Publications by authors named "Faiyaz Notta"

38 Publications

MYC levels regulate metastatic heterogeneity in pancreatic adenocarcinoma.

Cancer Discov 2021 Sep 22. Epub 2021 Sep 22.

Ontario Institute for Cancer Research.

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1826DOI Listing
September 2021

The value of GATA6 immunohistochemistry and computer-assisted diagnosis to predict clinical outcome in advanced pancreatic cancer.

Sci Rep 2021 Jul 22;11(1):14951. Epub 2021 Jul 22.

Laboratory Medicine Program, University Health Network, 200 Elizabeth Street, Toronto, ON, M5G 2C4, Canada.

Combination chemotherapy, either modified FOLFIRINOX (mFFX) or gemcitabine-nabpaclitaxel, are used in the treatment of most patients with advanced pancreatic ductal adenocarcinoma (PDAC), yet robust biomarkers of outcome are currently lacking to guide regimen selection. Here, we tested GATA6 immunohistochemistry (IHC) as a putative biomarker in advanced PDAC. GATA6 is a transcription factor in normal pancreas development. Two pathologists, blinded to clinical and molecular data, independently assessed GATA6 IHC in biopsy specimens of 130 patients with advanced PDAC, in 2 distinct phases (without and with computer assistance using the open source software QuPath). Low GATA6 IHC expression was associated with shorter overall survival [median OS 6.2 months for patients with GATA6 low tumors vs. 11.5 months for patients with GATA6 high tumors, HR 1.66 (95% CI 1.15-2.40), P = 0.007]. Progression appears to be higher in GATA6-low tumors compared to GATA6-high tumors in patients treated with mFFX (P = 0.024) but not in patients treated with gemcitabine regimens. GATA6 IHC expression was significantly associated with molecular subtypes (P = 0.0003). Digital assistance markedly improved interrater concordance (Cohen's kappa scores of 0.32 vs. 0.95). Our results provide strong evidence that GATA6 IHC can be used as a single biomarker in the clinic to predict clinical outcome in advanced PDAC, warranting further investigation in prospective clinical trials. These results provide the basis for an improved classification of PDAC and future biomarker design using digital pathology workflow.
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http://dx.doi.org/10.1038/s41598-021-94544-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298486PMC
July 2021

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4.

Sci Adv 2021 May 7;7(19). Epub 2021 May 7.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces HO metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.
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http://dx.doi.org/10.1126/sciadv.abf7114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104867PMC
May 2021

PTHrP Drives Pancreatic Cancer Growth and Metastasis and Reveals a New Therapeutic Vulnerability.

Cancer Discov 2021 Jul 15;11(7):1774-1791. Epub 2021 Feb 15.

Herbert Irving Comprehensive Cancer Center, Division of Digestive and Liver Diseases, Department of Medicine, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York.

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by ) is frequently amplified as part of the amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. , which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of , a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292165PMC
July 2021

Genomic Features and Classification of Homologous Recombination Deficient Pancreatic Ductal Adenocarcinoma.

Gastroenterology 2021 05 30;160(6):2119-2132.e9. Epub 2021 Jan 30.

PanCuRx Translational Research Initiative, Ontario Institute for Cancer Research, Toronto, Ontario, Canada; Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Surgery, University of Toronto, Toronto, Ontario, Canada; Hepatobiliary/Pancreatic Surgical Oncology Program, University Health Network, Toronto, Ontario, Canada. Electronic address:

Background And Aims: Homologous recombination deficiency (HRD) in pancreatic ductal adenocarcinoma (PDAC), remains poorly defined beyond germline (g) alterations in BRCA1, BRCA2, and PALB2.

Methods: We interrogated whole genome sequencing (WGS) data on 391 patients, including 49 carriers of pathogenic variants (PVs) in gBRCA and PALB2. HRD classifiers were applied to the dataset and included (1) the genomic instability score (GIS) used by Myriad's MyChoice HRD assay; (2) substitution base signature 3 (SBS3); (3) HRDetect; and (4) structural variant (SV) burden. Clinical outcomes and responses to chemotherapy were correlated with HRD status.

