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Miriam Reuschenbach,
Katinka Kansy,
Kira Garbe,
Svetlana Vinokurova,
Christa Flechtenmacher,
Csaba Toth,
Elena-Sophie Prigge,
Oliver C Thiele,
Siegmar Reinert,
Jürgen Hoffmann, Magnus von Knebel Doeberitz,
Kolja Freier
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ABSTRACT: OBJECTIVES: The aim of the present study was to identify HPV-attributable SCC of the oral cavity (OSCC) in a cohort of patients from southern Germany. MATERIALS AND METHODS: A sensitive PCR-enzyme immunoassay (EIA) was followed by a more specific in situ hybridization (ISH) to detect high risk human papillomavirus (HPV). An immunohistochemical dual-staining for p16(INK4a) and the proliferation marker Ki-67 was used to assess whether co-expression of p16(INK4a)/Ki-67 is a better surrogate marker for HPV in OSCC than p16(INK4a) alone, based on the hypothesis that combined p16(INK4a) and Ki-67 expression might specifically discriminate oncogene-induced p16(INK4a) expression from cell-cycle arrest-inducing senescence-associated p16(INK4a) expression. RESULTS: HPV-DNA by PCR-EIA could be detected in 25.1% (69/275) of the tumors, but ISH was negative in all of them. Diffuse p16(INK4a) overexpression was detected in 11 HPV PCR-positive tumors, but also in 6 HPV PCR-negative tumors. p16(INK4a)-expressing cells in diffusely positive tumors co-expressed Ki-67, irrespective of the HPV status. Neither the sole HPV status nor combined HPV/p16(INK4a) status nor the sole p16(INK4a) status was significantly associated with disease free or overall survival, however a trend towards better overall survival of patients whose tumor expressed p16(INK4a) in a focal pattern (=p16(INK4a)-positive/Ki-67-negative cells) compared to no p16(INK4a) expression (p=0.09) was observed. CONCLUSION: Viral DNA can be detected in some tumors by a sensitive PCR, but absence of ISH signals indicates that the HPV-attributable fraction is smaller than estimated from PCR positivity. p16(INK4a)/Ki-67 co-expression is detectable in a fraction of OSCC irrespective of the HPV status.
Oral Oncology 04/2013; · 2.86 Impact Factor
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ABSTRACT: The differentiation between hereditary and sporadic microsatellite-unstable (MSI-H) colorectal cancer is a crucial step in Lynch syndrome diagnostics. Within MSI-H colorectal cancers, the BRAF V600E mutation is strongly associated with sporadic origin. We here asked whether BRAF V600E-specific immunohistochemistry (clone VE1) is helpful in separating sporadic from Lynch syndrome-associated MSI-H colorectal cancers. To that end, we performed VE1 immunohistochemistry and BRAF sequencing in a series of 91 MSI-H colorectal cancer specimens from patients tested for Lynch syndrome. Concordance of VE1 immunohistochemistry and molecular BRAF mutation status was observed in 90 out of 91 (98.9%) MSI-H samples. All eleven tumors classified as BRAF V600E mutation-positive by Sanger sequencing were immuno-positive, and 79 (98.8%) out of 80 tumors classified as BRAF wild type showed negative staining. All VE1-positive tumors were MLH1 and PMS2-negative by immunohistochemistry. None of the tumors from MMR gene germline mutation carriers (n=28) displayed positive VE1 staining, indicating that BRAF V600E mutation-specific immunostaining has a low risk of excluding Lynch syndrome patients from germline mutation analysis. In conclusion, implementation of VE1 immunohistochemistry was able to detect BRAF mutated MSI-H colorectal cancers with a sensitivity of 100% and a specificity of 98.7%. Among MLH1-negative colorectal cancers, the rate of VE1-positive lesions was 21%, offering the exclusion of these patients from MMR germ line testing. We therefore suggest the integration of VE1 immunohistochemistry into the diagnostic panel of Lynch syndrome. © 2013 Wiley Periodicals, Inc.
