published_or_final_version / Dentistry / Master / Master of Dental Surgery

Current treatment options for malignant brain tumors not only frequently fail to cure the disease due to local recurrence, but also may severely compromise quality of remaining life even when tumor mass is reduced in large part because they interfere with mechanisms of neuroplasticity and function of bystander tissue. The aims of this dissertation are to: (a) assess neurological impairments associated with rapid focal cortical tissue displacement; (b) evaluate the specific impact of conventional and novel treatments on neurorestoration while controlling tissue compression without the confound of related events linked to tumor physiology; (c) identify the behavioral change pattern during brain tumor progression and investigate the stealth nature of brain tumors; (d) demonstrate how anti-cancer treatments affect brain function especially when administered in the silent stages of brain tumors; and (e) develop treatment strategies that might improve therapeutic effectiveness and brain function. We adopted a new focal mass compression model providing rapid displacement of tissue in the underlying sensorimotor cortex, as well as the traditional rat and mouse glioma xenograft models that exhibit prominent tumor growth and invasion, given the varied aims and contexts of our different studies. Various conventional and novel brain tumor treatments were employed in this dissertation, including local and systemic chemotherapy, antiangiogenic agents, photodynamic therapy, and a glutamate antagonist. A neurorestorative therapy with atorvastatin was evaluated in its effects on functional recovery after photodynamic therapy. Functional outcomes were measured with an array of behavioral tests, which are sensitive to mild focal insults to the sensorimotor cortex and can detect recovery of function. Histopathological assessments consisted of Nissl staining, hematoxylin-andeosin (H&E) staining, and immunohistochemistry, depending on varied purposes, used in conjunction with a computer imaging analysis system. In clinical trials, functional outcome is as critical to gauging the success of a treatment as is patient survival time. Both preclinical screening of anti-cancer interventions for the ability to shrink tumors effectively with minimal disturbance of neuroplasticity and developing combination therapy with neurorestorative regimens following neurotoxic cancer treatments should allow for optimal promotion of plastic mechanisms in the remaining normal brain tissue.

Thirty six cases of human endometrium (9 normals, 9 adenomatous hyperplasias and 18 cancers) were established as finite monolayer tissue cultures. All were evaluated for the presence of estrogen binding sites (EBS) by an indirect immunofluorescent (IF) technique. Positive EBS were identified in 33% of normals, 67% of hyperplasias and 62% of cancers. Serial subpassage EBS evaluation was performed in fourteen cases. In was observed that the stability of EBS positivity in vitro was related to type of endometrium rather than culture longevity (2 of 2 normals, 2 of 4 hyperplasias and 1 of 8 cancers remained positive throughout the period of study). Twelve of the cancers were studied for estrogen receptor by cytosol assay and 11 (91.6%) correlated with the IF marker method. Thirty-eight cases of breast cancer were studied for EBS by a direct cytochemical and immunofluorescent technique. Evaluation by the direct method proved to be consistent and easy in performance. Morphologic positivity was 60% with the indirect method and 94.7% with the direct method. Correlation with the chemical cytosol was 77% with the indirect method and 86.8% with the direct method. These results confirm and compare favorably with other reported studies of morphologic methods. It was suggested that attention should be directed to cellular localization of receptors as a possible means for predicting endocrine responsiveness of neoplasms. Cancers from tissues presumed to be target-variants for estrogen stimulation were investigated with the direct cytochemical technique to determine if EBS were present. Forty-eight randomly selected tumors from multiple organ systems were assessed for EBS. Appropriate control tests were used to determine specificity. A total of 23 of the 48 cases were interpreted as positive for EBS. These sites were localized to cytoplasmic, nuclear and nucleolar cellular compartments. Estrogen and progesterone receptors in patients with breast carcinoma are of value in the selection of patients for hormonal adjuvant therapy. Whether this will prove to be true for EBS in a variety of neoplasms is currently unknown and is worthy of investigation.

No description available.

