Publications by authors named "Marjo van Puijenbroek"

26 Publications

  • Page 1 of 1

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

Eur J Hum Genet 2011 Mar 8;19(3):307-12. Epub 2010 Dec 8.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ejhg.2010.187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061992PMC
March 2011

Early onset MSI-H colon cancer with MLH1 promoter methylation, is there a genetic predisposition?

BMC Cancer 2010 May 5;10:180. Epub 2010 May 5.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Background: To investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer.

Methods: We studied a set of 46 MSI-H colon tumors cases with MLH1 promoter methylation which was enriched for patients with an age of onset below 50 years (n=13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: BRAF, KRAS, GADD45A and the MLH1 -93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for GADD45A mutations and germline MLH1 methylation. SNP array analysis was performed on a subset of tumors.

Results: We identified two cases (33 and 60 years) with MLH1 germline promoter methylation. BRAF mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to BRAF mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. GADD45A sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity.

Conclusion: Somatic or germline GADD45A mutations did not explain sporadic MSI-H colon cancer. Although germline MLH1 methylation was found in two individuals, locus-specific somatic MLH1 hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of BRAF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-10-180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2880297PMC
May 2010

Comprehensive genetic analysis of seven large families with mismatch repair proficient colorectal cancer.

Genes Chromosomes Cancer 2010 Jun;49(6):539-48

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Approximately 40% of colorectal cancer (CRC) families with a diagnosis of hereditary nonpolyposis CRC on the basis of clinical criteria are not a consequence of mismatch repair (MMR) deficiency. Such families provide supporting evidence for the existence of a hitherto unidentified highly penetrant gene mutation. To gain further understanding of MMR-competent familial colorectal cancer (FCC), we studied seven large families with an unexplained predisposition for CRC to identify genetic regions that could harbor CRC risk factors. First, we conducted a genome-wide linkage scan using 10K single-nucleotide polymorphism (SNP) arrays to search for disease loci. Second, we studied the genomic profiles of the tumors of affected family members to identify commonly altered genomic regions likely to harbor tumor suppressor genes. Finally, we studied the possible role of recently identified low-risk variants in the familial aggregation of CRC in these families. Linkage analysis did not reveal clear regions of linkage to CRC. However, our results provide support linkage to 3q, a region that has previously been linked to CRC susceptibility. Tumor profiling did not reveal any genomic regions commonly targeted in the tumors studied here. Overall, the genomic profiles of the tumors show some resemblance to sporadic CRC, but additional aberrations were also present. Furthermore, the FCC families did not appear to have an enrichment of low-risk CRC susceptibility loci. These data suggest that factors other than a highly penetrant risk factor, such as low or moderate-penetrance risk factors, may explain the increased cancer risk in a subset of familial CRCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/gcc.20763DOI Listing
June 2010

Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.

J Mol Diagn 2010 Jan 3;12(1):27-34. Epub 2009 Dec 3.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2353/jmoldx.2010.090028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797715PMC
January 2010

Colorectal carcinomas in MUTYH-associated polyposis display histopathological similarities to microsatellite unstable carcinomas.

BMC Cancer 2009 Jun 15;9:184. Epub 2009 Jun 15.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Background: MUTYH-associated polyposis (MAP) is a recessively inherited disorder which predisposes biallelic carriers for a high risk of polyposis and colorectal carcinoma (CRC). Since about one third of the biallelic MAP patients in population based CRC series has no adenomas, this study aimed to identify specific clinicopathological characteristics of MAP CRCs and compare these with reported data on sporadic and Lynch CRCs.

Methods: From 44 MAP patients who developed > or = 1 CRCs, 42 of 58 tumours were analyzed histologically and 35 immunohistochemically for p53 and beta-catenin. Cell densities of CD3, CD8, CD57, and granzyme B positive lymphocytes were determined. KRAS2, the mutation cluster region (MCR) of APC, p53, and SMAD4 were analyzed for somatic mutations.

Results: MAP CRCs frequently localized to the proximal colon (69%, 40/58), were mucinous in 21% (9/42), and had a conspicuous Crohn's like infiltrate reaction in 33% (13/40); all of these parameters occurred at a higher rate than reported for sporadic CRCs. Tumour infiltrating lymphocytes (TILs) were also highly prevalent in MAP CRCs. Somatic APC MCR mutations occurred in 14% (5/36) while 64% (23/36) had KRAS2 mutations (22/23 c.34G>T). G>T transversions were found in p53 and SMAD4, although the relative frequency compared to other mutations was low.

