[show abstract][hide abstract] ABSTRACT: 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.
European journal of human genetics: EJHG 03/2011; 19(3):307-12. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many hereditary nonpolyposis colorectal cancers (CRCs) cannot be explained by Lynch syndrome. Other high penetrance genetic risk factors are likely to play a role in these mismatch repair (MMR)-proficient CRC families. Because genomic profiles of CRC tend to vary with CRC susceptibility syndromes, our aim is to analyze the genomic profile of MMR-proficient familial CRC to obtain insight into the biological basis of MMR-proficient familial CRC. We studied 30 MMR-proficient familial colorectal carcinomas, from 15 families, for genomic aberrations, including gains, physical losses, and copy-neutral loss of heterozygosity LOH (cnLOH) using SNP array comparative genomic hybridization. In addition, we performed somatic mutation analysis for KRAS, BRAF, PIK3CA and GNAS. The frequency of 20q gain (77%) is remarkably increased when compared with sporadic CRC, suggesting that 20q gain is involved in tumor progression of familial CRC. There is also a significant increase in the frequency of cnLOH and, as a consequence, a reduced frequency of physical loss compared with sporadic CRC. The most frequent aberrations observed included gains of 7p, 7q, 8q, 13q, 20p and 20q as well as physical losses of 17p, 18p and 18q. Most of these changes are also observed in sporadic CRC. Mutations in KRAS were identified in 37% of the MMR-proficient CRCs, and mutations in BRAF were identified in 16%. No mutations were identified in PIK3CA or chromosome 20 candidate gene GNAS. We show that the patterns of chromosomal instability of MMR-proficient familial CRC are clearly distinct from those from sporadic CRC. Both the increased gain on chromosome 20 and the increased levels of cnLOH suggest the presence of yet undiscovered germline defects that can, in part, underlie the cancer risk in these families.
International Journal of Cancer 03/2011; 130(4):837-46. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Genes Chromosomes and Cancer 03/2010; 49(6):539-48. · 3.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
The Journal of molecular diagnostics: JMD 12/2009; 12(1):27-34. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
The Journal of Pathology 04/2009; 219(1):69-76. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Cancer Research 01/2009; 68(24):10333-40. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Familial Cancer 05/2008; 7(4):319-30. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
European Journal of HumanGenetics 05/2008; 16(10):1235-9. · 4.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic instability is known to drive colorectal carcinogenesis. Generally, a distinction is made between two types of genetic instability: chromosomal instability (CIN) and microsatellite instability (MIN or MSI). Most CIN tumours are aneuploid, whereas MSI tumours are considered near-diploid. However, for MUTYH-associated polyposis (MAP) the genetic instability involved in the carcinogenesis remains unclear, as near-diploid adenomas, aneuploid adenomas and near-diploid carcinomas have been reported. Remarkably, our analysis of 26 MAP carcinomas, using SNP arrays and flow sorting, showed that these tumours are often near-diploid (52%) and mainly contain chromosomal regions of copy-neutral loss of heterozygosity (LOH) (71%). This is in contrast to sporadic colon cancer, where physical loss is the main characteristic. The percentage of chromosomal gains (24%) is comparable to sporadic colorectal cancers with CIN. Furthermore, we verified our scoring of copy-neutral LOH versus physical loss in MAP carcinomas by two methods: fluorescence in situ hybridization, and LOH analysis using polymorphic markers on carcinoma fractions purified by flow sorting. The results presented in this study suggest that copy-neutral LOH is an important mechanism in the tumorigenesis of MAP.
The Journal of Pathology 04/2008; 216(1):25-31. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
Clinical Cancer Research 01/2008; 14(1):139-42. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
Clinical Cancer Research 11/2007; 13(19):5729-35. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Parathyroid carcinoma remains difficult to diagnose. Recently, it has been shown that mutations in the HRPT2 gene (encoding parafibromin) are associated with the development of parathyroid carcinoma. Although MEN1 is not typically thought to be involved in carcinoma formation, parathyroid carcinoma may be an extremely rare feature of the multiple endocrine neoplasia type 1 (MEN1) syndrome. We recently concluded that loss of heterozygosity (LOH) of the MEN1 gene is present in a relatively large number of parathyroid carcinomas, often in combination with LOH at the HRPT2 locus. The aim of this study was to evaluate the role of MEN1 and HRPT2 mutations in sporadic parathyroid tumours fulfilling histological criteria for malignancy.
Formalin-fixed, paraffin-embedded (FFPE) parathyroid carcinoma tissue from 28 cases identified in the period 1985-2000 in the Netherlands was studied. HRPT2 (27/28 cases) and MEN1 (23/28 cases) were analysed by direct sequencing.
Somatic MEN1 mutations were found in three of 23 (13%) sporadic parathyroid carcinoma cases; these consisted of one missense and two frameshift mutations. One of the latter two cases displayed lymph-node and lung metastases during follow-up. Six HRPT2 mutations were found in 4/27 cases (15%): five were truncating mutations and one was a missense mutation. Consistent with previously published reports, we found double mutations (2x) and germline mutations (2x) in apparently sporadic parathyroid carcinomas.
These results suggest that not only HRPT2 but also MEN1 mutations may play a role in sporadic parathyroid cancer formation.
[show abstract][hide abstract] ABSTRACT: 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.
Familial Cancer 02/2007; 6(1):43-51. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: Bi-allelic germline mutations in the MUTYH gene give rise to multiple adenomas and an increased incidence of colorectal cancer. In addition, duodenal adenomas and other extra-colonic manifestations have been described in MUTYH-associated polyposis (MAP) patients. We describe two patients with bi-allelic MUTYH gene mutations with duodenal carcinoma. The tumour in Patient A was detected during evaluation of non-specific abdominal complaints. Patient B was already diagnosed with tens of adenomas and a colon carcinoma, when a duodenal neoplasm was detected. The identification of somatic G>T mutations in codon 12 of the K-RAS2 gene provides evidence that the duodenal lesions were induced by MUTYH deficiency. Studies in larger series of MAP patients are needed to investigate the risk of upper-gastro-intestinal malignancies and to determine further guidelines for endoscopical surveillance.
Journal of Clinical Pathology 11/2006; 59(11):1212-5. · 2.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: hMLH1 and hMSH2 can be considered tumor suppressor genes, as both alleles must be inactivated in order to lose the mismatch repair (MMR) function. In this regard, it has been proposed that LOH at MMR loci is a common Knudson's second-hit mechanism in HNPCC patients. However, experimental evidence supporting this view is scarcely found in the literature. We have performed a comprehensive analysis of LOH in 45 HNPCC tumors carrying a germline alteration in MMR loci. Overall, we have detected LOH at MMR loci in 56% of the cases. However, up to 40% of the LOH events targeted the mutant allele, arguing against a second-hit role in these tumors. Interestingly, the age at diagnosis was significantly older in these patients. To explain this and previous data, we propose a dual role for LOH at MMR loci in HNPCC.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.