The Potential Application of Reovirus to the Treatment of Oral Squamous Cell Carcinoma: A Novel yet Putative Treatment Modality for the Practicing D.D.S.
Prepared by our late founder, George Chatzis.
Prepared for Dr. Mara.
Oral cancer ranks among the worst forms of cancer in mortality and morbidity and accounted for 274 000 cases worldwide in 2002. Although significant progress has been made in early detection, diagnosis, and treatment, the 5-year survival rates for patients with oral cancer have not improved much in the past 25 years and remain approximately 50%. Part of this increase in detection is due in part to the Dentist’s role in cancer screening of the oral cavity in their patient population (oralcancerfoundation.org). This high lethality is in part due to the advanced state of malignancy exhibited by oral cancers when they are detected. However, when detected early, they can be cured in 80-90% of the cases, using current treatments (Khuri and Jain, 2004).
Much of the research in oral cancer is aimed at unraveling the molecular mechanisms involved in initiation and progression to malignancy of oral cancer which helps to improve the prognosis of the disease and can be targeted to develop novel therapeutic strategies. It is well known that proto oncogenes arenormal genes that can become oncogenes, either after mutation or increased expression. They code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein product. Upon activation of a proto oncogene, it (or its protein product) becomes a tumor inducing agent, an oncogene.
Currently, there are new developments in cancer treatment by utilizing oncolytic viruses. Many biotech companies are researching oncolytic viruses and some are currently in Phase II clinical trials in Canada (Oncolytics Biotech Inc using Reovirus).
An oncolytic virus is one that requires a specific overactive signal transduction pathway in order to replicate and enter a lytic phase of viral infection. In other words the goal of using oncolytic viruses in cancer treatment is to inject the patient with a virus that exhibits selective replicating ability in tumor cells with subsequent lysis/cell death while leaving the normal cells unscathed. A unique element in this cancer treatment is that the drug, namely the virus, replicates itself in tumor cells and releases millions of viral copies to infect adjacent tumor cells. Simply put the tumors become factories for the production of the drug/virus that kills them. In the media, the term “Tumor Grenades” and “Smart Bomb Drugs” have been used to describe the use of oncolytic viruses to the treatment of cancer (Maclean’s, January 2005, Scientific American, October 2003).
The goal of this paper is to discuss the potential use of oncolytic Reovirus as applied to treatment of oral cancers such as squamous cell carcinoma, including a novel, putative role of the dentist working in concert with oncologists in the administering of oncolytic therapy.
Wild-type Reovirus is currently being developed by Oncolytics Biotech Inc in Calgary for use in cancer therapy under the trade name REOLYSIN. They are currently running numerous clinical trials on humans (initial phase I, phase I prostate, phase I/II glioblastoma, Phase I combination radiation therapy, Phase I/II Recurrent Malignant Gliomas, Phase I systemic delivery trial, and new Phases announced in collaboration with the National Cancer Institute in the US .
Reoviridae (respiratory enteric orphan viridae) is a family of naked double-stranded RNA viruses. Depending on the specific species, Reoviruses have 10, 11, or 12 genomic segments contained in two or three concentric icosahedral capsids (Wagner et al., 2004). The nine genera of reoviridae infect a wide range of hosts including vertebrates, invertebrates, and plants. Four of these nine genera have viruses that infect humans. Human rotavirus and Colorado tick fever virus are the most well-known human Reoviruses.
As mentioned previously, Reovirus usurps the activated Ras-signaling pathway of the host tumour cell for its own replication and targets malignant tumour cells with activated Ras (Strong et. al., 1998) The restriction of Reovirus replication in normal cells is due to the activation of the double-stranded RNA-activated protein kinase (PKR) by early viral transcripts, which inhibits the translation of viral proteins (translation block). Activated Ras (or an activated element of the Ras pathway) inhibits PKR activation and allows viral protein synthesis and a lytic infection to occur. Although only about 30% of all human tumours have mutations in the ras gene, the fact that the Ras pathway can be activated by other elements in the absence of mutation in ras itself suggests that a significantly higher percentage of human cancers could be susceptible to Reovirus oncolysis (Bos, J.L., 1989). Indeed, research has demonstrated that Reovirus infects a variety of established human cancer cell lines derived from many cancer types including breast, brain, colon, ovarian, pancreatic and prostate cancer (Wilcox et. al., 2001, Hirasawa et. al., 2002 and Norman et. al., 2002)..
