Virus-negative Tumors Research Articles

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402-restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402-restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients.

Human papilloma virus: A new risk factor in a subset of head and neck cancers

Head and neck cancer is the sixth most common malignancy worldwide. Tobacco smoking and alcohol consumption are two well known behavioral risk factors associated with head and neck cancer. Recently, evidence is mounting that infection with human papilloma virus, most commonly human papilloma virus-16 is responsible for a subset of head and neck squamous cell carcinoma especially tumors of tonsillar origin. The molecular pathway used by human papilloma virus to trigger malignant transformation of tissue is different from that of other well known risk factors, i.e. smoking and alcohol, associated with squamous cell carcinoma. Apparently, these subsets of patients with human papilloma virus positive tumor are more likely to have a better prognosis than human papilloma virus negative tumor. Considering this fact, the human papilloma virus infection should be determined in all oropharyngeal cancers since it can have a major impact on the decision making process of the treatment.

Challenges of integrating chemotherapy and targeted therapy with radiation in locally advanced head and neck squamous cell cancer

The curative treatment of locally advanced head and neck squamous cell cancer has advanced greatly in recent years, with the establishment of standard of care indications for chemoradiation (CRT). At the same time, there have been advances in each modality, including intensity-modulated radiation therapy, sequential chemotherapy and more tailored combination therapies. However, with new therapies come new challenges. This review will discuss some of the novel approaches to treating head and neck squamous cell cancer, particularly the introduction of biological agents into treatment paradigms, and some of the challenges arising as the field advances. A number of recent clinical trials have focused on reducing the disadvantages of concurrent CRT, specifically acute toxicity, lack of compliance and potential for late effects affecting quality of life and function. In particular, the use of biological agents as radiosensitizers has led to the investigation of new combination therapies, such as epidermal growth factor receptor inhibitors administered concurrently with CRT. These new therapies have potential for improving overall survival and lowering locoregional recurrence rates. Combination therapies hold promise for improving outcomes of patients with head and neck squamous cell cancer, both human papilloma virus-associated and human papilloma virus-negative tumors. The introduction of intensity modulated radiation therapy and biological agents into CRT treatment approaches may reduce some of the disadvantages of more traditional radiation and CRT treatments. Although many challenges remain, the possibility of improving survival with reduced toxicity through treatment selection based on risk stratification and prognostic biomarkers is incrementally evolving.

Epstein–Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells

The B cell lymphomas associated with Epstein–Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus–tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

58 Evidence for a Novel Role for Histamine Regulation of Cholangiocarcinoma Growth By An Autocrine Mechanism

correlated these data with the simultaneous presence of hepatitis B and C (HBV and HCV) infection.Methods: We quantified methylation levels at 19 CpG loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC, CDH1, RUNX3, RIZ1, SFRP2, MINT31, COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) using combined bisulfite restriction analysis (COBRA). In total, 81 advanced tumors (well-differentiated HCCs >2 cm in size or moderately-poorly differentiated HCC), 12 early stage tumors (dysplastic nodules or well-differentiated HCC 2 cm size showing denser methylation compared with early tumors at these CpG loci. Additionally, HCV-related tumors tended to carry higher methylation levels at Group3 loci compared with HBV-related and virus-negative tumor, in both early and advanced tumors. Conclusions: Aberrant methylation is a frequent event in the liver and sequentially progresses from non-cancerous liver to early stage cancers and finally to advanced HCC. The cumulative differences in methylation levels among various liver tissues (precancerous livers, early and advanced tumors) suggest that aberrant methylation is an early event in HCC that progresses with the advancing stage. Lastly, simultaneous HCV infection may accelerate the carcinogenetic process in HCC.

P16INK4A Hypermethylation Is Associated with Hepatitis Virus Infection, Age, and Gender in Hepatocellular Carcinoma

The tumor suppressor gene p16INK4A is mainly inactivated by an epigenetic change involving promoter hypermethylation in hepatocarcinogenesis. The possible clinical impact of p16INK4A methylation and the potential risk factors for this epigenetic alteration have not been thoroughly investigated. We studied the methylation status and mRNA and protein expression of p16INK4A in 50 hepatocellular carcinomas and corresponding nonneoplastic liver lesions using methylation-specific PCR, reverse transcription-PCR, and immunohistochemical techniques. p16INK4A hypermethylation was observed in 58% (29 of 50) of the hepatocellular carcinomas and 16% (6 of 38) of the corresponding chronic hepatitis and cirrhosis tissue samples. p16INK4A methylation was significantly associated with mRNA and protein expression (P <0.001 and P=0.003, respectively). All of the p16INK4A-methylated tumors were positive for hepatitis B virus or hepatitis C virus markers, but none of the virus-negative tumors exhibited p16INK4A methylation (P=0.006). The frequency of p16INK4A hypermethylation tended to be higher in hepatitis C virus-related tumors (23 of 32, 72%) than in hepatitis B virus-related tumors (6 of 13, 46%; P=0.1). Aberrant methylation of p16INK4A was also related significantly to increasing age, female gender, and normal levels of serum PIVKA-II (P=0.02, 0.04, and 0.04, respectively). No statistically significant difference in survival was observed between patients with p16INK4A hypermethylation and those without. Our observations suggest that p16INK4A hypermethylation may contribute to hepatocarcinogenesis from an early stage and that multiple risk factors, such as viral infections, age, and gender, may be associated with p16INK4A hypermethylation in hepatocarcinogenesis.

