[The two-in-one hit model of the short-cut carcinogenesis of colorectal carcinomas in MLH1-associated Lynch syndrome]. / Das Two-in-one-hit-Modell der beschleunigten Genese von kolorektalen Karzinomen beim MLH1-assoziierten Lynch-Syndrom.

In a recently published study a new genetic hypothesis was established that explained the existence of CTNNB1 mutations in Lynch syndrome-associated colorectal carcinomas (MLH1-LS-CRC). This hypothesis states that a mitotic recombination on chromosome 3p simultaneously leads to inactivation of the mismatch repair gene MLH1 and to the activation of CTNNB1. This explains the increased frequency of CTNNB1 mutations in MLH1-LS-CRC compared with other colon carcinomas. To test this hypothesis, various experiments were carried out that show that the first phase of recombination occurs in non-cancerous tissues, which favours the development of CTNNB1 mutations. This mechanism could explain the rapid tumour progression in MLH1-LS-CRC. The results highlight the importance of mitotic recombination in carcinogenesis and provide an insight into the genetic basis of colorectal carcinoma in the context of Lynch syndrome.

European guidelines from the EHTG and ESCP for Lynch syndrome: an updated third edition of the Mallorca guidelines based on gene and gender.

BACKGROUND: Lynch syndrome is the most common genetic predisposition for hereditary cancer but remains underdiagnosed. Large prospective observational studies have recently increased understanding of the effectiveness of colonoscopic surveillance and the heterogeneity of cancer risk between genotypes. The need for gene- and gender-specific guidelines has been acknowledged. METHODS: The European Hereditary Tumour Group (EHTG) and European Society of Coloproctology (ESCP) developed a multidisciplinary working group consisting of surgeons, clinical and molecular geneticists, pathologists, epidemiologists, gastroenterologists, and patient representation to conduct a graded evidence review. The previous Mallorca guideline format was used to revise the clinical guidance. Consensus for the guidance statements was acquired by three Delphi voting rounds. RESULTS: Recommendations for clinical and molecular identification of Lynch syndrome, surgical and endoscopic management of Lynch syndrome-associated colorectal cancer, and preventive measures for cancer were produced. The emphasis was on surgical and gastroenterological aspects of the cancer spectrum. Manchester consensus guidelines for gynaecological management were endorsed. Executive and layperson summaries were provided. CONCLUSION: The recommendations from the EHTG and ESCP for identification of patients with Lynch syndrome, colorectal surveillance, surgical management of colorectal cancer, lifestyle and chemoprevention in Lynch syndrome that reached a consensus (at least 80 per cent) are presented.

Lifestyle factors and risk of sporadic colorectal cancer by microsatellite instability status: a systematic review and meta-analyses.

Introduction: The association of lifestyle factors with molecular pathological subtypes of colorectal cancer (CRC), such as microsatellite instability (MSI), could provide further knowledge about the colorectal carcinogenic process. The aim of this review was to evaluate possible associations between lifestyle factors and risk of sporadic CRC by MSI status. Methods: PubMed and Web of Science were searched for studies investigating the association between alcohol, body mass index, dietary fiber, hormone replacement therapy (HRT), non-steroidal anti-inflammatory drugs, physical activity, red meat, smoking, or statin use, with MSI-high (MSI-H) and microsatellite stable (MSS) CRC. Meta-analyses were carried out to calculate summary relative risks (sRR). Results: Overall, 31 studies reporting on the association between lifestyle factors and CRC according to MSI status were included in this review. Ever smoking was associated with MSI-H (sRR = 1.62; 95% CI: 1.40-1.88) and MSS/MSI-low CRC (sRR = 1.10; 95% CI: 1.01-1.20), but the association was significantly stronger for MSI-H CRC. The use of HRT was associated with a 20% decrease (sRR = 0.80; 95% CI: 0.73-0.89) in the risk of MSS CRC, but was not associated with MSI-H CRC. An increase in body mass index per 5 kg/m2 was equally associated with MSS and MSI-H CRC (sRR = 1.22, in both cases), but was statistically significant for MSS CRC only (95% CI: 1.11-1.34 and 0.94-1.58, respectively). Limited evidence for associations between other lifestyle factors and CRC by MSI status exists. Conclusions: Lifestyle factors, such as HRT and smoking are differentially associated with the risk of MSI-H and MSS CRC. Further research on associations of lifestyle factors and CRC subtypes is necessary to provide a better understanding of the CRC disease pathway.

Functional characterization of the tumor-suppressor MARCKS in colorectal cancer and its association with survival.

