Annette H. J. T. Bröcker-Vriends

Clinical findings with implications for genetic testing in families with clustering of colorectal cancer.

BACKGROUND Germ-line mutations in DNA mismatch-repair genes (MSH2, MLH1, PMS1, PMS2, and MSH6) cause susceptibility to hereditary nonpolyposis colorectal cancer. We assessed the prevalence of MSH2 and MLH1 mutations in families suspected of having hereditary nonpolyposis colorectal cancer and evaluated whether clinical findings can predict the outcome of genetic testing. METHODS We used denaturing gradient gel electrophoresis to identify MSH2 and MLH1 mutations in 184 kindreds with familial clustering of colorectal cancer or other cancers associated with hereditary nonpolyposis colorectal cancer. Information on the site of cancer, the age at diagnosis, and the number of affected family members was obtained from all families. RESULTS Mutations of MSH2 or MLH1 were found in 47 of the 184 kindreds (26 percent). Clinical factors associated with these mutations were early age at diagnosis of colorectal cancer, the occurrence in the kindred of endometrial cancer or tumors of the small intestine, a higher number of family members with colorectal or endometrial cancer, the presence of multiple colorectal cancers or both colorectal and endometrial cancers in a single family member, and fulfillment of the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer (at least three family members in two or more successive generations must have colorectal cancer, one of whom is a first-degree relative of the other two; cancer must be diagnosed before the age of 50 in at least one family member; and familial adenomatous polyposis must be ruled out). Multivariate analysis showed that a younger age at diagnosis of colorectal cancer, fulfillment of the Amsterdam criteria, and the presence of endometrial cancer in the kindred were independent predictors of germ-line mutations of MSH2 or MLH1. These results were used to devise a logistic model for estimating the likelihood of a mutation in MSH2 and MLH1. CONCLUSIONS Assessment of clinical findings can improve the rate of detection of mutations of DNA mismatch-repair genes in families suspected of having hereditary nonpolyposis colorectal cancer.

Multiplicity in polyp count and extracolonic manifestations in 40 Dutch patients with MYH associated polyposis coli (MAP)

Objective: To investigate the contribution of MYH associated polyposis coli (MAP) among polyposis families in the Netherlands, and the prevalence of colonic and extracolonic manifestations in MAP patients. Methods: 170 patients with polyposis coli, who previously tested negative for APC mutations, were screened by denaturing gradient gel electrophoresis and direct sequencing to identify MYH germline mutations. Results: Homozygous and compound heterozygous MYH mutations were identified in 40 patients (24%). No difference was found in the percentage of biallelic mutation carriers between patients with 10–99 polyps or 100–1000 polyps (29% in both groups). Colorectal cancer was found in 26 of the 40 patients with MAP (65%) within the age range 21 to 67 years (median 45). Complete endoscopic reports were available for 16 MAP patients and revealed five cases with gastro-duodenal polyps (31%), one of whom also presented with a duodenal carcinoma. Breast cancer occurred in 18% of female MAP patients, significantly more than expected from national statistics (standardised morbidity ratio = 3.75). Conclusions: Polyp numbers in MAP patients were equally associated with the attenuated and classical polyposis coli phenotypes. Two thirds of the MAP patients had colorectal cancer, 95% of whom were older than 35 years, and one third of a subset of patients had upper gastrointestinal lesions. Endoscopic screening of the whole intestine should be carried out every two years for all MAP patients, starting from age 25–30 years. The frequent occurrence of additional extraintestinal manifestations, such as breast cancer among female MAP patients, should be thoroughly investigated.

Microsatellite Instability, Immunohistochemistry, and Additional PMS2 Staining in Suspected Hereditary Nonpolyposis Colorectal Cancer