Conclusion: MAP CRCs show some similarities to micro-satellite unstable cancers, with a preferential proximal location, a high rate of mucinous histotype and increased presence of TILs. These features should direct the practicing pathologist towards a MAP aetiology of CRC as an alternative for a mismatch repair deficient cause. High frequent G>T transversions in APC and KRAS2 (mutated in early tumour development) but not in P53 and SMAD4 (implicated in tumour progression) might indicate a predominant MUTYH effect in early carcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-9-184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706846PMC
June 2009

MUTYH-associated polyposis carcinomas frequently lose HLA class I expression - a common event amongst DNA-repair-deficient colorectal cancers.

J Pathol 2009 Sep;219(1):69-76

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Human leukocyte antigen (HLA) class I expression defects frequently occur in colorectal cancers bearing mismatch repair (MMR) deficiencies and are interpreted as immune evasion mechanisms to avoid cancer cell recognition and elimination by the immune system. MMR-deficient tumours are thought to be more prone to lose HLA class I expression, due to their frequent generation of aberrant peptides which can stimulate a cytotoxic T-cell-mediated response. MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome caused by defects in the MUTYH DNA repair enzyme. Impairment of MUTYH activity could lead to a surplus of mutated peptides which would be presented to cytotoxic T-cells through the HLA class I molecules. We have studied the frequency of HLA class I expression defects in MAP carcinomas and have compared it to those observed in MMR-deficient and -proficient colorectal tumours. Immunohistochemical detection of the expression of HLA class I, beta2-microglobulin (beta2m), and antigen-processing machinery molecules was performed in 37 primary MAP carcinomas and nine metastases resected from 29 MAP patients. Furthermore, we sequenced the beta2m, TAP1, and TAP2 genes. Defects in HLA class I expression were detected in 65% of primary MAP carcinomas, affecting 72% of patients. HLA class I expression abnormalities were often concomitant with beta2m expression loss and mutations in the beta2m gene. Loss of HLA class I expression is thus a frequent event in MAP carcinomas, similarly to MMR-deficient colorectal tumours. The extensive mutagenic background of these tumours most likely triggers a strong selective pressure, exerted by the immune system on the tumour, which favours the outgrowth of tumour cell clones with an immune evasive phenotype. Our data provide additional evidence for a link between DNA repair deficiencies and altered HLA class I phenotypes in colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.2569DOI Listing
September 2009

Genome-wide allelic state analysis on flow-sorted tumor fractions provides an accurate measure of chromosomal aberrations.

Cancer Res 2008 Dec;68(24):10333-40

Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands.

Chromosomal aberrations are a common characteristic of cancer and are associated with copy number abnormalities and loss of heterozygosity (LOH). Tumor heterogeneity, low tumor cell percentage, and lack of knowledge of the DNA content impair the identification of these alterations especially in aneuploid tumors. To accurately detect allelic changes in carcinomas, we combined flow-sorting and single nucleotide polymorphism arrays. Cells derived from archival cervical and colon cancers were flow-sorted based on differential vimentin and keratin expression and DNA content and analyzed on single nucleotide polymorphism arrays. A new algorithm, the lesser allele intensity ratio, was used to generate a molecular measure of chromosomal aberrations for each case. Flow-sorting significantly improved the detection of copy number abnormalities; 31.8% showed an increase in amplitude and 23.2% were missed in the unsorted fraction, whereas 15.9% were detected but interpreted differently. Integration of the DNA index in the analysis enabled the identification of the allelic state of chromosomal aberrations, such as LOH ([A]), copy-neutral LOH ([AA]), balanced amplifications ([AABB]), and allelic imbalances ([AAB] or [AAAB], etc.). Chromosomal segments were sharply defined. Fluorescence in situ hybridization copy numbers, as well as the high similarity between the DNA index and the allelic state index, which is the average of the allelic states across the genome, validated the method. This new approach provides an individual molecular measure of chromosomal aberrations and will likely have repercussions for preoperative molecular staging, classification, and prognostic profiling of tumors, particularly for heterogeneous aneuploid tumors, and allows the study of the underlying molecular genetic mechanisms and clonal evolution of tumor subpopulations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-08-2665DOI Listing
December 2008

Genome-wide copy neutral LOH is infrequent in familial and sporadic microsatellite unstable carcinomas.

Fam Cancer 2008 15;7(4):319-30. Epub 2008 Apr 15.

Department of Pathology, Leiden University Medical Center, Building L1Q, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.

Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10689-008-9194-8DOI Listing
January 2009

Frequent mutations in the 3'-untranslated region of IFNGR1 lack functional impairment in microsatellite-unstable colorectal tumours.

Eur J Hum Genet 2008 Oct 16;16(10):1235-9. Epub 2008 Apr 16.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Microsatellite repeats are frequently found to be mutated in microsatellite-instable colorectal tumours. This suggests that these mutations are important events during tumour development. We have observed frequent mutations in microsatellite-instable (MSI-H) tumours and cell lines of a conserved A14 repeat within the 3'-untranslated region of the interferon-gamma receptor 1 gene (IFNGR1). The repeat was mutated in 59% (41 of 70) of colon carcinomas and in all four MSI-H colon cancer cell lines tested. In-vitro analysis of these cell lines did not show a decreased responsiveness to standard IFNgamma concentrations when compared to microsatellite-stable colon cancer cell lines. A functional consequence of the frequently found microsatellite instability in IFNGR1 is therefore not evident.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ejhg.2008.81DOI Listing
October 2008

Identification of patients with (atypical) MUTYH-associated polyposis by KRAS2 c.34G > T prescreening followed by MUTYH hotspot analysis in formalin-fixed paraffin-embedded tissue.

Clin Cancer Res 2008 Jan;14(1):139-42

Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands.

Purpose: To assess the feasibility of identifying patients with (atypical) MUTYH-associated polyposis (MAP) by KRAS2 c.34G > T prescreening followed by MUTYH hotspot mutation analysis in formalin-fixed paraffin-embedded tissue (FFPE).

Methods: We collected 210 colorectal FFPE tumors from 192 individuals who presented with <10 adenomas or familial mismatch repair proficient colorectal carcinomas with <10 concomitant adenomas. The tissues were tested for somatic KRAS2 mutations and for three Dutch hotspot MUTYH germ line mutations (p.Tyr165Cys, p.Gly382Asp, and p.Pro391Leu) by sequencing analysis.

Results: The c.34G > T, KRAS2 transversion was detected in 10 of 210 tumors. In one of these 10 cases, a monoallelic p.Gly382Asp MUTYH mutation was found and a full MUTYH analysis in leukocyte DNA revealed an unclassified variant p.Met269Val. This was in a 61-year-old patient with a cecum carcinoma and three adenomas. After further requests, her family case history revealed that her brother had had between 10 and 15 adenomas and turned out to carry both MUTYH germ line mutations. MUTYH hotspot mutation screening in 182 patients without the somatic c.34G > T KRAS2 mutation led to the detection of three monoallelic germ line MUTYH mutation carriers.

Conclusion: KRAS2 c.34G > T somatic prescreening, followed by MUTYH hotspot mutation analysis when positive, can identify patients with (atypical) MAP. If heterozygous hotspot MUTYH mutations are identified, a complete germ line MUTYH mutation screening should be carried out if possible. Immediate MUTYH hotspot mutation analysis is a practical alternative in patients with >10 adenomas or in cases of multiple colorectal carcinomas in one generation for which only FFPE tissue is available.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-07-1705DOI Listing
January 2008

Molecular analysis of colorectal cancer tumors from patients with mismatch repair proficient hereditary nonpolyposis colorectal cancer suggests novel carcinogenic pathways.

Clin Cancer Res 2007 Oct;13(19):5729-35

Laboratory of Molecular Oncology, Hospital Clínico San Carlos, Madrid, Spain.

Purpose: A subset of colorectal cancers (CRC) arises in families that, despite fulfilling clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC), do not show evidence of a mismatch repair (MMR) deficiency. The main objective of this study was to characterize these tumors at the molecular level.

Experimental Design: After comprehensive germ line mutation scanning, microsatellite analysis, and MMR protein expressions, we selected a well-defined cohort of 57 colorectal tumors with no evidence of MMR defects. In this group of tumors, we analyzed KRAS, BRAF, and APC somatic mutations, as well as methylguanine methyltransferase (MGMT) and beta-catenin expression. We correlated these alterations with clinicopathologic data and explored the relationship between KRAS G > A transitions and lack of MGMT expression.

Results: The mutation profile at the RAS/RAF/MAPK pathway mimics sporadic microsatellite-stable CRCs. We found an average age of diagnosis 10 years older in KRAS-mutated patients (P = 0.001). In addition, we show that KRAS G > A transitions are actively selected by tumors, regardless of MGMT status. Similarities with HNPCC high-microsatellite instability tumors are observed when APC data are analyzed. The APC mutation rate was low and small insertions/deletions accounted for 70% of the alterations. In addition, we found a low frequency of beta-catenin nuclear staining. Finally, we did not find evidence of tumors arising in individuals from the same family sharing molecular features.

