[Human papillomavirus and squamous cell cancer of the head and neck region : Prognostic, therapeutic and prophylactic implications]. / Humane Papillomviren bei Plattenepithelkarzinomen der Kopf- und Halsregion : Relevanz für Prognose, Therapie und Prophylaxe.

Human papilloma viruses (HPV) are responsible for approximately half of all oropharyngeal squamous cell carcinomas (OPSCC) and incidence rates of HPV-associated OPSCC continue to increase substantially. The defined viral carcinogenesis permits development of specific diagnostic, therapeutic, and prophylactic approaches. Laboratory identification of HPV-associated OPSCC may be achieved by p16(INK4a) immunohistochemistry combined with HPV DNA detection by polymerase chain reaction (PCR) using tumor tissue. Patients with HPV-associated OPSCC have a relatively good prognosis; therefore, the HPV status plays an important role in patient guidance. Due to the relatively favorable prognosis, ongoing studies are evaluating whether less rigorous therapy for HPV-positive patients results in equally good cure rates. The criteria for patient selection are, however, still uncertain. Particularly markers for detection of HPV-positive patients with a high risk of treatment failure are lacking. Besides tumor stage and comorbidities, distinct genomic, epigenetic, and immunologic alterations are prognostically relevant for HPV-associated OPSCC, and might be of predictive value. Furthermore, the characteristic molecular alterations suggest the possibility of novel vigilant and specific therapy approaches. These may be inhibitors of the phosphatidylinositol 3­kinase (PI3K) pathway, which is frequently activated in HPV-associated OPSCC, and immunotherapeutic methods, e. g., therapeutic vaccination. Although prophylactic HPV vaccinations may also prevent development of HPV-associated OPSCC, foreseeable effects on OPSCC incidence will be low, given the low vaccination rates in Germany. This highlights the fact that interdisciplinary research networks should enhance the necessary activities related to HPV-associated OPSCC.

GHSR DNA hypermethylation is a common epigenetic alteration of high diagnostic value in a broad spectrum of cancers.

Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Here, we report a DNA methylation mark that is characteristic of seven studied malignancies, namely cancers of lung, breast, prostate, pancreas, colorectum, glioblastoma and B cell chronic lymphocytic leukaemia (CLL) (n = 137). This mark was defined by substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) through bisulfite pyrosequencing. The degree of aberrant methylation was capable of accurate discrimination between cancer and control samples. The highest sensitivity and specificity of cancer detection was achieved for cancers of pancreas, lung, breast and CLL yielding the area under the curve (AUC) values of 1.0000, 0.9952, 0.9800 and 0.9400, respectively. Narrowing to a single CpG site within the gene's promoter or four consecutive CpG units of the highest methylation levels within the first exon improved the detection power. GHSR hypermethylation was detected already at the early stage tumors. The accurate performance of this marker was further replicated in an independent set of pancreatic cancer and control samples (n = 78). These findings support the candidature of GHSR methylation as a highly accurate pan-cancer marker.

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.

[HPV-associated carcinomas of the female genital tract. Molecular mechanisms of development]. / HPV-assoziiertes Karzinom des weiblichen Genitaltrakts. Molekulare Mechanismen der Entstehung.

Infections with human papillomaviruses (HPV) are a common occurrence in both men and women. In contrast HPV-associated neoplasias are relatively rare and occur only in certain areas of the body. The virus has obviously developed efficient mechanisms for its persistence without inducing too much damage to the host. The formation of neoplasia seems to be more an exception. Epigenetic mechanisms play an important role in the regulation of viral gene expression. Investigations have indicated that exactly the transition from the permissive infection stage to a transformation stage, where neoplastic alterations can occur due to expression of the viral oncogenes, is associated with certain methylation patterns of the viral genome which promote the expression of the oncogenes E6 and E7. The transforming stage is seen as the actual carcinogenic event and can be immunohistochemically detected by the biomarker p16(INK4a).

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.

[Human papillomaviruses in the pathogenesis of intraepithelial neoplasia (AIN) and carcinoma of the anus]. / Humane Papillomviren in der Pathogenese der intraepithelialen Neoplasien (AIN) und Karzinome des Anus.