Results: Biallelic tumor inactivation of gBRCA or PALB2 was evident in 43 of 49 germline carriers identifying HRD-PDAC. HRDetect (score ≥0.7) predicted gBRCA1/PALB2 deficiency with highest sensitivity (98%) and specificity (100%). HRD genomic tumor classifiers suggested that 7% to 10% of PDACs that do not harbor gBRCA/PALB2 have features of HRD. Of the somatic HRDetect cases, 69% were attributed to alterations in BRCA1/2, PALB2, RAD51C/D, and XRCC2, and a tandem duplicator phenotype. TP53 loss was more common in BRCA1- compared with BRCA2-associated HRD-PDAC. HRD status was not prognostic in resected PDAC; however in advanced disease the GIS (P = .02), SBS3 (P = .03), and HRDetect score (P = .005) were predictive of platinum response and superior survival. PVs in gATM (n = 6) or gCHEK2 (n = 2) did not result in HRD-PDAC by any of the classifiers. In 4 patients, BRCA2 reversion mutations associated with platinum resistance.

Conclusions: Germline and parallel somatic profiling of PDAC outperforms germline testing alone in identifying HRD-PDAC. An additional 7% to 10% of patients without gBRCA/PALB2 mutations may benefit from DNA damage response agents.
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http://dx.doi.org/10.1053/j.gastro.2021.01.220DOI Listing
May 2021

Subtype-Discordant Pancreatic Ductal Adenocarcinoma Tumors Show Intermediate Clinical and Molecular Characteristics.

Clin Cancer Res 2021 01 13;27(1):150-157. Epub 2020 Oct 13.

Pancreas Centre BC, Vancouver, British Columbia, Canada.

Purpose: RNA-sequencing-based subtyping of pancreatic ductal adenocarcinoma (PDAC) has been reported by multiple research groups, each using different methodologies and patient cohorts. "Classical" and "basal-like" PDAC subtypes are associated with survival differences, with basal-like tumors associated with worse prognosis. We amalgamated various PDAC subtyping tools to evaluate the potential of such tools to be reliable in clinical practice.

Experimental Design: Sequencing data for 574 PDAC tumors was obtained from prospective trials and retrospective public databases. Six published PDAC subtyping strategies (Moffitt regression tools, clustering-based Moffitt, Collisson, Bailey, and Karasinska subtypes) were used on each sample, and results were tested for subtype call consistency and association with survival.

Results: Basal-like and classical subtype calls were concordant in 88% of patient samples, and survival outcomes were significantly different ( < 0.05) between prognostic subtypes. Twelve percent of tumors had subtype-discordant calls across the different methods, showing intermediate survival in univariate and multivariate survival analyses. Transcriptional profiles compatible with that of a hybrid subtype signature were observed for subtype-discordant tumors, in which classical and basal-like genes were concomitantly expressed. Subtype-discordant tumors showed intermediate molecular characteristics, including subtyping gene expression ( < 0.0001) and mutant allelic imbalance ( < 0.001).

Conclusions: Nearly 1 in 6 patients with PDAC have tumors that fail to reliably fall into the classical or basal-like PDAC subtype categories, based on two regression tools aimed toward clinical practice. Rather, these patient tumors show intermediate prognostic and molecular traits. We propose close consideration of the non-binary nature of PDAC subtypes for future incorporation of subtyping into clinical practice.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2831DOI Listing
January 2021

Delving into Early-onset Pancreatic Ductal Adenocarcinoma: How Does Age Fit In?

Clin Cancer Res 2021 01 21;27(1):246-254. Epub 2020 Sep 21.

BC Cancer, Vancouver, British Columbia, Canada.

Purpose: With the rising incidence of early-onset pancreatic cancer (EOPC), molecular characteristics that distinguish early-onset pancreatic ductal adenocarcinoma (PDAC) tumors from those arising at a later age are not well understood.

Experimental Design: We performed bioinformatic analysis of genomic and transcriptomic data generated from 269 advanced (metastatic or locally advanced) and 277 resectable PDAC tumor samples. Patient samples were stratified into EOPC (age of onset ≤55 years; = 117), intermediate (age of onset 55-70 years; = 264), and average (age of onset ≥70 years; = 165) groups. Frequency of somatic mutations affecting genes commonly implicated in PDAC, as well as gene expression patterns, were compared between EOPC and all other groups.

