of microsatellite instability (MSI-H) often with abnormal expression of MMR proteins is key to the diagnosis of Lynch Syndrome, but is also observed in ～15% of sporadic CRC.     

Tumour-specific methylation of PTPRG intron 1 locus in sporadic and Lynch syndrome colorectal cancer

DNA methylation is a hallmark in a subset of right-sided colorectal cancers. Methylation-based screening may improve prevention and survival rate for this type of cancer, which is often clinically asymptomatic in the early stages. We aimed to discover prognostic or diagnostic biomarkers for colon cancer by comparing DNA methylation profiles of right-sided colon tumours and paired normal colon mucosa using an 8.5 k CpG island microarray. We identified a diagnostic CpG-rich region, located in the first intron of the protein-tyrosine phosphatase gamma gene (PTPRG) gene, with altered methylation already in the adenoma stage, that is, before the carcinoma transition. Validation of this region in an additional cohort of 103 sporadic colorectal tumours and 58 paired normal mucosa tissue samples showed 94% sensitivity and 96% specificity. Interestingly, comparable results were obtained when screening a cohort of Lynch syndrome-associated cancers. Functional studies showed that PTPRG intron 1 methylation did not directly affect PTPRG expression, however, the methylated region overlapped with a binding site of the insulator protein CTCF. Chromatin immunoprecipitation (ChIP) showed that methylation of the locus was associated with absence of CTCF binding. Methylation-associated changes in CTCF binding to PTPRG intron 1 could have implications on tumour gene expression by enhancer blocking, chromosome loop formation or abrogation of its insulator function. The high sensitivity and specificity for the PTPRG intron 1 methylation in both sporadic and hereditary colon cancers support biomarker potential for early detection of colon cancer.     

An alternative approach to establishing unbiased colorectal cancer risk estimation in Lynch syndrome

PurposeBiallelic pathogenic variants in the mismatch repair (MMR) genes cause a recessive childhood cancer predisposition syndrome known as constitutional mismatch repair deficiency (CMMRD). Family members with a heterozygous MMR variant have Lynch syndrome. We aimed at estimating cancer risk in these heterozygous carriers as a novel approach to avoid complicated statistical methods to correct for ascertainment bias.MethodsCumulative colorectal cancer incidence was estimated in a cohort of PMS2- and MSH6-associated families, ascertained by the CMMRD phenotype of the index, by using mutation probabilities based on kinship coefficients as analytical weights in a proportional hazard regression on the cause-specific hazards. Confidence intervals (CIs) were obtained by bootstrapping at the family level.ResultsThe estimated cumulative colorectal cancer risk at age 70 years for heterozygous PMS2 variant carriers was 8.7% (95% CI 4.3–12.7%) for both sexes combined, and 9.9% (95% CI 4.9–15.3%) for men and 5.9% (95% CI 1.6–11.1%) for women separately. For heterozygous MSH6 variant carriers these estimates are 11.8% (95% CI 4.5–22.7%) for both sexes combined, 10.0% (95% CI 1.83–24.5%) for men and 11.7% (95% CI 2.10–26.5%) for women.ConclusionOur findings are consistent with previous reports that used more complex statistical methods to correct for ascertainment bias. These results underline the need for MMR gene–specific surveillance protocols for Lynch syndrome.     

Underutilization of Lynch syndrome screening in a multisite study of patients with colorectal cancer

PurposeThe aim of this study was to examine Lynch syndrome screening of patients with metastatic colorectal cancer in integrated health-care-delivery organizations.MethodsWe determined the availability of Lynch syndrome screening criteria and actual Lynch syndrome screening in the medical records of 1,188 patients diagnosed with metastatic colorectal cancer between 2004 and 2009 at seven institutions in the Cancer Research Network.ResultsWe found infrequent use of Lynch syndrome screening (41/1,188). Family history was available for 937 of the 1,188 patients (79%). There was sufficient information to assess Lynch syndrome risk using family history–based criteria in 719 of the 937 patients (77%) with family history documentation. In 391 individuals with a family history of a Lynch syndrome–associated cancer, 107 (27%) could not be evaluated due to missing information such as age of cancer onset. Eleven percent of patients who met the Bethesda criteria and 25% of individuals who met the Amsterdam II criteria were screened for Lynch syndrome. Recommended guidelines were adhered to during screening, but no testing method was preferred.ConclusionThe information required for Lynch syndrome screening decisions is routinely collected but seldom used. There is a critical gap between collection of family history and its use to guide Lynch syndrome screening, which may support a case for implementation of universal screening guidelines.Genet Med15 12, 933–940.     

Three new mutations in hereditary nonpolyposis colorectal cancer (Lynch syndrome II) in Uruguay

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary form of colorectal cancer (CRC). Our purpose is to describe three extended HNPCC families, each of which manifests novel germline mutations in Uruguay, a small country that is a study model for cancer investigation given its high cancer incidence and mortality rate. This is a study of three extended HNPCC families in which extensive genealogic information, medical history, and pathology findings are critically reviewed. DNA testing was performed for evidence of HNPCC mutations. The findings reveal three novel germline mutations, namely MLH1, with a deletion resulting in a frameshift and a premature stop codon (codon 228) in one of the families; in the second family, MSH2 exon 1, codon 61 at nucleotide 181, which results in immediate stop of translation; and in the third family, a mutation in MSH2 at exon 3: the amino acid at nucleotide 530, codon 117, causing a frameshift and a premature stop codon eight base pairs later. We conclude that it is important to study HNPCC mismatch repair genes because of emerging evidence for genotypic and phenotypic heterogeneity, which will harbor the potential to eventually translate this knowledge into specific screening and management protocols. Future projections for such mutations could even contribute to the emergence of molecular-based designer drugs developed through advances in genomics, proteomics, high-throughput screening, and bioinformatics, which would be effective therapeutically for these high-cancer risk patients.     

ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome,familial adenomatous polyposis,and MYH-associated polyposis)

Lynch syndrome, familial adenomatous polyposis, and Mut Y homolog (MYH)-associated polyposis are three major known types of inherited colorectal cancer, which accounts for up to 5% of all colon cancer cases. Lynch syndrome is most frequently caused by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2 and is inherited in an autosomal dominant manner. Familial adenomatous polyposis is manifested as colonic polyposis caused by mutations in the APC gene and is also inherited in an autosomal dominant manner. Finally, MYH-associated polyposis is caused by mutations in the MUTYH gene and is inherited in an autosomal recessive manner but may or may not be associated with polyps. There are variants of both familial adenomatous polyposis (Gardner syndrome—with extracolonic features—and Turcot syndrome, which features medulloblastoma) and Lynch syndrome (Muir–Torre syndrome features sebaceous skin carcinomas, and Turcot syndrome features glioblastomas). Although a clinical diagnosis of familial adenomatous polyposis can be made using colonoscopy, genetic testing is needed to inform at-risk relatives. Because of the overlapping phenotypes between attenuated familial adenomatous polyposis, MYH-associated polyposis, and Lynch syndrome, genetic testing is needed to distinguish among these conditions. This distinction is important, especially for women with Lynch syndrome, who are at increased risk for gynecological cancers. Clinical testing for these genes has progressed rapidly in the past few years with advances in technologies and the lower cost of reagents, especially for sequencing. To assist clinical laboratories in developing and validating testing for this group of inherited colorectal cancers, the American College of Medical Genetics and Genomics has developed the following technical standards and guidelines. An algorithm for testing is also proposed.Genet Med16 1, 101–116.