Approximately 40% of men and women in the United States will be diagnosed with at least one form of cancer in their lifetime, with cancer being implicated in one in four deaths. While great strides have been made in early diagnosis and treatment using standard regimens of chemotherapy and radiation, resulting in an overall decrease in cancer mortality, tumor initiation, growth and metastasis continue to evade control. The continued search for effective and targeted drugs has been hindered by the high failure rate of costly clinical trials, highlighting a need for more accurate preclinical models of disease, not only for pharmaceutical testing, but also biological research and assay development. The dominant role of the tumor microenvironment in regulating tumor initiation, progression, and metastasis has been well documented, driving the application of tissue engineering strategies in cancer biology. In vitro models that recapitulate clinically-relevant features of native tumors with greater fidelity than monolayer tissue cultures have the potential to yield discovery of novel therapeutic targets and regimens while also providing critical insights into mechanisms of tumor resistance. This thesis describes a tissue engineering strategy for generating an in vitro tumor model of human conventional chondrosarcoma using a custom biomimetic scaffold, and characterizes the effect of the biomaterial on cancer cell phenotype. Together with a previously validated and published in vitro model of human Ewing’s sarcoma tumors, we further investigated the effect of microenvironmental factors including matrix stiffness, niche composition, and three-dimensionality on the expression of a key cell cycle regulator and tumor suppressor mutated or lost in a wide variety of cancers, p53. A transcription factor nicknamed the “guardian of the genome,” p53 is activated in normal tissues in response to stress and triggers cellular responses including cell cycle arrest and apoptosis, or induces transcription of DNA repair enzymes to promote cell survival. The unifying hypothesis of this thesis was that the tumor microenvironment does in fact influence expression of tumor suppressors like p53, ultimately contributing to the progression of tumors toward metastasis and chemoresistance, and that these effects can be probed in vitro using disease-specific engineered tumor models to identify novel druggable targets and biomarkers with prognostic significance.

Previous work (Thorgaard, G. H. et al., Aquatic Toxicology 46:121-126, 1999) showed triploid rainbow trout (0. mykiss) given embryonic carcinogen bath exposures had significant reduction of induced tumors relative to diploids. In the present study, trout were made triploid by thermal shock after fertilization. At age of 5 months they were given dietary carcinogen: aflatoxin B1 (AFB₁) or 7,12-dimethylbenz[a]anthracene (DMBA) for 30 or 120 days. The dietary exposures were at known tumorigenic levels (100, 200 and 300 ppb AFB₁; 250, 500 and 850 ppm DMBA). At about 16 months after fertilization the fish were sacrificed and tumor incidence and multiplicity were assessed. At all levels of carcinogen and in all tumorous organs tumor incidence was lower in the triploid fish. For DMBA-fed fish it was seen that the diploid:triploid incidence ratios ranged from 2.0 to 9.0 and for AFB₁ from 3.1 to 6.0. Weight class analyses dissociated the tumor incidence effects of growth from the effects of triploidy. Weight classes plotted against logit tumor incidence at all doses and durations showed parallel logistic lines. In every case the triploid curve was substantially lower than the diploid curve, showing the independent suppressive effect of triploidy. Fifteen triploid DMBA liver tumors were examined by direct cycle-sequencing of p53 PCR products across the exons 5, 7 and 8 known to contain nearly all human tumor p53 mutations. There were no p53 mutations seen at, or above, the present threshold of detection, (for radiolabeled manual sequencing, under 5% of mutant in normal). Fluorescent sequencing of 15 stomach tumors, also showed no p53 mutations in the hotspot-containing exons. Mutation detection by sequencing the trout Ki-ras1 gene, ortholog human KRAS2, showed codons 12, and 61 mutations in DMBA-fed trout liver and stomach tumors. The DMBA liver tumor Ki-ras1 mutation incidence showed no change by ploidy. There was a significant reduction in Ki-ras1 exon 1 mutations in triploid stomach tumors (5% in triploids v. 33% in diploids, Fisher's Exact test p<O.O5). AFB₁ liver tumors showed Ki-ras1 mutation incidence of 75% (9/12) in diploids and 90% (9/10) triploids, nearly all in exon 1, this mutation difference with respect to ploidy did not reach significance. / Graduation date: 2001

No description available.