Results: EOPC tumors showed significantly lower frequency of somatic single-nucleotide variant (SNV)/insertions/deletions (indel) in ( = 0.0017), and were more likely to achieve biallelic mutation of through homozygous copy loss as opposed to heterozygous copy loss coupled with a loss-of-function SNV/indel mutation, the latter of which was more common for tumors with later ages of onset ( = 1.5e-4). Transcription factor forkhead box protein C2 () was significantly upregulated in EOPC tumors ( = 0.032). Genes significantly correlated with in PDAC samples were enriched for gene sets related to epithelial-to-mesenchymal transition (EMT) and included ( = 1.8e-8), ( = 6.5e-5), and ( = 2.4e-2).

Conclusions: Our comprehensive analysis of sequencing data generated from a large cohort of PDAC patient samples highlights a distinctive pattern of biallelic mutation in EOPC tumors. Increased expression of in EOPC, with the correlation between and EMT pathways, represents novel molecular characteristics of EOPC..
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1042DOI Listing
January 2021

Clinical and genomic characterisation of mismatch repair deficient pancreatic adenocarcinoma.

Gut 2021 Oct 15;70(10):1894-1903. Epub 2020 Sep 15.

Ontario Institute for Cancer Research, Toronto, Ontario, Canada

Objective: To describe the clinical, pathological and genomic characteristics of pancreatic cancer with DNA mismatch repair deficiency (MMRD) and proficiency (MMRP).

Design: We identified patients with MMRD and MMRP pancreatic cancer in a clinical cohort (N=1213, 519 with genetic testing, 53 with immunohistochemistry (IHC)) and a genomic cohort (N=288 with whole-genome sequencing (WGS)).

Results: 12 out of 1213 (1.0%) in the clinical cohort were MMRD by IHC or WGS. Of the 14 patients with Lynch syndrome, 3 (21.4%) had an MMRP pancreatic cancer by IHC, and 4 (28.6%) were excluded because tissue was unavailable for testing. MMRD cancers had longer overall survival after surgery (weighted HR after coarsened exact matching 0.11, 95% CI 0.02 to 0.78, p=0.001). One patient with an unresectable MMRD cancer has an ongoing partial response 3 years after starting treatment with PD-L1/CTLA-4 inhibition. This tumour showed none of the classical histopathological features of MMRD. 9 out of 288 (3.1%) tumours with WGS were MMRD. Despite markedly higher tumour mutational burden and neoantigen loads, MMRD cancers were significantly less likely to have mutations in usual pancreatic cancer driver genes like and , but more likely to have mutations in genes that drive cancers with microsatellite instability like and . MMRD tumours were significantly more likely to have a basal-like transcriptional programme and elevated transcriptional markers of immunogenicity.

Conclusions: MMRD pancreatic cancers have distinct clinical, pathological and genomic profiles. Patients with MMRD pancreatic cancer should be considered for basket trials targeting enhanced immunogenicity or the unique genomic drivers in these malignancies.
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http://dx.doi.org/10.1136/gutjnl-2020-320730DOI Listing
October 2021

Intraductal Transplantation Models of Human Pancreatic Ductal Adenocarcinoma Reveal Progressive Transition of Molecular Subtypes.

Cancer Discov 2020 10 23;10(10):1566-1589. Epub 2020 Jul 23.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated; however, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present a xenotransplantation model of PDAC in which neoplasms originate from patient-derived organoids injected directly into murine pancreatic ducts. Our model enables distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively. Parameters that influence PDAC subtype specification in this intraductal model include cell plasticity and hyperactivation of the RAS pathway. Finally, through intratumoral dissection and the direct manipulation of gene dosage, we identify a suite of -regulated secreted and membrane-bound proteins that may represent potential candidates for therapeutic intervention in patients with PDAC. SIGNIFICANCE: Accurate modeling of the molecular subtypes of pancreatic cancer is crucial to facilitate the generation of effective therapies. We report the development of an intraductal organoid transplantation model of pancreatic cancer that models the progressive switching of subtypes, and identify stochastic and RAS-driven mechanisms that determine subtype specification...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664990PMC
October 2020

GATA6 Expression Distinguishes Classical and Basal-like Subtypes in Advanced Pancreatic Cancer.