The myristoylated alanine-rich C-kinase substrate (MARCKS) acts as a tumor suppressor in a variety of human neoplasms. In colorectal cancers (CRCs), MARCKS has been shown to be a preferential target of mutational inactivation in tumors following the microsatellite instability (MSI-H) pathway but little is known about its impact on intestinal carcinogenesis. To investigate the relevance of MARCKS inactivation in more detail, we analyzed 926 MSI-typed CRCs for MARCKS expression by immunohistochemistry and studied the functional consequences of MARCKS depletion in colorectal cancer cell lines. We found that loss of MARCKS expression was not restricted to MSI-H cancers but also occurred in microsatellite stable (MSS) tumors, where it was associated with an adverse outcome regarding overall survival, cancer-specific and disease-free survival (P=0.002, P=0.0018, P=0.0001, respectively; univariate analysis). In MARCKS-positive MSS colon cancer cell lines (SW480 and SW707) small interfering RNA (siRNA)-mediated knockdown of MARCKS conferred resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. This was accompanied by the downregulation of the TRAIL receptors DR4 and DR5 at the cell surface and activation of AKT signaling. Inhibition of AKT signaling and transient overexpression of wild-type MARCKS, but not of MARCKS lacking the effector domain (ED), abolished the anti-apoptotic effect. In conclusion, our data show that inactivation of MARCKS is common in CRCs and is associated with adverse outcome in MSS cancers. The finding that MARCKS acts as a mediator of apoptosis in MSS CRC cells adds a novel tumor-suppressing function to the so far established roles of MARCKS in cell motility and proliferation and can explain the prognostic effect of MARCKS depletion in MSS CRC.

[Pathogenesis of microsatellite-unstable colorectal cancer. Evaluation of new diagnostic and therapeutic options]. / Pathogenese mikrosatelliteninstabiler kolorektaler Karzinome. Evaluierung neuer diagnostischer und therapeutischer Optionen.

The molecular pathogenesis of colorectal cancer is heterogeneous. Whereas the majority of colorectal cancers follow the classical adenoma-carcinoma sequence and display chromosomal instability, a subset of approximately 15 % of colorectal cancers show a deficiency of the DNA mismatch repair system. These carcinomas present with numerous mutations at repetitive DNA stretches, a phenotype termed high-level microsatellite instability (MSI-H). The pathogenesis of MSI-H cancers is driven by mismatch repair deficiency-induced insertion/deletion mutations affecting microsatellites located in the coding region of tumor suppressor genes, such as TGFBR2. The MSI-induced mutations of tumor suppressor genes not only lead to functional inactivation but also to shifts of the translational reading frame and consequently to the generation of frameshift peptides (FSPs). These FSPs can be recognized as foreign by the host immune system. It could be shown that in the majority of MSI-H colorectal cancer patients, FSP-specific T cell-mediated immune responses can be detected. These tumor antigen-specific immune responses are regarded as a major reason for the dense local lymphocyte infiltration which is typical of MSI-H colorectal cancer. A further characteristic feature of MSI-H cancers is the occurrence of alterations affecting the cellular antigen presentation mechanism where beta2-microglobulin (B2M) mutations that directly result from DNA mismatch repair deficiency represent the most common mechanism. It could be demonstrated that B2M mutations are associated with M0 stage and a very favorable prognosis. The characterization of the particular immunological properties of MSI-H tumors have paved the way for the initiation of a clinical trial in which FSP vaccination is currently being clinically evaluated in patients with MSI-H colorectal cancer.

Microsatellite instability and Beta2-Microglobulin mutations as prognostic markers in colon cancer: results of the FOGT-4 trial.

BACKGROUND: High-level microsatellite instability (MSI-H) has been reported as a prognostic marker in colon cancer. We here analysed the prognostic significance of MSI and mutations of the Beta2-Microglobulin (B2M) gene, which occur in about 30% of MSI-H colon cancer, in the cohort of the prospective FOGT-4 (Forschungsruppe Onkologie Gastrointestinale Tumoren, FOGT) trial. METHODS: Microsatellite instability status was determined using standard protocols (NCI/ICG-HNPCC panel and CAT25) in 223 colon cancer lesions. Beta2-Microglobulin mutation status was evaluated by exon-wise sequencing in all MSI-H lesions. RESULTS: Patients with MSI-H (n=34) colon cancer presented with a significantly lower risk of relapse after 12 months of follow-up compared with MSS (n=189) colon cancer patients (5 year time to relapse: MSI-H 0.82 vs MSS 0.66, P=0.03). No significant difference in overall survival was detected. Beta2-Microglobulin mutations were identified in 10 (29.4%) out of 34 MSI-H colon cancers and were associated with a complete absence of disease relapse or tumour-related death events (P=0.09). CONCLUSION: The risk of late disease relapse was significantly lower in patients with MSI-H compared with MSS colon cancer. Moreover, B2M mutations may contribute to the favourable outcome of MSI-H colon cancer patients and should therefore be evaluated as a potential prognostic marker in future clinical trials.

