In recent years, more data is becoming available for historical oncology case analysis. A large dataset that describes over 500 patient cases of Head and Neck Squamous Cell Carcinoma is a potential goldmine for finding ways to improve oncological decision support. Unfortunately, the best approaches for finding useful inferences are unknown. With so much information, from DNA and RNA sequencing to clinical records, we must use computational learning to find associations and biomarkers. The available data has sparsity, inconsistencies, and is very large for some datatypes. We processed clinical records with an expert oncologist and used complex modeling methods to substitute (impute) data for cases missing treatment information. We used machine learning algorithms to see if imputed data is useful for predicting patient survival. We saw no difference in ability to predict patient survival with the imputed data, though imputed treatment variables were more important to survival models. To deal with the large number of features in RNA expression data, we used two approaches: using all the data with High Performance Computers, and transforming the data into a smaller set of features (sparse principal components, or SPCs). We compared the performance of survival models with both datasets and saw no differences. However, the SPC models trained more quickly while also allowing us to pinpoint the biological processes each SPC is involved in to inform future biomarker discovery. We also examined ten processed molecular features for survival prediction ability and found some predictive power, though not enough to be clinically useful.

Radiation treatment for head and neck cancer has devastating effects on swallowing ability. Prophylactic swallowing exercises are often recommended. However, the evidence for these exercises is equivocal and information regarding critical components of an exercise program is lacking. The purpose of this systematic review was to examine the evidence regarding lingual strengthening exercises as a component of a prophylactic swallowing program.

Title: Inhibition of interleukin-6 trans-signaling by sgp130Fc is anti-tumorigenic in head and neck squamous cell carcinoma. Background: Head and neck squamous cell carcinoma (HNSCC) is a highly inflammatory cancer type, and interleukin-6 (IL-6) is associated with this phenotype. Elevated expression of IL-6 is linked to tumor progression, recurrence, metastasis, and resistance to therapy in HNSCC. However, targeting IL-6 or IL-6 receptor (IL-6R) has demonstrated little to no clinical efficacy. IL-6 signals through a classical signaling pathway via membrane IL-6R or a trans-signaling pathway via soluble IL-6R (sIL-6R). Recent evidence suggests that classical signaling induces acute, transient inflammation, eventually resulting in homeostasis; whereas trans-signaling may induce chronic, pro-tumorigenic inflammation. Therefore we propose that IL-6 trans-signaling is associated with the pro-inflammatory phenotype observed in HNSCC. We wanted to determine whether inhibition of IL-6 trans-signaling by sgp130Fc would better demonstrate anti-tumor efficacy and increase HNSCC tumor response to radiation, chemotherapy, and targeted therapy (cetuximab) compared to global IL-6 pathway inhibition. Method/Results: Baseline levels of IL-6, IL-6R, sIL-6R, and sgp130 proteins in HNSCC cells were determined using ELISA and flow cytometry. Cisplatin, radiation, and cetuximab treatments each induced HNSCC cell secretion of IL-6 and sIL-6R in vitro, yet adding sgp130Fc to those treatments did not further reduce clonogenic survival. Sgp130Fc treatment significantly suppressed SQ20B tumor growth in nude mice, whereas global IL-6 pathway inhibition by IL-6R antagonist tocilizumab did not; however, cetuximab reduced the efficacy of sgp130Fc in this animal model. Sgp130Fc also sensitized SQ20B xenograft tumors to radiation and chemotherapy in nude mice and suppressed SCCVII tumor growth in male but not female C3H/HeJ mice. Conclusion: Inhibition of IL-6 trans-signaling by sgp130Fc displayed significant anti-tumor effects as a single therapy and sensitized resistant HNSCC tumors to radiation and chemotherapy in vivo; however, sgp130Fc did not reduce survival of HNSCC cells in vitro. These results suggest that the efficacy of sgp130Fc relies on targeting another part of the microenvironment instead of tumor cells directly. Sgp130Fc has promise both as a single therapy and potentially as combined therapy with radiation and chemotherapy in HNSCC.