HPV infections have been implicated in the pathogenesis of anal cancers. The mode of infection and subsequent transformation resembles very much the pathogenesis of cervical and other HPV-associated cancers. The molecular dissection of individual steps required to achieve cellular transformation within an HPV-infected cell led to the identification of novel biomarkers that make it possible to identify HPV-transformed cells with substantially higher precision in comparison to conventional methods. Since effective antiretroviral therapy allows for possible long-term survival of HIV-infected individuals who are at very high risk to develop HPV-associated cancers in the anogenital tract, these new developments have become increasingly relevant for practicing dermatologists and proctologists. We here briefly review the basic concepts and some clinical applications of this recent research.

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.

[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.

Overexpression of a member of the pentraxin family (PTX3) in human soft tissue liposarcoma.

A unique feature of human soft tissue liposarcoma is a stable (12;16)(q13;p11) translocation observed mainly in myxoid and roundcell liposarcomas. This translocation results in FUS/CHOP fusion transcripts with a corresponding oncogenic protein. We hypothesised that genes downstream of FUS/CHOP might serve as attractive candidates for novel tumour associated antigens. Among a panel of analysed genes, only pentraxin related gene (PTX3) demonstrated high expression in liposarcomas as compared to normal tissues. The analysis of RNA and protein expression demonstrated concordant results. However, the level of RNA and protein overexpression did not correlate in all cases. Finally, PTX3 expression was not related to presence of a FUS/CHOP fusion transcript within the liposarcoma tissues. PTX3 has been associated with adipocyte differentiation and now, additionally, is characterised by a markedly increased expression in human soft tissue liposarcoma. This finding mandates further research efforts to clarify the exact role of PTX3 in liposarcoma oncogenesis.

The p53 codon 72 variation is associated with the age of onset of hereditary non-polyposis colorectal cancer (HNPCC).

The polymorphic variants at codon 72 of the p53 gene were shown to be functionally distinct in vitro, whereby the arginine (arg) variant induces apoptosis more efficiently than the proline (pro) variant. From the evidence that the DNA mismatch repair system and p53 interact to maintain genomic integrity, we hypothesized that the codon 72 variation may influence the age of onset of disease in HNPCC patients. We tested 538 patients for p53 codon 72 variants, including 167 unrelated patients with pathogenic germline mutations in MSH2 or MLH1 and colorectal carcinoma as first tumour, 126 patients with sporadic microsatellite stable colorectal cancers, and 245 healthy controls. The median age of onset was 41, 36, and 32 years for MSH2 or MLH1 mutation carriers with arg/arg, arg/pro, and pro/pro genotypes, respectively. The log rank test revealed significant differences in the age of onset between arg/arg and pro/pro individuals (p = 0.0002) and in arg/pro versus arg/arg and pro/pro individuals (p = 0.0026 and p = 0.0217, respectively). A Cox regression model indicated an additive mode of inheritance. No significant differences in age of onset were observed among different genotype carriers with microsatellite stable tumours. Our results suggest that p53 codon 72 genotypes are associated with the age of onset of colorectal carcinoma in a mismatch repair deficient background in a dose dependent manner. These findings may be relevant for preventive strategies in HNPCC.

[Molecular pathogenesis of cervical cancer and its first steps]. / Molekulare Pathogenese des Zervixkarzinoms und seiner Vorstufen.

Specific types of the human papillomaviruses (high risk human papillomaviruses) play an essential role in the pathogenesis of cervical cancer. Although infections by these viruses are very common in the general population, only few result in clinically relevant lesions. Continuous and deregulated expression of two viral oncoproteins E6 and E7 in basal or parabasal cells are required to induce and maintain neoplastic growth. In the course of an acute HPV-infection these genes are exclusively expressed in cell cycle arrested, terminally differentiated cells in the intermediate or superficial layers of the epithelium. Accidental activation of these genes in proliferating cells in the basal or parabasal cell layers results in interference with the cell cycle regulation, disturbances of the mitotic spindle apparatus and centrosome functions. This results in numerical and structural chromosome aberrations, chromosomal instability, increasing aneuploidy and initiates cervical carcinogenesis. The deregulated expression of the viral oncogene E7 is indicated by strong over-expression of the cellular p16(INK4a) gene product. This finding may have significant influence on novel strategies in cervical cancer diagnosis and screening. This review summarizes the basic molecular mechanisms of how papillomaviruses contribute to cellular transformation and how this can influence future diagnostic applications.

