Distinct merkel cell polyomavirus molecular features in tumour and non tumour specimens from patients with merkel cell carcinoma.

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.

Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma.

To investigate whether integration of HPV DNA in cervical carcinoma is responsible for structural alterations of the host genome at the insertion site, a series of 34 primary cervical carcinomas and eight cervical cancer-derived cell lines were analysed. DNA copy number profiles were assessed using the Affymetrix GeneChip Human Mapping 250K Sty array. HPV 16, 18 or 45 integration sites were determined using the DIPS-PCR technique. The genome status at integration sites was classified as follows: no change, amplification, transition normal/gain, normal/loss or gain/LOH. A single HPV integration site was found in 34 cases; two sites were found in seven cases; and three sites in one case (51 sites). Comparison between integration sites and DNA copy number profiles showed that the genome status was altered at 17/51 (33%) integration sites, corresponding to 16/42 cases (38%). Alterations detected were amplification in nine cases, transition normal/loss in four cases, normal/gain in three cases, and gain/LOH in one case. A highly significant association was found between genomic rearrangement and integration of HPV DNA (p < 10(-10)). Activation of the replication origin located in viral integrated sequences in a cell line derived from one of the primary cervical carcinomas induced an increase of the amplification level of both viral and cellular DNA sequences flanking the integration locus. This mechanism may be implicated in the triggering of genome amplification at the HPV integration site in cervical carcinoma. Structural alterations of the host genome are frequently observed at the integration site of HPV DNA in cervical cancer and may act in oncogenesis.

Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis.

Merkel cell carcinoma (MCC), a skin tumour with neuroendocrine features, was recently found to be associated with a new type of human polyomavirus, called Merkel cell virus (MCV). We investigated the specificity of this association as well as a causal role of MCV in oncogenesis. DNA and RNA from ten cases of MCC were analysed using PCR and RT-PCR. DNA from 1241 specimens of a wide range of human tumours was also analysed. The DIPS technique was used to identify the integration locus of viral DNA sequences. Array CGH was performed to analyse structural alterations of the cell genome. MCV DNA sequences were found in all ten cases of MCC and in none of the 1241 specimens of other tumour types. Clonal integration of MCV into the host genome was seen in all MCC cases and was checked by FISH in one case. A recurrent pattern of conserved viral sequences which encompassed the replication origin, the small tumour (ST), and the 5' part of the large tumour (LT) antigen DNA sequences was observed. Both ST and LT viral sequences were found to be significantly expressed in all MCCs. Neither recurrent site of integration nor alteration of cellular genes located near the viral sequences was observed. The tight association of MCV with MCC, the clonal pattern of MCV integration, and the expression of the viral oncoproteins strongly support a causative role for MCV in the tumour process. This information will help the development of novel approaches for the assessment and therapy of MCC and biologically related tumours.

Identification of a proliferation gene cluster associated with HPV E6/E7 expression level and viral DNA load in invasive cervical carcinoma.

Specific HPV DNA sequences are associated with more than 90% of invasive carcinomas of the uterine cervix. Viral E6 and E7 oncogenes are key mediators in cell transformation by disrupting TP53 and RB pathways. To investigate molecular mechanisms involved in the progression of invasive cervical carcinoma, we performed a gene expression study on cases selected according to viral and clinical parameters. Using Coupled Two-Way Clustering and Sorting Points Into Neighbourhoods methods, we identified a 'cervical cancer proliferation cluster' composed of 163 highly correlated transcripts. Most of these transcripts corresponded to E2F pathway genes controlling cell division or proliferation, whereas none was known as TP53 primary target. The average expression level of the genes of this cluster was higher in tumours with an early relapse than in tumours with a favourable course (P = 0.026). Moreover, we found that E6/E7 mRNA expression level was positively correlated with the expression level of the cluster genes and with viral DNA load. These findings suggest that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV expression level may thus provide a biological marker useful for prognosis assessment and specific therapy of the disease.

Increased risk of systemic relapses associated with bone marrow micrometastasis and circulating tumor cells in localized ewing tumor.