International Journal of Cancer 03/2013; · 5.44 Impact Factor
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ABSTRACT: HPV 58 is detected commonly in cervical cancer in East Asian countries. To evaluate the HPV 58 physical state, the amplification of papillomavirus oncogene transcripts (APOT) and hybridisation assays were established. Episome- and integrate-derived transcripts were confirmed by direct sequencing. Twenty-nine HPV 58 positive samples from various cervical lesions were used. The results showed that the episome-derived transcripts were recognised as two major specific amplified products (1,040 and 714bp). Two splice donor sites were mapped to the 5́ splice site of the E1 gene on SD898 and SD899 and spliced to the 3́ acceptor site of the E4 gene on SA3353, SA3356 and SA3365. The episome-derived transcripts were found 100% in normal cervical epithelia and low-grade lesions (9/9 cases) while the integrate-derived transcripts were detected in 13.3% of high-grade lesions (2/15 cases) and in 20% of carcinomas (1/5 cases). HPV 58 integration sites were found on chromosomes 4q21, 12q24 and 18q12. Using the established APOT assay, the results revealed not only novel information on the HPV 58 transcription patterns of episomal transcripts, but also integration site. The APOT assay is a reliable and useful tool for the detection of the HPV 58 physical state and its oncogene expression.
Journal of virological methods 03/2013; · 2.13 Impact Factor
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Christoph Engel,
Markus Loeffler,
Verena Steinke,
Nils Rahner,
Elke Holinski-Feder,
Wolfgang Dietmaier,
Hans K Schackert,
Heike Goergens, Magnus von Knebel Doeberitz,
Timm O Goecke, [......],
Encarna Gómez García,
Frederik J Hes,
Nicoline Hoogerbrugge,
Fred H Menko,
Theo A M van Os,
Rolf H Sijmons,
Anja Wagner,
Irma Kluijt,
Peter Propping,
Hans F A Vasen
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ABSTRACT: PURPOSEPatients with Lynch syndrome are at high risk for colon and endometrial cancer, but also at an elevated risk for other less common cancers. The purpose of this retrospective cohort study was to provide risk estimates for these less common cancers in proven carriers of pathogenic mutations in the mismatch repair (MMR) genes MLH1, MSH2, and MSH6. PATIENTS AND METHODS
Data were pooled from the German and Dutch national Lynch syndrome registries. Seven different cancer types were analyzed: stomach, small bowel, urinary bladder, other urothelial, breast, ovarian, and prostate cancer. Age-, sex- and MMR gene-specific cumulative risks (CRs) were calculated using the Kaplan-Meier method. Sex-specific incidence rates were compared with general population incidence rates by calculating standardized incidence ratios (SIRs). Multivariate Cox regression analysis was used to estimate the impact of sex and mutated gene on cancer risk.ResultsThe cohort comprised 2,118 MMR gene mutation carriers (MLH1, n = 806; MSH2, n = 1,004; MSH6, n = 308). All cancers were significantly more frequent than in the general population. The highest risks were found for male small bowel cancer (SIR, 251; 95% CI, 177 to 346; CR at 70 years, 12.0; 95% CI, 5.7 to 18.2). Breast cancer showed an SIR of 1.9 (95% CI, 1.4 to 2.4) and a CR of 14.4 (95% CI, 9.5 to 19.3). MSH2 mutation carriers had a considerably higher risk of developing urothelial cancer than MLH1 or MSH6 carriers. CONCLUSION
The sex- and gene-specific differences of less common cancer risks should be taken into account in cancer surveillance and prevention programs for patients with Lynch syndrome.
Journal of Clinical Oncology 10/2012; · 18.37 Impact Factor
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ABSTRACT: Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to 4 E2 binding sites (E2BS 1 - 4) in the viral upstream regulatory region (URR). Modification of E2 functions for example by methylation of E2BSs are hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BS 1 - 4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared to lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes. © 2012 Wiley Periodicals, Inc.