Conclusions: We show evidence that CRC tumors arising in HNPCC families without MMR alterations have distinctive molecular features. Overall, our work shows that systematic analysis of somatic alterations in a well-defined subset of CRCs is a good approach to provide new insights into the mechanisms of colorectal carcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-06-2996DOI Listing
October 2007

HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression.

BMC Cancer 2007 Feb 22;7:33. Epub 2007 Feb 22.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Abnormalities in Human Leukocyte Antigen (HLA) class I expression are common in colorectal cancer. Since HLA expression is required to activate tumor antigen-specific cytotoxic T-lymphocytes (CTL), HLA class I abnormalities represent a mechanism by which tumors circumvent immune surveillance. Tumors with high microsatellite instability (MSI-H) are believed to face strong selective pressure to evade CTL activity since they produce large amounts of immunogenic peptides. Previous studies identified the prevalence of HLA class I alterations in MSI-H tumors. However, those reports did not compare the frequency of alterations between hereditary and sporadic MSI-H tumors neither the mechanisms that led to HLA class I alterations in each subgroup.

Methods: To characterize the HLA class I expression among sporadic MSI-H and microsatellite-stable (MSS) tumors, and HNPCC tumors we compared immunohistochemically the expression of HLA class I, beta2-microglobulin (beta2m), and Antigen Processing Machinery (APM) components in 81 right-sided sporadic and 75 HNPCC tumors. Moreover, we investigated the genetic basis for these changes.

Results: HLA class I loss was seen more frequently in MSI-H tumors than in MSS tumors (p < 0.0001). Distinct mechanisms were responsible for HLA class I loss in HNPCC and sporadic MSI-H tumors. Loss of HLA class I expression was associated with beta2m loss in HNPCC tumors, but was correlated with APM component defects in sporadic MSI-H tumors (p < 0.0001). In about half of the cases, loss of expression of HLA class I was concordant with the detection of one or more mutations in the beta2m and APM components genes.

Conclusion: HLA class I aberrations are found at varying frequencies in different colorectal tumor types and are caused by distinct genetic mechanisms. Chiefly, sporadic and hereditary MSI-H tumors follow different routes toward HLA class I loss of expression supporting the idea that these tumors follow different evolutionary pathways in tumorigenesis. The resulting variation in immune escape mechanisms may have repercussions in tumor progression and behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-7-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1808468PMC
February 2007

The natural history of a combined defect in MSH6 and MUTYH in a HNPCC family.

Fam Cancer 2007 ;6(1):43-51

Department of Pathology, Leiden University Medical Center, Building L1Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

In the inherited syndromes, MUTYH-associated polyposis (MAP) and hereditary nonpolyposis colorectal cancer (HNPCC), somatic mutations occur due to loss of the caretaker function that base-repair (BER) and mismatch repair (MMR) genes have, respectively. Recently, we identified a large branch from a MSH6 HNPCC family in which 19 family members are heterozygous or compound heterozygous for MUTYH germ line mutations. MSH6/MUTYH heterozygote mutation carriers display a predominant HNPCC molecular tumour phenotype, with microsatellite instability and underrepresentation of G>T transversions. A single unique patient is carrier of the MSH6 germline mutation and is compound heterozygote for MUTYH. Unexpectedly, this patient has an extremely mild clinical phenotype with sofar only few adenomas at age 56. Four out of five adenomas show characteristic G>T transversions in APC and/or KRAS2, as seen in MUTYH associated polyposis. No second hit of MSH6 is apparent in any of the adenomas, due to retained MSH6 nuclear expression and a lack of microsatellite instability. Although this concerns only one case, we argue that the chance to find an additional one is extremely small and currently a mouse model with this genotype combination is not available. Moreover, the patients brother who is also compound heterozygous for MUTYH but lacks the MSH6 germline mutation presented with a full blown polyposis coli. In conclusion, these data would support the notion that abrogation of both MSH6 DNA mismatch repair and base repair might be mutually exclusive in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10689-006-9103-yDOI Listing
September 2007

High-resolution analysis of HLA class I alterations in colorectal cancer.