[Chromosomal instability, microsatellite instability and CpG island methylator phenotype: roles in small intestinal carcinogenesis]. / Chromosomale Instabilität, Mikrosatelliteninstabilität und "CpG island methylator phenotype": Rolle in der Dünndarmkarzinogenese.

AIMS: Intestinal carcinogenesis is associated with genetic instability affecting either the chromosomal level (CIN) or microsatellite DNA sequences (MIN). In addition, epigenetic alterations, such as aberrant CpG island methylation (CIMP) may contribute to tumor development. While these single genetic alterations have frequently been addressed in intestinal carcinogenesis little is known about the interaction of the epigenetics and genetics in tumorigenesis. This investigation therefore aimed to define the synergistic effects of CIN, MSI and CIMP in small bowel adenocarcinomas. METHODS: A total of 37 primary small bowel adenocarcinomas were investigated for CIN, MSI, CIMP, KRAS and BRAF mutations. The results showed that CIN was found in 22 out of 37 (57%) tumors (3 out of 9 microsatellite instable and 19 out of 28 microsatellite stable carcinomas) and 9 carcinomas (24%) were microsatellite and chromosomally stable. Aberrant CIMP was detected in 16% of chromosomal instable tumors and in 44% of both microsatellite instable and microsatellite and chromosomally stable carcinomas, KRAS was mutated in 55%, 0% and 10% of chromosomal instable, microsatellite instable and microsatellite and chromosomal stable tumors, respectively, while BRAF mutations occurred in 6% of chromosomal instable and 22% of both microsatellite instable and microsatellite and chromosomal stable carcinomas. CONCLUSION: Chromosomal instable carcinomas of the small intestine are distinguished from microsatellite instable and microsatellite and chromosomal stable tumors by a high frequency of KRAS mutations and low frequencies of CIMP and BRAF mutations. In microsatellite instable and microsatellite and chromosomally stable cancers, CIMP and BRAF/KRAS mutations are similarly distributed indicating common mechanisms of tumor initiation or progression in the molecular pathogenesis.

High-risk human papillomavirus in non-melanoma skin lesions from renal allograft recipients and immunocompetent patients.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) can be detected in a proportion of non-melanoma skin cancers. Data on prevalence are inconclusive, but are essential to estimate the relevance of HR-HPV, particularly with regard to prophylactic HPV vaccines for skin cancer prevention. METHODS: High-risk human papillomavirus DNA was investigated in 140 non-melanoma skin lesions from 54 immunocompetent patients and 33 immunosuppressed renal allograft recipients. Expression of p16(INK4a), a marker for HR-HPV oncogene expression in the uterine cervix, and of p53 and pRB was evaluated immunohistochemically. RESULTS: The highest prevalence of HR-HPV was found in squamous cell cancer (SCC) (46.2% (6 out of 13) in immunosuppressed and 23.5% (4 out of 17) in immunocompetent patients). High-risk human papillomavirus positivity was accompanied by diffuse p16(INK4a) expression in most SCC (P<0.001) and basal cell cancers (P=0.02), while almost all SCC in situ were p16(INK4a) positive irrespective of HR-HPV presence (P=0.66). Diffuse p16(INK4a) expression was associated with lack of pRB expression (P=0.001). p53 was strongly expressed in 40.0% (56 out of 140) of the lesions irrespective of HR-HPV presence. CONCLUSION: High-risk human papillomavirus can be detected in lesions of keratinised squamous epithelia. The association of HR-HPV with diffuse p16(INK4a) expression might indicate HR-HPV oncogene expression in a proportion of lesions. Overexpression of p53 suggests p53 pathway alterations in HR-HPV-positive and -negative lesions.

High density of FOXP3-positive T cells infiltrating colorectal cancers with microsatellite instability.