Purpose: Immunohistochemistry (IHC) and microsatellite instability (MSI) analysis can be used to identify patients with a possible DNA mismatch repair defect [hereditary nonpolyposis colorectal carcinoma (HNPCC)]. The Bethesda criteria have been proposed to select families for determination of MSI. The aims of this study were to assess the yield of MSI analysis in families suspected for HNPCC, to compare the results of immunohistochemical staining and MSI analysis, and to assess the additional value of PMS2 staining. Experimental Design: Clinical data and tumors were collected from 725 individuals from 631 families with suspected HNPCC. MSI analysis was performed using eight markers including the 5 National Cancer Institute markers. Four immunohistochemical staining antibodies were used (MLH1, MSH2, MSH6 and PMS2). Results: A MSI-H (tumors with instability for >30% of the markers) phenotype in colorectal cancers (CRCs) was observed in 21–49% of families that met the various Bethesda criteria. In families with three cases of CRC diagnosed at age > 50 years, families with a solitary case of CRC diagnosed between ages 45 and 50 years, and families with one CRC case and a first-degree relative with a HNPCC-related cancer, one diagnosed between ages 45 and 50 years (all Bethesda-negative families), the yield of MSI-H was 10–26%. Immunohistochemical staining confirmed the MSI results in 93% of the cases. With IHC, adding PMS2 staining led to the identification of an additional 23% of subjects with an hMLH1 germ-line mutation (35 carriers were tested). Conclusions: The Bethesda guidelines for MSI analysis should include families with three or more cases of CRC diagnosed at age > 50 years. The age at diagnosis of CRC in the original guidelines should be raised to 50 years. Routine IHC diagnostics for HNPCC should include PMS2 staining.

Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients

Instability of microsatellite repeat sequences has been observed in colorectal carcinomas and in extracolonic malignancies, predominantly endometrial tumours, occurring in the context of hereditary non‐polyposis colorectal cancer (HNPCC). Microsatellite instability (MSI) as a feature of human DNA mismatch repair (MMR)‐driven tumourigenesis of the uterine mucosa has been studied primarily in sporadic tumours showing predominantly somatic hypermethylation of MLH1. The present study shows that all endometrial carcinomas (n=12) from carriers of MLH1 and MSH2 germline mutations demonstrate an MSI‐high phenotype involving all types of repeat markers, while in endometrial carcinomas from MSH6 mutation carriers, only 36% (4 out of 11) demonstrate an MSI‐high phenotype. Interestingly, an MSI‐high phenotype was found in endometrial hyperplasias from MSH2 mutation carriers, in contrast to hyperplasias from MLH1 mutation carriers, which exhibited an MSI‐stable phenotype. Instability of only mononucleotide repeat markers was found in both endometrial carcinomas and hyperplasias from MSH6 mutation carriers. In 29 out of 31 (94%) endometrial tumour foci, combined MSI and immunohistochemical analysis of MLH1, MSH2, and MSH6 could predict the identified germline mutation. The observation of MSI in endometrial hyperplasia and of altered protein staining for the MMR genes supports the idea that inactivation of MMR genes is an early event in endometrial tumourigenesis. A correlation was found between the variation in the extent and level of MSI and the age of onset of carcinoma, suggesting differences in the rate of tumour progression. A high frequency of MSI in hyperplasias, found only in MSH2 mutation carriers, might indicate a more rapid tumour progression, correlating with an earlier age of onset of carcinoma. The present study indicates that assessment of altered protein staining combined with MSI analysis of endometrial tumours might direct the mutational analysis of MMR genes. Copyright

Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.

Immunohistochemistry (IHC) of mismatch repair (MMR) proteins in colorectal tumors together with microsatellite analysis (MSI) can be helpful in identifying families eligible for mutation analysis. The aims were to determine sensitivity of IHC for MLH1, MSH2, and MSH6 and MSI analysis in tumors from known MMR gene mutation carriers; and to evaluate the use of tissue microarrays for IHC (IHC-TMA) of colon tumors in its ability to identify potential carriers of MMR gene mutations, and compare it with IHC on whole slides. IHC on whole slides was performed in colorectal tumors from 45 carriers of a germline mutation in one of the MMR genes. The TMA cohort consisted of 129 colon tumors from (suspected) hereditary nonpolyposis colorectal cancer (HNPCC) patients. Whole slide IHC analysis had a sensitivity of 89% in detecting MMR deficiency in carriers of a pathogenic MMR mutation. Sensitivity by MSI analysis was 93%. IHC can also be used to predict which gene is expected to harbor the mutation: for MLH1, MSH2, and MSH6, IHC on whole slides would have correctly predicted the mutation in 48%, 92%, and 75% of the cases, respectively. We propose a scheme for the diagnostic approach of families with (suspected) HNPCC. Comparison of the IHC results based on whole slides versus TMA, showed a concordance of 85%, 95%, and 75% for MLH, MSH2, and MSH6, respectively. This study therefore shows that IHC-TMA can be reliably used to simultaneously screen a large number of tumors from (suspected) HNPCC patients, at first in a research setting.

Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 andMLH1. Recently, mutations in another MMR gene, MSH6 (also known asGTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related toMSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 andMLH1.  Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from aMSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 andMLH1 carriers (32% by the age of 80v >80%).  Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers).  Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 yearsv 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 yearsv 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect.  The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.

