David Huntsman


Relevant Thesis-Based Degree Programs


Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

The proteomic and metabolomic characterization of clear cell ovarian cancer : towards better management strategies (2023)

Clear cell ovarian cancer (CCOC) is a molecularly unique subtype of epithelial ovarian cancer for which treatment options are still limited. Patients with late stage CCOC do not respond to gold-standard platinum and taxane based chemotherapeutics, and effective targeted therapies for this cancer are still lacking. Due to its relative rarity, the molecular landscape of this ovarian cancer subtype has not been fully deciphered. In this thesis, I used global screening techniques to elucidate the proteomic and metabolomic landscapes of CCOC, compared to other common epithelial ovarian cancer subtypes. I found that CCOC is a unique entity compared to its other epithelial ovarian cancer counterparts in both its proteomic and metabolomic landscapes. I reported proteomic diversity within CCOC cases presented as subgroups of distinct molecular signatures. Moreover, through integrated proteometabolomic analysis, I identified aberrancies in purine metabolism, cysteine/glutathione metabolism, as well as glucose metabolism. I further demonstrated that available CCOC cell lines reflect the proteomic and metabolomic signatures in CCOC clinical samples. The heterogenous biology seen in clinical samples and cell lines suggests that the comprehensive understanding of this cancer require appropriate in-vitro models to represent its diverse molecular phenotypes. Lastly, our proteomic understanding led to the identification of the lack of an arginine synthesizing enzyme, arginosuccinate synthase, in CCOC and other rare ovarian cancer subtypes including small cell ovarian carcinomas hypercalcemic type. Cancers lacking this enzyme has been shown to be sensitive to a molecular agent depriving extracellular arginine. I show that this therapeutic agent was effective in curbing the growth of arginosuccinate synthase-negative rare ovarian cancers in-vitro and in in-vivo murine models, thus identifying a potential therapy for these very aggressive subtypes. My research provides the largest reported proteomic landscape of CCOC cases, in addition, I report the first metabolic landscape of CCOC and other ovarian cancer subtypes. Both will serve as valuable resources for further research into CCOC biology and therapeutic development. Furthermore, I demonstrate of how proteomic and metabolomic understanding can accelerate therapeutic development in rare ovarian cancers by using arginosuccinate synthase deficiency as an example.

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Adult-type granulosa cell tumour of the ovary: a FOXL2-centric disease (2022)

Adult-type granulosa cell tumours (AGCT) represent 3-5% of ovarian cancers. AGCT is typically diagnosed at an early stage and is treated by surgery. However, tumour recurrence occurs in one-third of patients, and approximately 50% of relapsed patients succumb to their disease. A somatic missense mutation (c.402C>G; pC134W) in FOXL2 was identified in over 95% of AGCTs. Although this FOXL2 mutation enhanced the ability to accurately diagnose challenging cases, there is still a major clinical challenge in identifying patients that are at risk of relapse and how the FOXL2 C134W mutation contributes to tumourigenesis remains to be elucidated. Objectives: 1) Characterize the genomic and transcriptomic features of AGCT and 2) Develop novel model systems to study the FOXL2 C134W mutation in AGCTHypotheses: 1) Secondary mutations and/or transcriptional alterations explain the clinical and biological diversity in AGCT and 2) An appropriate cell context and microenvironment is required to understand the role of FOXL2 C134W in AGCT. Methods: Whole genome and targeted sequencing were performed to identify secondary mutations. RNA-Sequencing was performed to describe the transcriptional landscape. Transduction of FOXL2 C134W mutant and hTERT into primary granulosa cells was investigated for the establishment of a de novo transformation model. Development of tumour-derived cell lines and patient-derived xenografts were explored. Results: AGCT is not a disease that cannot be sub-stratified by genomic features. Besides the AKT1 (c.49G>A; pE17K) hotspot mutation identified at a low frequency (2/130, 1.5%) in AGCT, no actionable mutations were identified. TERT C228T promoter mutations were identified in primary AGCTs (51/229, 22%) and recurrent AGCTs (24/58; 41%) suggesting that these mutations play a role in tumour initiation and progression. Transcriptome analysis did not separate AGCTs based on disease status. The transduction of FOXL2 C134W mutant and ectopic expression of TERT into primary granulosa cells was not sufficient for oncogenic transformation. Immortalization of AGCT-derived patient cells and development of patient-derived xenografts were unsuccessful. Conclusions: AGCT represents a specific clinical entity driven by the FOXL2 C402G mutation. Therapeutic strategies focusing on targeting the FOXL2 C134W protein or its downstream consequences are the most promising approach to developing novel treatments for AGCT.

