212 research outputs found
Kras mutation in eutopic endometrium and development of endometriosis : a novel mouse model
No full textEndometriosis is a gynaecological condition that affects 10-15% of people with a uterus and is defined when endometrial-like tissue forms lesions in ectopic environments, most commonly within the pelvic cavity [1, 2]. The reflux of eutopic endometrium into the pelvic cavity, via retrograde menstruation, is the most common explanation for endometriosis development; however, more factors (such as gene expression alterations and immune dysregulation) are likely to contribute to the development of endometriotic lesions [3, 4]. It has recently been discovered that patients with deep infiltrating endometriosis, an invasive subtype of endometriosis, tend to have a mutation in KRAS codon 12 confined to the epithelium of the endometriosis lesions and within the eutopic endometrium [5]. To achieve a deeper understanding of the roles of the KRAS mutation in the development of endometriosis, this thesis has two aims: 1) compare the morphology and expression profiles between Kras-mutant and Kras-wildtype eutopic endometrium and 2) develop a novel mouse model for endometriosis with epithelium-restricted Kras mutation. In Aim 1, I found significant downregulation of genes related to the extracellular matrix and upregulation of genes involved in inflammation in Kras-mutant compared to Kras-wildtype uterine tissue. Furthermore, differential regulation of genes involved in epithelial-mesenchymal transition, decidualization, and ferroptosis were also identified. The differential expression of various genes between Kras-mutant and wildtype uterine tissue may partially explain the development of endometriosis and associated symptoms. Aim 2 investigates this potential role of Kras mutations in the development of endometriosis; however, lesion establishment was unsuccessful. Nonetheless, this model provided a good framework to inform the development of future endometriosis mouse models. The characterization of differentially regulated genes between mutated and wildtype endometrial tissue could have large implications in future preclinical studies that focus on the role of somatic mutations in the proliferation and growth of the invasive tissue, with the end goal of developing novel personalized treatments suitable for patients affected by endometriosis.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat
Central sensitization and somatic activating KRAS mutations in endometriosis-associated pain
No full textEndometriosis is the presence of endometrial-like tissue growing abnormally outside the uterus and affects 10% of women and an unknown number of gender diverse people. There are 3 anatomical types of endometriosis: superficial peritoneal (SUP), ovarian endometrioma (OMA), and deep infiltrating endometriosis (DIE). Pelvic pain is a cardinal symptom in endometriosis. The etiology of pelvic pain in endometriosis is multifactorial, meaning that multiple factors may play a role in this pain, such as central nervous system sensitization and peripheral factors (e.g., somatic mutations in endometriosis lesions).
Central nervous system amplification of pain pathways (central sensitization) has been found in people with endometriosis and pelvic pain, which manifests as non-gynecologic pain contributors (central sensitivity syndromes (CSS)). In Chapter 2, I hypothesized that the Central Sensitization Inventory (CSI) could be used to identify centrally sensitized people in an endometriosis population and predict surgical outcomes. My results indicated that a CSI cut-off of 40 or higher could identify a person with 3 or more CSS with good sensitivity and specificity.
Additionally, recent findings showed that 25% of DIE (without concurrent cancer) harbour somatic activating mutations of the KRAS oncogene in endometriosis epithelial cells. In Chapter 3, I explored the prevalence of somatic activating KRAS codon 12 mutations in endometriosis lesions and their association with clinical variables (e.g., pain scores). My results indicated that OMA samples had the highest KRAS mutation prevalence compared to the other anatomical types. Additionally, KRAS mutations were associated with more severe anatomic disease (i.e., higher stage and more invasive endometriosis).
