Despite improvements in treatment, the prognosis for patients with advanced head and neck squamous cell carcinomas (HNSCC) has only improved to a minor degree. To raise the success rate and minimize morbidity further developments in diagnostics are highly desired. Radioimmunodiagnosis could offer a more specific and sensitive diagnostic method. Herein, we have evaluated different radioimmunoconjugates directed against CD44v6 and epidermal growth factor receptor (EGFR) for imaging of HNSCC. The studies were performed in a murine HNSCC xenograft model. Initially, the 111In-labeled anti CD44v6 chimeric monoclonal antibody U36 (cMAb U36) was evaluated. The novel radioimmunoconjugate showed high and accumulating tumor uptake. Since small molecules might be advantageous for imaging, due mainly to their shorter circulation half-life in the bloodstream, we then investigated antibody fragments F(ab’)2 and Fab’ derived from cMAb U36. The highest tumor-to-blood ratio was achieved with the dimeric antibody fragment F(ab’)2, compared with both the intact anti-body and monomeric Fab’. Furthermore, the possibility of improving EGFR-targeted imaging was explored by pre-blocking EGFR. The liver uptake of injected labeled human epidermal growth factor (hEGF) was significantly reduced when an excess of unlabeled hEGF was injected 30 minutes in advance. However, as hEGF stimulates cell proliferation it may be inadvisable to treat cancer patients with large amounts. Alternatively, pre-blocking with an anti-EGFR Affibody molecule (ZEGFR:955)2 demonstrated similar decrease in liver uptake as unlabeled hEGF. Finally, (ZEGFR:955)2 was compared with other Affibody molecules with higher affinity to EGFR, ZEGFR:1907 and (ZEGFR:1907)2, as pre-blocking agents. In addition, a novel hEGF radioimmunoconjugate, [67Ga]Ga-NOTA-Bn-NCS-hEGF was used for EGFR targeting. The dimeric (ZEGFR:1907)2 showed greatest reduction in non-tumor uptake, and highest tumor-to-organ ratio in EGFR expressing organs, when injected in advance of the radioimmunoconjugate. To summarize, the results presented here demonstrate how different radioimmunoconjugates as well as pre-blocking EGFR can improve the radioimmunodiagnosis of head and neck squamous cell carcinomas.