One of the main objectives of combining radiation treatment and chemotherapy is to obtain a therapeutic gain by an improved tumour control with less or no enhancement of normal tissue toxicity. The optimal schedule for the combined treatment of cisplatin-radiation is still under investigation. Neither the optimal time interval, nor the most adequate sequence of administration of cisplatin and radiation are known. The results of the trials are also inconclusive. Some trials showed a supra-additive effect from the administration of cisplatin before radiotherapy, others, on contrary, from the injection of drug after radiotherapy. The present work encompasses the major challenges brought by the combined modality treatment: cisplatin-radiotherapy. The major goal of this work was to investigate the optimal treatment sequencing between cisplatin and radiotherapy and also the optimal schedule for head and neck carcinomas. Therefore, a computer-based tumour model with literature-given biological parameters has been developed which has allowed the simulation of treatment with radiation and chemotherapy. Radiotherapy has been simulated on the virtual tumour and the effects of radiotherapy on tumour regression and regrowth have been analyzed. Also, the mechanisms of cisplatin's action on tumour have been implemented, and the phenomena of drug resistance and tumour repopulation during chemotherapy studied. Finally, the combined modality treatment has been simulated, and the effect of drug-radiation interaction on tumour behaviour evaluated. The current investigation has shown that cisplatin administered immediately before radiation gives similar tumour control to the post-radiation sequencing of the drug. Furthermore, the killing effect of the combined modality treatment on tumour increases with the increase in cell recruitment. The individual cell kill produced by cisplatin and radiation leads to an additive-only tumour response when the treatments are given concurrently, and for a synergistic effect cisplatin must potentiate the effect of radiation. The final conclusion, by which cisplatin administered on a daily basis leads to a better tumour control than cisplatin administered weekly, is in accordance with the latest trial results on head and neck cancers. Therefore, treatment regimens that correlate better with the pharmacokinetics and the radiobiological properties of the therapeutic agents result in better outcomes. / Thesis (Ph.D.)--School of Chemistry and Physics, 2004.

Aim: The overall aim of this thesis was to acquire knowledge about daily life with focus on eating problems during the trajectory of care for patients with head and neck cancer treated with radiotherapy. Method: The data in study I were gained from medical and nursing records of 50 patients. Documented parameters of eating problems, their causes and consequences, and undertaken interventions were collected before treatment, during radiotherapy, and one, six, and twelve months after completion of treatment, using a study-specific audit instrument. Data were analysed with descriptive and inferential non-parametric statistics. In study II eight patients were interviewed during the radiotherapy treatment period with focus on experiences of eating problems. In study III nine patients were interviewed six to twelve weeks after treatment with the focus on experiences of daily life during the trajectory of care having eating problems. In study IV twelve patients were interviewed about their conceptions of the significance of a supportive nursing care clinic during the whole trajectory of care. Data were analysed with interpretative phenomenology (II, III) and phenomenography (IV). Findings: The four studies showed that being a patient in the trajectory of care often meant that life was disturbed and threatened. This was partly due to the eating problems and their consequences, which could occur during the whole trajectory of care (I, III, IV) but was experienced as most intense and severe during radiotherapy (II) and the nearest weeks after completion of radiotherapy (III, IV). The disturbances and threats experienced due to eating problems could affect the whole person as they were physical (I-IV), psychological, social and existential (II, III). The experiences of eating problems due to the tumour and its treatment and the experience of having cancer per se were strongly connected as one phenomenon, which disturbed and threatened the informants’ daily life. The other part that disturbed the patients’ life was the waiting in suspense. A long and trying waiting in uncertainty was experienced due to lack of knowledge and support, practical as well as emotional. This was most pronounced during pauses in radiotherapy (III) and after completion of the treatment when the lack of support from the health care was obvious (I, II, III). The patients were then most often left to their own devices. In order to endure, they needed both inner strength, described as own coping strategies, and strength from outside, described as support from family, friends and health care professionals (II, III). The nurse clinic was found to give a hand to hold during the whole trajectory of care (IV). It could meet these patients’ needs of knowledge, care and support, both concerning practical measures related to the eating problems and other side-effects of the treatment, and concerning their emotional needs. In addition the nurse clinic could support the relatives in their worries and anxiety (IV). Conclusion: This thesis showed the necessity of continuous assessment, treatment and evaluation of patients’ problems, and the patients’ needs of information and support throughout the trajectory of care.

Development of a simple, specific, sensitive and rapid RNA microchip for detection of Head and Neck Cancer (HNC) mRNA, pathogenic bacteria and dengue virus (DENV) RNA is reported. By use of nucleases and polymerases specific RNAs are selectively labeled and detected without separation, reverse transcription and or polymerase chain reaction. This is accomplished by designing specific Hybrid probes consisting of DNA-2’-O-Me-RNA-DNA regions to target the RNA of interest. Upon hybridization with the target RNA, RNase H digestion is used to remove the 3’- RNA sequences which exposes the template for Klenow extension with reporter molecules such as hapten or fluorophore labels. This novel RNA microchip is fast (ca. 1 h detection time), selective as individual RNAs are detected in a synthetic mixture and total RNA mixtures, specific for single nucleotide polymorphisms (SNPs) discrimination and sensitive up to attomole level for chemiluminescence detection and lower femtomole for gold nanoparticles (AuNPs) and silver staining method. Using chemiluminescence, HNC biomarkers, VCAM1 and IL8 are specifically labeled and detected in the presence of thousands of other mRNAs in cancer cell lines and human colon cancer total RNA without interference. Furthermore, the method is highly specific as shown with DENV SNPs discrimination. Moreover, we report rapid (ca 1hour), selective, specific multi-marker detection of pathogenic mRNAs and HNC mRNAs using AuNPs-silver staining on the RNA microchip. Streptavidin gold nanoparticles technology has a potential in the analysis of specific mRNAs in a wide array of field including infectious diseases diagnosis, viral infections, food safety, gene expression profiling and cancer detection. A simple and rapid NaOH RNA extraction procedure was developed for E. coli total RNA extraction with specific results on the RNA microchip using both chemiluminescence and AuNPs silver staining. This extraction avoids the use of commercial RNA purification kits thus reducing the cost. Furthermore, visual detection on the RNA microchip is simple, does not require electricity or special equipment, and therefore is a good candidate for field diagnostics with minimum resources.