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Functional investigations of Capicua (CIC) and ATAXIN-1-Like (ATXN1L) in cancer (2021)

Mutations in the Capicua (CIC) gene, located on chromosome 19q, were first identified in up to 70% of 1p19q-codeleted, IDH-mutated Oligodendrogliomas (ODGs), a subtype of diffuse low grade glioma (DLGG). Since then, several studies have highlighted that loss or dysregulation of CIC is associated with tumour progression, worse prognosis, and treatment resistance in multiple cancer types. One CIC binding partner, Ataxin-1-Like (ATXN1L), has been implicated as an important regulator of CIC function in murine development, but little is known in the context of cancer. In this thesis, we characterize the functional interaction between CIC and ATXN1L and their relationship in regulating pathways involved in tumourigenesis.The function of ATXN1L in relation to CIC was investigated by assessing and comparing the transcriptomic consequences of ATXN1L knockout (KO) and CIC KO using isogenic cell lines. Loss of either CIC or ATXN1L led to concordant dysregulation of gene sets in each context which converged upon several pathways involved in differentiation and activation of the mitogen activated protein kinase (MAPK) pathway as a result of decreased CIC-DNA binding. Further analysis of cancers harboring deletions in CIC and ATXN1L, namely, stomach adenocarcinoma, prostate adenocarcinoma, and astrocytoma, resulted in convergent dysregulation of several pathways involved in cell proliferation and growth.In addition to decreased CIC-DNA binding, loss of ATXN1L resulted in increased CIC protein instability as a result of proteasomal degradation which was found to be independent of ERK activity, the canonical CIC degradation pathway. Instead, loss of ATXN1L was found to promote CIC instability through ubiquitination by the E3-ligase TRIM25, a novel CIC interactor. Transcriptomic analyses of breast carcinomas with TRIM25 amplification and liver hepatocellular carcinomas with high TRIM25 expression revealed dysregulation of genes and pathways reminiscent of CIC loss, supporting its role in the post-translational regulation of CIC.The results of these studies illuminate our understanding and the intricacy of mechanisms which regulate normal CIC function in cancer; and further supports the role of CIC as a potent tumour suppressor in a multitude of cancer types.

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Molecular characterization and genetic diagnosis of cancer predisposition syndromes using genome and transcriptome sequencing (2020)

Genetic variation makes important and often uncharacterized contributions to both rare syndromes and more common complex diseases. Around 8% of all cancers are caused by high- and moderate-penetrance deleterious germline variants that are present in an individual from birth and predispose to specific cancer types throughout life. Such cancer predisposition genes associated with moderate to high lifetime cancer risks, conventionally defined as greater than twofold and fivefold increases, respectively, are involved in various biological pathways required for regulating cellular proliferation, maintaining genome integrity, and mediating inter- and intracellular signaling. Clinical use of multigene next-generation sequencing panels has improved molecular diagnosis of cancer predisposition syndromes, demonstrating both genetic heterogeneity and phenotypic variability amongst carriers. However, many individuals with a strong personal or family cancer history receive uninformative results from clinical genetic testing that may lead to increased health anxiety and missed opportunities for increased cancer screening, cancer prevention or use of targeted therapies. Therefore, the main objective of my dissertation was to characterize the biological significance, functional impact, and heterogeneity of genetic variation underlying high-penetrance cancer predisposition syndromes to improve rates of genetic diagnosis. Using genome and transcriptome sequencing, I explored molecular characteristics associated with inactivation of high-penetrance cancer predisposition genes across advanced cancers and in an organoid model system. Tissue-specific molecular signatures provided insights into the aetiology of site-specific tumour development in carriers, allowing opportunities for carrier ascertainment and differential genetic diagnosis of suspected hereditary cancer families. Based on findings that structural variants account for 10% of causal variants, I investigated the utility of long-read sequencing in the clinical interpretation of germline structural variants that were undetected or unresolved through next-generation sequencing. Short- and long-read genome sequencing improved genetic diagnosis in known and suspected carriers for autosomal dominant cancer syndromes, demonstrating incomplete penetrance and phenotypic heterogeneity in population-based and disease-specific cancer cohorts. The research presented here thus supports a broader understanding of the contributions of germline variation to cancer susceptibility and disease progression, ultimately informing guidelines for screening, variant interpretation, and clinical management.