ABSTRACT There is an increasing incidence of neoplasms in the kidney and a poor prognosis for patients who are diagnosed with advanced or metastatic kidney cancer. Renal cell carcinoma (RCC) constitutes the most prevalent form of kidney neoplasm in the adult population. Although surgery or cryoablation are successful curative treatments for localized RCC, improved diagnostic methods facilitating early detection and characterization of renal tumours may enable more effective use of less invasive treatments, especially for metastases. Currently there are no suitable tumour markers available for diagnostic, prognostic or predictive purposes. By increasing our knowledge of the underlying molecular characteristics of RCC, we may be able to identify molecular pathways involved in tumour growth and metastasis, and this knowledge may expedite the development of targeted therapies, and may identify useful markers of RCC development and progression. This thesis aimed to identify new genes involved in resistance to cancer therapy in RCC and to analyse their incidence and test their function in human RCC tissue, using immunohistochemistry in a large cohort of patients with RCC and paired normal tissue, and in vitro models. The lack of induction of apoptosis in RCCs by conventional cancer therapies such as chemotherapy, immunotherapy or radiation is central to their resistance to treatment. If apoptotic pathways that are activated in successful treatments were identified, they might be used for targets in future therapies. The hypothesis tested in this thesis was that genes or proteins involved in the molecular control of apoptosis, identified from RCC cell culture models and RNA microarray, will be useful for molecular profiling in human RCCs of different subtypes, as markers of those specific subtypes of the RCCs, as indicators of prognosis of the diagnosis, or as targets for future therapy regimens. The broad aims of this project were: To establish a model in which significantly increased apoptosis in RCCs in cell culture could be correlated with alterations in apoptotic pathway genes, to investigate the functional significance of some of those genes; and to maximise information gained from these basic experimental laboratory studies on new apoptosis genes in RCC development and progression by determining their expression patterns in tissue microarrays (TMAs) generated from different subtypes of human RCCs. The specific aims of the project were: 1) To establish an RCC cell culture model with high levels of induced apoptosis for RNA analyses using microarray to identified apoptotic genes that are novel in RCC investigations; 2) To describe the role and functional significance of some of the novel RCC apoptosis pathway genes using molecular investigations, including silencing RNA techniques; and 3) To use TMAs and immunohistochemistry to determine whether RCC subtypes can be distinguished by protein expression profiles of selected new apoptosis pathway genes. The thesis is presented as a literature review (Chapter 1), materials and methods (Chapter 2), four original research segments (Chapters 3 to 6), and finally a segment that summarises the results and presents future directions (Chapter 7). The first of the original research Chapters (Chapter 3) addressed a question “can apoptosis in RCCs be induced in cell culture to such a level that apoptotic pathways may be analysed?” Two RCC cell lines (ACHN and SN12K-1) were treated with IFN-a (500IU/mL), radiotherapy (20 Gy) or dual therapy of these two treatments. Apoptosis was quantified using microscopy and morphological characteristics and verified using enzymatic labelling of cells. The ACHN cell line, treated with the dual therapy and analysed at 24 hours, had a significantly increased level of apoptosis (p<0.05) compared with the non-therapy treated controls, and negligible mitosis. The increased expression or activation of at least some known apoptotic pathway genes (Bcl-2, Bax, caspase-3, 8 and 9, and p53) was verified in this model. The ACHN/dual therapy model was selected for further study. The second of the original research Chapters (Chapter 4) addressed the question “what apoptosis-regulating genes are significantly different in the apoptotic ACHN cells?” An RNA microarray assay (112 apoptosis-related genes) was carried out using RNA extracted from treated and non-treated ACHN cells and analysed for alterations of at least 1.9-fold in transcript levels. 21 genes had upregulated transcript levels in the treated cells, and one had down-regulated transcript levels. A search of the literature revealed three gene families with altered transcript levels in the treated RCCs that were novel: the TNF receptor-associated factor (TRAF), caspase recruitment domain (CARD) and cell death-inducing DFF-45 effector (CIDE) gene families. Representative members of these families were then investigated for protein expression alterations. The results for one particular gene, TRAF1, indicated it might be worthy of further study in modulation experiments (Chapter 6). The next research chapter (Chapter 5) asked the question “since the ACHN cells express TRAF-1 and have increased TRAF-1 with increasing apoptosis, what happens to the levels of mitosis and apoptosis in these cells when TRAF-1 expression is knocked down?” Silencing RNA (siRNA) techniques were used to knock down TRAF-1 in the ACHN RCC cell line using the same model as was described in Chapter 3. Successful knock down was gained after 72 hrs of transfection with a commercially-available siRNA against TRAF-1. These cells were then treated with the radiation, IFN-α or dual therapy treatments. In the cells with TRAF-1 knock down, there was significantly less apoptosis and more mitosis than was seen in the non-transfected cells. These results indicate that TRAF-1 does play a functional role in induction of apoptosis in RCCs and is worth investigating further in targeted therapies. The final of the research Chapters (Chapter 6) looked at the molecular distinctions among ccRCC, papillary, collecting duct, chromophobe and unclassified types of RCC. Molecules that might distinguish one from another would be valuable clinically, and it would be especially valuable if these molecules could also be targeted for therapeutic benefit. By knowing the action of these molecules in the apoptotic pathways used by RCCs when they do regress or die during cancer therapies, we might be able to devise new targeted therapies for RCCs. This research Chapter asked the question “could molecular profiling with an array of apoptotic pathway genes, novel to investigations in RCCs, provide information that would distinguish the subtypes?” TMAs prepared from 121 RCC and paired normal patient samples, where available, were investigated. Most RCC samples were ccRCC. Antibodies against selected members of the TRAF), CARD and CIDE gene families were selected from the RNA microarray data. These genes (TRAF1, TRAF3, TRAF4, inhibitor of caspase-activated DNAase/ICAD and nucleolar protein-3/NOL3) were analysed in TMAs using immunohistochemistry and digital scanning or bright field microscopy and graded scales of intensity and distribution, blinded to sub-type of RCC. After microscopy, scores for subtypes of RCC were compared with their normal tissue. Significant differences were found for TRAF1 and NOL3. The results indicate TRAF1 and NOL3 have potential for improving outcome or diagnosis in RCCs. In summary, there are no effective treatments against metastatic RCC, and no suitable grade or stage-defining biomarker for metastatic RCC subtypes. One of the main reasons for therapy resistance in RCCs is their inability to use or activate apoptotic pathway molecules. By investigating the reasons for RCC resistance to cancer therapies, we may be able to improve both diagnosis and treatment strategies. By defining the mechanisms and pathways of resistance to therapy-induced apoptosis, and developing methods to manipulate the pathways to cell death to defeat therapy resistance, we will have a better chance to develop successful markers of RCC subtypes and also design new and successful RCC therapies.