BMC Cancer 2006 Oct 2;6:233. Epub 2006 Oct 2.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Previous studies indicate that alterations in Human Leukocyte Antigen (HLA) class I expression are frequent in colorectal tumors. This would suggest serious limitations for immunotherapy-based strategies involving T-cell recognition. Distinct patterns of HLA surface expression might conceal different immune escape mechanisms employed by the tumors and are worth further study.

Method: We applied four-color multiparameter flow cytometry (FCM), using a large panel of alloantigen-specific anti-HLA-A and -B monoclonal antibodies, to study membranous expression of individual HLA alleles in freshly isolated colorectal cancer cell suspensions from 21 patients.

Results: Alterations in HLA class I phenotype were observed in 8 (38%) of the 21 tumors and comprised loss of a single A or B alleles in 4 cases, and loss of all four A and B alleles in the other 4 cases. Seven of these 8 tumors were located on the right side of the colon, and those showing loss of both HLA-A and -B membranous expression were all of the MSI-H phenotype.

Conclusion: FCM allows the discrimination of complex phenotypes related to the expression of HLA class I. The different patterns of HLA class I expression might underlie different tumor behavior and influence the success rate of immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-6-233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1599746PMC
October 2006

Heterozygous mutations in PMS2 cause hereditary nonpolyposis colorectal carcinoma (Lynch syndrome).

Gastroenterology 2006 Feb;130(2):312-22

Center for Human and Clinical Genetics, Leiden University Medical Center, The Netherlands.

Background & Aims: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating PMS2 mutations have been reported in HNPCC-suspected families. Our aim was to further assess the role of PMS2 in HNPCC.

Methods: We performed Southern blot analysis in 112 patients from MLH1-, MSH2-, and MSH6-negative HNPCC-like families. A subgroup (n = 38) of these patients was analyzed by denaturing gradient gel electrophoresis (DGGE). In a second study group consisting of 775 index patients with familial colorectal cancer, we performed immunohistochemistry using antibodies against MLH1, MSH2, MSH6, and PMS2 proteins. In 8 of 775 tumors, only loss of PMS2 expression was found. In these cases, we performed Southern blot analysis and DGGE. Segregation analysis was performed in the families with a (possibly) deleterious mutation.

Results: Seven novel mutations were identified: 4 genomic rearrangements and 3 truncating point mutations. Three of these 7 families fulfill the Amsterdam II criteria. The pattern of inheritance is autosomal dominant with a milder phenotype compared with families with pathogenic MLH1 or MSH2 mutations. Microsatellite instability and immunohistochemical analysis performed in HNPCC-related tumors from proven carriers showed a microsatellite instability high phenotype and loss of PMS2 protein expression in all tumors.

Conclusions: We show that heterozygous truncating mutations in PMS2 do play a role in a small subset of HNPCC-like families. PMS2 mutation analysis is indicated in patients diagnosed with a colorectal tumor with absent staining for the PMS2 protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2005.10.052DOI Listing
February 2006

Mass spectrometry-based loss of heterozygosity analysis of single-nucleotide polymorphism loci in paraffin embedded tumors using the MassEXTEND assay: single-nucleotide polymorphism loss of heterozygosity analysis of the protein tyrosine phosphatase receptor type J in familial colorectal cancer.

J Mol Diagn 2005 Nov;7(5):623-30

Leiden University Medical Center, Department of Pathology, Building L1Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

As the number of identified single-nucleotide polymorphisms (SNPs) increases, high-throughput methods are required to characterize the informative loci in large patient series. We investigated the feasibility of MassEXTEND LOH analysis using Sequenom's MassArray RT software, a mass spectrometry method, as an alternative to determine loss of heterozygosity (LOH). For this purpose, we studied the c.827A>C SNP (1176A>C p.Gln276Pro) in protein tyrosine phosphatase receptor type-J (PTPRJ), which is frequently deleted in human cancers. In sporadic colorectal cancer (CRC), c.827A>C showed allele-specific LOH of the c.827A allele, which is important because LOH of PTPRJ may be an early event during sporadic CRC. To elucidate the impact of this low-penetrance gene on familial CRC, we studied c.827A>C in 222 familial CRC cases and 156 controls. In 6.2% of the A/C genotyped CRC samples, LOH of c.827A was observed with MassEXTEND LOH analysis and confirmed by conventional sequencing. Furthermore, a case with LOH of c.827A showed no LOH in 22 synchronously detected adenomas, including one with malignant transformation. The importance of the PTPRJ- c.827A>C SNP appears to be limited in familial CRC. We conclude that MassEXTEND LOH analysis (using Sequenom's MassARRAY RT software) is a sensitive, high-throughput, and cost-effective method to screen SNP loci for LOH in formalin-fixed paraffin-embedded tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867548PMC
http://dx.doi.org/10.1016/S1525-1578(10)60596-XDOI Listing
November 2005

Homozygosity for a CHEK2*1100delC mutation identified in familial colorectal cancer does not lead to a severe clinical phenotype.