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Hereditary diffuse gastric cancer: cancer risk and the personal cost of preventive surgery (2017)

Germline CDH1 mutation carriers are at risk for early-onset diffuse gastric cancer and female carriers have an additional risk of lobular breast cancer. Reliable estimates of cancer risk are essential for genetic counselling and clinical management of mutation carriers. Prophylactic total gastrectomy (PTG) to eliminate the gastric cancer risk is an option for mutation carriers. Current information on post-surgical outcomes and quality of life is limited. The objectives of this research were 1) To improve the evidentiary basis of genetic counselling by deriving reliable estimates of cancer risk in CDH1 mutation carriers; 2) To catalogue a comprehensive list of all novel and previously reported germline mutations to date; and 3) To provide data on post-surgical clinical outcomes and to describe the impact of the surgery on participants’ quality of life. Methods: Penetrance was derived from 67 mutation-positive families comprising 4031 individuals (350 affected with gastric cancer and 99 with breast cancer). Participants were recruited through multiple sources for clinical outcomes and quality of life study. Hospital records provided information on clinical outcomes. All participants were asked to complete validated questionnaires measuring generic and condition specific QOL (PROMIS, EORTC and SF 36v.II) at a single point. Results: By age 75 years, the cumulative incidence of gastric cancer was 70% (95% confidence interval [CI], 40%-94%) for males and 56% (95% CI, 27%-90%) for females. The risk of breast cancer for females was 42% (95% CI, 23%-68%) by 75 years. The mutational landscape of CDH1 did not reveal mutational hotspots but several shared mutations are seen in unrelated families. The 53 participants who had undergone PTG reported frequent symptoms of fatigue (59%), abdominal pain (55%), and diarrhea (45%). Cognitive, role and social function plus the symptoms anxiety, pain, taste, dyspnea and diarrhea were significant predictor variables for quality of life (p
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PPP2R1A Mutations in Gynaecologic Cancers: Functional Characterization and Use in the Genomic Classification of Tumours (2015)

Endometrial carcinoma is the most common gynaecological cancer in developed countries. The current endometrial pathologic classification system lacks reproducibility, which has hampered the development of new treatments for these cancers. The PP2A phosphatase complexes are responsible for regulating many cellular pathways, and may play a role in the deregulation of endometrial cancer-associated pathways. In this thesis, the role of PPP2R1A mutations in the subtype-specific classification of gynaecological tumours was investigated. Additionally, mutational profiles will be used to improve the classification of the subtypes of endometrial carcinomas. Lastly, the functional effect of mutant PPP2R1A on PP2A-subunit protein interactions will be determined, in the context of endometrial cancer cell lines. Next-generation and Sanger sequencing was used to determine the presence of mutations in endometrial and ovarian carcinomas. PPP2R1A isogenic endometrial-specific cell lines were generated using somatic cell gene knockout by homologous recombination. Co-immunoprecipitation and mass spectrometry was used to determine effects of the PPP2R1A W257L mutation on its ability to interact with PP2A subunits. Subtype-specific somatic PPP2R1A mutations were identified in endometrial serous carcinomas. Low-grade endometrial endometrioid carcinomas were defined by mutations in the genes: ARID1A, PTEN, PIK3CA, CTNNB1, and KRAS, whereas high-grade endometrioid also harbor TP53 mutations. Endometrial serous carcinomas harbor mutations in PPP2R1A, FBXW7, PIK3CA and TP53. Consequently, the molecular profiles proved useful in assisting classification of tumours with overlapping morphological features that cause irreproducibility in diagnoses. Proteomic analysis of isogenic cell lines determined that the PPP2R1A W257L mutation disrupts interaction with PPP2R5C and PPP2R5D subunits. In addition, PPP2R1A mutated protein caused an increased interaction with the endogenous PP2A inhibitor SET/I2PP2A. The integration of mutational profiles and other genomic features will be used to improve clinical and pathological classification in endometrial tumours that are difficult to diagnose. PPP2R1A mutations are likely playing a role in the transformation of gynaecological carcinoma, by disrupting PP2A subunit interactions with tumour suppressor functions. Increased interaction of mutant PPP2R1A with SET/I2PP2A adds another layer of complexity to the tumour suppressive role of PP2A. In the future, targeting the PP2A complex with novel therapeutics could provide an alternative method for treating gyneacological cancers with poor outcomes.