Context: Squamous cell carcinoma (SCC) is the second most common skin cancer. Ultraviolet radiation (UVR) exposure, its most important risk factor, has mostly been investigated in cross-sectional study designs. This study presents a comprehensive and longitudinal examination of determinants of SCC, including photoageing. Objective: To examine the determinants of SCC and its precursor condition of photoageing. Above all, the objective was to investigate the interplay of phenotypic traits; occupation and leisure-time sun exposure patterns; and personal exposures, in particular, tobacco smoking and life course sun exposure, upon the risk of SCC and photoageing. Setting and Design: This investigation formed part of a large community-based, long-term cohort study of skin cancer. The Nambour Skin Cancer Study (forthwith, the Nambour Study) began in 1986 and concluded in 2007. In 1986 a random sample of 2095 people (aged 20-69 years) from Nambour, Queensland participated in a skin cancer survey. In 1992, a 5 year field trial to assess the preventive actions of sunscreen and beta-carotene was initiated (n=1621). Regular full skin examinations were conducted to determine the presence of skin cancer and actinic skin damage. In 1994, participants detailed their life course sun exposure (n=1290). After the trial ended in 1996, participants continued to complete regular questionnaires and ascertainment of skin cancers continued to 2007. Participants: The participants were 1339 unselected adults aged 25 to 75 years who had taken part in the Nambour Study in 1992 and consented to the follow-up study. Methods: Life course sun exposure hours were estimated from questionnaires and the approximate UVR exposure for Nambour (latitude 26S) was determined. Descriptive analyses examined patterns of exposure within the population. Factors influencing the severity of photoageing were also investigated. Informed by these analyses, relative risks were calculated for determinants of SCC and population attributable risk percentage (PAR%) for key modifiable risk factors. To investigate tobacco smoking as a risk factor for SCC, systematic review and meta-analysis were performed. Exposure measures: Pigmentary phenotype, estimated UVR exposure, tobacco smoking, sun-related behaviours, e.g. sunscreen use. Outcome measures: Incident and histologically proven SCC of the skin from 1992 to 2007 was the main outcome assessed. Photoageing, assessed clinically and micro-topographically (Beagley and Gibson scale), was an intermediate outcome measure and an objective measure of cumulative sun exposure in the final SCC analysis. Results: Examination of self-reported UVR revealed mean annual exposures were highest in early life and older adulthood (older than 60 years.) Women reported spending less time in the sun than men in all stages of life (p<0.05) and the more sun-sensitive the person’s skin type, the less sun exposure was reported at each life stage. The role of tobacco smoking in cutaneous SCC was reviewed in the published literature and a small positive association was noted in the meta-analysis. However, few studies had adjusted, or adjusted adequately, for sun exposure. Within the Nambour Study, with adjustment for age, sex, skin phenotype, lifetime sun exposure, current and former smoking had no association with SCC (RR 1.2, 95%CI 0.7, 2.0 and RR 1.1, 95%CI 0.8, 1.5, respectively compared with lifelong non-smokers). In this study population, with moderate to severe photoageing at study baseline, increasing age, male sex, a sun-sensitive phenotype were found to increase the odds of more severe actinic damage (p<0.05). High or very high UVR exposure in adulthood predicted a greater severity (OR 2.2, 95%CI 1.3, 4.0). Finally, the determinants of SCC were examined. Increasing age (4% increase per year of life, 95%CI 3% to 5%), male sex (RR 1.4, 95%CI 1.1, 1.9) and fair skin (RR 4.7 95%CI 2.0, 11.4) were associated with SCC. Having light eye colour and fair or red hair also significantly increased SCC risk. Recalled life course sun exposure overall was not found to be associated with SCC. Signs of actinic damage at baseline were, however, very strongly associated with SCC. Recent sun exposure, defined as that reported in the period (1-2 years) before the occurrence of SCC or for those unaffected at the end of the study, was also examined. A strong positive association was found between high recent exposure and SCC (RR 2.1, 95%CI 1.3, 3.3). PAR% estimates of prominent modifiable risk factors for SCC suggested considerable potential for reduction in incidence for at-risk populations if recent sun exposure were reduced. Conclusions: Subjective measures of solar UVR exposure and objective measures of photoageing varied according to personal and phenotypic factors. The interplay between risk factors observed here highlight the need to control for confounding in investigating solar factors as causes of skin cancer. Although SCC occurred on the background of high cumulative UVR exposure, which was best determined with objective rather than recalled measures, recent UVR exposure was also important. Self-reported recent exposure being less subject to recall bias than reported life course exposure may have partly influenced this, but the impact of UVR acting as a tumour initiator and promoter is also likely to explain the relation of SCC to sun exposure in the recent past.

