Oncolytic viral therapy for prostate cancer: efficacy of reovirus as a biological therapeutic.

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Morbidity in immunosuppressed (SCID/NOD) mice treated with reovirus (dearing 3) as an anti-cancer biotherapeutic.

Specific viral oncolysis of cancer cells has aroused great interest as a potential anti-cancer therapy. Reovirus was proposed as an anti-cancer biotherapeutic several years ago, as it elicits virus-mediated death of human cancer cells both in vitro and in mouse model systems. A common model system for reovirus oncolysis is the NOD/ LtSz-scid/scid (SCID/NOD) immunocomprimised mouse. While human tumour xenografts are effectively killed by intra-tumour injections of reovirus, the mice often exhibit discoloration and necrosis of extremities including feet, distal leg, tail and ears several weeks after injection. This phenomenon never occurs in sham-injected mice, nor is it observed in wild type or nude mice. The pathogenesis of this "Black Foot" lesion has not yet been described, but may be of relevance for future human studies of biotherapeutics. Examination of SCID/NOD mice was performed at various time points following intratumoral injection of reovirus. Immunohistological evaluation of tissues reveals infection of cardiac myocytes and venous endothelial cells at approximately 2 days post infection. Over time, venules and veins showed a mixed inflammatory vasculitis and thrombus formation. Synchronously, the heart showed diffuse myocyte death, with dystrophic calcification. The results indicate that the "Black Foot" syndrome is likely due to venous vasculitis secondary to reovirus infection, on a background of reovirus myocarditis and heart failure. The rationale for the selective susceptibility of venous over arterial endothelium to reovirus infection is currently unknown. The results of this study may be relevant to the use of oncolytic viruses, particularly reovirus, in the anti-cancer therapy of immunosuppressed patients.

Reovirus oncolysis as a novel purging strategy for autologous stem cell transplantation.

Hematologic stem cell rescue after high-dose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro, in vivo, and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. Human monocytic and myeloma cell lines as well as enriched ex vivo lymphoma, myeloma, and Waldenström macroglobulinemia patient tumor specimens were used in an experimental purging model. Viability of the cell lines or purified ex vivo tumor cells of diffuse large B-cell lymphoma, chronic lymphocytic leukemia, Waldenström macroglobulinemia, and small lymphocytic lymphoma was significantly reduced after reovirus treatment. Further, [35S]-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cellular proteins demonstrated reovirus protein synthesis and disruption of host cell protein synthesis as early as 24 hours. Admixtures of apheresis product with the abovementioned tumor cells and cell lines treated with reovirus showed complete purging of disease. In contrast, reovirus purging of enriched ex vivo multiple myeloma, Burkitt lymphoma, and follicular lymphoma was incomplete. The oncolytic action of reovirus did not affect CD34+ stem cells or their long-term colony-forming assays even after granulocyte colony-stimulating factor (G-CSF) stimulation. Our results indicate the ex vivo use of an unattenuated oncolytic virus as an attractive purging strategy for autologous stem cell transplantations.