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Characterization of hereditary cancer syndrome (2013)

Hereditary cancer syndromes predispose to early-onset or multiple cancers in a person or family, follow Mendelian inheritance patterns and demonstrate stereotyped patterns of tumor development. Genotype-phenotype correlations direct clinical genetic testing and provide guidance for hereditary cancer management. This thesis began by examining the association of lobular breast cancer with germline mutations in CDH1, the gene encoding the epithelial cell-cell adhesion molecule E-cadherin and tested whether CDH1 represented a high-frequency breast cancer susceptibility gene, apart from its association with hereditary diffuse gastric cancer. In addition it examined other genotype-phenotype correlations including the association between granular cell tumors and a multiple congenital anomaly syndrome, the specific correlation between a recurrent somatic mutation in a transcription factor and adult-type granulosa cell tumors and the strong association of germline BRCA1 and BRCA2 mutations with high-grade serous epithelial ovarian cancer. These candidate gene analyses were performed using low-throughput molecular technologies. With the advent of cheaper DNA-sequencing capabilities, the application of these new technologies to novel Mendelian disease gene discovery and hereditary cancer management became the subsequent focus of the thesis. Objectives: To determine the frequency of germline CDH1 mutations in women with lobular breast cancer unselected for familial gastric cancer; to define the associations between several alternative genotype-phenotype correlations; and, to apply next-generation sequencing to determine the basis of a Mendelian disorder, in order to determine its utility as a potential familial cancer gene discovery and clinical tool. Selected Methods: Single amplicon mutation screening and sequence analysis of a large cohort of women with early-onset or familial lobular breast cancer. Next-generation sequencing analysis of a family with multiple individuals cosegregating spondyloepiphyseal dysplasia and retinitis pigmentosa. Results: Without a selective history of diffuse gastric cancer, potentially pathogenic germline mutations in CDH1 occured in women with early-onset or hereditary lobular breast cancer in less than two percent of individuals. Diagnosis of a Mucolipidosis type III gamma was possible using new sequencing technologies. Conclusion: There is utility in understanding genotype-phenotype correlations in order to direct genetic testing and novel gene identification. Next-generation sequencing technologies can succesfully be applied to Mendelian disorders with clear phenotypes for gene discovery.

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Clear cell ovarian carcinoma and emergence of the novel tumour suppressor gene Arid1A (2013)

Clear cell carcinomas (CCCs) are a subtype of ovarian cancer that is understudied, and whichdoes not respond well to conventional therapeutic strategies. There is a desperate need toclarify the genetic mechanisms of CCC to allow for the development of subtype specifictherapeutics. To determine genetic changes responsible for the development of CCC, wholetranscriptomes of 18 ovarian CCCs and one ovarian CCC cell line were sequenced. Somaticmutations were found in ARID1A in 6 samples. ARID1A encodes BAF250a, a key componentof the SWI/SNF chromatin remodeling complex. ARID1A was sequenced in an additional210 ovarian carcinomas and a second CCC cell line, and BAF250a expression was measuredby immunohistochemistry (IHC) in an additional 455 ovarian carcinomas and over 3000 nonovarianmalignancies. Overall, ARID1A mutations were seen in 46% of CCCs and 30% ofendometrioid carcinomas (EC) implicating ARID1A as a tumour suppressor frequentlydisrupted in CCC and EC. Loss of the BAF250a protein correlated strongly with the presenceof ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expressionwere evident in the tumour and contiguous atypical endometriosis, implicating ARID1A lossas an early event in tumourigenesis. Screening 3000 cases of different malignancies by IHCshowed loss of BAF250a was is most frequent in cancers of endometrial origin. Reversephase protein array (RPPA) for tumour samples with known ARID1A mutation statusrevealed a notable change pAKT-Thr³⁰⁸ (FDR
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The kisspeptin and GPR54 ligand-receptor pair in autocrine and endocrine signalling in cancer (2009)