Diffuse Midline Glioma (DMG) are universally fatal, primarily pediatric malignancies affecting the midline structures (i.e., pons, thalamus, and spinal cord) of the central nervous system. Despite decades of clinical trials, no drugs have emerged as effective against this disease, and treatment remains limited to palliative radiation therapy. Primary treatment challenges include: A) Well-stablished, yet non-actionable, genetic alterations; B) significant intratumoral heterogeneity, and C) blood-brain barrier (BBB) drug permeability. Here, we address the former two challenges by leveraging network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulators (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct DMG cell states. We reverse engineered the first DMG gene regulatory network from 122 publicly available DMG RNA-seq profiles with ARACNe and inferred sample-specific MR protein activity with VIPER based on the differential expression of their targets. Nine of the top 25 most active MRs (i.e., FOXM1, CENPF, TOP2A, ASF1B, E2F2, TIMELESS, MYBL2, CENPK, TRIP13) comprise a well-characterized MR block (MRB2), frequently activated across aggressive tumors, and found to be enriched in DMG patient MR signatures (Fisher’s Exact Test p = 3.96x10-16). A pooled CRISPR/Cas9-mediated knockout (KO) screen across three DMG patient cell lines targeting 1,433 genes identified a set of 73 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p = 0.000034). FOXM1 emerged as a highly essential MR, significantly activated across virtually all patients. We then generated drug-induced differential protein activity from RNA-seq profiles following perturbation with 372 oncology drugs in two DMG cell lines that together recapitulate DMG patient MR and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Department of Health approved OncoTreat algorithm OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and tyrosine kinase inhibitors in subsets of patients, overlapping with published DMG drug screens. Importantly, 80% of OncoTreat-predicted drugs (p < 10-5) from three DMG patient tumor biopsies showed in vitro sensitivity in cultured tumor cells from the respective patients, with overall 68% accuracy among 223 drugs evaluated by both OncoTreat and in vitro drug screen (Fisher’s Exact Test p = 0.0449). Given known resistance in DMG to single-agent therapy, we further interrogated single-cell DMG regulatory networks generated by ARACNe with gene expression signatures from 3,039 tumor cells previously published across six patients using VIPER to infer single-cell regulatory protein activity. Unsupervised clustering of cells by protein activity defined 7 patient-independent cell states with distinct MR profiles reflecting known glial lineage markers (OPC-like-S1, OPC-like-S2, OC-like-S1, OC-like-S2, Cycling, AC-like, and AC/OPC-like). We identified drugs that invert the MR activity profiles of the individual cell states by using OncoTarget (inhibitors of individual MRs) or OncoTreat using the drug-induced differential protein activity we previously generated. Predicted drugs were distinct across the previously defined cell states with bulk RNA-seq recapitulating predictions seen in the more prevalent OPC-like stated, but failing to recapitulate the MRs and drug predictions for the smaller AC-like stated. We selected five drugs targeting the OPC/cycling-like cells (Trametinib, Dinaciclib, Avapritinib, Mocetinostat, and Etoposide), and four drugs targeting the AC-like cells (Ruxolitinib, Venetoclax, Napabucasin, Larotrectinib) for further validation as these states comprised most tumor cells across patients. We then generated single-cell RNA-seq for 95,687 cells after 5 days of treatment with either vehicle control (n = 4) or candidate drug (n = 2-3/drug) in subcutaneous SU-DIPG-XVII patient cell line-derived mouse models. We show this model recapitulates DMG cell states seen in patients, and confirm reduction in tumor growth and significant depletion of either OPC/cycling-like cells or AC-like cells in line with our drug predictions for 8/9 candidate drugs (Chi-square p<0.01). We further treated a syngeneic (DIPG4423) orthotopic DMG model with each drug and demonstrate significant differences in survival with Avapritinib, Dinaciclib, and Trametinib. Notably, the combination of drugs targeting OPC/cycling-like and AC-like cells (i.e. Trametinib+Ruxolitinib, Dinaciclib+Ruxolitinib, Avapritinib+Venetoclax, etc.) showed significantly lower tumor volumes after 2 weeks of treatment as compared to vehicles or each drug alone, and significant survival differences for some of the combinations. This work provides a precision medicine platform to nominate much-needed novel drug combinations addressing DMG tumor heterogeneity for further study to improve outcomes in this devastating disease.

