Dianne Chadwick

A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

Specimen Quality Evaluation in Canadian Biobanks Participating in the COEUR Repository

Human biological specimens are important for translational research programs such as the Canadian Ovarian Experimental Unified Resource (COEUR) funded by the Terry Fox Research Institute. Sample quality is an important consideration, as it directly impacts the quality of ensuing research. The aim of the present study was to determine the quality of tissues collected from different sites contributing to the COEUR cohort. Samples from high-grade serous ovarian tumors (fresh frozen and corresponding paraffin-embedded tissues) were provided by nine participating Canadian biobanks. All samples were shipped to a central site using a Standard Operating Protocol (SOP). DNA and RNA extraction was conducted by the quality control division of the Canadian Tumor Repository Network (CTRNet). DNA quality was determined by ß-globin gene PCR amplification, and RNA quality by the RNA integrity number (RIN), as measured by the Agilent BioAnalyzer. DNA of acceptable quality had at least three bands of ß-globin amplified from DNA (n=115/135), and a RIN number ≥7 was considered very good for RNA (n=80/135). Sample preparation and storage time had little effect on RNA or DNA quality. Protein expression was assessed on tissue microarray by immunohistochemistry with antibodies against p53, WT1, E-cadherin, CK-7, and Ki67 from formalin fixed-paraffin embedded (FFPE) tissues. As seen with a nonhierarchical clustering statistical method, there was no significant difference in immunostaining of paraffin tissues among specimens from different biobanks. Interestingly, patients with worse outcome were highly positive for p53 and weak for WT1. In conclusion, while there was no common SOP for retrospectively collected material across Canadian biobanks, these results indicate that specimens collected at these multiple sites are of comparable quality, and can serve as an adequate resource to create a national cohort for the validation of molecular biomarkers in ovarian cancer.

Genomics-Driven Precision Medicine for Advanced Pancreatic Cancer: Early Results from the COMPASS Trial.

Purpose: To perform real-time whole genome sequencing (WGS) and RNA sequencing (RNASeq) of advanced pancreatic ductal adenocarcinoma (PDAC) to identify predictive mutational and transcriptional features for better treatment selection. Experimental Design: Patients with advanced PDAC were prospectively recruited prior to first-line combination chemotherapy. Fresh tumor tissue was acquired by image-guided percutaneous core biopsy for WGS and RNASeq. Laser capture microdissection was performed for all cases. Primary endpoint was feasibility to report WGS results prior to first disease assessment CT scan at 8 weeks. The main secondary endpoint was discovery of patient subsets with predictive mutational and transcriptional signatures. Results: Sixty-three patients underwent a tumor biopsy between December 2015 and June 2017. WGS and RNASeq were successful in 62 (98%) and 60 (95%), respectively. Genomic results were reported at a median of 35 days (range, 19–52 days) from biopsy, meeting the primary feasibility endpoint. Objective responses to first-line chemotherapy were significantly better in patients with the classical PDAC RNA subtype compared with those with the basal-like subtype (P = 0.004). The best progression-free survival was observed in those with classical subtype treated with m-FOLFIRINOX. GATA6 expression in tumor measured by RNA in situ hybridization was found to be a robust surrogate biomarker for differentiating classical and basal-like PDAC subtypes. Potentially actionable genetic alterations were found in 30% of patients. Conclusions: Prospective genomic profiling of advanced PDAC is feasible, and our early data indicate that chemotherapy response differs among patients with different genomic/transcriptomic subtypes. Clin Cancer Res; 24(6); 1344–54. ©2017 AACR.

LSD1-Mediated Epigenetic Reprogramming Drives CENPE Expression and Prostate Cancer Progression

Androgen receptor (AR) signaling is a key driver of prostate cancer, and androgen-deprivation therapy (ADT) is a standard treatment for patients with advanced and metastatic disease. However, patients receiving ADT eventually develop incurable castration-resistant prostate cancer (CRPC). Here, we report that the chromatin modifier LSD1, an important regulator of AR transcriptional activity, undergoes epigenetic reprogramming in CRPC. LSD1 reprogramming in this setting activated a subset of cell-cycle genes, including CENPE, a centromere binding protein and mitotic kinesin. CENPE was regulated by the co-binding of LSD1 and AR to its promoter, which was associated with loss of RB1 in CRPC. Notably, genetic deletion or pharmacological inhibition of CENPE significantly decreases tumor growth. Our findings show how LSD1-mediated epigenetic reprogramming drives CRPC, and they offer a mechanistic rationale for its therapeutic targeting in this disease. Cancer Res; 77(20); 5479-90. ©2017 AACR.