Kisspeptins and their receptor, GPR54, mediate sex hormone release through stimulation of the hypothalamic-pituitary-gonadal axis and have been implicated as metastasis suppressors. Expression of kisspeptin and GPR54 has been associated with less invasive cancers as determined by RNA expression, and a multitude of in vitro studies has consistently shown that overexpression of either ligand or receptor in malignant cell lines results in a less invasive phenotype. We hypothesized that expression of GPR54/kisspeptin in epithelial malignancies is predictive of disease outcome and altering endogenous GPR54 signalling in malignant breast and ovarian epithelial cells could alter their metastatic properties. We have determined by immunohistochemistry that kisspeptin and GPR54 are independent favourable prognostic markers for ovarian carcinoma and are specific for the clear cell cancer subtype; the least characterized of the subtypes. Additionally, loss of GPR54 is associated with poor prognosis in node positive breast cancer patients and is also lost in prostate cancer and testicular germ cell nonseminomas as compared to more benign disease. Moreover, secreted kisspeptin is elevated above physiological levels in the plasma of women with gynaecological cancers, including ovarian cancer. We evaluated GPR54 expression across a panel of breast and ovarian cancer cell lines to create an in vitro model system with which to knockdown GPR54 expression using RNA interference. However, we discovered that endogenous GPR54 was internalized rather than localized to the plasma membrane of these cancer cell lines. Consequently, internal GPR54 was unable to signal through its canonical Gαq pathway. To discover novel genes involved in kisspeptin-GPR54 signalling, we assessed gene expression differences between the Gpr54 and Kiss1 knockout mice as compared to wildtype mice. Our novel candidate list provides insight into physiological signalling in the hypothalamus that can then be applied to epithelial anti-metastatic signalling. Our results also support the sex hormone negative feedback effect on kisspeptin expression as reported in the current literature.In summary, we have confirmed kisspeptin and GPR54 as favourable prognostic markers, are the first to report the intracellular localization of GPR54 in endogenously expressing cancer cell lines, and we have introduced a list of novel genes involved in signalling.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Investigating molecular mechanisms of dedifferentiation and mutations in dedifferentiated endometrial carcinoma (2023)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Investigation into the early pathogenesis of lynch syndrome associated endometrial cancer (2023)

Endometrial cancer is the most common gynecologic malignancy in Canada with increasing rates of both incidence and mortality. Historically, histopathological subtyping of endometrial cancer was employed but was determined to be less than accurate at patient risk stratification. Recent advances in molecular subtyping have revealed mismatch repair deficient endometrial cancer as the second most deadly subtype. Mismatch repair deficient endometrial cancers may be acquired through somatic or germline aberrations to the four mismatch DNA repair genes: MLH1, PMS2, MSH2, and MSH6. Germline mutations to mismatch DNA repair genes results in Lynch Syndrome, an autosomal dominant hereditary cancer predisposition syndrome which increases the risk of endometrial cancer in affected individuals, however, the pathogenesis of Lynch Syndrome associated endometrial cancer has yet to be fully elucidated. To investigate the early pathogenic events following the introduction of MLH1 and PMS2 deficiencies, both in vivo and ex vivo models were utilized. A cohort (n = 6) of archival FFPE tissues, taken from prophylactic hysterectomies performed for Lynch Syndrome patients with germline MLH1 and PMS2 mutations, were immunohistochemically profiled for mismatch repair deficient endometrial glands. MLH1 deficient benign endometrial glands were identified in four cases with a novel report of zonal MLH1 deficient benign endometrial glands. In one case with zonal MLH1 deficient endometrial glands, multiplex immunohistochemical staining based panel was used to quantify endometrial epithelial cell differentiation by examination of secretory, ciliated, and proliferative cell types in both MLH1 proficient and deficient endometrial glands where no significant difference in cell type proportion was observed. MLH1 and PMS2 deficient endometrial organoid models were established using benign hysterectomy tissues and CRISPR-Cas9 mediated gene editing. Single cell RNA sequencing of MLH1 and PMS2 deficient endometrial organoids revealed no significant alterations in MLH1 deficient organoid cells and slight alterations to epithelial mesenchymal transition genes in PMS2 deficient organoids. Altogether, this data suggests that mismatch repair deficiency does not result in immediate changes to gene expression or cell differentiation in endometrial glands and that progression towards endometrial cancer is reliant on the accumulation of mutations.