In Chapter 4, I addressed the gap between evidence and practice by translating research findings in a one-day workshop and an accessible video format to patients with endometriosis and their loved ones. My results indicated that a one-day workshop on sexual pain and endometriosis showed promise at improving confidence in self-managing sexual pain and improving knowledge on endometriosis, as well as intent for behavioral changes. Overall, my dissertation work may inform a future clinical decision aid to identify the primary factors contributing to endometriosis pain and suggest specific therapies to treat those factors and reduce unnecessary procedures.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat
HGSOC subtypes and treatment interaction : implications for clinical implementation of the PrOTYPE classifier
No full textHigh-grade serous ovarian cancer (HGSOC) represents approximately 70% of cases of ovarian carcinoma. Investigation into its molecular features led to the discovery of four subtypes of HGSOC, which differ from each other in their gene expression, cellular composition and survival outcomes. However, methods of whole-genome expression clustering have limited the reproducibility of existing subtype-specific research findings as well as being unsuitable for use in individual cases, such as in a clinical setting. PrOTYPE has been developed as a clinical-grade tool for determining the subtype of HGSOC tumours according to expression of a select panel of genes. To investigate the potential utility of determining HGSOC subtype using PrOTYPE in the clinical decision-making process, this thesis has two aims: 1) explore subtype-specific differences in survival outcomes of HGSOC patients treated with the anti-angiogenic drug bevacizumab, to determine whether subtype could be used to inform treatment decisions, and 2) evaluate the subtypes of paired samples collected before and after treatment to platinum and taxane chemotherapy to determine whether exposure to chemotherapy induces changes in subtype. In Aim 1, I found that cases belonging to the immunoreactive C2.IMM subtype generally had improved overall and progression-free survival when treated with bevacizumab. Results also suggested that the C5.PRO subtype may be associated with poorer survival when treated with bevacizumab compared to if treated with platinum and taxane chemotherapy alone. However, these findings could not be validated in independent cohorts; stratification of patients by subtype-specific treatment response in a clinical setting would require further investigation before being implemented. Additionally, a search for novel genes associated with differential treatment outcomes with bevacizumab identified several genes that may be useful in future research on targeted approaches to bevacizumab treatment. In Aim 2, I found that change in subtype from before to after exposure to neoadjuvant chemotherapy occurred in the majority (11/16) of the cases studied; this indicates that pre-chemotherapy subtype cannot be determined from post-chemotherapy samples. Clinical and prognostic significance of HGSOC subtype after chemotherapy remains an area for future investigation. Subtyping using PrOTYPE could be used in the clinical context to guide treatment decisions and evaluate patient prognosis.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat
Characterization of the role of HACE1 in Wilms' tumours
No full textOur research group recently identified the novel ubiquitin-protein ligase gene, HACE1
(HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1), on Chr. 6 near a
unique t(6;15)(q21;q21) translocation in a Wilms' tumour. HACE1 levels are reduced in ~75%
of Wilms' tumours and a number of other malignancies compared to patient-matched normal
tissue. Furthermore, aberrant methylation patterns within CpG islands upstream of the HACE1
gene, in Wilms' tumours, correlate increased levels of methylation with lower expression
(60%). This has led us to hypothesize that HACE1 functions as a tumour suppressor gene. To
explore this hypothesis, I have established two retroviral model systems. First, I have observed
that Wilms' tumour derived cell lines grown in soft agar show reduced colony number and size
when cells were engineered to stably over-express HACE1 compared with empty vector or a
non-functional HACE1 mutant. In vivo tumour formation in nude mice injected with a Wilms'
tumour cell line overexpressing HACE1 is also highly attenuated compared with empty vector
control. Second, I have identified a number of small interfering RNA (siRNA) sequences that
functionally knock-down HACE1 expression. These siRNAs have been used in a stable
lentiviral system to reduce HACE1 expression by as much as 80% in a number of cell lines,
mimicking the low level of HACE1 observed in Wilms' tumours. Knocking down HACE1
levels in HEK293 cells resulted in an increase in both the number and size of soft agar colonies.
HACE1 appears to affect several different growth and survival related pathways. Although
direct targets of HACE1 are not yet validated, changes in AKT and GSK3β activation and the
levels of cyclin D1 are consistently affected by HACE1 expression. Taken together, these data
support a role for HACE1 as a novel tumour suppressor whose reduced expression can
contribute to malignant progression.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat
Abstract AP09: GENOMIC ALTERATIONS IN ENDOMETRIOSIS
No full textAbstract
BACKGROUND: Endometriosis affects ~10% of reproductive–aged women and is characterized by extra–uterine biphasic growth of uterine endometrial epithelium and stroma. Endometriosis is widely considered to be a hormonally–dependent inflammatory condition and is classified into three anatomically defined types: deep infiltrating endometriosis that locally invades pelvic organs; ovarian endometriotic cysts, and superficial peritoneal endometriosis. A clonal “precursor–cancer” link to co–occurring clear cell and endometrioid ovarian cancers has been established and somatic mutations occur in these cancer–associated endometriotic cysts, including distant benign–appearing lesions. The spectrum of genetic alteration in non–cancer associated endometriosis is not known.
METHODS: We screened a pilot series of 10 deep–infiltrating [pelvic] endometriosis lesions, without co–occurrence or history of cancer, for somatic alterations using laser captured endometrial stroma and epithelium. Two overlapping amplicon panels and digital PCR were used to validate mutations.
RESULTS: In four of ten cases we validated the presence of activating codon 12 KRAS mutations. Mutations were restricted to the epithelium and no evidence of any alterations was found in the stromal fraction.
CONCLUSIONS: We provide incontrovertible evidence of activating KRAS mutations in 40% of deep infiltrating endometriosis. This frequency of KRAS mutations is substantially higher than what has been observed in clear cell (CCOC) or endometrioid (ENOC) ovarian cancers and the occurrence of non–ovarian primary CCOC/ENOC is largely anecdotal. Our evidence suggests that these mutations represent core features of endometriosis itself, rather than a risk of malignant transformation. Overall we propose this as an opportunity to fundamentally shift the study of endometriosis to a spectrum of neoplasms. This may lead to the generation of a molecularly informed classification system and ultimately to improved prognostication and treatment.
