10.1245/ASO.2006.08.035
Annals of Surgical Oncology 13:1078-1084 (2006)
© 2006 Society of Surgical Oncology
Pilot Study of Oncolytic Viral Therapy Using Mutant Herpes Simplex Virus (HF10) Against Recurrent Metastatic Breast Cancer
Hideto Kimata, MD, PhD1,
Tsuneo Imai, MD, PhD1,
Toyone Kikumori, MD, PhD1,
Osamu Teshigahara, MD, PhD1,
Tetsuro Nagasaka, MD, PhD2,
Fumi Goshima, MD, PhD3,
Yukihiro Nishiyama, MD, PhD3 and
Akimasa Nakao, MD, PhD1
1 Department of Surgery II, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
2 Department of Histopathology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
3 Department of Virology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
Correspondence: Address correspondence and reprint requests to: Akimasa Nakao, MD, PhD; E-mail: nakaoaki{at}med.nagoya-u.ac.jp.
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ABSTRACT
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Background: An oncolytic herpes simplex virus type 1 mutant (HF10) has been isolated and evaluated for antitumor efficacy in a syngeneic immunocompetent mouse model, where it was effective against cancer and conferred resistance to rechallenge with tumor cells in all surviving mice. Several studies have shown that HF10 is effective and safe for use against localized or peritoneally disseminated nonneuronal malignant tumors in animals.
Methods: A pilot study using HF10 was initiated in six patients with cutaneous or subcutaneous metastases from breast cancer. For each patient, .5 mL of HF10 suspension containing various viral doses was injected into one nodule; .5 mL of sterile saline was injected into another. All patients were monitored for local and systemic adverse effects. Nodules were excised 14 days after injection for histopathologic studies.
Results: All patients tolerated the intratumoral injection of HF10. No adverse effects occurred, and histopathological evaluation revealed 30% to 100% cancer cell death.
Conclusions: This pilot study found HF10 to be safe and effective against metastatic breast cancer.
Key Words: Oncolytic viral therapy • Herpes virus therapy • Breast cancer • Clinical study • Cancer • UL56
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INTRODUCTION
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Breast cancer is one of the most common and most feared cancers in women. In particular, the prognosis of patients with advanced or recurrent breast cancer remains poor despite refinements in multimodality therapies involving chemotherapeutic and hormonal agents. In Japan, the number of breast cancer patients is increasing gradually, possibly reflecting changes in aspects of lifestyle, such as diet and stress. Accordingly, new curative approaches are required.
We previously established a syngeneic immunocompetent mouse model of disseminated peritoneal colon carcinoma by intraperitoneal (IP) injection of colon 26 cells to evaluate an oncolytic herpes simplex virus (HSV) type 1 mutant, HF10,1–3 as a novel agent for cancer therapy. We previously examined the feasibility of using HSV in treatment of IP tumor dissemination and defined the biological determinants of its antitumor efficacy.4,5 The survival rate of animals treated with a single IP injection of 107 plaque-forming units (pfu) of HF10 was 83.3%, whereas that of a control group treated with the same volume of saline was 33.3%.5 Animals treated with three IP injections of HF10 at 107 pfu each showed 100% survival, whereas in a control group treated with three saline injections, the survival rate was 28.6%.5 HSV antigen–positive cells were detected in several peritoneal nodules on day 5, particularly surrounding degenerated and necrotic areas.4,5 Other organs, such as liver, spleen, intestine, kidney, pancreas, and brain, showed no HSV antigen–positive cells after IP treatment with HF10.4,5 When we examined the effect of HF10 treatment of peritoneal dissemination of colonic carcinoma cells in mice previously immunized against HSV, the immunized mice still had 100% survival.5 Thus, antibodies acquired against HF10 did not interfere with the therapeutic effect of viral treatment against tumor cells with HF10. Our data supported the potential use of HSV oncolytic therapy for humans with preexisting immunity to HSV. Our finding that mice rescued by HSV treatment were resistant to rechallenge with colon 26 cells4,5 indicated that the viral induction of an antitumor immune response could reduce tumor recurrence.