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Human fallopian tube organoid models of high grade serous ovarian cancer initiation (2021)

Ovarian cancer is the sixth most lethal cancer in women. Out of all ovarian cancers – the high grade serous (HGSC) subtype is the most common and deadliest. This cancer originates from fallopian tube secretory cells and has several putative precursor lesions that have been described in the fallopian tube. These precursors show a spectrum of morphology from normal to abnormal and have been identified in patients with and without cancer. Genetically, HGSC is characterized by widespread genomic instability and ubiquitous TP53 mutation. A variety of model systems have attempted to interrogate how carcinogenesis occurs in the fallopian tube, but few examine the effect of TP53 mutation alone, and virtually all use other species to model human disease. In this thesis I describe the development, validation, and characterization of normal human fallopian tube derived organoids that have had exogenous mutations in TP53 introduced by the targeted cutting of the Cas9 nuclease. Analysis of these organoids was undertaken by live-cell imaging, histochemistry, and single-cell genomic sequencing. Relative to controls, mutant organoids showed normal morphology and growth but abnormal p53 immunohistochemistry. Single-cell sequencing further validated the strength of this model system and revealed comparable genomic stability between TP53 mutants and controls. These findings make sense in light of p53 signature precursor lesions’ normal morphology and low proliferative index; and support the idea that TP53 mutation alone is not sufficient to cause carcinogenesis in the fallopian tube. Reagents and theoretical approaches to interrogating the effects of TP53 missense mutations and homologous recombination deficiency pathway members have also been designed. This research supports genetic perturbation of patient-derived normal organoids as a robust and promising avenue of research.

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LINE-1 retrotranspositions in epithelial ovarian cancer: can we use DNA parasites for good purpose? (2018)

High grade serous ovarian cancer (HGSC), endometrioid ovarian cancer (ENOC) and clear cell ovarian cancer (CCOC) are the three most common subtypes of epithelial ovarian cancers (EOC). While HGSC arise from serous tubal intraepithelial carcinomas (STIC) lesions in the fallopian tube, ENOC and CCOC share a common precursor lesion, endometriosis (ectopic growth of uterine lining). Effective biomarkers of early cancer development and recurrence are lacking. We performed whole genome sequencing (WGS) and observed highly recurrent retrotransposition events originating from an active LINE-1 retrotransposon (L1) in the TTC28 gene in one-third of our ENCO and CCOC cohort. L1s are mobile genetic elements that encodes their own protein machineries to “copy-and-paste” their sequences into random genomic loci. A process called 3’ transduction occur when L1s insert the unique downstream DNA sequences along with their own sequences. All these processes may fuel genomic instability, as such L1s are epigenetically silenced in normal tissues, but are found to be re-activated in cancers and cancer precursor lesions. We hypothesize that L1s activate early in EOC tumorigenesis and that TTC28-L1 3’ transductions could be used as markers of tumor development and progression.Using conventional and multiplex PCR on formalin-fixed paraffin-embedded (FFPE) tumor tissues, we found that TTC28-L1 3’ transductions occurred early and preceded many somatic mutations. We developed a probe-based target capture sequencing method that could identify novel TTC28-L1 3’ transductions in frozen tumor and FFPE tissues, and potentially in circulating tumor DNA. Using immunohistochemistry (IHC), we observed high L1 protein expressions in HGSC and its precursor lesions. Our results suggest that TTC28-L1 events occur early in EOC development and L1 protein expressions may reflect pre-malignant transformations. The use of L1 protein IHC and our target capture assay could be explored as a potential method to track such development.