Clin Cancer Res 2020 09 10;26(18):4901-4910. Epub 2020 Mar 10.

Division of Pathology, University Health Network, Toronto, Ontario, Canada.

Purpose: To determine the impact of basal-like and classical subtypes in advanced pancreatic ductal adenocarcinoma (PDAC) and to explore GATA6 expression as a surrogate biomarker.

Experimental Design: Within the COMPASS trial, patients proceeding to chemotherapy for advanced PDAC undergo tumor biopsy for RNA-sequencing (RNA-seq). Overall response rate (ORR) and overall survival (OS) were stratified by subtypes and according to chemotherapy received. Correlation of with the subtypes using gene expression profiling, hybridization (ISH) was explored.

Results: Between December 2015 and May 2019, 195 patients (95%) had enough tissue for RNA-seq; 39 (20%) were classified as basal-like and 156 (80%) as classical. RECIST response data were available for 157 patients; 29 basal-like and 128 classical where the ORR was 10% versus 33%, respectively ( = 0.02). In patients with basal-like tumors treated with modified FOLFIRINOX ( = 22), the progression rate was 60% compared with 15% in classical PDAC ( = 0.0002). Median OS in the intention-to-treat population ( = 195) was 9.3 months for classical versus 5.9 months for basal-like PDAC (HR, 0.47; 95% confidence interval, 0.32-0.69; = 0.0001). expression by RNA-seq highly correlated with the classifier ( < 0.001) and ISH predicted the subtypes with sensitivity of 89% and specificity of 83%. In a multivariate analysis, GATA6 expression was prognostic ( = 0.02). In exploratory analyses, basal-like tumors, could be identified by keratin 5, were more hypoxic and enriched for a T-cell-inflamed gene expression signature.

Conclusions: The basal-like subtype is chemoresistant and can be distinguished from classical PDAC by GATA6 expression..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3724DOI Listing
September 2020

A Four-Chemokine Signature Is Associated with a T-cell-Inflamed Phenotype in Primary and Metastatic Pancreatic Cancer.

Clin Cancer Res 2020 04 21;26(8):1997-2010. Epub 2020 Jan 21.

The Research Institute of the McGill University Health Centre, Montréal, Québec, Canada.

Purpose: The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC.

Experimental Design: Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing.

Results: Among all known human chemokines, a coregulated set of four (, and ) was strongly associated with CD8 T-cell infiltration ( < 0.001). Expression of this "4-chemokine signature" positively correlated with transcriptional metrics of T-cell activation (, and ), cytolytic activity ( and ), and immunosuppression (, and ). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell-inflamed phenotype across primary PDAC and PDAC liver metastases.

Conclusions: A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2803DOI Listing
April 2020

Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.

Nat Genet 2020 02 13;52(2):231-240. Epub 2020 Jan 13.

Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada.

Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.
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http://dx.doi.org/10.1038/s41588-019-0566-9DOI Listing
February 2020

Altered Gene Expression along the Glycolysis-Cholesterol Synthesis Axis Is Associated with Outcome in Pancreatic Cancer.

Clin Cancer Res 2020 01 3;26(1):135-146. Epub 2019 Sep 3.

Pancreas Centre BC, Vancouver, British Columbia, Canada.

Purpose: Identification of clinically actionable molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcome. Intertumoral metabolic heterogeneity contributes to cancer survival and the balance between distinct metabolic pathways may influence PDAC outcome. We hypothesized that PDAC can be stratified into prognostic metabolic subgroups based on alterations in the expression of genes involved in glycolysis and cholesterol synthesis.

Experimental Design: We performed bioinformatics analysis of genomic, transcriptomic, and clinical data in an integrated cohort of 325 resectable and nonresectable PDAC. The resectable datasets included retrospective The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) cohorts. The nonresectable PDAC cohort studies included prospective COMPASS, PanGen, and BC Cancer Personalized OncoGenomics program (POG).

Results: On the basis of the median normalized expression of glycolytic and cholesterogenic genes, four subgroups were identified: quiescent, glycolytic, cholesterogenic, and mixed. Glycolytic tumors were associated with the shortest median survival in resectable (log-rank test = 0.018) and metastatic settings (log-rank test = 0.027). Patients with cholesterogenic tumors had the longest median survival. and -amplified tumors had higher expression of glycolytic genes than tumors with normal or lost copies of the oncogenes (Wilcoxon rank sum test = 0.015). Glycolytic tumors had the lowest expression of mitochondrial pyruvate carriers and . Glycolytic and cholesterogenic gene expression correlated with the expression of prognostic PDAC subtype classifier genes.