J Pathol 2005 Jun;206(2):198-204

Department of Pathology, Leiden University Medical Centre, The Netherlands.

It has recently been suggested that the frequency of the germline CHEK2*1100delC mutation is higher among breast cancer families with colorectal cancer, although the mutation does not seem to be significantly associated with familial colorectal cancer. Five hundred and sixty-four familial colorectal tumours were studied for expression of CHEK2 using tissue microarrays and an antibody against the NH2-terminal SQ regulatory domain of the CHEK2 protein. Normal colonic tissue from patients whose tumours showed loss of CHEK2 expression was investigated further using fragment and sequence analysis for the presence of a CHEK2*1100delC mutation and five other (R117G, R137Q, R145W, I157T, and R180H) known germline variants in CHEK2. Twenty-nine tumours demonstrated loss of expression for CHEK2. Analysis of matched normal colonic tissue from these patients revealed germline CHEK2*1100delC mutation in three cases. In two of these, the mutation was heterozygous but, interestingly, the third patient proved to be homozygous for the deletion, using six different primer pair combinations. None of the other tested germline variants were identified. No CHEK2*1100delC mutations were found in patients whose tumours stained positive. Homozygosity for the CHEK2*1100delC mutation appears not to be lethal in humans. No severe clinical phenotype was apparent, although the patient died from colonic carcinoma at age 52 years. This observation is in line with recent knockout mouse models, although in the latter, cellular defects in apoptosis and increased resistance to irradiation seem to exist. It is also concluded that CHEK2 protein abrogation is not caused by the CHEK2 germline variants R117G, R137Q, R145W, I157T, and R180H in familial colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.1764DOI Listing
June 2005

Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype.

Cancer Res 2004 Oct;64(20):7405-11

Laboratory of Cancer Genetics and Bioinformatics Special Program, Van Andel Research Institute, Grand Rapids, Michigan, USA.

Parathyroid tumors are heterogeneous, and diagnosis is often difficult using histologic and clinical features. We have undertaken expression profiling of 53 hereditary and sporadic parathyroid tumors to better define the molecular genetics of parathyroid tumors. A class discovery approach identified three distinct groups: (1) predominantly hyperplasia cluster, (2) HRPT2/carcinoma cluster consisting of sporadic carcinomas and benign and malignant tumors from Hyperparathyroidism-Jaw Tumor Syndrome patients, and (3) adenoma cluster consisting mainly of primary adenoma and MEN 1 tumors. Gene sets able to distinguish between the groups were identified and may serve as diagnostic biomarkers. We demonstrated, by both gene and protein expression, that Histone 1 Family 2, amyloid beta precursor protein, and E-cadherin are useful markers for parathyroid carcinoma and suggest that the presence of a HRPT2 mutation, whether germ-line or somatic, strongly influences the expression pattern of these 3 genes. Cluster 2, characterized by HRPT2 mutations, was the most striking, suggesting that parathyroid tumors with somatic HRPT2 mutation or tumors developing on a background of germ-line HRPT2 mutation follow pathways distinct from those involved in mutant MEN 1-related parathyroid tumors. Furthermore, our findings likely preclude an adenoma to carcinoma progression model for parathyroid tumorigenesis outside of the presence of either a germ-line or somatic HRPT2 mutation. These findings provide insights into the molecular pathways involved in parathyroid tumorigenesis and will contribute to a better understanding, diagnosis, and treatment of parathyroid tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-04-2063DOI Listing
October 2004

Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance.

Gastroenterology 2004 Jul;127(1):17-25

Center of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Background & Aims: Hereditary nonpolyposis colorectal carcinoma (HNPCC) is caused by a mutated mismatch repair (MMR) gene. The aim of our study was to determine the cumulative risk of developing cancer in a large series of MSH6 mutation carriers.

Methods: Mutation analysis was performed in 20 families with a germline mutation in MSH6. We compared the cancer risks between MSH6 and MLH1/MSH2 mutation carriers. Microsatellite instability (MSI) analysis and immunohistochemistry (IHC) were performed in the available tumors.