Moreover, when a mouse breast cancer cell line, MM102-TC, and two human breast cancer cell lines, MCF-7 and YMB-1, were injected into the flanks of athymic nude mice, all cancers arising from cells sensitive to HF10 showed tumor regression and prolonged host survival.6 Such antitumor effects also have been observed for other malignant cells, including bladder cancer and sarcoma cells.4
We concluded that the efficacy of HF10 would depend on both the extent of the intratumoral viral replication and the induction of a host antitumor immune response, independently of tumor type. Because HF10 is replication competent but highly attenuated, showing greatly decreased neurovirulence, adverse effects should be rare. Even when adverse events occur, HF10 is extremely sensitive to antiherpetic drugs such as acyclovir or ganciclovir; thus, it can be administered safely to humans. In this pilot study, we present the results of intratumoral injection of HF10 into cutaneous or subcutaneous nodules of recurrent breast cancer in six patients.
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MATERIALS AND METHODS
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Genomic Structure of HF10
The HF10 strain of HSV-1 was an unselected clone derived from our stock of HF that was known to cause extensive membrane fusion in infected cells. The viruses were grown in embryonic chick cells and stored in aliquots at –80°C until use. Viral titers assayed in chick embryo cells were expressed as pfu per milliliter. The genomic structure of HF10, shown in Fig. 1
, has two main features: (1) a 3832-base pair (bp) deletion between nucleotides 116514 and 120347, resulting in a complete open reading frame of Infected Cell Polypeptide (ICP) 0 but a partial deletion in the UL56 open reading frame and complete loss of its promoter and (2) the deletion of sequences between nucleotides 6025 and 8319 from the terminal repeats long, with the 6027-bp DNA sequence from nucleotides 110488 to 116514 inserted with an inverted orientation. As a result, HF10 contained two complete copies of UL53, UL54, and UL55; one complete and one partial copy of UL52; and two incomplete copies of UL56 without a promoter.4 The role of the UL56 gene may be important in understanding the molecular basis of HF10 attenuation in vivo. Our observations are consistent with previous reports7,8 that lack of the UL56 product markedly reduces the neuroinvasiveness of HSV-1 without affecting viral replication in most cultured cell types. HF10 was avirulent in mice after IP inoculation. We recently have shown9 that the UL56 protein of HSV-2 is a tail-anchored type II membrane protein localized to the Golgi apparatus and endosomes. This possible participation of UL56 in vesicular trafficking suggests that it may be involved in the anterograde axonal transport of viral envelope glycoproteins.9
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FIG. 1. A schematic representation of the genetic genome structure of HF10. Locations of deletions and insertions in the genome of HF10 are shown. Derivations of the nucleotide positions are indicated in the text. An expansion indicates the position of genes within the deletion and insertion regions. Arrows indicate the position and orientation of the genes with expansions.bp, base pairs. TRL: Terminal Repeat Long, TRS: Terminal Repeat Short, US: Unique Short, IRS: Internal Repeat Short.
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Patients
All six patients were female, aged 48 to 76 years, serum HSV antibody-positive, and had a diagnosis of breast cancer. All had undergone mastectomy and had recurrences treated with chemotherapy, endocrine therapy, surgical therapy, and/or radiotherapy. Despite such treatment, however, these recurrent foci showed progression and metastasized to the cutaneous or subcutaneous region, where the lesions were proven pathologically to represent recurrent breast cancer. Profiles of patients are shown in Table 1.
Methods
In this pilot study we performed a preliminary investigation of toxicity and also possible efficacy in six patients <80 years old with cutaneous or subcutaneous recurrent breast cancer, which was considered to be progressive disease by traditional systemic therapy, to assess the therapeutic potential of HF10 in human disease. We chose patients with cutaneous or subcutaneous metastases so that virus or vehicle could easily be injected into lesions.
Prior therapy for each of the patients was as follows (Table 1
). Patient 1 had a left mastectomy; hormonal therapy with medroxyprogesterone acetate; chemotherapy with CMF (cyclophosphamide [CPA], methotrexate, and 5-fluorouracil), CEF (CPA, epirubicin, and 5-fluorouracil), paclitaxel (TXL), docetaxel, and trastuzumab; and radiotherapy to the left chest wall (twice), neck, right breast (twice), brain, sternum, and left axilla. Patient 2 had undergone left mastectomy; chemotherapy with CMF, CEF, TXL, and docetaxel; and radiotherapy to the neck, parasternal region, and chest wall. Patient 3 had received only mastectomy and subsequent chest wall resection. Patient 4 had had a right mastectomy, hormone therapy (anastrozole), and chemotherapy with TXL, CPA, and 5-FU. Patient 5 had received a mastectomy and hormone therapy with tamoxifen and chemotherapy with oral 5-FU and had undergone three local resection procedures. Patient 6 had undergone mastectomy; hormonal therapy with medroxyprogesterone acetate, toremifen, and tamoxifen; and chemotherapy with CEF, CPA, and 5-FU. Patient 3 refused all traditional systemic therapy for recurrence and consented only to the clinical trial of HF10.
