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Oncolytic viruses are a promising method of cancer therapy, even for advanced malignancies. HF10, a spontaneously mutated herpes simplex type 1, is a potent oncolytic agent. The interaction of oncolytic herpes viruses with the tumor microenvironment has not been well characterized. We injected HF10 into tumors of patients with recurrent breast carcinoma, and sought to determine its effects on the tumor microenvironment. Six patients with recurrent breast cancer were recruited to the study. Tumors were divided into two groups: saline-injected (control) and HF10-injected (treatment). We investigated several parameters including neovascularization (CD31) and tumor lymphocyte infiltration (CD8, CD4), determined by immunohistochemistry, and apoptosis, determined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Median apoptotic cell count was lower in the treatment group (P ¼ 0.016). Angiogenesis was significantly higher in treatment group (P ¼ 0.032). Count of CD8-positive lymphocytes infiltrating the tumors was higher in the treatment group (P ¼ 0.008). We were unable to determine CD4-positive lymphocyte infiltration. An effective oncolytic viral agent must replicate efficiently in tumor cells, leading to higher viral counts, in order to aid viral penetration. HF10 seems to meet this criterion; furthermore, it induces potent antitumor immunity. The increase in angiogenesis may be due to either viral replication or the inflammatory response.

Mouse T-cell antigens Rt6.1 and Rt6.2 are glycosylphosphatidylinositol-anchored arginine-specific adenosine diphosphate (ADP)-ribosyltransferases. In the present study, we obtained evidence that an arginine-specific ADP-ribosyltransferase activity liberated from BALB/c mouse splenocytes by phosphatidylinositol-specific phospholipase C increased fivefold in the presence of dithiothreitol and that the activity was immunoprecipitated by polyclonal antibodies generated against recombinant rat RT6.1. When mouse Rt6.1 was expressed as a recombinant protein, the transferase activity of Rt6.1 was stimulated by dithiothreitol, and inhibited by N-ethylmaleimide, while activities of recombinant mouse Rt6.2 and the Glu-207 mutant of rat RT6.1 [Hara, N., Tsuchiya, M., and Shimoyama, M. (1996) J. Biol. Chem. 271, 29552-29555] were unaffected by either agent. In addition to four cysteine residues conserved among mouse Rt6 and rat RT6 antigens, Rt6.1 has two extra cysteine residues at positions 80 and 201. To investigate a contribution of these extra cysteines in mouse Rt6.1 to thiol dependency of Rt6.1 transferase activity, Cys-80 and Cys-201 of Rt6.1 were replaced with serine and phenylalanine, respectively, the corresponding residues of mouse Rt6. 2 and rat RT6.1. Transferase activity of the Phe-201 mutant of Rt6.1 lost thiol dependency while that of the Ser-80 mutant remained thiol-dependent. Thus, we conclude that mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase, and that Cys-201 confers thiol dependency on Rt6.1 transferase. Our study indicates that arginine-specific ADP-ribosyltransferase activity detected on BALB/c mouse splenocytes is attributed to Rt6.1 and that Rt6.1 differs from Rt6.2 in enzymatic property of the transferase and perhaps in immunoregulatory functions.

F i g. 2 Ab d o mi n a l C T. A：P l a i n C T s h o we d a s o l i d l e s i o n , 3. 0 c m i n d i a me t e r , i n t h e n e c k o f t h e g a l l b l ad d e r (a r r o w). B ：Th e s o l i d l e s i o n wa s n o t e n h a n c e d. F i g. 3 E R C s h o we d n o f l o w o f t h e c o n t r a s t me d i u m i n t o t h e g a l l b l a d d e r .

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Aim: HF10 is a naturally mutated oncolytic virus subtype of Herpesviridae which has a potent antitumor activity. There is no study evaluating the mechanism of action of the virus on the tumor tissue in actual clinical setting. Present study is unique in evaluating the impact of HF-10 treatment on tumor microenvironment from actual clinical setting of patients with recurrent breast cancers. Patients and Methods: 6 patients with recurrent breast cancer were recruited to clinical part of the study in order to evaluate the efficacy of HF10 therapy. The virus was delivered in various doses: single dose injection of 104 pfu/0.5 ml to patient 1, single dose injection of 105pfu/0.5ml to patient 2, 3 dose injection of 105pfu/0.5ml to patient 3, single dose injection of 5x105pfu/0.5 ml to patient 4 and 5, 3 doses injection of 5x105pfu/0.5 ml to patient 6. In the present study specimens from 5 patients were eligible for evaluation. The patient 6 had a fibrotic tissue remnant therefore was excluded from histopathological analysis. The specimens were divided into 2 groups; saline injected control group and HF10 injected treatment group. Investigated parameters included neovascularization (CD31 antigen) and tumor lymphocyte infiltration (CD8 and CD4 antigen) determined by immunohistochemistry and apoptosis determined by TUNEL assay. Results: Median apoptotic cell count was 25.6/hpf(x100) in treatment group where as 50.0/hpf(x100) in the control group (p=0.016). On the other hand CD31 traced neovascularization were significantly higher in treatment group when compared to the control group [medians 30.0/hpf(x100) versus 12.0/hpf (x100); p=0.032]. Median CD8 positive lymphocyte count infiltrating the tumors in the treatment and control groups were 75.0/hpf (x100) and 42.0/hpf (x100); respectively (p=0.008). We were unable to detect CD4 positive cells in both groups. Conclusions: Our results suggest that reduced apoptosis by HF10 results in a prominent oncolytic activity because apoptosis is a host cell defense mechanism that limits viral infection by shutting down the cellular machinery necessary for viral production. Reduced apoptosis causes efficient intratumoral propagation of HF10 and viral cycle leads to enhanced oncolysis. Wild-type herpes simplex virus type 1 virus cause some of its pathology by increasing the vascularity of infected tissue but uptil now we have encountered no data regarding impact HF10 on angiogenesis in human tissue. Most probable explanation would be the inflammatory process enhancing angiogenesis. Nevertheless this must be carefully addressed in following clinical trials to improve safety of the treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 450.

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