David Huntsman

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
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)

No abstract available.

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 (2010 - 2018)
LINE-1 retrotranspositions in epithelial ovarian cancer: can we use DNA parasites for good purpose? (2018)

No abstract available.

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)

No abstract available.

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