Whole Genomes Define Concordance of Matched Primary, Xenograft, and Organoid Models of Pancreas Cancer

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among solid malignancies and improved therapeutic strategies are needed to improve outcomes. Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 ‘trios’ of matched primary tumour, PDX, and PDO. We developed a new pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Comparison of genomic events in the tumours and matched disease models displayed single-gene concordance across major PDAC driver genes, and genome-wide similarities of copy number changes. Genome-wide and chromosome-centric analysis of structural variation (SV) events revealed high variability across tumours and disease models, but also highlighted previously unrecognized concordance across chromosomes that demonstrate clustered SV events. Our approach and results demonstrate that PDX and PDO recapitulate PDAC tumourigenesis with respect to simple somatic mutations and copy number changes, and capture major SV events that are found in both resected and metastatic tumours.

Abstract 167: Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background: Despite recent advances in personalized cancer therapeutics, little impact has been made on curing advanced melanomas over the last several decades. Patients with metastatic melanoma generally have a poor prognosis; survival is limited and typically the 5-year survival rate is less than 15% in patients with metastatic disease. Dysregulation of BRAF signaling has been shown to be one of these key drivers of the pathogenesis of disease in a large subpopulation (∼40%) of patients. Although targeted therapy with BRAF and MEK inhibitors is the mainstay of therapy for recurrent/metastatic melanomas, this treatment modality provides a temporary respite. Often resistant tumor clones(carrying mutations in oncogenes) emerge, followed by a rapid progression, causing the patients to succumb to the disease. As such, in this study, we aim to understand inter- and intra- tumor heterogeneity that leads to resistance to therapy. Methods: We conducted deep exome sequence analysis (250X coverage) of a collection of 8 metastatic melanoma tumor samples collected by rapid autopsy from a patient who became resistant to Vemurafinib despite harboring sensitizing BRAF p. V600E mutations. We used whole exome sequence analysis to detect somatic mutations and copy number variants in every metastatic tumor. To infer subclonal populations present within each metastasis, we applied a combination of clonal inference tools to determine subclonal structures based on somatic point mutations (PyClone) and copy number alterations (TITAN). Subsequently, we applied clustering algorithms to the inferred populations to construct inter-tumor phylogenies. Results: We found that 89% of somatic point mutations were shared across all metastatic sites and were primarily clonal. Conversely, the cellular prevalence inferred from copy number alterations showed a greater degree of subclonal evolution. We found a clear relationship between the physical locations of tumors and shared subclonal patterns, whereby metastases from lung, brain and omentum metastases clustered distinctly from those in rectum, small intestine, peritoneum, diaphragm and mesentery. To look for shared patterns of subclonal variation underlying metastatic disease and resistance to therapy, we are currently extending our analysis to an additional six patients, each with tissues from 7-9 metastatic sites collected by rapid autopsy. Conclusion: Our analysis of multiple metastatic sites from a single patient by deep exome sequence analysis found most somatic mutations were shared across tumor sites and subclonal evolution appears to be driven primarily by acquisition of copy number alterations. Tumors in near proximity shared greater genomic similarity compare to distant sites. Ongoing analysis of additional patients is required to uncover recurrent mechanisms of resistance to targeted therapies in this disease. Citation Format: Soroush Samadian, Prashant Bavi, Dianne Chadwick, Arnavaz Danesh, Mark Dowar, Tiantian Li, Madura Siva, Michael Roehl, Anthony m. Joshua, Trevor J. Pugh. Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 167.