Head and neck cancers account for 3% of all newly diagnosed cancers, of which 90% are squamous cell carcinomas (SCC). Improvements in surgery, radiotherapy and chemotherapy have done little to improve the mortality of this disease over the past 20 years while current clinicopathological predictors of disease outcome are sub-optimal. Identifying molecular targets of prognostic and therapeutic significance in head and neck squamous cell carcinomas (HNSCC) may help direct novel therapies to patients whom it is most likely to benefit. Accrued knowledge of the biology of HNSCC has highlighted specific aberrations in pRb and p53 pathways which warrant further study. An immunohistochemical analysis (IHC) in a cohort of 145 patients with SCC of the anterior tongue was performed. Protein expression of the pRb and p53 pathways and related molecules that directly or indirectly influence cell cycle progression at the G1/S phase checkpoint was assessed. We determined that over-expression of E2F-1 occurred in &gt35% of these cancers and associated with improved overall survival on univariate analysis. The strongest multivariate model included: regional lymph node status, tumour grade, p16INK4A, cyclin D1 and p14ARF. This is the first study to determine that p14ARF is an independent marker of both improved diseasefree survival and overall survival in a cohort of SCC of the anterior tongue. Unrecognized molecular heterogeneity is thought to account for the unpredictable clinical response to ZD1839, an EGFR tyrosine kinase inhibitor. We explored the anti-proliferative effects following ZD1839 treatment alone or in combination with radiotherapy in cyclin D1 and E2F-1 over-expressing SCC9 HNSCC cells. SCC9 cells over-expressing cyclin D1 or E2F-1 were highly resistant to ZD1839 treatment, while E2F-1 clones were also radioresistant. Combined therapy in SCC9 controls had a greater anti-proliferative effect than each individual treatment. These data showed that cyclin D1 and E2F-1 may have utility as markers of ZD1839 resistance. The data in this thesis contribute to our knowledge of the clinical behaviour and molecular pathology of HNSCC. Specifically the molecular data identifies novel markers of outcome in SCC of the anterior tongue such as p14ARF, and therapeutic response to ZD1839 such as cyclin D1 and E2F-1. This study addresses in part, the current issues and limitations of management in HNSCC and has the potential to contribute to strategies that may be developed to improve the outcome for patients who develop HNSCC in the future.

Head and neck cancers account for 3% of all newly diagnosed cancers, of which 90% are squamous cell carcinomas (SCC). Improvements in surgery, radiotherapy and chemotherapy have done little to improve the mortality of this disease over the past 20 years while current clinicopathological predictors of disease outcome are sub-optimal. Identifying molecular targets of prognostic and therapeutic significance in head and neck squamous cell carcinomas (HNSCC) may help direct novel therapies to patients whom it is most likely to benefit. Accrued knowledge of the biology of HNSCC has highlighted specific aberrations in pRb and p53 pathways which warrant further study. An immunohistochemical analysis (IHC) in a cohort of 145 patients with SCC of the anterior tongue was performed. Protein expression of the pRb and p53 pathways and related molecules that directly or indirectly influence cell cycle progression at the G1/S phase checkpoint was assessed. We determined that over-expression of E2F-1 occurred in &gt35% of these cancers and associated with improved overall survival on univariate analysis. The strongest multivariate model included: regional lymph node status, tumour grade, p16INK4A, cyclin D1 and p14ARF. This is the first study to determine that p14ARF is an independent marker of both improved diseasefree survival and overall survival in a cohort of SCC of the anterior tongue. Unrecognized molecular heterogeneity is thought to account for the unpredictable clinical response to ZD1839, an EGFR tyrosine kinase inhibitor. We explored the anti-proliferative effects following ZD1839 treatment alone or in combination with radiotherapy in cyclin D1 and E2F-1 over-expressing SCC9 HNSCC cells. SCC9 cells over-expressing cyclin D1 or E2F-1 were highly resistant to ZD1839 treatment, while E2F-1 clones were also radioresistant. Combined therapy in SCC9 controls had a greater anti-proliferative effect than each individual treatment. These data showed that cyclin D1 and E2F-1 may have utility as markers of ZD1839 resistance. The data in this thesis contribute to our knowledge of the clinical behaviour and molecular pathology of HNSCC. Specifically the molecular data identifies novel markers of outcome in SCC of the anterior tongue such as p14ARF, and therapeutic response to ZD1839 such as cyclin D1 and E2F-1. This study addresses in part, the current issues and limitations of management in HNSCC and has the potential to contribute to strategies that may be developed to improve the outcome for patients who develop HNSCC in the future.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 109-124).

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 111-127). Also available on the Internet.

Thesis (M.D.)--University of Hong Kong, 1995. / "September 1994." Includes bibliographical references.

Thesis (doctoral)--Uppsala University, 1981. / At head of title: From the Department of Oncology, Division of Gynecologic Oncology, University Hospital, Uppsala, Sweden. Bibliography: p. 29-34.

Diss. med. dent. Zürich. / Literaturverz.