Results: A total of 146 MSH6 mutation carriers were identified. In these carriers, the cumulative risk for colorectal carcinoma was 69% for men, 30% for women, and 71% for endometrial carcinoma at 70 years of age. The risk for all HNPCC-related tumors was significantly lower in MSH6 than in MLH1 or MSH2 mutation carriers (P = 0.002). In female MSH6 mutation carriers, the risk for colorectal cancer was significantly lower (P = 0.0049) and the risk for endometrial cancer significantly higher (P = 0.02) than in MLH1 and MSH2 mutation carriers. In male carriers, the risk for colorectal cancer was lower in MSH6 mutation carriers, but the difference was not significant (P = 0.0854). MSI analysis in colorectal tumors had a sensitivity of 86% in predicting a MMR defect. IHC in all tumors had a sensitivity of 90% in predicting a mutation in MSH6.

Conclusions: We recommend starting colonoscopic surveillance in female MSH6 mutation carriers from age 30 years. Prophylactic hysterectomy might be considered in carriers older than 50 years. MSI and IHC analysis are sensitive tools to identify families eligible for MSH6 mutation analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2004.03.068DOI Listing
July 2004

Microsatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer.

Clin Cancer Res 2004 Feb;10(3):972-80

Department of Gastroenterology, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, the Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.ccr-0956-3DOI Listing
February 2004

The role of mismatch repair gene defects in the development of adenomas in patients with HNPCC.

Gastroenterology 2004 Jan;126(1):42-8

Department of Gastroenterology, Leiden University Medical Center, Leiden, Netherlands.

Background And Aims: The adenoma-carcinoma sequence in hereditary nonpolyposis colorectal cancer (HNPCC) is accelerated. It remains unknown whether the mismatch repair (MMR) defect also promotes the development of adenomas. The aim of this study was to compare the risk of developing colorectal adenoma and carcinoma in HNPCC carriers and noncarriers (controls) and to compare the features of adenomas in both groups.

Methods: Eighty-six families with a known MMR gene mutation from the Dutch HNPCC Registry were analyzed. Subjects with known mutation status with colonoscopies performed for the purpose of surveillance were selected for this study. Information on the surveillance examinations was obtained from medical reports. The histology of all adenomas was confirmed. Immunohistochemistry was performed in a subgroup of adenomas.

Results: We identified 249 carriers and 247 controls. The proportion of subjects free of an adenoma at the age of 60 years was 29.7% for carriers and 70.8% for controls (P < 0.05). The adenomas in carriers were larger, and a higher proportion had villous components and/or high-grade dysplasia (P < 0.05, all analyses). The adenomas and carcinomas of the carriers were located predominantly in the proximal colon. Most adenomas showed absent staining of the MMR proteins.

Conclusions: This study indicates that the MMR defect is involved in the early stages of development of adenomas. We recommend immunohistochemical staining of large adenomas with high-grade dysplasia in young patients (younger than 50 years) to identify patients with suspected HNPCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2003.10.043DOI Listing
January 2004

Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma.

J Pathol 2004 Jan;202(1):86-94

Department of Pathology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Malignant transformation of parathyroid tumours is rare. Nevertheless, this small subset of malignant tumours often creates diagnostic and therapeutic problems. In this work, the morphological characteristics of 26 primary parathyroid carcinomas and seven metastases have been studied. Furthermore, immunohistochemical expression profiles for the calcium sensing receptor (CASR), cyclin D1 (CCND1), and Ki-67 were determined for parathyroid carcinomas and compared with adenomas and hyperplasias using a tissue microarray. Loss of heterozygosity (LOH) of the chromosome 1q region containing the HRPT2 gene and chromosome 11q (MEN1) was determined in the carcinomas. In contrast to the adenomas and hyperplasias, 31% of carcinomas demonstrated down-regulation of CASR. A significant correlation was found between CASR expression and the Ki-67 proliferation index. Chromosome 1q and chromosome 11q LOH were found in 12 of 22 (55%) and 11 of 22 (50%) carcinomas tested, respectively. Combined 1q and 11q LOH was seen in 8 of 22 (36%) carcinomas, in contrast to the low percentage of LOH reported in both regions in adenomas. In conclusion, this study demonstrates that combined 1q and 11q LOH in parathyroid tumours is suggestive of malignant behaviour. Strong down-regulation of the CASR protein is seen in a proportion of parathyroid carcinomas with a high proliferation index.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.1489DOI Listing
January 2004

Pancreatic carcinoma in carriers of a specific 19 base pair deletion of CDKN2A/p16 (p16-leiden).