Citation Format: Michael S. Anglesio, Tayyebeh M. Nazeran, Hugo M. Horlings, Vivian Lac, Amy Lum, Janine Senz, Julie Ho, Amy Wang, Fontayne Wong, Catherine Allaire, Christina Williams, C. Blake Gilks, Paul J. Yong, and David G. Huntsman. GENOMIC ALTERATIONS IN ENDOMETRIOSIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP09.</jats:p
Abstract DPOC-014: BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT
No full textAbstract
PURPOSE: Women with endometriosis, a painful condition caused by displaced endometrial tissue, have a 3-fold increased risk of developing endometrioid ovarian cancer (ENOC) and clear cell ovarian cancer (CCOC). How two distinct cancers arise from the same precursor lesion is unknown. Sensitive biomarkers are needed to identify women with endometriosis at risk of developing cancer. We performed whole genome sequencing on 29 ENOC and 36 CCOC cases and observed a highly frequent insertion event originating from an active LINE-1 (L1) retrotransposon in the TTC28 gene. L1 retrotransposons are mobile genetic elements that can take downstream DNA pieces and insert them into random genomic locations in a process called 3' transduction. L1s are epigenetically silenced in normal tissues, but are known to become activated in a variety of cancers. A recent study showed a stepwise loss of methylation across various L1 loci between normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and ENOC/CCOC tissues. We hypothesize that TTC28 L1 retrotransposon is an early event in the transformation of endometriosis into ENOC and CCOC and such events could be used as biomarkers for endometriosis with high cancer risk.
METHODS: We compared the presence of TTC28 L1 3' transductions to six SNVs and frame shifts mutations in normal, endometriosis, and tumor tissues from different anatomical sites in four ENOC and four CCOC cases. PCR followed by Sanger sequencing was used to detect TTC28 L1 insertions, and micro-fluidic PCR assay followed by MiSeq sequencing used to detect SNV/frameshift mutations. To broaden the analysis we will use a target capture sequencing method to track novel TTC28 and other L1 transductions. In these experiments probes tiling 1kbp downstream of L1s will be used to capture DNA fragments containing L1 transductions; the captured fragments will be sequenced on the MiSeq. We will assess the difference in TTC28 L1 methylation status between normal, endometriosis, and tumor tissues via the sequencing of bi-sulfite treated DNA.
RESULTS: TTC28 L1 retrotransposition insertion is present at all 5 tumor sites in 75% (6/8) of cases, and is present in 3/5 or 4/5 tumor sites in the remaining cases. Analysis shows that TTC28 L1 insertion preceded some SNV and/or frameshift mutations. Preliminary results show that TTC28 L1 promoters are unmethylated in tumors with L1 insertions. Future experiments involving additional cases with endometriosis tissues will be performed. We expect to see L1 promoter hypomethylation and L1 transductions in endometriosis tissues.
CONCLUSION: TTC28 L1 promoter hypomethylation and TTC28 L1 transductions may be early events in the transformation of endometriosis to cancer that can be explored as a method to predict tumor development. The development of a target capture assay to detect novel L1 transductions will be crucial for investigating cases without whole genome sequencing data. Ultimately, we hope to detect L1 insertions in plasma samples, and use L1 insertions as a biomarker to identify high-risk endometriosis cases.
Citation Format: Zhouchunyang Xia, Dawn Cochrane, Michael S Anglesio, Tayyebeh M Nazeran, Janine Senz, Amy Lum, Ali Bashashati, Yi Kan Wang, Sohrab P Shah, David Huntsman. BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-014.</jats:p
You won't believe this old test … that does cheap single-cell mutation detection.
No full textDetecting mutations in single cells from cancer specimens is now a major area of translational research. In a recent article in this journal, Khalique et al validated an immunohistochemistry assay for ARID1A that reliably identifies loss of function mutations in single cells in tissue sections. This work exemplifies best practice for developing and orthogonally validating immunohistochemical assays to provide clearly interpretable mutational results with spatial context
Profiling the Cancer Genome
No full text Cancer profiling studies have had a profound impact on our understanding of the biology of cancers in a number of ways, including providing insights into the biological heterogeneity of specific cancer types, identification of novel oncogenes and tumor suppressors, and defining pathways that interact to drive the growth of individual cancers. Several large-scale genomic studies are underway that aim to catalog all biologically significant mutational events in each cancer type, and these findings will allow researchers to understand how mutational networks function within individual tumors. The identification of molecular predictive and prognostic tools to facilitate treatment decisions is an important step for individualized patient therapy and, ultimately, in improving patient outcomes. Whereas there are still significant challenges to implementing genomic testing and targeted therapy into routine clinical practice, rapid technological advancements provide hope for overcoming these obstacles. </jats:p
Clear cell carcinoma of the ovary: A report from the first Ovarian Clear Cell Symposium, June 24th, 2010
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