Conclusions: Metabolic classification specific to glycolytic and cholesterogenic pathways provides novel biological insight into previously established PDAC subtypes and may help develop personalized therapies targeting unique tumor metabolic profiles..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1543DOI Listing
January 2020

Morphological classification of pancreatic ductal adenocarcinoma that predicts molecular subtypes and correlates with clinical outcome.

Gut 2020 02 14;69(2):317-328. Epub 2019 Jun 14.

Anatomical Pathology, Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.

Introduction: Transcriptional analyses have identified several distinct molecular subtypes in pancreatic ductal adenocarcinoma (PDAC) that have prognostic and potential therapeutic significance. However, to date, an indepth, clinicomorphological correlation of these molecular subtypes has not been performed. We sought to identify specific morphological patterns to compare with known molecular subtypes, interrogate their biological significance, and furthermore reappraise the current grading system in PDAC.

Design: We first assessed 86 primary, chemotherapy-naive PDAC resection specimens with matched RNA-Seq data for specific, reproducible morphological patterns. Differential expression was applied to the gene expression data using the morphological features. We next compared the differentially expressed gene signatures with previously published molecular subtypes. Overall survival (OS) was correlated with the morphological and molecular subtypes.

Results: We identified four morphological patterns that segregated into two components ('gland forming' and 'non-gland forming') based on the presence/absence of well-formed glands. A morphological cut-off (≥40% 'non-gland forming') was established using RNA-Seq data, which identified two groups (A and B) with gene signatures that correlated with known molecular subtypes. There was a significant difference in OS between the groups. The morphological groups remained significantly prognostic within cancers that were moderately differentiated and classified as 'classical' using RNA-Seq.

Conclusion: Our study has demonstrated that PDACs can be morphologically classified into distinct and biologically relevant categories which predict known molecular subtypes. These results provide the basis for an improved taxonomy of PDAC, which may lend itself to future treatment strategies and the development of deep learning models.
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http://dx.doi.org/10.1136/gutjnl-2019-318217DOI Listing
February 2020

Childhood cerebellar tumours mirror conserved fetal transcriptional programs.

Nature 2019 08 1;572(7767):67-73. Epub 2019 May 1.

Computational Biology Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada.

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood.
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http://dx.doi.org/10.1038/s41586-019-1158-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675628PMC
August 2019

Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases.

Cancer Cell 2019 02 24;35(2):267-282.e7. Epub 2019 Jan 24.

Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.
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http://dx.doi.org/10.1016/j.ccell.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398439PMC
February 2019

Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer.

Cancer Discov 2018 09 31;8(9):1112-1129. Epub 2018 May 31.

Memorial Sloan Kettering Cancer Center, New York, New York.

Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. .
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http://dx.doi.org/10.1158/2159-8290.CD-18-0349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125219PMC
September 2018

Mutations in Mitochondrial DNA From Pancreatic Ductal Adenocarcinomas Associate With Survival Times of Patients and Accumulate as Tumors Progress.

Gastroenterology 2018 05 29;154(6):1620-1624.e5. Epub 2018 Mar 29.

Informatics Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada. Electronic address:

Somatic mutations have been found in the mitochondria in different types of cancer cells, but it is not clear whether these affect tumorigenesis or tumor progression. We analyzed mitochondrial genomes of 268 early-stage, resected pancreatic ductal adenocarcinoma tissues and paired non-tumor tissues. We defined a mitochondrial somatic mutation (mtSNV) as a position where the difference in heteroplasmy fraction between tumor and normal sample was ≥0.2. Our analysis identified 304 mtSNVs, with at least 1 mtSNV in 61% (164 of 268) of tumor samples. The noncoding control region had the greatest proportion of mtSNVs (60 of 304 mutations); this region contains sites that regulate mitochondrial DNA transcription and replication. Frequently mutated genes included ND5, RNR2, and CO1, plus 29 mutations in transfer RNA genes. mtSNVs in 2 separate mitochondrial genes (ND4 and ND6) were associated with shorter overall survival time. This association appeared to depend on the level of mtSNV heteroplasmy. Non-random co-occurrence between mtSNVs and mutations in nuclear genes indicates interactions between nuclear and mitochondrial DNA. In an analysis of primary tumors and metastases from 6 patients, we found tumors to accumulate mitochondrial mutational mutations as they progress.
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http://dx.doi.org/10.1053/j.gastro.2018.01.029DOI Listing
May 2018

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

Clin Cancer Res 2018 03 29;24(6):1344-1354. Epub 2017 Dec 29.