Clin Cancer Res 2003 Sep;9(10 Pt 1):3598-605

Departments of Gastroenterology, Leiden University Medical Center, Leiden PB 9600, the Netherlands.

Purpose: The purpose is to document the clinical, pathological, and genetic features of pancreatic carcinoma (PC) in carriers of a specific p16-Leiden mutation (a 19-bp deletion in exon 2 of the CDKN2A gene).

Experimental Design: Clinical data and paraffin embedded tissue were obtained from 12 patients of p16-Leiden-positive families with PC. Because of the known 19-bp germ-line deletion, we could specifically analyze the genotype of the wild-type allele for loss of heterozygosity. K-ras codon 12 mutations were determined and immunohistochemical testing for p16, Tp53, Smad4, and cyclooxygenase 2 was performed.

Results: The average age of subjects that developed PC (8 males) was 58 years (range, 43-74 years). Histology was considered as conventional ductal adenocarcinoma in 11 of 12 and neuroendocrine carcinoma (1 of 12). The carcinomas were located in the head (10 of 12), corpus (1 of 12), and tail (1 of 12) of the pancreas. The specific p16-Leiden mutation was confirmed in the tissue of all subjects. Loss of heterozygosity of the wild-type allele was present in 2 of 7 tumors analyzed. Immunostaining for p16 was negative in 10 of 10. Tp53 mutations were detected in 5 of 12. Smad4 was negative in 5 of 12 and cyclooxygenase 2 was overexpressed in 11 of 12. K-ras codon 12 mutations were present in 9 of 10 and in three precursor lesions even before abrogation of p16 protein expression was seen (one of three).

Conclusions: The p16-Leiden deletion was associated with progression toward conventional ductal adenocarcinomas in all cases but one. Our observations might support the feasibility of early diagnosis of PC in p16-Leiden mutation carriers and might also indicate that chemoprevention needs consideration.
View Article and Find Full Text PDF

Download full-text PDF

Source
September 2003

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene.

Am J Hum Genet 2003 May 25;72(5):1088-100. Epub 2003 Mar 25.

Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180263PMC
http://dx.doi.org/10.1086/373963DOI Listing
May 2003

Down-regulation of HLA-A expression correlates with a better prognosis in colorectal cancer patients.

Lab Invest 2002 Dec;82(12):1725-33

Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands.

To evaluate the prognostic impact of human leukocyte antigen class I (HLA-I) expression on immune surveillance in colorectal cancer, we studied 88 curatively resected tumors for HLA-A and HLA-B/C expression and correlated these data to clinical and histopathological parameters. HLA-A was normal (all tumor cells had HLA expression) in 32%, reduced (HLA-negative and -positive tumor cells coexisted) in 56%, or absent (no tumor cells expressed HLA) in 12% of evaluable cases. HLA-B/C was normal in 47%, reduced in 47%, and absent in 7% of the cases. Considering both markers, total HLA-I expression was normal in 27%, reduced in 63%, absent in 7%, and could not be evaluated in 3% of the cases due to absent HLA-A expression in tumor and normal cells. Down-regulation of HLA-A expression significantly correlated with a lower tumor stage (p = 0.005), mucinous tumors (p = 0.05), a lower incidence of recurrences (p = 0.03), and a longer disease-free survival (p = 0.02). Down-regulation of HLA-B/C expression correlated with a lower tumor stage (p < 0.001) and a longer disease-free survival (p = 0.04). In multivariate analysis, HLA-A down-regulation was the only prognostic factor correlated with a longer disease-free survival (p = 0.02). Six tumors were negative for HLA-A and -B/C and did not recur during follow-up. Therefore, we analyzed microsatellite instability (MSI) in these cases. Three of these six tumors indeed showed down-regulation of MLH-1, MSH-2, or MSH-6, indicating a MSI-high phenotype. Beta-2-microglobulin protein expression was lost in five of six of the HLA-I-negative cases, but frame shift mutations in three repetitive sequences in beta2-microglobulin were absent. In contrast, loss of MLH-1, MSH-2, and MSH-6-protein expression was only observed in two of nine matched controls with reduced or normal HLA-A and -B/C expression. Our data showed that HLA-I was down-regulated in 72% of colorectal cancers and provided independent prognostic information for a longer disease-free survival. The better prognosis may be caused by elimination of HLA-negative cells by natural killer cells or by an attenuated tumor aggressiveness, as is seen in tumors with a MSI-high phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.lab.0000043124.75633.edDOI Listing
December 2002