Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Evaluation of regional and distant metastases in head and neck squamous cell carcinoma patients with special reference to the assessment of regional lymph nodes in oral cancer

For optimal treatment planning, a thorough assessment of the metastatic status of mucosal squamous cell carcinoma of the head and neck (HNSCC) is required. Current imaging methods do not allow the recognition of all patients with metastatic disease. Therefore, elective treatment of the cervical lymph nodes is usually given to patients in whom the risk of subclinical metastasis is estimated to exceed 15-20%. The objective of this study was to improve the pre-treatment evaluation of patients diagnosed with HNSCC. Particularly, we aimed at improving the identification of patients who will benefit from elective neck treatment. Computed tomography (CT) of the chest and abdomen was performed prospectively for 100 patients diagnosed with HNSCC. The findings were analysed to clarify the indications for this examination in this patient group. CT of the chest influenced the treatment approach in 3% of patients, while CT of the abdomen did not reveal any significant findings. Our results suggest that CT of the chest and abdomen is not indicated routinely for patients with newly diagnosed HNSCC but can be considered in selected cases. Retrospective analysis of 80 patients treated for early stage squamous cell carcinoma of the oral tongue was performed to investigate the potential benefits of elective neck treatment and to examine whether histopathological features of the primary tumour could be used in the prediction of occult metastases, local recurrence, or/and poor survival. Patients who had received elective neck treatment had significantly fewer cervical recurrences during the follow-up when compared to those who only had close observation of the cervical lymph nodes. Elective neck treatment did not result in survival benefit, however. Of the histopathological parameters examined, depth of infiltration and pT-category (representing tumour diameter) predicted occult cervical metastasis, but only the pT-category predicted local recurrence. Depth of infiltration can be used in the identification of at risk patients but no clear cut-off value separating high-risk and low-risk patients was found. None of the histopathological parameters examined predicted survival. Sentinel lymph node (SLN) biopsy was studied as a means of diagnosing patients with subclinical cervical metastases. SLN biopsy was applied to 46 patients who underwent elective neck dissection for oral squamous cell carcinoma. In addition, SLN biopsy was applied to 13 patients with small oral cavity tumours who were not intended to undergo elective neck dissection because of low risk of occult metastasis. The sensitivity of SLN biopsy for finding subclinical cervical metastases was found to be 67%, when SLN status was compared to the metastatic status of the rest of the neck dissection specimen. Of the patients not planned to have elective neck dissection, SLN biopsy revealed cervical metastasis in 15% of the patients. Our results suggest that SLN biopsy can not yet entirely replace elective neck dissection in the treatment of oral cancer, but it seems beneficial for patients with low risk of metastasis who are not intended for elective neck treatment according to current treatment protocols.

Injection laryngoplasty with autologous fascia for treatment of unilateral vocal fold paralysis

The purpose of this dissertation was to study the applicability of minced autologous fascia graft for injection laryngoplasty of unilateral vocal fold paralysis (UVFP). Permanence of augmentation and host versus graft tissue reactions were of special interest. The topic deals with phonosurgery, which is a subdivision of the Ear, Nose and Throat-speciality of medicine. UVFP results from an injury to the recurrent or the vagal nerve. The main symptom is a hoarse and weak voice. Surgery is warranted for patients in whom spontaneous reinnervation and a course of voice therapy fails to improve the voice. Injection laryngoplasty is a widespread surgical technique which aims to restore glottic closure by augmenting the atrophied vocal muscle, and also by turning the paralyzed vocal fold towards midline. Currently, there exists a great diversity of synthetic, xenologous, homologous, and autologous substances available for injection. An autologous graft is perfect in terms of biocompatibility. Free fascia grafts have been successfully used in the head and neck surgery for decades, but fascia had not been previously applied into the vocal fold. The fascia is harvested from the lateral thigh under local anesthesia and minced into paste by scissors. Injection of the vocal fold is performed in laryngomicroscopy under general anesthesia. Three series of clinical trials of injection laryngoplasty with autologous fascia (ILAF) for patients with UVFP were conducted at the Department of Otorhinolaryngology of the Helsinki University Central Hospital. The follow-up ranged from a few months to ten years. The aim was to document the vocal results and possible morbidity related to graft harvesting and vocal fold injection. To address the tissue reactions and the degree of reabsoprtion of the graft, an animal study with a follow-up ranging from 3 days to 12 months was performed at the National Laboratory Animal Center, University of Kuopio. Harvesting of the graft and injection was met with minor morbidity. Histological analysis of the vocal fold tissue showed that fascia was well tolerated. Although some resorption or compaction of the graft during the first months is evident, graft volume is maintained well. When injected deep and laterally into the vocalis muscle, the fascia graft allows normal vibration of the vocal fold mucosa to occur during phonation. Improvement of voice quality was seen in all series by multiple objective parameters of voice evaluation. However, the vocal results were poor in cases where the nerve trauma was severe, such as UVFP after chest surgery. ILAF is most suitable for correction of mild to moderate glottic gaps related to less severe nerve damage. Our results indicate that autologous fascia is a feasible and safe new injection material with good and stable vocal results. It offers a practical solution for surgeons who treat this complex issue.

Prognostic markers in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.