PURPOSE: The presence of metastasis is a major prognostic factor in Ewing tumor (ET). The relapse pattern of patients with localized tumors has long indicated that cases with disseminated ET cells escape detection at diagnosis. ET cells are characterized by specific gene fusions that can be detected with high sensitivity and specificity by reverse transcriptase polymerase chain reaction (RT-PCR). PATIENTS AND METHODS: RT-PCR targeting EWS-FLI-1 or EWS-ERG transcripts was used to search for occult tumor cells in peripheral blood (PB) and bone marrow (BM) at diagnosis in 172 patients with ET, and the prognostic significance of this parameter was assessed. RESULTS: As we suggested previously in a smaller series of patients, RT-PCR positivity of the BM was correlated with a high risk of adverse outcome in the overall study population (P =.007). More interestingly, among patients with otherwise localized tumors, BM micrometastasis also predicted significantly poorer disease-free survival rates (P =.043). The presence of circulating tumor cells (CTC) was more frequently observed in patients with large tumors (P =.006). CTC were associated with a poor outcome among patients with clinically localized disease (P =.045). Patients with clinically localized disease and peripheral occult tumor cells as evidenced by BM and/or PB RT-PCR positivity had axial or proximal tumors and experienced relapses at a systemic rather than at a local level. CONCLUSION: Patients with localized ET and BM micrometastasis or CTC are comparable to patients with metastases in terms of the localization of the primary tumor, outcome, and relapse pattern.

Molecular detection of the synovial sarcoma translocation t(X;18) by real-time polymerase chain reaction in paraffin-embedded material.

The t(X;18) translocation is known to be a useful marker for the diagnosis of synovial sarcoma. In this study, the authors describe a new real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect SYT/SSX fusion transcripts using paraffin-embedded and frozen tumor specimens. A series of 38 soft tissue sarcomas were analyzed. Diagnosis was based on clinical, histologic, and immunohistochemical examination. The fusion transcripts were detected in 16 of 17 synovial sarcoma samples (the 17th sample was not suitable for molecular analysis). No t(X;18)-fusion transcript was PCR-amplified in the 21 nonsynovial sarcoma mesenchymal tumors. Therefore, real-time PCR amplification appears to be a powerful, rapid, specific, and sensitive technique that can be used routinely to diagnose the synovial sarcoma t(X;18) translocation. In addition, the t(X;18) can be detected not only on frozen but also on paraffin-embedded tumor samples.

Human papillomavirus mutational insertion: specific marker of circulating tumor DNA in cervical cancer patients.

INTRODUCTION: In most cases of cervical cancers, HPV DNA is integrated into the genome of carcinoma cells. This mutational insertion constitutes a highly specific molecular marker of tumor DNA for every patient. Circulating tumor DNA (ctDNA) is an emerging marker of tumor dynamics which detection requires specific molecular motif. To determine whether the sequence of the cell-viral junction could be used in clinical practice as a specific marker of ctDNA, we analyzed a series of cervical cancer patient serums. METHODS AND FINDINGS: Serum specimens of 16 patients diagnosed with HPV16/18-associated cervical cancer, and for which the viral integration locus had been previously localized, were analyzed. Sequential serum specimens, taken at different times during the course of the disease, were also available for two of these cases. ctDNA was found in 11 out of 13 patients with tumor size greater than 20 mm at diagnosis, and analysis of sequential serum specimens showed that ctDNA concentration in patients serum was related to tumor dynamics. CONCLUSIONS: We report that HPV mutational insertion constitutes a highly specific molecular marker of ctDNA in HPV-associated tumor patients. Using this original approach, ctDNA was detected in most cervical cancer patients over stage I and ctDNA concentration was found to reflect tumor burden. In addition to its potential prognostic and predictive value, HPV mutation insertion is likely to constitute a new molecular surrogate of minimal residual disease and of subclinical relapse in HPV-associated tumor. This is of major importance in the perspective of specific anti-HPV therapy.

High-resolution mapping of amplicons of the short arm of chromosome 1 in two neuroblastoma tumors by microarray-based comparative genomic hybridization.

Deletion of chromosome arm 1p is one of the most frequent genetic alterations in neuroblastoma. However, using conventional comparative genomic hybridization, we have observed amplifications on 1p in 2 neuroblastoma tumors at bands 1p34.2 and 1p36.3, respectively. Using a medium-resolution genomic array containing 178 PACs/BACs from 1p and then 2 high-resolution arrays containing contigs of overlapping PACs/BACs from the amplified regions, we could precisely map and delineate both amplicons. The 1p34.2 amplicon appeared as a homogeneous amplification unit, whereas the 1p36.3 amplicon had a more complex structure, with 2 noncontiguous, highly amplified regions and several moderate amplification units. In this case, fluorescence in situ hybridization analysis confirmed the amplification of several clones and indicated that the 2 highest amplification units corresponded to 2 populations of double minute chromosomes, one of which also contained the MYCN locus. This is the first report of 1p amplifications in primary neuroblastomas.