Nearly all hereditary paragangliomas in The Netherlands are caused by two founder mutations in the SDHD gene

Hereditary paragangliomas or glomus tumors are usually benign slow‐growing tumors in the head and neck region. The inheritance pattern of hereditary paraganglioma is autosomal dominant with imprinting. Recently, we have identified the SDHD gene encoding subunit D of the mitochondrial respiratory chain complex II as one of the genes involved in hereditary paragangliomas. Here, we demonstrate that two founder mutations, Asp92Tyr and Leu139Pro, are responsible for paragangliomas in 24 and 6 of the 32 independently ascertained Dutch paraganglioma families, respectively. These two mutations were also detected among 20 of 55 isolated patients. Ten of the isolated patients had multiple paragangliomas, and in eight of these SDHD germline mutations were found, indicating that multicentricity is a strong predictive factor for the hereditary nature of the disorder in isolated patients. In addition, we demonstrate that the maternally derived wild‐type SDHD allele is lost in tumors from mutation‐carrying patients, indicating that SDHD functions as a tumor suppressor gene.

Diagnostic Approach and Management of Lynch Syndrome (Hereditary Nonpolyposis Colorectal Carcinoma): A Guide for Clinicians

The patient with a family history for colorectal carcinoma constitutes a complicated diagnostic challenge involving many clinicians. The diagnostic workup of familial colorectal cancer is an elaborate and time consuming process in which the family and several medical specialists closely collaborate. However, establishing a diagnosis can be very rewarding. If a mutation is detected in the family, a satisfactory explanation can be provided for an accumulation of tumors at young age, and often of untimely death. Appropriate presymptomatic testing can be offered to reduce mortality among at‐risk family members, and relatives not at risk can avoid uncertainty and needlessly intensive surveillance.

Family system characteristics and psychological adjustment to cancer susceptibility genetic testing: a prospective study.

This study examined prospectively the contribution of family functioning, differentiation to parents, family communication and support from relatives to psychological distress in individuals undergoing genetic susceptibility testing for a known familial pathogenic BRCA1/2 or Hereditary nonpolyposis colorectal cancer‐related mutation. Family functioning, differentiation to parents, hereditary cancer‐related family communication and perceived support from relatives were assessed in 271 participants for genetic testing before test result disclosure. Hereditary cancer distress (assessed by the Impact of Event Scale) and cancer worry (assessed by the Cancer Worry Scale) were assessed before, 1 week after, and 6 months after test result disclosure. Participants reporting more cancer‐related distress over the study period more frequently perceived the communication about hereditary cancer with relatives as inhibited, the nuclear family functioning as disengaged‐rigid or enmeshed‐chaotic, the support from partner as less than adequate and the relationship to mother as less differentiated. Especially, open communication regarding hereditary cancer and partner support may be important buffers against hereditary cancer distress. Identifying individuals with insufficient sources of support and addressing the family communication concerning hereditary cancer in genetic counseling may help the counselee to adjust better to genetic testing.

Bannayan-Riley-Ruvalcaba syndrome: further delineation of the phenotype and management of PTEN mutation-positive cases.

Bannayan–Riley–Ruvalcaba syndrome (BRRS) is characterised by macrocephaly, intestinal hamartomatous polyps, lipomas, pigmented maculae of the glans penis, developmental delay and mental retardation. The syndrome follows an autosomal dominant pattern of inheritance. In 1997 reports on two BRRS patients with a deletion at 10q23.2–q24.1 were published. In the same year, the first two families with BRRS and a mutation of the PTEN gene were reported. Mutations in the PTEN gene have also been demonstrated in patients with Cowden syndrome (CS), which shows partial clinical overlap with BRRS, and in families with cases both of BRRS and CS. PTEN mutation positive BRRS and CS are likely to be different phenotypic presentations of the same syndrome. If BRRS and CS are one single condition, the question arises whether patients with BRRS should be screened for malignant tumours, since patients with Cowden syndrome have an increased risk of breast, endometrial, thyroid and renal cancer. We present two isolated cases and one family and confirm that BRRS and CS are allelic. Furthermore, we review the PTEN mutation positive BRRS cases, to further delineate the phenotype and to compare the cases with a genomic deletion with the cases with a point mutation. We recommend offering BRRS cases with a mutation in PTEN the same surveillance protocol for (malignant) tumours as is currently recommended for CS. In addition, we propose a yearly haemoglobin test from early infancy for the early detection of intestinal hamartomas, which are likely to give severe complications, especially in BRRS cases.