Introduction: Radiofrequency ablation (RFA) is a popular method of treating unresectable liver tumours by the use of a high frequency, alternating electrical current that heats and destroys tumour cells. The size of the ablation is limited by localised charring of adjacent tissue that prevents further conduction of the radiofrequency current. In the clinical setting, this results in increased rates of local recurrence in tumours that are greater than 3 cm in diameter as multiple, overlapping ablations need to be performed to treat the one tumour. To overcome this problem, a modified form of RFA called Bimodal Electric Tissue Ablation (BETA) has been created. BETA adds a direct electrical current to the alternating radiofrequency current, thus establishing its bimodal character. When direct currents are used in biological tissues, water is transferred from anode to cathode by a process called electro-osmosis. By attaching the cathode to the radiofrequency electrode, water is attracted to the area thus preventing tissue desiccation and charring. The BETA circuit has been constructed and tested using a porcine model. The aims of the studies are to confirm that larger ablations can be produced with the BETA system and that it is safe to use in an animal model. Three studies have been performed to test these aims in porcine liver. Methods: The first study was designed to compare sizes of the ablation produced between standard RFA and the BETA circuit. This was followed by a long-term study to assess associated changes to liver function and pathological changes within the liver as well as identifying any other treatment related morbidity. The third study assessed the difference in ablation size and safety aspects when the positive electrode of the direct current circuitry was moved from small surface area under the skin to a large surface area on the skin. Results: Ablations with significantly larger diameters are created with the BETA circuit using a multi-tine needle (49.55 mm versus 27.78 mm, p<0.001). This finding was confirmed in the third experiment using a straight needle (25 mm versus 15.33 mm, p<0.001). Ablations produced by the BETA circuit induce coagulative necrosis within the treated liver and the injury heals by fibrosis in a manner similar to other thermal therapies. Significant rises in some serum liver enzymes are seen within 24 hours of treatment but these return to normal within 4 days. An electrolytic type injury can be produced at the site of the positive electrode. By increasing the surface area of this electrode, the risk of tissue damage is decreased but ablations are significantly smaller (18 mm versus 25 mm, p<0.001). Conclusions: The BETA circuit consistently produces significantly larger ablations than RFA. The treatment appears safe but positioning of the positive electrode of the direct current requires careful consideration. Injuries produced behave like other thermal therapies with coagulative necrosis followed by fibrotic healing. As larger ablations are consistently produced, it is hypothesised that with further refinements, tumours greater than 3 cm in diameter could be treated with lower rates of recurrence. / Thesis (M.S.) -- University of Adelaide, School of Medicine, 2008

Introduction: Radiofrequency ablation (RFA) is a popular method of treating unresectable liver tumours by the use of a high frequency, alternating electrical current that heats and destroys tumour cells. The size of the ablation is limited by localised charring of adjacent tissue that prevents further conduction of the radiofrequency current. In the clinical setting, this results in increased rates of local recurrence in tumours that are greater than 3 cm in diameter as multiple, overlapping ablations need to be performed to treat the one tumour. To overcome this problem, a modified form of RFA called Bimodal Electric Tissue Ablation (BETA) has been created. BETA adds a direct electrical current to the alternating radiofrequency current, thus establishing its bimodal character. When direct currents are used in biological tissues, water is transferred from anode to cathode by a process called electro-osmosis. By attaching the cathode to the radiofrequency electrode, water is attracted to the area thus preventing tissue desiccation and charring. The BETA circuit has been constructed and tested using a porcine model. The aims of the studies are to confirm that larger ablations can be produced with the BETA system and that it is safe to use in an animal model. Three studies have been performed to test these aims in porcine liver. Methods: The first study was designed to compare sizes of the ablation produced between standard RFA and the BETA circuit. This was followed by a long-term study to assess associated changes to liver function and pathological changes within the liver as well as identifying any other treatment related morbidity. The third study assessed the difference in ablation size and safety aspects when the positive electrode of the direct current circuitry was moved from small surface area under the skin to a large surface area on the skin. Results: Ablations with significantly larger diameters are created with the BETA circuit using a multi-tine needle (49.55 mm versus 27.78 mm, p<0.001). This finding was confirmed in the third experiment using a straight needle (25 mm versus 15.33 mm, p<0.001). Ablations produced by the BETA circuit induce coagulative necrosis within the treated liver and the injury heals by fibrosis in a manner similar to other thermal therapies. Significant rises in some serum liver enzymes are seen within 24 hours of treatment but these return to normal within 4 days. An electrolytic type injury can be produced at the site of the positive electrode. By increasing the surface area of this electrode, the risk of tissue damage is decreased but ablations are significantly smaller (18 mm versus 25 mm, p<0.001). Conclusions: The BETA circuit consistently produces significantly larger ablations than RFA. The treatment appears safe but positioning of the positive electrode of the direct current requires careful consideration. Injuries produced behave like other thermal therapies with coagulative necrosis followed by fibrotic healing. As larger ablations are consistently produced, it is hypothesised that with further refinements, tumours greater than 3 cm in diameter could be treated with lower rates of recurrence. / Thesis (M.S.) -- University of Adelaide, School of Medicine, 2008