High-level microsatellite instability (MSI-H) in colorectal cancer accounts for about 12% of colorectal cancers and is typically associated with a dense infiltration with cytotoxic CD8-positive lymphocytes. The role of regulatory T cells that may interfere with the host's antitumoural immune response in MSI-H colorectal cancers has not been analysed yet. Using an antibody directed against the regulatory T-cell marker transcription factor forkhead box P3 (FOXP3), regulatory T cells were examined in 70 colorectal cancers with known MSI status (MSI-H, n=37; microsatellite stable, n=33). In MSI-H colorectal cancers, we found a significantly higher intraepithelial infiltration with FOXP3-positive cells (median: 8.5 cells per 0.25 mm(2) vs 3.1 cells per 0.25 mm(2) in microsatellite stable, P<0.001), and a significantly elevated ratio of intraepithelial to stromal infiltration (0.05 vs 0.01 in microsatellite stable, P<0.001). CD8-positive cell counts were related positively to the number of FOXP3-positive cells (Spearman's rho=0.56 and 0.55, respectively). Our results show that the elevated number of CD8-positive lymphocytes found in MSI-H colorectal cancers is paralleled by an enhanced infiltration with CD8-negative FOXP3-positive cells. These data suggest that FOXP3-positive cells may play a role in the regulation of the immune response directed against MSI-H colorectal cancers at the primary tumour site.

Psychosocial outcome following genetic risk counselling for familial colorectal cancer. A comparison of affected patients and family members.

Few studies have reported prospective data on psychosocial outcomes after genetic counselling in families with suspected hereditary non-polyposis colorectal cancer (HNPCC). This prospective study examines the impact of multidisciplinary risk counselling on the psychosocial outcome of 139 affected cancer patients and 233 family members without cancer at risk for HNPCC. Participants completed questionnaires specific to HNPCC before and 8 weeks after attending the familial cancer clinic. Affected patients' levels of distress were closely related to their health status and exceeded that of unaffected individuals, as did worry regarding their relatives' risk. A significant reduction in general anxiety (Hospital Anxiety and Depression Scale), distress specific to familial CRC (Impact of Events Scale) and general cancer worry (Distress Hereditary Disorder) was demonstrated after counselling in both affected patients and unaffected individuals. Reduction in distress was more pronounced in affected patients given a high risk of HNPCC compared with those at intermediate risk. Among unaffected individuals, distress declined regardless of what clinical risk they were assigned. Their perceptions of risk and cancer-related threat declined, while confidence in effective surveillance increased. These results suggest the beneficial effects of multidisciplinary counselling even when high-risk information is conveyed. A patient's previous cancer experience is likely to contribute to clinically relevant distress (15% of those patients), indicating the need for appropriate counselling.

[Molecular pathogenesis. Its importance in targeted therapy in colorectal cancer]. / Molekulare Pathogenese. Ihre Bedeutung für die zielgerichtete Therapie beim kolorektalen Karzinom.

Colorectal cancer has the second highest mortality of all cancers in Germany. In spite of advances in surgical and chemotherapeutic treatment, efficient new therapies need to be developed. In recent years, advances have been achieved by novel targeted therapies that are specifically directed against altered signaling pathways of malignant cells. Colorectal cancers represent a heterogeneous tumor entity, and response to targeted therapies varies individually. About 15% of colorectal carcinomas are characterized by a deficient DNA mismatch repair system and microsatellite instability (MSI). These MSI cancers apparently have a decreased sensitivity to chemotherapy and frequently show evidence of a pronounced anti-tumoral immune response of the host. This immune response is likely to be mediated by a high number of tumor-specific antigens generated during MSI tumorigenesis. Interventions specifically targeting these antigens may be the basis for novel therapeutic strategies in MSI colorectal cancer and will be evaluated in clinical trials.

Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases.

Colorectal carcinomas (CRCs) with high microsatellite instability (MSI-H) share clinicopathological features distinctly different from their microsatellite stable (MSS) counterparts. Unlike MSS cancers, MSI-H CRCs occur predominantly in the right-sided colon and are often characterised by a strong lymphocyte infiltration. A poor differentiation pattern is found in most MSI-H CRCs, even though patients with MSI-H carcinomas seem to have a significantly longer survival after surgical resection. To clarify which factors contribute to the obvious paradoxon of a more favourable prognosis of MSI tumours, several clinical and histopathological features as well as the microsatellite status were evaluated in 120 colorectal cancer cases fulfilling clinical criteria (Bethesda) indicative for familial colorectal cancer. Microsatellite instability status and lymphocyte infiltration were related to tumour stage and patients' follow-up. Statistical analysis confirmed well-known relations, such as enhanced lymphocyte infiltration accompanied by Crohn's like reaction (CLR) in MSI-H cancers (CLR+ in 27 out of 47 MSI-H vs 14 out of 71 MSS CRCs, P<0.001). However, after stratification for depth of local invasion and penetration of the primary tumour, T3 tumours displaying MSI had a significantly lower rate of distant metastases (M1 in four out of 35 MSI-H vs 20 out of 41 MSS CRCs, P<0.001). A similar tendency was observed for CLR-positive CRCs (M1 in six out of 29 CLR+ vs 17 out of 45 CLR- CRCs, P=0.13). In a logistic regression model, the MSI-H phenotype and the presence of CLR were independent predictors of a low UICC stage (P=0.006 and 0.04, respectively). These data, together with the recent definition of highly immunogenic neo-antigens expressed in MSI-H tumour cells, suggest that MSI-H CRCs elicit a protective host response that may prevent metastasis formation.