International Journal of Cancer 10/2012; · 5.44 Impact Factor
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ABSTRACT: Current diagnostic approaches for primary cervical cancer screening, work-up of equivocal or positive screening results or follow-up after treatment of precancerous lesions primarily rely on the morphologic interpretation of squamous epithelial cells (Pap cytology), in some setting accompanied by the detection of human papillomavirus DNA and have largely contributed to remarkable reduction of disease incidence in countries with implemented screening programs. However, these approaches are limited by a poor sensitivity and reproducibility of Pap cytology and low specificity for high grade cervical intraepithelial neoplasia of HPV DNA detection assays. Early detection might be improved by complementing or even replace these tests by markers which are more directly related to molecular events triggering HPV-induced carcinogenesis and thereby might deliver more accurate diagnostic performance. The delineation of molecular changes which occur during different stages of HPV infections and the identification of changes which induce neoplastic alterations allow for the detection of markers that specifically highlight the transforming stage of the infection where viral oncogenes are overexpressed and therefore allow for a more specific diagnosis of lesions that require treatment. The evaluation of such markers in clinical studies revealed that some indeed show an improved diagnostic performance compared to Pap cytology or HPV DNA tests only.
Current pharmaceutical design 09/2012; · 4.41 Impact Factor
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Kathrin Bauer,
Nina Nelius,
Miriam Reuschenbach,
Moritz Koch,
Jürgen Weitz,
Gunnar Steinert,
Jürgen Kopitz,
Philipp Beckhove,
Mirjam Tariverdian, Magnus von Knebel Doeberitz,
Matthias Kloor
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ABSTRACT: High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.
Cancer Immunology and Immunotherapy 06/2012; · 3.70 Impact Factor
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ABSTRACT: The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta-analysis to assess the accuracy of cyclin-dependent kinase inhibitor 2A (p16(INK4a) ) immunocytochemistry compared with high-risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC-US) or low-grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta-analysis. Seventeen studies were included in the meta-analysis. The pooled sensitivity of p16(INK4a) to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%-88.2%) and 83.8% (95% CI, 73.5%-90.6%) in ASC-US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%-76.4%) and 65.7% (95% CI, 54.2%-75.6%), respectively. Eight studies provided both HC2 and p16(INK4a) triage data. p16(INK4a) and HC2 had similar sensitivity, and p16(INK4a) has significantly higher specificity in the triage of women with ASC-US (relative sensitivity, 0.95 [95% CI, 0.89-1.01]; relative specificity, 1.82 [95% CI, 1.57-2.12]). In the triage of LSIL, p16(INK4a) had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81-0.94]; relative specificity, 2.74 [95% CI, 1.99-3.76]). The published literature indicated the improved accuracy of p16(INK4a) compared with HC2 testing in the triage of women with ASC-US. In LSIL triage, p16(INK4a) was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.
Cancer Cytopathology 06/2012; 120(5):294-307. · 3.33 Impact Factor
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ABSTRACT: Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes. Mutation carriers have a high risk of developing colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa.
Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients.
20 crypt foci with no MMR protein expression were detected in 20·1 cm(2) of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm(2) of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm(2), set 2: 4·8 cm(2)). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance.
We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination.
German Cancer Aid and German Research Foundation.
The lancet oncology 04/2012; 13(6):598-606. · 14.47 Impact Factor
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ABSTRACT: Biomarkers indicating the initiation of neoplastic transformation processes in human papillomavirus (HPV)-infected epithelial cells are moving into the focus of cancer prevention research, particularly for anogenital cancer, including cancer of the uterine cervix. Based on the in-depth understanding of the molecular events leading to neoplastic transformation of HPV-infected human cells, the cyclin-dependent kinase inhibitor p16(INK4a) turned out to be substantially overexpressed in virtually all HPV-transformed cells. This finding opened novel avenues in diagnostic histopathology to substantially improve the diagnostic accuracy of cervical cancer and its precursor lesions. Furthermore, it provides a novel technical platform to substantially improve the accuracy of cytology-based cancer early-detection programs. Here, we review the molecular background and the current evidence for the clinical utility of the p16(INK4a) biomarker for HPV-related cancers, and cervical cancer prevention in particular.