PanCuRx Translational Research Initiative, Ontario, Institute for Cancer Research, Toronto, Ontario, Canada.

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. 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. 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 ( = 0.004). The best progression-free survival was observed in those with classical subtype treated with m-FOLFIRINOX. expression in tumor measured by RNA 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. Prospective genomic profiling of advanced PDAC is feasible, and our early data indicate that chemotherapy response differs among patients with different genomic/transcriptomic subtypes. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968824PMC
March 2018

A genetic roadmap of pancreatic cancer: still evolving.

Gut 2017 12 9;66(12):2170-2178. Epub 2017 Oct 9.

Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

A diagnosis of pancreatic ductal adenocarcinoma (PDA) is often fatal. PDA is widely recognised as one of the 'incurable cancers' because therapies against this tumour type are generally ineffective. The fatal nature of this tumour is due to its aggressive clinical course. Pancreatic cancer commonly presents at the metastatic stage; even in cases where tumours are localised to the pancreas at diagnosis, metastatic seeds have often been invariably been spawned off, frustrating surgical attempts to cure the cancer. The key principles of pancreatic cancer mutational development were outlined nearly two decades ago using the genetics of precursor lesions to position the various stages of tumour progression. Since then, there has been a cavalcade of new data. How these recent studies impact the classical perceptions of pancreatic cancer development is a work in progress. Given that significant improvements in patient outcomes are not in sight for this disease, it is likely that broadening the current perspectives and acquiring deeper biological insights into the morphogenetic route of tumour development will be needed to foster new strategies for more effective cancer control.
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http://dx.doi.org/10.1136/gutjnl-2016-313317DOI Listing
December 2017

Association of Distinct Mutational Signatures With Correlates of Increased Immune Activity in Pancreatic Ductal Adenocarcinoma.

JAMA Oncol 2017 Jun;3(6):774-783

PanCuRx Translational Research Initiative, Ontario Institute for Cancer Research, Toronto, Ontario, Canada.

Importance: Outcomes for patients with pancreatic ductal adenocarcinoma (PDAC) remain poor. Advances in next-generation sequencing provide a route to therapeutic approaches, and integrating DNA and RNA analysis with clinicopathologic data may be a crucial step toward personalized treatment strategies for this disease.

Objective: To classify PDAC according to distinct mutational processes, and explore their clinical significance.

Design, Setting, And Participants: We performed a retrospective cohort study of resected PDAC, using cases collected between 2008 and 2015 as part of the International Cancer Genome Consortium. The discovery cohort comprised 160 PDAC cases from 154 patients (148 primary; 12 metastases) that underwent tumor enrichment prior to whole-genome and RNA sequencing. The replication cohort comprised 95 primary PDAC cases that underwent whole-genome sequencing and expression microarray on bulk biospecimens.

Main Outcomes And Measures: Somatic mutations accumulate from sequence-specific processes creating signatures detectable by DNA sequencing. Using nonnegative matrix factorization, we measured the contribution of each signature to carcinogenesis, and used hierarchical clustering to subtype each cohort. We examined expression of antitumor immunity genes across subtypes to uncover biomarkers predictive of response to systemic therapies.