Human Herpesvirus 8–Associated Solid Lymphomas that Occur in AIDS Patients Take Anaplastic Large Cell Morphology

Human herpesvirus type 8 (HHV-8; Kaposi's sarcoma–associated herpesvirus) is a recently isolated human herpesvirus frequently identified in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Here we report three cases of HHV-8–bearing solid lymphomas that occurred in AIDS patients (Cases 1–3). All three patients were homosexual men presenting extranodal masses in the lungs (Case 1) or skin (Cases 2 and 3), together with the presence of Kaposi's sarcoma (Case 1), primary effusion lymphoma (Case 2), or multicentric Castleman's disease (Case 3). These solid lymphomas exhibited anaplastic large cell morphology and expressed CD30, corresponding to the recent diagnostic criteria of anaplastic large cell lymphoma (ALCL). The chromosomal translocation t(2;5)-associated chimeric protein p80NPM/ALK was not observed in any of these cases. HHV-8 was detected in all of these cases by polymerase chain reaction, immunohistochemistry of HHV-8–encoded ORF73 protein, and in situ hybridization of T1.1. Epstein-Barr virus was detected only in Cases 2 and 3 by in situ hybridization. It is interesting that inoculation of a cell line obtained from a primary effusion lymphoma cell in Case 2 to severe combined immunodeficiency mice produced HHV-8–positive and Epstein-Barr virus–negative tumors in inoculated sites. These tumor cells exhibited phenotypes of ALCL that were identical to the subcutaneous tumor cells of this particular patient. These findings clearly show that HHV-8 can associate with solid lymphomas and that it can take anaplastic large cell morphology. Those lymphomas should be distinguished from the classical ALCL as were defined by the revised European-American classification of lymphoid neoplasms even though morphology and a part of immunophenotype mimic that of classical ALCL.

Cancer genes generated by rare chromosomal rearrangements rather than activation of oncogenes.

The 20 known transforming onc genes of retroviruses are defined by sequences that are transduced from cellular genes, termed proto-oncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox since such genes are clearly undesirable. The hypothesis predicts (i) that viral onc genes and proto-onc genes are isogenic, (ii) that expression of proto-onc genes induces tumors, (iii) that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes, and (iv) that diploid tumors exist that differ from normal cells only in transcriptionally or mutationally activated proto-onc genes. As yet, none of these predictions is confirmed. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore the hypothesis, that proto-onc genes are latent cellular oncogenes, appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germline configuration of cellular genes, generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumor cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumors are initiated with, and possibly by, these abnormalities as predicted by Boveri in 1914 (Zur Frage der Entstehung maligner Tumoren, Jena, Fischer).

Cancer genes: rare recombinants instead of activated oncogenes (a review).

The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts that viral onc genes and proto-onc genes are isogenic; that expression of proto-onc genes induces tumors; that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: Structural comparisons between viral onc genes, essential retroviral genes, and proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. Proto-onc genes are frequently expressed in normal cells. To date, not one activated proto-onc gene has been isolated that transforms diploid cells. Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumor cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumors are initiated with, and possibly by, these abnormalities, as predicted by Boveri in 1914.

Are activated proto-onc genes cancer genes?

The main objective of cancer molecular biology is to identify cancer genes. Despite fierce efforts, this objective has still not been met [1–3]. As yet the only known cancer genes are the transforming onc genes of retroviruses. Typically these viruses initiate and maintain cancers with autonomous transforming genes that are dominant in susceptible cells [5]. The discovery of Single gene determinants of cancer in retroviruses has become a precedent that has infected cancer gene research. It has made retroviral onc genes the favorite models of cellular oncogenes, although the relevance of single-gene models to virus-negative tumors is as yet unknown. Fortunately, onc genes are either detrimental or at least useless to the viability of the virus and thus are not maintained by retroviruses. They are the produets of rare, genetic accidents, generated by illegitimate recombinations between retroviruses and cellular genes, termed proto-onc genes.KeywordsBurkitt LymphomaTransforming GeneCellular OncogeneMurine Sarcoma VirusAvian Erythroblastosis VirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Many murine tumor models associated with murine leukemia virus(es) (MuLV) have been successfully treated by passive administration of antiviral antibodies. There is a large body of virus-negative tumors, however, which are lowly antigenic and thus refractory to such approaches. Therefore, an investigation was done for determination of whether such tumors could be rendered susceptible to passive serum therapy by introduction of MuLV antigens onto the tumor cell surface. For this purpose a 3-methylcholanthrene-induced fibro-sarcoma from inbred C57BL/6J mice was chosen. Following infection of the tumor in vitro with Friend murine leukemia virus (F-MuLV), the tumor was found to be susceptible to treatment with a high-titered heterologous anti-F-MuLV gp71 antiserum. The specificity of the treatment was determined by conduction of the therapy on the uninfected parental tumor, in which case there was no effect. In addition, therapy could be initiated at time points when demonstrable tumors were present and successfully treated animals were resistant to rechallenge with the infected tumor. Thus conversion of a lowly antigenic virus-negative tumor to an MuLV-positive antigenic tumor rendered such growths susceptible to immunologic management.