Expert Review of Proteomics 04/2012; 9(2):149-63. · 3.68 Impact Factor
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ABSTRACT: Germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene lead to silencing of MSH2 and cause Lynch syndrome. We have recently reported that lack of EPCAM expression occurs in many, but not all tumors from Lynch syndrome patients with EPCAM germline deletions. The differences in EPCAM expression were not related to the localization of EPCAM germline deletions. We therefore hypothesized that the type of the second somatic hit, which leads to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. To test this hypothesis and to evaluate whether lack of EPCAM expression can already be detected in Lynch syndrome-associated adenomas, we analyzed four carcinomas and two adenomas from EPCAM germline deletion carriers for EPCAM protein expression and allelic deletion status of the EPCAM gene region by multiplex ligation-dependent probe amplification. In four out of six tumors we observed lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results demonstrate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of the second somatic hit that inactivates MSH2. Moreover, we report lack of EPCAM protein expression in a colorectal adenoma, suggesting that EPCAM immunohistochemistry may detect EPCAM germline deletions already at a precancerous stage.
Modern Pathology 03/2012; 25(6):911-6. · 4.79 Impact Factor
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Journal of Investigative Dermatology 02/2012; 132(2):491-3. · 6.31 Impact Factor
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Niels Halama,
Sara Michel,
Matthias Kloor,
Inka Zoernig,
Axel Benner,
Anna Spille,
Thora Pommerencke, Doeberitz Magnus von Knebel,
Gunnar Folprecht,
Birgit Luber,
Nadine Feyen,
Uwe M Martens,
Philipp Beckhove,
Sacha Gnjatic,
Peter Schirmacher,
Esther Herpel,
Juergen Weitz,
Niels Grabe,
Dirk Jaeger
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ABSTRACT: Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.
Cancer Research 08/2011; 71(17):5670-7. · 7.86 Impact Factor
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ABSTRACT: High level microsatellite instability (MSI-H) is a hallmark of Lynch syndrome-associated colorectal cancer (CRC). MSI-H CRC express immunogenic tumour antigens as a consequence of DNA mismatch repair deficiency-induced frameshift mutations. Consequently, frameshift antigen-specific immune responses are commonly observed in patients with Lynch syndrome-associated MSI-H CRC. Dendritic cells (DC) and macrophages play a crucial role in the induction and modulation of immune responses. We here analysed DC and macrophage infiltration in MSI-H and microsatellite-stable CRC. Sixty-nine CRC (MSI-H, n = 33; microsatellite-stable, n = 36) were examined for the density of tumour-infiltrating DC, Foxp3-positive regulatory T cells, and CD163-positive macrophages. In MSI-H lesions, S100-positive and CD163-positive cell counts were significantly higher compared to microsatellite-stable lesions (S100: epithelium P = 0.018, stroma P = 0.042; CD163: epithelium P < 0.001, stroma P = 0.046). Additionally, numbers of CD208-positive mature DC were significantly elevated in the epithelial compartment of MSI-H CRC (P = 0.027). High numbers of tumour-infiltrating Foxp3-positive T cells were detected in tumours showing a low proportion of CD208-positive, mature DC among the total number of S100-positive cells. Our study demonstrates that infiltration with DC, mature DC, and macrophages is elevated in MSI-H compared to microsatellite-stable CRC. The positive correlation of Foxp3-positive Treg cell density with a low proportion of mature DC suggests that impaired DC maturation may contribute to local immune evasion in CRC. Our results demonstrate that DC and macrophages in the tumour environment likely play an important role in the induction of antigen-specific immune responses in Lynch syndrome. Moreover, impaired DC maturation might contribute to local immune evasion in CRC.