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.

[Viral carcinogenesis of head and neck tumors]. / Virale Kanzerogenese von Kopf-Hals-Tumoren.

Approximately 15% of malignant diseases are caused by infectious agents. Human papilloma viruses (HPV) can be frequently found in oral carcinomas, especially tonsillar cancer. A group of HPV-infected tumors shows clear signs for a virally induced transformation process: high-risk HPVs can be detected in all tumor cells, the viral oncogenes E6 and E7 are constantly expressed and lead to upregulation of cellular p16(INK4a), a cyclin-dependent kinase inhibitor. The patients frequently lack typical risk factors associated with head and neck cancers such as drinking and smoking. Epstein-Barr viruses (EBV) are associated with lymphoproliferative disorders and cause nasopharyngeal carcinoma (NPC). NPC has a high incidence in some East Asian countries. In this review, the molecular pathogenesis of HPV- and EBV-associated malignancies are described and the clinical relevance of the presented findings is discussed.

Hematogenous tumor cell dissemination during colonoscopy for colorectal cancer.

BACKGROUND: It has long been suspected that mechanical influences may enhance the release of viable colorectal cancer cells into the circulation. The objective of this study was to determine the extent of hematogenous tumor cell spread in colorectal cancer patients during colonoscopy. METHODS: Peripheral venous blood samples were taken before and after colonoscopy from 44 patients with colorectal cancer. Blood samples were examined using a reverse-transcriptase polymerase chain reaction assay to amplify cytokeratin 20 transcripts. RESULTS: Eleven patients with colorectal cancer displayed circulating tumor cells before and after colonoscopy (25%), whereas tumor cells were detected in six of 44 patients (14%) only after the procedure (p = 0.03, McNemar's test: tumor cell detection before after colonoscopy). All control samples consistently tested negative. CONCLUSIONS: Mechanical forces may result in enhanced release of viable colorectal cancer cells into the circulation; however, the clinical significance of these results needs to be clarified.

Detection of disseminated tumour cells as a potential surrogate-marker for monitoring palliative chemotherapy in colorectal cancer patients.

In a pilot study the effect of palliative chemotherapy on the detection rates of circulating tumour cells in peripheral venous blood of stage IV colorectal cancer patients was investigated. The results indicate a recruitment of tumour cells during chemotherapy and suggest a poorer survival for tumour cell positive patients. Circulating tumour cells have been shown to be a potential prognostic factor in patients who undergo curative resection for colorectal cancer. The effect of chemotherapy on the detection rates of disseminated tumour cells in blood has not yet been adequately investigated. Objective of this pilot-study was to analyze circulating tumour cells in peripheral venous blood of colorectal cancer patients undergoing chemotherapy in order to evaluate its potential value as a surrogate-marker for predicting clinical outcome after chemotherapy. Our hypothesis was that chemotherapy results in a reduction of the detection rates of circulating tumour cells in colorectal cancer patients. Forty-two Stage IV patients were examined at three different time points before and during palliative chemotherapy for the presence of disseminated tumour cells, using a previously described RT-PCR-assay for cytokeratin 20. 80.1% of the patients showed disseminated tumour cells at least once. Before chemotherapy, patients with multi-organ metastases were positive in 62.5%, patients with locally limited disease in only 14.3%. After the first chemotherapy, the detection rates in the latter group increased to 62.5%, for all patients in the same time from 46.3% to 57.5%. Clinical therapy responders showed an increase in the detection rates from 28.5% before to 71.4% after chemotherapy. In contrast, chemotherapy had no effect on tumour cell detection rates of patients with progressive disease (57% before vs. 60% after therapy). Patients with detected circulating tumour cells showed a shorter overall survival than patients without circulating tumour cells (83 vs. 53 weeks). Clinical therapy responders on average lived only 3 weeks longer than non-responders. In contrast to the original hypothesis, our data suggest a recruiting of circulating tumour cells during chemotherapy in advanced colorectal cancer. Further investigations are needed to clarify the potential role of circulating tumour cells for monitoring chemotherapy in these patients.

New markers for cervical dysplasia to visualise the genomic chaos created by aberrant oncogenic papillomavirus infections.