[Molecular pathology in hereditary colorectal cancer. Recommendations of the Collaborative German Study Group on hereditary colorectal cancer funded by the German Cancer Aid (Deutsche Krebshilfe)]. / Molekularpathologische Diagnostik beim erblichen Dickdarmkarzinom. Empfehlungen und Resultate aus dem Verbundprojekt der Deutschen Krebshilfe "Krebsvorsorge und Krebsfrüherkennung bei Familiärem Darmkrebs".

Although twin studies indicate that inherited genetic factors contribute to about 35% of colorectal cancers (CRC), the exact genetic background has currently been elucidated in only 5-10% of cases. These comprise several hereditary cancer predisposition syndromes that present with a high number of syn- or metachronous neoplasms within an affected person and/or family. Many of these tumors exhibit typical histopathological changes. In general, one should discriminate between cancer syndromes associated with adenomatous and non-adenomatous (i.e., hamartomatous) polyps, the latter being quite rare. The patient's age often serves as a substantial hint to hereditary cancer. The next step of diagnostic work-up includes analysis of microsatellite instability (MSI) together with immunohistochemical detection of a loss of expression in one of the most frequently affected mismatch repair genes (MSH2, MSH6; MLH1, PMS2). Finally, the molecular demonstration of a gene mutation in the blood or germline is the most expensive and tedious procedure. This requires a signed informed consent from the patient after appropriate genetic counseling.

Significant increase of a specific variant TSG101 transcript during the progression of cervical neoplasia.

The human tumour susceptibility gene TSG101 has recently been identified on chromosomal locus 11p15.1-15.2 which is frequently affected by genetic alterations in neoplastic lesions of the uterine cervix. Aberrant transcripts of the TSG101 gene have been reported in various tumour entities, including breast, ovarian and prostate cancers, but also in several non-neoplastic tissues. We analysed TSG101 transcription by reverse transcription-polymerase chain reaction (RT-PCR) in a total of 139 clinical samples of cervical tissues and in cervical carcinoma cell lines. Variant transcripts were observed in all cell lines, in 69 of 122 (57%) cervical dysplasia and carcinoma samples and in five of 17 (29%) normal cervical tissues. One specific variant TSG101 transcript (delta 154-1054) was detected with a significantly increased frequency in advanced preneoplastic cervical lesions. However, the relative abundance of variant TSG101 transcripts appeared to be generally low, as only wild-type, but no variant transcripts were detectable in Northern blot analyses of cervical carcinoma cell lines. These data point to a progressive loss of stringent splice control functions or to extended alternative splicing in advanced dysplasia and neoplasia. The relative amounts of variant transcripts do not support a major functional role of TSG101 variants in cervical carcinogenesis.

Identical variant TSG101 transcripts in soft tissue sarcomas and various non-neoplastic tissues.

Inactivation of the TSG101 gene was recently shown to induce malignant transformation of NIH/3T3 fibroblasts. Abnormal TSG101 transcription profiles were observed in various human cancers, and large intragenic deletions of the TSG101 gene were reported for a series of human breast cancer specimens, pointing to a potential tumor-suppressive activity of TSG101. However, subsequent more detailed studies on a large panel of breast carcinoma samples did not confirm the tumor-associated genomic deletions. Here we analyzed the transcription patterns of the TSG101 gene in soft-tissue sarcomas and non-neoplastic human tissues. Forty-five of 71 soft tissue sarcoma samples (63%) displayed variant transcripts; however, identical aberrant transcripts were also detected in seven of 15 non-neoplastic control tissues. Restriction fragment length polymorphism analysis of the TSG101 gene excluded major genomic rearrangements in the soft tissue sarcoma samples. Northern blot analysis revealed a very low abundance of variant transcripts as compared with the wild-type TSG101 transcript. These data point to aberrant splicing of the TSG101 mRNA in normal and transformed human mesenchymal tissues rather than tumor specific alterations of the TSG101 gene. In summary, this analyses does not support a pathogenic role for altered TSG101 expression in human soft tissue sarcomas.