Familial Cancer 05/2011; 10(3):557-65. · 1.30 Impact Factor
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ABSTRACT: Diffuse overexpression of p16(INK4a) in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus-mediated transformation. Focal p16(INK4a) expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16(INK4a) triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16(INK4a) , i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16(INK4a) -positive cells in epithelia displaying focal p16(INK4a) expression pattern do not coexpress proliferation-associated Ki-67 protein, while p16(INK4a) -positive cells in lesions with diffuse p16(INK4a) expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16(INK4a) and Ki-67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16(INK4a) , and in all of these lesions p16(INK4a) -positive cells were found to be negative for Ki-67 expression. Diffuse expression of p16(INK4a) was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki-67 immunoreactivity in a large proportion of p16(INK4a) -positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16(INK4a) and Ki-67. Coexpression of Ki-67 and p16(INK4a) in the same cell is entirely restricted to cervical lesions displaying diffuse p16(INK4a) expression, whereas in lesions with focal p16(INK4a) expression, p16(INK4a) -expressing cells are negative for Ki-67. Thus, diffuse expression of p16(INK4a) reflects lesions with proliferation-competent cells, while p16(INK4a) -expressing cells associated with focal expression patterns are cell cycle arrested.
International Journal of Cancer 03/2011; 130(2):388-94. · 5.44 Impact Factor
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Karin Hardt,
Sven Boris Heick,
Beate Betz,
Timm Goecke,
Haniyeh Yazdanparast,
Robin Küppers,
Kati Servan,
Verena Steinke,
Nils Rahner,
Monika Morak,
Elke Holinski-Feder,
Christoph Engel,
Gabriela Möslein,
Hans-Konrad Schackert, Magnus von Knebel Doeberitz,
Christian Pox,
Johannes H Hegemann,
Brigitte Royer-Pokora
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ABSTRACT: Missense mutations of the DNA mismatch repair gene MLH1 are found in a significant fraction of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) and their pathogenicity often remains unclear. We report here all 88 MLH1 missense variants identified in families from the German HNPCC consortium with clinical details of these patients/families. We investigated 23 MLH1 missense variants by two functional in vivo assays in yeast; seven map to the ATPase and 16 to the protein interaction domain. In the yeast-2-hybrid (Y2H) assay three variants in the ATPase and twelve variants in the interaction domain showed no or a reduced interaction with PMS2; seven showed a normal and one a significantly higher interaction. Using the Lys2A (14) reporter system to study the dominant negative mutator effect (DNE), 16 variants showed no or a low mutator effect, suggesting that these are nonfunctional, three were intermediate and four wild type in this assay. The DNE and Y2H results were concordant for all variants in the interaction domain, whereas slightly divergent results were obtained for variants in the ATPase domain. Analysis of the stability of the missense proteins in yeast and human embryonic kidney cells (293T) revealed a very low expression for seven of the variants in yeast and for nine in human cells. In total 15 variants were classified as deleterious, five were classified as variants of unclassified significance (VUS) and three were basically normal in the functional assays, P603R, K618R, Q689R, suggesting that these are neutral.