Head and neck squamous cell carcinomas (HNSCCs) most commonly develop in older patients (≥60 years of age) with a history of tobacco and alcohol use. However, young individuals (≤45 years of age) can also develop HNSCC, often without common risk factors. Increasing evidence shows that Human Papillomavirus (HPV) infection is associated with particular HNSCC sites (e.g. oropharynx). We assessed the Roche Linear Array HPV Genotyping Test in several lesions and then examined the prevalence of HPV in HNSCCs from young and older patients. HPV infection was most prevalent in oropharyngeal cancers (16/22, 73%), rarely found in oral cavity cancers (2/53, 4%), and other head and neck sites (1/17, 6%). HPV positive tumors were associated with patients that were >40 and <60 years old (p=0.02). The absence or shortened time of carcinogen exposure from common risk factors and the development of oral squamous cell carcinoma (OSCC) at an early age suggest aberrant genetic events that are different than those in OSSCs from older patients. We used Affymetrix SNP 6.0 arrays to genomically profile oral tumors from young and older patients. Tumors from young patients showed different regions/genes of copy number alterations than those from older patient tumors. An increase of regions of loss of heterozygosity (LOH) in tumors from older patients was observed, and there was a high prevalence of copy number neutral LOH on chromosome 9 in tumors from young and older patients. These data suggest different genetic mechanisms in these patient groups. We have previously shown that HNSCCs from younger patients exhibited a high incidence of microsatellite instability (MSI), a marker of defective mismatch repair (MMR). Deregulated mRNA levels of hPMS1, hPMS2 and hMLH1 were observed and absent/low expression of hPMS1, hPMS2 and hMLH1 protein levels were observed in >50% of OSCCs. No mutations were observed in hPMS1 and hPMS2 and no significant differences of MSI or LOH were observed across genomic loci between tumors of young and older patients. The role of these genetic mechanisms in oral cancer appears complex; studies such as ours should further improve our knowledge of the molecular mechanisms leading to early-onset oral carcinomas.

Objective: Evaluate a patient-reported-oral mucositis scale (PROMS) on its own and in relation to existing measures of mucositis. Methods: 50 patients with head and neck cancer receiving radiotherapy were examined before cancer treatment, twice weekly during 6-7 weeks of therapy and post-therapy. Oral mucositis (OM) signs were evaluated clinically using NCI-CTCAE v.3, OMAS criteria and Total VAS-OMAS score. OM symptoms were recorded on PROMS-VAS questionnaires. Albumin and polymorphonuclear neutrophils were measured in saline rinses. The PROMS data were subjected to Spearman rank correlations versus the other clinical and biomarker data. Results: 33 participants completed the study. Significant correlations (p<.001) were seen between PROMS scores and other clinical and biomarker indicators of OM at a group level. Significant variations were seen between individuals. Conclusion: The PROMS tool demonstrates good correlation with other clinical indicators of OM and adds novel dimensions to currently available methods of assessments used for quantification of OM.

Objective: Evaluate a patient-reported-oral mucositis scale (PROMS) on its own and in relation to existing measures of mucositis. Methods: 50 patients with head and neck cancer receiving radiotherapy were examined before cancer treatment, twice weekly during 6-7 weeks of therapy and post-therapy. Oral mucositis (OM) signs were evaluated clinically using NCI-CTCAE v.3, OMAS criteria and Total VAS-OMAS score. OM symptoms were recorded on PROMS-VAS questionnaires. Albumin and polymorphonuclear neutrophils were measured in saline rinses. The PROMS data were subjected to Spearman rank correlations versus the other clinical and biomarker data. Results: 33 participants completed the study. Significant correlations (p<.001) were seen between PROMS scores and other clinical and biomarker indicators of OM at a group level. Significant variations were seen between individuals. Conclusion: The PROMS tool demonstrates good correlation with other clinical indicators of OM and adds novel dimensions to currently available methods of assessments used for quantification of OM.

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.