Magnus von Knebel-Doeberitz

Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC

Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCR-screening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli

Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild‐type allele. Inactivation of APC leads to impaired degradation of β‐catenin, thereby promoting continuous cell‐cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ‐line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second‐hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of β‐catenin to the mutational status of the APC gene. Exon 3 of the β‐catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated β‐catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of β‐catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP‐associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients.

Comprehensive Genetic Counseling for Families At Risk for HNPCC: Impact on Distress and Perceptions

The aim of the study was to explore distress and health beliefs before and after comprehensive interdisciplinary counseling in families at risk for hereditary non-polyposis colorectal cancer (HNPCC). Results reported here were derived from a consecutive sample of 65 counselees [31 patients with colorectal cancer (CRC) and 34 unaffected at-risk persons] who participated in interdisciplinary counseling provided by human geneticists, surgeons, and psycho-oncologists before genetic testing. Data were collected from self-administered questionnaires before, as well as 4-6 weeks after, counseling. Distress and perceptions specific to HNPCC were assessed at both timepoints using standardized as well as author-derived instruments. Distress declined after counseling, as did worries related to HNPCC. An increase was found in personal belief in control of cancer risk, for instance, in the perceived efficacy of early detection of CRC. We also observed a trend toward greater anticipated ability to cope with a positive gene test after counseling. Changes after counseling were generally more pronounced for persons at risk, as compared to patients with cancer. The decrease in distress was partly attributable to an increase in personal self-confidence. One-third of the sample reported enhanced communication specific to hereditary disease within the family after counseling. A substantial minority, however, said they experienced increased worry and physical symptoms after counseling. Overall, counselees demonstrated less stress and perceived cancer threat as well as enhanced beliefs regarding personal control over cancer, suggesting an overall beneficial impact of comprehensive counseling. Further research is needed to identify those individuals most at risk for increased fear and worry related to HNPCC so that they may be most appropriately counseled.

Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: A comprehensive analysis of 3,671 families

Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time‐consuming, clinical criteria and tumor‐tissue analysis are widely used as pre‐screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor‐tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability‐high (MSI‐H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI‐H small‐bowel cancer (p < 0.001). No MMR mutations were found among patients under 40‐years‐old with only colorectal adenoma. Familial clustering of Lynch syndrome‐related tumors, early age of onset, and familial occurrence of small‐bowel cancer were clinically relevant predictors for Lynch syndrome.

Cervical cytology biobanking in Europe

A cervical cytology biobank (CCB) is an extension of current cytopathology laboratory practice consisting in the systematic storage of Pap smears or liquid-based cytology samples from women participating in cervical cancer screening with the explicit purpose to facilitate future scientific research and quality audit of preventive services. A CCB should use an internationally agreed uniform cytology terminology, be integrated in a national or regional screening registry, and be linked to other registries (histology, cancer, vaccination). Legal and ethical principles concerning personal integrity and data safety must be respected strictly. Biobank-based studies require approval of ethical review boards. A CCB is an almost inexhaustible resource for fundamental and applied biological research. In particular, it can contribute to answering questions on the natural history of HPV infection and HPV-induced lesions and cancers, screening effectiveness, exploration of new biomarkers, and surveillance of the short- and long-term effects of the introduction of HPV vaccination. To understand the limitations of CCB, more studies are needed on the quality of samples in relation to sample type, storage procedures, and duration of storage.

p16INK4a immunocytochemistry versus HPV testing for triage of women with minor cytological abnormalities: A systematic review and meta-analysis

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 (p16INK4a) 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 p16INK4a 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 p16INK4a triage data. p16INK4a and HC2 had similar sensitivity, and p16INK4a 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, p16INK4a 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 p16INK4a compared with HC2 testing in the triage of women with ASC‐US. In LSIL triage, p16INK4a was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012.

Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene

Abstract Germline mutations in the adenomatous polyposis coli gene cause familial adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5′-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.

Effective Antitumoral Immune Responses Are Not Induced by Cytosine Deaminase Suicide Gene Transfer in a Syngeneic Rat Pancreatic Carcinoma Model

Background: Experimental gene transfer can make tumors more immunogenic, leading to local regression and inducing immunological memory sufficient to permit resistance to a tumor rechallenge. However, this rarely had any significant impact on large established tumors. Methods: To analyze potential immunological effects, we used weakly immunogenic pancreatic carcinomas in syngeneic, immunocompetent Lewis rats and performed in situ adenoviral mediated cytosine deaminase (CD) gene transfer followed by administration of the prodrug, 5-fluorocytosine (5FC). In order to reflect the clinical situation, such treated tumors were surgically resected and animals were rechallenged with parental DSL6A pancreatic tumor cells. Tumor growth and cytotoxic activity of immune cells were determined. Results: CD/5FC treatment of the DSL6A cells revealed significant induction of apoptosis in vitro and slowed down tumor progression in syngeneic hosts. Furthermore, we observed neither significant change in tumor growth nor protective immunity in the rechallenged animals. Analysis of T lymphocytes showed no specific cytotoxic activity against DSL6A cells. There was only a trend towards a minor NK cell activation. Conclusions: Albeit the present study failed to induce protective antitumor immunity, the initial finding of reduced tumor growth argues for the development of multimodal therapeutic options to overcome negative impacts of advanced malignant disease or chemotherapy-related anergy and immunosuppression.

Expression profiling of cervical cancer biopsies using high-resolution dual-label imaging of 33 P and 3 H on microarrays

Expression profiling of cervical cancer biopsies using high-resolution dual-label imaging of 33 P and 3 H on microarrays

In vivo adenovirus mediated gene transfer of the Eschericha coli cytosine meaminase gene to pancreatic tumours induces chemosensitivity to 5-fluorocytosine

IN VIVO ADENOVIRUS MEDIATED GENE TRANSFER OF THE ESCHERICHA COLI CYTOSINE DEAMINASE GENE TO PANCREATIC TUMOURS INDUCES CHEMOSENSITIVITY TO 5-FLUOROCYTOSINE. Dalibor Antolovic, Sven C. Eisold, Michael Linnebacher, Geeske C. Meyer, Susanne Dihlmann, Jan Schmidt, Ernst KIar, Christian Herfarth, Magnus von Knebel-Doeberitz, Univ of Heidelberg, Dept Surg, Heidelberg, Germany; Univ of Heidelberg, Heidelberg, Germany.