Results: The discovery cohort was 53% male (n = 79) and had a median age of 67 (interquartile range, 58-74) years. The replication cohort was 50% male (n = 48) and had a median age of 68 (interquartile range, 60-75) years. Five predominant mutational subtypes were identified that clustered PDAC into 4 major subtypes: age related, double-strand break repair, mismatch repair, and 1 with unknown etiology (signature 8). These were replicated and validated. Signatures were faithfully propagated from primaries to matched metastases, implying their stability during carcinogenesis. Twelve of 27 (45%) double-strand break repair cases lacked germline or somatic events in canonical homologous recombination genes-BRCA1, BRCA2, or PALB2. Double-strand break repair and mismatch repair subtypes were associated with increased expression of antitumor immunity, including activation of CD8-positive T lymphocytes (GZMA and PRF1) and overexpression of regulatory molecules (cytotoxic T-lymphocyte antigen 4, programmed cell death 1, and indolamine 2,3-dioxygenase 1), corresponding to higher frequency of somatic mutations and tumor-specific neoantigens.

Conclusions And Relevance: Signature-based subtyping may guide personalized therapy of PDAC in the context of biomarker-driven prospective trials.
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http://dx.doi.org/10.1001/jamaoncol.2016.3916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824324PMC
June 2017

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

Nature 2016 Oct 12;538(7625):378-382. Epub 2016 Oct 12.

Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota 55905, USA.

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.
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http://dx.doi.org/10.1038/nature19823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446075PMC
October 2016

Senescent Carcinoma-Associated Fibroblasts Upregulate IL8 to Enhance Prometastatic Phenotypes.

Mol Cancer Res 2017 01 27;15(1):3-14. Epub 2016 Sep 27.

Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.

Carcinoma-associated fibroblasts (CAF) represent a significant component of pancreatic cancer stroma and are biologically implicated in tumor progression. However, evidence of both cancer-promoting and -restraining properties amongst CAFs suggests the possibility of multiple phenotypic subtypes. Here, it is demonstrated that senescent CAFs promote pancreatic cancer invasion and metastasis compared with nonsenescent control CAFs using in vitro Transwell invasion models and in vivo xenograft mouse models. Screening by gene expression microarray and cytokine ELISA assays revealed IL8 to be upregulated in senescent CAFs. Experimental modulation through IL8 overexpression or receptor inhibition implicates the IL8 pathway as a mediator of the proinvasive effects of senescent CAFs. In a cohort of human pancreatic cancer cases, more abundant stromal senescence as indicated by p16 immunohistochemistry correlated with decreased survival in patients with early-stage disease. These data support senescent fibroblasts as a pathologically and clinically relevant feature of pancreatic cancer. The inhibition of senescent stroma-cancer signaling pathways has the potential to restrain pancreatic cancer progression.

Implications: Findings show that senescent cancer-associated fibroblasts secret excess IL8 to promote pancreatic cancer invasion and metastasis; thus, senescent CAFs represent a phenotypic subtype, challenging conventional assumptions that CAFs are a homogeneous population. Mol Cancer Res; 15(1); 3-14. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0192DOI Listing
January 2017

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.

Science 2016 Jan 5;351(6269):aab2116. Epub 2015 Nov 5.

Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.
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http://dx.doi.org/10.1126/science.aab2116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816201PMC
January 2016

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

Cancer Cell 2015 Sep 27;28(3):343-56. Epub 2015 Aug 27.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2C4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada.

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.
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http://dx.doi.org/10.1016/j.ccell.2015.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573904PMC
September 2015

Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.

Science 2013 Feb 13;339(6119):543-8. Epub 2012 Dec 13.

Campbell Family Institute, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
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http://dx.doi.org/10.1126/science.1227670DOI Listing
February 2013

Hematopoiesis: a human perspective.

Cell Stem Cell 2012 Feb;10(2):120-36

Division of Stem Cell and Developmental Biology, Campbell Family Institute for Cancer Research/Ontario Cancer Institute, Toronto, ON M5G 1L7, Canada.

Despite its complexity, blood is probably the best understood developmental system, largely due to seminal experimentation in the mouse. Clinically, hematopoietic stem cell (HSC) transplantation represents the most widely deployed regenerative therapy, but human HSCs have only been characterized relatively recently. The discovery that immune-deficient mice could be engrafted with human cells provided a powerful approach for studying HSCs. We highlight 2 decades of studies focusing on isolation and molecular regulation of human HSCs, therapeutic applications, and early lineage commitment steps, and compare mouse and humanized models to identify both conserved and species-specific mechanisms that will aid future preclinical research.
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http://dx.doi.org/10.1016/j.stem.2012.01.006DOI Listing
February 2012
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