For study of each patient, at least two tumor nodules were required so that HF10 could be injected into one nodule and, as a control, saline into the other. All 6 patients had >10 cutaneous or subcutaneous nodules. First, a test nodule approximately 1 cm in diameter was injected with HF10 suspended in diluent at various doses as follows: patient 1, 104 pfu/.5 mL; patient 2, 105 pfu/.5 mL; patient 3, 105 pfu/.5 mL for 3 days; patient 4, 5 x 105 pfu/.5 mL; and patients 5 and 6, 5 x 105 pfu/.5 mL for 3 days. To infiltrate HF10 into the entire nodule, HF10 was injected three or four separate times at different sites. The second nodule, which was >5 cm from the first nodule, was injected with .5 mL of sterile saline for the same number of days as the first nodules were injected with virus. All patients were then monitored for local and systemic adverse effects, including body temperature, local heat, and erythema. Nodules were examined for changes in size and inflammatory signs. Blood tests were performed to determine the complete blood count, anti-HSV immunoglobulin (Ig)G, natural killer cells, interleukin (IL)-10, IL-12, interferon (IFN)-
, and IFN-ß on days 0, 1, 3, 7, 10, and 21 after viral injection. Two nodules injected with HF10 or sterile saline were excised 14 days after injection for histopathologic examination.
Histopathologic Examination
Histopathologic examination was performed with conventional hematoxylin and eosin (H&E) staining and by immunofluorescence examination with anti–herpes virus type 1 antibody (DAKO Corporation, Glostrup, Denmark). In assessing the therapeutic response, we used the histopathologic criteria for assessment of therapeutic response in breast cancer established by the Committee for Production of Histopathological Criteria of the Japanese Breast Cancer Society, 10 as shown in Table 2.
All patients gave written informed consent. This study was approved by the local ethics committee and by the institutional review board of our hospital.
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RESULTS
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All six patients tolerated the clinical trial well. Although no adverse effects from treatment occurred, cancer cell death and 30% to 100% tumor regression were seen histopathologically (Table 3).
Macroscopic Findings
The macroscopic size of nodules did not show obvious regression in five of six patients. In patient 3, who was injected with HF10 at 105 pfu/.5 mL for 3 days, macroscopically, the height of the recurrent nodule decreased. Figure 2A shows the preinjection ultrasonogram of patient 3; Fig. 2B shows the same lesion 10 days after injection. Compared with Fig. 2A, the height decreased by 30%.
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FIG. 2. Ultrasonography findings. (A) Subcutaneous metastasis of the breast cancer. (B) The same lesion 10 days after HF10 injection. Its height decreased approximately 30% compared with (A).
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Blood Tests
Signs of local and systemic adverse events, including the toxicity of HF10 injection, were monitored by blood tests (such as white blood cell count, HSV DNA, HSV IgG, and HSV IgM), body temperature, and blood pressure; no toxicity or side effect was observed. Laboratory data, including white blood cell count, HSV IgG, natural killer cells, IL-10, IL-12, IFN-, and IFN-ß, did not change.
Histopathology
In Fig. 3A, an excised saline-injected nodule from patient 3 stained with H&E showed invasive ductal carcinoma with no cell death. Figure 3B shows a nodule excised 14 days after HF10 injection in the same patient, revealing a decrease in the number of malignant cells. Viral inclusion bodies could be seen within the breast cancer cells. Figure 3C shows an excised saline-injected nodule stained with H&E from patient 5, demonstrating mucinous carcinoma including malignant cells. Figure 3D shows the absence of malignant cells in another nodule from patient 5, 14 days after HF10 injection.