Familial Cancer 03/2011; 10(2):273-84. · 1.30 Impact Factor
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ABSTRACT: High risk human papillomaviruses are squamous epitheliotropic viruses that may cause cervical and other cancers. HPV replication depends on squamous epithelial differentiation. Transformation of HPV-infected cells goes along with substantial alteration of the viral gene expression profile and preferentially occurs at transformation zones usually at the uterine cervix. Methylation of the viral genome may affect regulatory features that control transcription and replication of the viral genome. Therefore, we analyzed the methylation pattern of the HPV16 upstream regulatory region (URR) during squamous epithelial differentiation and neoplastic transformation and analyzed how shifts in the HPV URR methylome may affect viral gene expression and replication. HPV 16 positive biopsy sections encompassing all stages of an HPV infection (latent, permissive and transforming) were micro-dissected and DNA was isolated from cell fractions representing the basal, intermediate, and superficial cell layers, each, as well as from transformed p16(INK4a)-positive cells. We observed fundamental changes in the methylation profile of transcription factor binding sites in the HPV16 upstream regulatory region linked to the squamous epithelial differentiation stage. Squamous epithelial transformation indicated by p16(INK4a) overexpression was associated with methylation of the distal E2 binding site 1 leading to hyper-activation of the HPV 16 URR. Adjacent normal but HPV 16-infected epithelial areas retained hyper-methylated HPV DNA suggesting that these viral genomes were inactivated. These data suggest that distinct shifts of the HPV 16 methylome are linked to differentiation dependent transcription and replication control and may trigger neoplastic transformation.
PLoS ONE 01/2011; 6(9):e24451. · 4.09 Impact Factor
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ABSTRACT: Lynch syndrome is an inherited tumor predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Recently, germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene located upstream of MSH2 were identified as a novel mutational mechanism causing Lynch syndrome by epigenetic inactivation of the respective MSH2 allele. Immunohistochemical analysis of MMR protein expression is a hallmark of Lynch syndrome diagnostics, but it cannot distinguish between EPCAM deletion carriers and MSH2 mutation carriers. We hypothesized that EPCAM protein expression might be altered in tumors from patients with a germline EPCAM deletion.
Immunohistochemistry was used to assess EPCAM expression in Lynch syndrome-associated MSH2-negative tumors (n = 26). Multiplex ligation-dependent probe amplification (MLPA) analysis was performed to detect germline deletions of the EPCAM and MSH2 gene loci.
In four MSH2-negative tumors, a concomitant lack of EPCAM expression was detected. MLPA analysis revealed heterozygous EPCAM deletions in all patients with EPCAM-negative tumors. In contrast, EPCAM expression was positive in all cancers from patients with germline alterations affecting MSH2 but not EPCAM. Two EPCAM deletions were detected in patients with an EPCAM-positive tumor.
These results indicate that loss of EPCAM protein expression is frequent in tumors from patients with EPCAM germline deletions. EPCAM immunohistochemistry therefore represents a promising novel tool for the identification of Lynch syndrome patients with EPCAM germline deletions.
Journal of Clinical Oncology 01/2011; 29(2):223-7. · 18.37 Impact Factor
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ABSTRACT: Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT-PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.
Nucleic Acids Research 12/2010; 38(22):7916-26. · 8.03 Impact Factor
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ABSTRACT: Celiac disease (CD) is an inflammatory disorder associated with an increased risk of small bowel adenocarcinoma. Recent studies have demonstrated aberrant CpG island methylation (CIM) in chronic inflammation, aging and cancer. We hypothesized that CIM may link CD to small bowel carcinogenesis. We determined microsatellite instability (MSI), CIM, and expression of MLH1 and MGMT in 3 CD-associated small bowel carcinomas and corresponding non-neoplastic mucosa. The results were compared to those of small bowel mucosa from CD patients without carcinoma and 20 small bowel carcinomas from a non-CD origin. A high level CIM/MSI phenotype was found in all of the 3 CD-associated carcinomas and was associated with loss of MLH1 expression due to hypermethylation of the MLH1 promoter. This phenotype was noted in only 2 of the 20 investigated non-CD-associated carcinomas. Low-level CIM was already detectable in 9 of the 12 non-neoplastic mucosa samples of CD patients and in non-CD-associated carcinomas of elderly patients. In conclusion, our data reveal that the high-level CIM/MSI pathway is typical of CD-associated small bowel carcinomas and indicate that aberrant CpG island methylation links CD and carcinogenesis. The data further suggest that CD should be considered in patients with small bowel adenocarcinoma, particularly when the tumors display MSI.
Oncology Reports 12/2010; 24(6):1535-9. · 1.84 Impact Factor