Metastatic cancer is the major cause of death in most cancers. In many patients, microscopic or clinically evident metastasis has already occurred by the time the primary tumour is diagnosed. Complete treatment of widely spread metastases in distant organs by surgery, radiation or chemotherapy is nearly impossible. Effective chemotherapeutic treatment is hindered by the fact that metastatic tumours are generally less sensitive to chemotherapy when compared with their corresponding primary tumours (Fidler et. al., 1994 and Dong et. al., 1994). In this regard, oncolytic viruses are valuable because as infectious agents they are able to replicate and amplify in remote tumour sites and if administered systemically virtually any site can be targeted. Furthermore, activated ras signaling is common in human metastatic cancers thus implicating reovirus as a putative therapeutic agent for targeting metastases. (Chambers et. al., 1993 and Webb et al, 1998).
Over 600,000 new cases of head and neck cancer worldwide each year with 90% of these being squamous cell carcinoma (Khuri and Jain, 2004). Typically, SSC presents as a persistent mass, nodule, or indurated ulcer. Colour changes are common and consist of red or red and white hues (Samer, et. al., 2005). Involvement of adjacent tissues is possible, though not necessary and represents local invasion of the tumor. Symptoms are uncommon in earlier stages of the disease but become more frequent with advanced local invasion. Parasthesia and anesthesia in the absence of a history of trauma are two symptoms that are highly suggestive of an invasive malignancy. Metastatic spread occurs through the submandibular, cervical and jugular lymphatic pathways and distant metastases most commonly spread to the lungs (Samer et. al., 2005). The presence of cervical metastasis in patients with oral SCC is the most important critical factor in determining survival (Shibin et. al.,2006). The 5-year survival rate can decrease below 20% when cervical metastasis is present (Kligerman et. al., 1994). The majority of intraoral SCCs originate from non-keratinized mucosa. The three most common sites of involvement are the tongue (30%), lip (17%) and floor of the mouth (14%). The three major risk factors are smoking, alcohol and solar and/or ionizing radiation exposure.
Aside from clinical inspection, adjunctive diagnostic modalities are available to facilitate the early detection of SCC, thus increasing the chances of a better prognosis. Some current examples are toluidine blue vital staining (OralScreen TM), measurement of autofluorescence (VELscopeTM-BC, Canada), brush biopsy (OralScan TM Laboratories), and chemiluminescence (ViziLite TM). Regardless of which adjunctive aid is used, the definitive diagnosis still requires a biopsy and histological examination under a microscope.
An investigation of the literature regarding the activity of the ras pathway in squamous cell carcinoma was performed to determine if Reovirus can at least in theory usurp the ras cascade for SCC tumor lysis. In a study by Caulin et. al., that claimed the development of a novel mouse model for oral cancer, inducible activation of oncogenic ras resulted in tumor formation in the oral cavity. Although the lesions were classified as benign squamous papillomas, they exhibited changes often observed in early stages of tumorigenesis in human premalignant oral tumors. In support of this mouse model, it has been demonstrated that smoking induced carcinogenesis results in gene mutations in p53, ras and p16 (Denissenko, M.F., et. al., 1998).
As mentioned previously, mutations in other upstream elements in the ras cascade can also result in activation of ras. One of these is the transmembrane protein epidermal growth factor receptor (EGFR) that contains tyrosine kinase activity in the cytoplasmic portion. Epidermal growth factor – stimulated ras activation involves specific interactions between the EGFR, the adaptor proteins Grb2 and Shc, and the nucleotide exchange factor Sos-1. A mutation in EGFR that results in prolonged autophosphorylation, will lead to activation of ras and potential tumorigenesis. Studies by Rojas et. al., have demonstrated that competition with a phosphorylated peptide corresponding to the autophosphorylation site on EGFR did indeed block ras activation and supports the EGFR-Grb2-Sos1-Ras activation complex. Therefore, if aberrant EGFR signaling exists in squamous cell carcinoma the potential for oncolytic infection with Reovirus exists. Interestingly, a recent study by Lee et. al., does support this hypothesis as their research showed that three mutations in EGFR were detected in cells from human SCC of the head and neck. Furthermore, all of the mutations were the same in-frame deletion mutation in exon 19. This research was done in light of a new drug application targeted to treatment of SCC and non-small cell lung cancer. The drug Gefitinib, a tyrosine kinase inhibitor is in Phase II clinical trials in the USA. The rationale for its use is based on the research that indicates aberrant EGFR (a tyrosine kinase) signaling exists in SCC and non-small cell lung cancer. Also, antisense therapy against EGFR combined with endostatin blocked angiogenesis and tumor growth completely in nude mice and immunocompetent C3H mice implanted with cells from human or murine head and neck SCC (Li et. al., 2002).
