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Tong et al. 2012

Created By: Kaylin Braden
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Introduction
Ovarian cancer is the sixth most prevalent cancer in women and the most lethal of the gynecologic malignancies.1,2 If caught at an early stage, the majority (approximately 90%) of ovarian cancer patients are cured. Approximately 90% of ovarian tumors are epithelial in origin, with the remainder being sex cord–stromal and germ cell tumors. These latter, rarer tumor types of the ovary and epithelial tumors that are confined to one or both ovaries (ie, stage 1A/B) are usually treated with surgery alone due to the early diagnosis and lower metastatic potential. Over three-quarters of ovarian tumors, however, are discovered at an advanced metastatic stage, when prognosis is poor.

Most other carcinomas follow a pathway of disease dissemination that involves intravasation into the bloodstream followed by extravasation at distant tissue sites, as well involvement of lymphatic spread.3 Metastasis of epithelial ovarian cancer is unique in that it typically spreads by direct dissemination or shedding of cancer cells from the primary tumor site into the ascites of the peritoneal space, followed by secondary tumor seeding by implantation onto the serosal surfaces of abdominal organs.4,5  [1] This implies that there are likely molecular and cellular properties of ovarian tumor cells that dictate this pattern of metastasis; unique properties, perhaps, that could be exploited for more efficacious therapeutic strategies.

Clinical applications
Clinical studies using OVs to treat ovarian cancer is still in its infancy. As of April 2012, results from three phase I clinical trials in ovarian cancer have been published, involving Onyx-015,185 Ad5-Δ24-RGD,186 and measles virus MV-CEA.149 A phase I clinical trial testing JX-594 in patients with different types of cancers including ovarian cancer has also been published.187 Ad5-D24-GMCSF and Ad5/3-D24-GMCSF have also been evaluated under compassionate use regulated by the Finnish Medicines Agency FIMEA.188,189  In addition, there are two ongoing trials with Reolysin in ovarian cancer
and one ongoing trial with attenuated VV GL-ONC1 for all peritoneal metastatic cancers including ovarian cancer, according to the NIH website ClinicalTrials.gov
. Although early phase trials mainly address the safety, maximum tolerated dose, and toxicity spectrum of OVs, antitumor efficacy, viral replication, and antibody responses are also analyzed. Clinical efficacies are assessed by Response Evaluation Criteria in Solid Tumor (RECIST) criteria and cancer antigen (CA)-125 response. CA-125, also known as mucin 16, is a protein biomarker for recurrence of ovarian cancer.190 In all studies, eligible ovarian cancer patients have persistent or recurrent ovarian cancer, fallopian tube cancer, or primary peritoneal cancer after prior treatment with chemotherapy. The enrollees are older than 18 years and have adequate organ function. Participating patients, except for those in the JX-594 trial, receive administration of OVs through an IP catheter. All these clinical studies have shown that OVs were well tolerated, and no maximum tolerated doses were reached in any of the trials. Encouraging antitumor responses were observed in all but the Onyx-015 trial. Summaries of the clinical studies are provided in Table 2.

Onyx-015
The first human OV trial for ovarian cancer was conducted with adenovirus Onyx-015 in 16 patients.185 Four dose levels of virus (1 × 109, 1 × 1010, 3 × 1010, and 1 × 1011 plaque-forming units [pfu]) were tested in this trial. For a particular dose level, Onyx-015 was administered to patients daily for 5 days (one cycle) every 4 weeks. These patients received a total of 35 cycle treatments, with a mean of two cycles per patient. Most patients experienced common toxicity criteria grade 1 or 2 flu-like syndrome and abdomen pain after virus infusion. Only one patient who received 1 × 1010 pfu exhibited common toxicity criteria grade 3 abdominal pain and grade 3 diarrhea, and the patient’s toxicity profile was considered dose-limiting. No grade 4 toxicity was noted in any patient. [2] This toxicity study indicates that Onyx-015 administration is safe at the highest dosage tested. Viral DNA was detected 10 days after the last dose of virus in five of eight patients who were subjected to polymerase chain reaction (PCR) testing of peritoneal specimens. No viral DNA was detected in the blood samples in these patients. Interestingly, viral DNA was still detected in one patient 354 days after final virus treatment. These data, however, were not sufficient to prove the presence of viral replication since an increase in viral genome copy number was not shown. Antiadenovirus antibody responses were evident in 12 of 13 patients that were examined. Unfortunately, there was no clear evidence of antitumor activities induced by Onyx-15 in this trial. Four of the 16 patients showed brief stable disease after more than two cycles of Onyx-015, but soon developed progressive disease. Eventually, all patients stopped virotherapy because of development of progressive disease, except one who was removed from the trial due to Onyx-015 dose-limiting toxicity.