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Small cell carcinoma of the ovary, hypercalcemic type: model development and preclinical drug testing (2018)

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive ovarian cancer that usually occurs in women in their 20s. As SCCOHT has a very low survival rate, an effective therapeutic solution is urgently needed. Genomic studies have demonstrated that concurrent dual loss of SMARCA4/A2, the two only ATPase of the SWI/SNF chromatin-remodeling complex, occurs in more than 90% SCCOHT cases, which opens the opportunity for developing targeted therapeutics for treating SCCOHT. One major challenge for preclinical studies is the discrepancy between preclinical and clinical outcomes. Although preclinical anti-cancer drugs are mostly tested in subcutaneous xenograft models, they do not provide a relevant environmental support for most cancer types. In contrast, orthotopical models potentially provides similar microenvironment for tumor development. However, the tumor growth monitoring is challenging for orthotopical tumours. To overcome these challenges, I developed SCCOHT cell lines stably expressing mKate2, a far-red fluorescent protein. The application of these SCCOHT cell lines in both subcutaneous and intrabursal models indicated that the fluorescent signals directly correlated to the tumor progression. Pathological analysis revealed intrabursal tumor contained histological features typical of SCCOHT, such as follicle-like structures. Metastasis and ascites were also observed in the intrabursal model. Thus, intrabursal model of SCCOHT mimics the microenvironment of SCCOHT tumors developed in patients; introducing a fluorescent signal provides a convenient monitoring method of the tumor development. Previous studies have shown an antagonism between the SWI/SNF complex components and the polycomb repressive complex 2 (PRC2) in regulating various gene expression. As dual loss of SMARCA4 and SMARCA2 is a definitive feature of SCCOHT, we hypothesize that pharmaceutical inhibition of the activity of PRC2 in SCCOHT may be a potential therapeutic approach. Accordingly, depletion of EZH2, the catalytic subunit of PRC2, or pharmacological inhibition of PRC2 by either GSK126 or EED226 suppressed the proliferation of SCCOHT cell lines. Furthermore, administration of 200 mg/kg EED226 twice daily significantly decreased tumor progression in mice bearing the fluorescent SCCOHT1 subcutaneous xenograft. Therefore, targeting PRC2 is a potential therapeutic strategy for treating SCCOHT and fluorescent imaging can be used to monitor tumor growth in preclinical studies.

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Somatic cancer-driver mutations in endometriosis: implications beyond malignancy (2018)

Introduction: Endometriosis is a chronic, inflammatory gynecological disease characterized by the ectopic growth of endometrial-like tissue. Previous studies have established endometriosis as the precursor to clear cell and endometrioid ovarian carcinomas. The presence of somatic driver mutations in endometriosis is believed to represent early events in transformation, however our group has recently described the presence of such mutations in nearly one-quarter of cases of deep infiltrating endometriosis (DE) – a form of endometriosis that rarely progresses to malignancy. These mutations may play a fundamental role in the pathogenesis of endometriosis outside of the context of cancer, however it is unclear whether they occur in other forms of endometriosis or the eutopic endometrium – the likely tissue of origin for endometriosis. The purpose of my study is to: 1) analyze and compare the mutational profiles of DE and incisional (iatrogenic; IE) endometriosis and 2) characterize somatic cancer-drivers that exist in the eutopic endometrium and determine whether the presence of such mutations reflect the aging of this tissue.Methods: I macrodissected endometriosis tissue from women with IE or DE. Extracted DNA was analyzed by targeted sequencing and mutations were orthogonally validated by droplet digital PCR. PTEN and ARID1A immunohistochemistry was also performed for each specimen. Using the same protocol, I also analyzed hysterectomy and endometrial biopsy specimens obtained from cancer-free women.Results: Overall, we detected the presence of somatic alterations in 27.5% and 36.1% of IE and DE cases respectively. These events affected canonical components of RAS/MAPK or PI3K-Akt signaling pathways. Furthermore, over 50% of cancer-free women also harboured similar somatic alterations in their eutopic endometrial tissue. The presence of somatic cancer-drivers in the eutopic endometrium are likely regional and are correlated with age (p = 0.048).Conclusions: My findings are consistent with a uterine origin of endometriosis. Somatic cancer-driver alterations are commonly found in both endometriosis and the eutopic endometrium of cancer-free women and may reflect the accumulation of DNA damage over time. These somatic alterations alone are insufficient for malignant transformation and should be interpreted with caution in the early diagnosis of gynecologic malignancies given their common occurrence in cancer-free women.

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Genetics of familial gastric cancer: Beyond E-cadherin (CDHI) locus (2014)

Importance. Familial aggregation occurs in approximately 10% of gastric cancers, which are generally sub-classified histologically as intestinal-type and diffuse gastric cancers. Though the genetic basis of familial intestinal-type gastric cancers is not known, in
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