The research clearly shows that SCC is a likely candidate for Reovirus mediated oncolysis via EGFR mediated activation of the Ras signaling cascade. In order to gain some insight on what results are to be expected with reovirus, one has to examine the results of the clinical trials. In the phase I trial of Reovirus, evidence of viral activity was detected in 11 of 18 patients (61%), with the tumour regression ranging from 32% to 100% as measured on day 28 after intratumoural injection of Reovirus. The test group had different types of progressive cancer that had failed to respond to conventional therapies. That being said, the tumours were not screened for activated Ras, yet the detection of virus activity in 60% of the patients correlates well with the level of ras activation expected (between 50% and 70%) in a randomly selected group of cancer patients (www.oncolyticsbiotech.com). It is important to note that the primary outcome of the trial was safety and Reovirus demonstrated an extremely positive safety profile. None of the patients receiving Reovirus experienced any serious adverse events related to the virus, nor were there any dose limiting toxicities detected in any patient. One of the most interesting outcomes of this study involved a patient who was diagnosed with a squamous primary tumor of unknown origin. The tumor appeared to measure 3-4 cm in diameter at the initiation of treatment with Reovirus and the reduction in size was graded as a partial response because the reduction in tumor size was greater than 50% but there was still detectable tumour (fig 1). This result was impressive since the dosage of Reovirus was not an optimal dose, and nothing was said in their results as to whether or not only one dose was given which, was the case for many of the patients in this trial.
A critical obstacle to complete tumour lysis with Reovirus is the host’s immune response. One would expect the duration of activity of the virus in a host with a normal immune system to be less than the time needed to effectively lyse all tumor cells. Moreover, if a patient with cancer has been previously exposed to Reovirus, the immunological memory will rid the system of Reovirus even more rapidly. It is thought that 60% of the average population will have been exposed to Reovirus in childhood (Hirasawa et. al., 2003). In light of this, it is reasonable to assume that intratumoural injection of Reovirus may require multiple and more frequent injections in patients with prior exposure or, their immune system will need to be suppressed to allow for substantial lytic infection to occur. Research has been done regarding this matter, with results indicating that immune-competent mice injected with Reovirus and with the immunosuppressants cyclosporine A or anti-CD4 and anti-CD8 monoclonal antibodies survived significantly longer than mice treated with Reovirus alone and the control mice (Hirasawa et. al., 2003). Specifically, 100% of the control mice died by day 32 and 100% of the Reovirus alone mice died by Day 48, after 4 systemic injections. The two groups of mice treated with Reovirus and the immunosuppressants Cyclosporin-A or monoclonal antibodies faired far better, as approximately 20% and 50% of the mice survived at day 60, respectively.
Fig 1. Pictures of a patient undergoing phase-I treatment with Reovirus diagnosed with a squamous unknown primary tumor; before and after treatment.
A more recent development involves combined Reovirus therapy and normal radiation therapy. The data suggests that Reovirus enhances radiation cytotoxicity in cell cultures and mice (Vidal et. al., 2005). Some hypotheses that help to explain this effect are: radiation may increase the ability of Reovirus to infect tumour cells, radiation induced changes in cell-cycle distribution of tumour cells may render them more permissive to Reovirus infection, radiation may reduce the high tumour interstitial pressure that represents a barrier to effective intratumoural spread of virus.
The research and clinical trials of Reovirus have demonstrated promising results regarding oncolysis of tumours via intratumoural or systemic injection. The potential use of combined Reovirus therapy with immunosuppressants and radiation therapy, in humans with cancer, appears to be on the horizon. In the event Reovirus is considered efficacious in the treatment of oral squamous cell carcinoma, the question arises as to the role of the dentist. We have increased our capacity to diagnose oral cancer by better educating current dentists in training and our knowledge and experience in the anatomy and structures of the oral cavity surpasses that of physicians. It follows that the expertise of a dentist may be taken advantage of in the case of injecting oral SCC with Reovirus. A team approach that utilizes oncologists, dentists and oral pathologists might provide for more efficient management of treatment of oral cancer patients by allowing administering of the virus in the dental office with monitoring of tumour regression being done by the oncologist at the local cancer center. Not only will this translate into more efficient use of the oncologists’ and oral pathologists’ expertise, this new role for the dentist will improve dentist-patient relationships as well as increase the trust and respect of the dental/oral healthcare profession in general.
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