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FIG. 3. Histological studies. (A) Hematoxylin and eosin (H&E)-stained section of the saline-injected subcutaneous neoplasm of patient 3. Histological studies revealed that these were metastases of invasive ductal carcinoma of the breast. (B) Another lesion of the same patient after HF10 injection; the tumor area decreased approximately two thirds compared with (A). (C) H&E-stained section of the subcutaneous neoplasm of patient 5. Histological studies revealed that they were metastases of mucinous carcinoma of the breast. Tumor cells can be seen around the mucin. (D) Another lesion of the same patient after HF10 injection whose tumor cells have totally disappeared in comparison with (C). (A and B are x1000; C and D are x100.)
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Death was observed among 30% to 100% of malignant cells overall after HF10 injection, whereas no cancer cell death was seen in saline-injected nodules from patients 1 to 5 (Table 3). In patient 6, cancer cells in the recurrent nodules were too few to evaluate efficacy of HF10.
Immunofluorescence Examination
Immunofluorescence examination of excised HF10-injected nodules disclosed labeling of HF10 antigen confined to breast cancer cells in all patients (Fig. 4), with no antigen staining in adjacent nonneoplastic tissues. Because each of these patients was seropositive for HSV before the viral injection, the ability of HF10 to replicate within the tumor cells was not blocked by the previous exposure to HSV, including any immune consequences.
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FIG. 4. Immunofluorescence examination. The excised HF10-injected nodules showed evidence of viral infection confined to breast cancer cells in all patients.
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DISCUSSION
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Although oncolytic viral therapy originally referred to virally delivered gene therapy, the term is used here to denote a novel, effective anticancer modality involving both intratumoral viral replication and induction of a host immune response against the tumor.11–14
We chose HSV for this therapy because of several attractive characteristics of this virus: its ability to infect almost all kinds of cells; stronger infectivity than that seen with other viruses, such as adenovirus and adenoassociated virus; the well-characterized basic sequence of the HSV genome; the ability of low viral doses to kill infected cells; and the effectiveness of available antiviral agents, such as ganciclovir and acyclovir, against HSV, should the clinical need arise.
At present, many HSV mutants have been engineered and evaluated worldwide for therapeutic potential as agents in the treatment of cancers. Such mutants include G207, NV1020, and MGH1.15–17 G207 has deletions at both gamma 34.5 (RL1) loci and insertion of the Escherichia coli lacZ gene at UL39, whereas NV1020 has a deletion involving one gamma 34.5 locus, a deletion of the internal repeated region, and a loss of UL24. Both of these were made avirulent by inactivating the gamma 34.5 loci, although we consider this gene necessary for effective antitumor activity. In fact, almost all clinical studies using NV1020 and G207 in the United States have been discontinued because of an insufficient effect against advanced malignant tumors.
Accordingly, our group focused on inactivation of other dispensable genes, including UL56, to attenuate the virus8 while maintaining its ability to kill tumor cells. HF10, which we selected and purified from our stock of HSV-1 strain HF, has a deletion of 3832 bp from the right end of the UL and the UL/IRL (Unique Long/Internal Repeat Long) junction, which is inserted inversely into the terminal repeats long to result in the loss of UL56 expression.5,6 Although the precise role of the gene product in replication and pathogenicity of HSV is not well established, a previous study9 suggested that it may be involved in vesicular trafficking in infected calls. In both in vitro and in vivo studies, HF10 has demonstrated marked efficacy against many types of malignant tumors, such as colon cancer, breast cancer, melanoma, and sarcoma.
No adverse effects occurred in our patients treated with HF10, and cancer cell death proved to be 30% to 100% histopathologically. These data suggest that HF10 would be a safe and effective anticancer agent. A full-scale clinical trial of oncolytic viral therapy with HF10 is warranted in the near future for intratumoral injection, IP injection for peritoneal carcinoma, or intravascular injection for liver metastasis.
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ACKNOWLEDGMENTS
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Supported by the Japan Society for the Promotion of Science (JSPS KAKENHI 16390358), a grant (04-23609) from the Princes Takamatsu Cancer Research Fund, and Takeda Printing Company Limited. The authors thank Yoko Nishikawa, Tama Tsuruguchi, and Eiko Iwata for their technical support and assistance.
Received for publication August 26, 2005.
Accepted for publication March 15, 2006.
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ABSTRACT
INTRODUCTION