Ad5-Δ24-RGD
The tropism-modified Ad5-Δ24-RGD virus has been engineered to improve cancer-targeting and the oncolytic activity of early adenovirus vectors, such as Onyx-015, in preclinical studies. Consistently, a phase I clinical trial with Ad5-Δ24-RGD has yielded promising antiovarian cancer responses.186 In this clinical trial, vector dosages of Ad5-Δ24-RGD ranging from 1 × 109 viral particles per day (vp/d) to 1 × 1012 vp/d, with increase of 1/2 log vp/d in each successive cohort tested in 21 patients, of whom 18 patients had recurrent ovarian cancer. Virus was delivered daily for 3 consecutive days, and the patients were followed up on days 0–3, 7, 14, and 28 to evaluate toxicity, virus replication, and antitumor efficacy. As for the clinical study with Onyx-015, Ad5-Δ24-RGD treatment did not cause significant toxicity. Although no partial or complete responses were observed, 15 patients (71%) were shown to have stable disease. In addition, seven patients (33%) had deceased CA-125 levels, and four of them had .20% reduction. RGD-specific viral DNA was detected in ascites in 16 of 21 patients by quantitative real-time (qRT) PCR after virus treatment. More importantly, increased viral DNA copy number was detected at various time points after day 3 of virus treatment in seven patients, suggesting viral replication in the cancer cells. Immunohistochemistry analysis of ascites from selected patients confirmed the infection of ovarian cancer cells by the virus. Dose-dependent antiadenovirus neutralizing antibody response was generally detected in ascites and serum in all patients. Unlike Onyx-015, Ad5-Δ24-RGD DNA was also found in serum (ten patients), saliva (ten patients), and urine (nine patients), probably due to high replication activity of Ad5-Δ24-RGD in the IP cavity, leading to the dissemination of the virus.

MV-CEA
MV-CEA has been tested in 21 patients with recurrent ovarian cancer in a phase I trial study.149 Patients were treated with seven escalating doses of MV-CEA (103–109 TCID50 at 1-log increments) every 4 weeks for up to six cycles. No dose-limiting toxicity was observed with MV-CEA. Most toxicities were grade 1 or 2 fever, fatigue, and abdominal pain. Fourteen of the 21 patients (67%) had stable disease with median duration of 92.5 days. Nine of the 14 patients with stable disease (64%) were in the three highest dose levels, indicating dose-dependent outcomes. CA-125 levels were demonstrated to decrease .30% in five patients. Median overall survival of the patients in this trial was 12.15 months, while in similar patient populations the median survival is expected to be 6 months. Viral DNA was detected in the blood in four patients by qRT-PCR, but no virus shedding was detected in saliva and urine in any patient. CEA levels were elevated in the peritoneal fluid in one patient in the 108-TCID50 cohort and two in the 109-TCID50 cohort, and in the serum in all three patients in the 109-TCID50 cohort. No antibody responses to MV-CEA were observed. This might have resulted from preexisting high baseline anti-measles antibody in enrolled patients who were required to be immunized with measles for safety consideration in this first-ever human virotherapy trial with MV. Since MV predominantly utilizes the CD46 receptor for cell entry, the authors also examined the expression of CD46 in tumor specimens in 15 patients whose tissues were available, attempting to investigate the effect of CD46 expression on the oncolytic activities of MV-CEA. Thirteen of the 15 patients showed high-level expression of CD46; however, no association of CD46 expression with clinical efficacy was observed. Due to the small patient sample size and different dosages used in this initial clinical trial, whether CD46 expression is associated with clinical efficacy remains to be identified.

Ad5-D24-GMCSF and Ad5/3-D24-GMCSF
Two phase I clinical trials have been conducted to test whether GM-CSF could facilitate induction of antitumor immunity in the context of oncolytic Ad vectors Ad5-Δ24 and Ad5/3-Δ24.188,189 Twenty patients with 15 different types of cancers (four patients with ovarian cancer) and 21 patients with twelve different types of cancers (four patients with ovarian cancer) were treated with Ad5-D24-GMCSF and Ad5/3-D24-GMCSF, separately. Viruses were administered using ultrasound-guided intratumoral injection or intracavity injection as in ovarian cancer patients, with one-fifth of the dose given IV. The starting dose of virus was 8 × 109 vp/d in the Ad5-D24-GMCSF trial, 8 × 1010 vp/d in the Ad5/3-D24-GMCSF trial, and escalated to 4 × 1011 vp/d in both trials. Both studies showed that virus treatments were well tolerated and induced antitumoral and antiviral immune responses, as measured by the activation of tumor- and virus-specific cytotoxic T lymphocytes. Clinical benefits were also observed in some patients, including four patients with ovarian cancer in the Ad5-D24-GMCSF trial and one of four patients in the Ad5/3-D24-GMCSF trial.