In Vivo Antigenic Modification of Tumor Cells. I. Introduction of Murine Leukemia Virus Antigens on Non-Virus-Producing Murine Sarcomas23

Murine oncovirus antigens represent excellent targets for immune recognition, and virus-associated tumors are generally susceptible to various immunotherapy protocols. Virus-negative tumors, however, are nonimmunogenic and refractory to immunologic control. Therefore, the feasibility of the introduction of antigens onto non-virus-expressing tumors in situ in inbred C57BL/6J mice by systemic administration of nononcogenic murine retroviruses was investigated. Two classes of murine fibrosarcomas were studied: a 3-methylcholanthrene-induced fibrosarcoma syngeneic to C57BL/6 mice (MCA-FS) and a Harvey murine sarcoma virus-transformed, nonproducer fibrosarcoma syngeneic to C57BL/6 mice (H-NP). Both were found to be devoid of infectious ecotropic murine leukemia virus (MuLV) or MuLV antigens. A single dose of Friend murine leukemia virus (F-MuLV) was used to superinfect MCA-FS- and H-NP-induced tumors in vivo and converted these tumors to a highly productive, virus-positive state. In vivo superinfected tumors were indistinguishable from their preinfected counterparts by competition radioimmunoassays for the virion's major envelope glycoprotein, gp71, and its group-specific antigen, p30, and by assays for infectious virus. Analysis of virus from tumor extracts proved that the antigenic specificity of the superinfected tumor was provided by F-MuLV administered systemically to the animals. Finally, an immunoperoxidase technique, applied to tumor cross sections, demonstrated the uniform appearance of viral antigens in the superinfected tumors.

Specificity of adoptive chemoimmunotherapy of established syngeneic tumors.

To examine the specificity of adoptive chemoimmunotherapy (ACIT) of established syngeneic tumors, two noncross-reactive C57BL/6 tumors were studied: a Friend virus-induced tumor (FBL-3) and a chemically induced virus-negative tumor EL-4(G-). In vitro studies confirmed that these tumors are antigenically distinct by demonstrating that the cytotoxic responses of spleen cells from mice immunized in vivo and reexposed to tumor in vitro are immunologically specific. Studies of ACIT with cells from mice immunized in vivo demonstrated similar specificity. Mice receiving 5 x 10(6) FBL-3 on day 0 all died by day 13. Treatment on day 5 with cyclophosphamide (CY), 180 mg/kg, prolonged the median survival time (MST) to day 23. Treatment on day 5 with CY plus 2 x 10(7) normal nonimmune C57BL/6 cells or CY plus cells sensitized to EL-4(G-) had no additional effect on survival whereas 2 x 10(7) C57BL/6 cells sensitized to FBL-3 in vivo prolonged MST to day 64 and cured 13 of 32 mice. Similarly, mice given 2 x 10(5) EL-4(G-) on day 0 all died by day 16, and CY on day 5 prolonged the MST to day 22. As an adjunct to CY, 2 X 10(7) normal cells or cells sensitized to FBL-3 had a modest effect, prolonging the MST to days 37 and 36, respectively. However, treatment with CY plus 2 x 10(7) cells immune to EL-4(G-) cured 22 of 32 mice. The results demonstrate the immunologic specificity of ACIT of syngeneic tumors treated with immune syngeneic cells.

Expression of feline type‐C virus in normal and tumor tissues of the domestic cat

Tumor and normal tissues from domestic cats were examined for FeLV and RD-114 specific nucleic acids and proteins. Virus-positive tissues showed a 100- to 200-fold higher level of FeLV and a 200- to 400-fold higher level of FeLV p30 than virus-negative tumors and normal cat tissues. In contrast, the levels of RD-114 viral RNA and p30 antigen were comparable in the majority of tumorous and normal tissues examined. When the DNA of these tissues was examined for FeLV-related sequences, the virus-positive tumors were found to contain extra copies of FeLV DNA. Virus-negative tumors contained DNA sequences homologous with FeLV but the copy number of these sequences could not be distinguished from normal tissues. The results indicate that in certain FeLV-induced type-C lymphoproliferative diseases, extra type-C sequences not present in normal tissues can be identified.