Extensive research over the past 20 years provided strong evidence that persistent infections with high risk type human papillomaviruses (HR-HPVs) cause cervical cancer. However, depending on their age, more than 20% of normal women are infected with these viruses and only very few develop clinically relevant dysplastic lesions or even cancer. During an acute HPV infection, expression of viral genes, in particular the viral E6 and E7 oncogenes is restricted to differentiated epithelial cells, which lost the capability to replicate their genomes and are therefore at no further risk for acquiring functionally relevant mutations upon genotoxic damage. High grade cervical dysplasia, however, is initiated by deregulated expression of viral oncogenes in replicating epithelial stem cells. Here, the E6-E7 gene products submerge control of the cell cycle and mitotic spindle pole formation through complex interactions with various cellular protein complexes and induce severe chromosomal instability. The detailed molecular analysis of these interactions allowed to define new biomarkers for dysplastic cervical cells. E7 for example induces increasing expression of the cyclin dependent kinase inhibitor p16(ink4a) in dysplastic cells. This can be used to identify dysplastic cells in histological slides, cytological smears or samples taken for biochemical analyses with an yet unmet fidelity. Detection of specific viral mRNAs derived from integrated HPV genomes in advanced precancers can be used to identify lesions with a particularly high risk for progression into invasive carcinomas (APOT assay). These new markers will result in a modified classification of cervical precancers and improved screening assays. Here, we review the basic concept and potential clinical applications of these new developments.

[Glioblastoma multiforme as a manifestation of Turcot syndrome]. / Glioblastoma multiforme als Manifestation des Turcot-Syndroms.

Turcot's syndrome, clinically characterized by the coincident occurrence of primary tumors of the colon and the central nervous system, can genetically be divided into two syndromes: familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon carcinoma (HNPCC). In the present case, a 60-year-old patient with glioblastoma multiforme and a history of hereditary malignomas is described as an example of a HNPCC-associated Turcot's syndrome. New molecular biological methods and results give deeper insight into clinical syndromes, and the better understanding improves diagnostics, therapy, and outcome estimations, even in rare diseases. In the present case, a new germinal mutation could be identified.

Evaluation of Bethesda guidelines in relation to microsatellite instability.

PURPOSE: The Bethesda guidelines were developed for selection of patients whose tumors should be tested for high microsatellite instability. This study examined the validity of the different Bethesda criteria in relation to microsatellite instability status to simplify their use in clinical practice. METHODS: A total of 164 patients with colorectal or hereditary nonpolyposis colorectal cancer-associated cancers were registered on the basis of the Amsterdam criteria without age limitations (11 cases), multiple tumors (2 cases), the accumulation of colorectal cancer in the family (no first-degree relatives affected or the index patient's age up to 50 years; 45 cases), an early age at onset up to 50 years (13 cases), morphologic and histopathologic manifestations (right-sided colorectal cancer, mucinous undifferentiated histology; 1 case), and the Bethesda criteria (92 cases). The microsatellite instability status of tumors was determined using the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer marker reference panel. RESULTS: When applying all Bethesda criteria, high microsatellite instability tumors were identified in our hereditary nonpolyposis colorectal cancer registry with a sensitivity of 87 percent. Twenty-nine percent (27/92) of the Bethesda-positive patients displayed high microsatellite instability compared with 6 percent of patients (4/72) not meeting these criteria (P < 0.001). Only Bethesda Criteria 1, 3, and 4 showed a significantly different distribution of the microsatellite instability status when compared with those of the remaining patients registered (P < or = 0.001). These three criteria detected high microsatellite instability tumors in 48 percent (10/21), 50 percent (18/36), and 31 percent (21/67) of patients, respectively. When applying these criteria only, a cumulative detection rate of 77 percent of all (24/31) high microsatellite instability cases was found, thereby identifying 89 percent of high microsatellite instability tumors among the Bethesda-positive patients. Patients matching Criteria 1, 3, and 4 frequently showed hMSH2 or hMLH1 germline mutations and tumor-specific loss of protein expression. CONCLUSION: In our hereditary nonpolyposis colorectal cancer registry the complete Bethesda criteria showed the highest sensitivity to identify patients with high microsatellite instability tumors. However, for general medical practice outside academic centers, three criteria are reasonably accurate for adequate high microsatellite instability tumor selection.