JX-594
[3] Taking the lead that VV has adapted to acquire stability in the bloodstream and is capable of rapid spread to distal tissues,191 a phase I clinical trial was designed to test whether JX-594 could target metastatic tumors via IV infusion. Escalating dosages (1 × 105–3 × 107 pfu/kg) were administered in 23 patients with nine different types of cancers, including two patients with recurrent ovarian cancer.187 Results from this study showed that JX-594 could selectively infect tumors after IV infusion in a dose-dependent manner. Viral infection of tumors was detected in all eight patients receiving the two highest doses of JX-594, but in only two out of 15 patients receiving lower doses. [4Antitumor activities were also observed and appeared to be dose-dependent. One of the two patients with ovarian cancers receiving a lower dose of JX-594 was virus-negative in tumor but showed stable disease for more than 4 weeks after treatment. The other patient receiving the second- highest dose was virus-positive in the tumor and had stable disease for more than 16 weeks. The most common virus-associated adverse side effect was grade 1/2 flu-like symptoms, indicating that it is safe to administer JX-594 via this IV route.

GL-ONC1
GL-ONC1 (also named GLV-1h68) is an attenuated Lister strain VV with insertion of Renilla luciferase-GFP fusion gene, lacZ and β-glucuronidase reporter genes in the F14.5L, J2R (TK), and A56R (hemagglutinin) loci of the viral genome.192 This OV is being tested in a phase I/II trial in patients with advanced peritoneal cancers, which include ovarian cancer patients.193

Reolysin
The two ongoing Reolysin clinical trials are (1) a phase I trial in patients that did not respond to platinum chemotherapy, and (2) a phase II trial to investigate the safety and efficacy of Reolysin in combination with paclitaxel compared with a paclitaxel regime alone.194,195 In the first clinical trial, patients were treated with Reolysin via both IV and IP routes. Among the patients that have been treated, viral replication could be detected in peritoneal and ovarian cancer cells following IV administration.196 This is the first to reveal that reovirus can reach peritoneal and ovarian cancer via systemic delivery.

Conclusions
[5] The use of live viruses specifically to kill cancer cells dates back as early as the beginning of the last century.197 However, the field of oncolytic virotherapy did not truly expand as a systematic inquiry until two decades ago, when genetic approaches were first applied to modify OVs in order specifically to target cancer cells.63,90 In 2005, Ad vector H101 was approved in China as the world’s first approved OV for treatment of head and neck cancer in combination with chemotherapy. [6] Currently two oncolytic viruses, Reolysin and OncoVEX, a herpex simplex type 1 virus–based oncolytic therapeutic agent, have entered pivotal phase III trials. JX-594 is also being tested in phase II clinical trials for liver cancer and metastatic colorectal cancer, and has shown great promise in preliminary results. Early last year, OncoVEX was acquired by Amgen in a deal that has been valued at up to US$1 billion. Although promising, we are still far from fulfilling the great potential of oncolytic virotherapy. One key challenge is our as-of-yet modest understanding of the multifactorial interactions between the tumor, its microenvironment, OVs, and the host immune responses to both the OV and the cancer cells.

In many ways, the current state of oncolytic virotherapy is similar to the situation with the cytotoxic chemotherapy drugs first developed over half a century ago, with one notable exception. Like the older cytotoxic drugs still being used routinely in the clinic today, many OVs also exhibit potent anticancer properties in the preclinical setting, but there is a limit to what can be learned in animal models, and their correct exploitation in cancer patients now requires appropriate clinical trials to teach oncologists how to exploit them effectively. In stark contrast to the classic chemotherapeutics that now comprise the standard of care for so many cancers, the single most notable characteristic of OV therapies tested to date is their extraordinary safety record. This should encourage the oncology field to be optimistic that exploiting the great potential of oncolytic virotherapy now needs to be conducted in the clinical arena. Despite the challenges ahead, advances in our understanding of tumorigenesis, antitumor immunity, and molecular biology and anticancer properties of OVs have helped and will continue to shape the translation of preclinical and clinical studies into significant clinical outcomes for cancer patients.
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