Abstract 3381: Standardizing the analysis of Ki-67 immunohistochemical assays

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Intro Immunohistochemical assays of the proliferation marker, Ki-67, have been associated with poorer clinical prognosis in breast cancer. However, a high degree of inconsistency in scores has been demonstrated in inter-laboratory and intra-laboratory Ki-67 positivity scorings, this has limited its potential in clinical practice. In this study, we aim to find a more consistent method for scoring Ki-67 positivity among malignant breast tumours. Methods Six Tissue Microarray (TMA) blocks were sectioned and immunohistochemistry was performed with Anti-Ki-67 antibody. Slides were then evaluated and Ki-67 positive cells in invasive breast carcinoma were scored as a percent positivity manually by a trained analyst with random sample quality assurance (QA) by trained pathologists. This was used as the standard benchmark for the experiment as it has been correlated successfully with clinical outcome. Successively, the same six slides were then annotated on Aperio ePathology software by two observers with different levels of pathology training and experience. The annotated regions were analyzed for Ki-67 positivity with Aperio ePathology software on UHN BioBank servers. The computer analyzed scores were compared to the manual benchmark scores. Results The difference between computer-analyzed and manual-scores were relatively large, Observer-A-annotated-computer-analyzed vs. analyst-manual-scores had a difference of 4.23% to 16.96%, while Observer-B-annotated-computer-analyzed vs. analyst-manual-scores had a difference of 7.13% to 15.03%. Interestingly, Observer-A-annotated-computer-analyzed vs. Observer-B-annotated-computer-analyzed scores only had a difference of 0.49% to 2.91%. Pearson Correlation was calculated for all samples on a case-by-case basis and we found there to be a linear correlation of 0.564, with a P-value of 3.6082×10-8, between the computer scores and the manual scores; suggesting significant correlation between the computer scores and the manual score. Conclusion A significant linear correlation has been observed between the computer score and the manual score. However, while the data does not seem to support the idea that a semi-automatic method of computer scoring will replace analyst manual scoring, most of the large contributing variables have been identified. We plan in the next steps of the project to continue to decrease the effects of such variables. It is interesting that the inter-observer computer score displayed a minimal amount of difference, again with the variables identified. This could signify a more consistent method of Ki-67 scoring. Further experiments will be conducted to continue to reduce the variables and optimize the system to gain similar performance as manual scoring. Hopefully in the near future, computerized immunohistochemical analysis can replace the tedious task of manual scoring. Citation Format: Tian Yu Liu, Trillium Chang, Adewunmi Adeoye, Willa Shi, Sheng-Ben Liang, Dianne Chadwick, Michael H.A. Roehrl, Naomi Miller, Fei-Fei Liu, Susan J. Done. Standardizing the analysis of Ki-67 immunohistochemical assays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3381. doi:10.1158/1538-7445.AM2015-3381

Abstract B91: Primary tumor xenograft establishment from pancreatic resection specimens.

Background: As part of Canada9s International Cancer Genome Consortium (ICGC) pancreatic cancer genome sequencing project and to develop a large number of patient-derived cancer models for functional studies, we initiated a program to systematically establish primary xenografts from patients who underwent pancreatic cancer resection. Methods: Informed consent was obtained from patients scheduled for resection of their pancreatic tumors. Tumor samples were harvested from the resected specimens and placed into transport medium within 30 minutes after the pancreatectomies. Samples were implanted into the subcutaneous and orthotopic (pancreatic) sites of nonobese diabetic (NOD) SCID mice. Tumor growths were recorded weekly and animals were sacrificed when the tumors reached 1.5 cm3 humane endpoint. Tumor fragments were processed for reimplantation into new hosts, serially for up to five generations, as well as for histopathology evaluation, cryopreservation, and snap-frozen tissue banking. Results: Between September 2008 and December 2011, primary tumors from 110 consented pancreatic cancer patients were obtained and implanted. The pathology reported pancreatic ductal adenocarcinoma in 91 of these patients. Xenograft tumors formed in 65 of 91 (71%) ductal cancers. Among 26 samples that failed to engraft, 2 were characterized as carcinoma in situ, and four samples were found to contain no tumor cells. The engraftment rate reached 76% (65/85) when tumor cells were present in the implanted samples. Xenograft tumor growth rates were highly variable with majority of the tumors reaching endpoint within 3 months after the implantation. Histological features were highly similar between xenograft tumors and their corresponding patient primary tumors. Conclusions: A great majority of invasive adenocarcinomas from pancreatic resection can form primary tumor xenografts in NOD SCID mice. Appropriate pathological sampling of the primary tumor is one of the crucial elements for the high success rate. Citation Format: Emin Ibrahimov, Nhu-An Pham, Fannong Meng, Mayleen Sukhram, Dianne Chadwick, Stefano Serra, Patricia Shaw, Corwyn Rowsell, Calvin Law, John McPherson, Steven Gallinger, Ming- Sound Tsao. Primary tumor xenograft establishment from pancreatic resection specimens. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B91.