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Global initiatives focused on whole genome and transcriptome analysis (WGTA) of metastatic pancreatic ductal adenocarcinoma (PDAC) tumor cohorts are driving progress in understanding the clinical impact of PDAC heterogeneity. This knowledge is key to continue the discovery of subsets of patients who could benefit from biomarker-informed targeted therapy and expand the currently limited PDAC treatment options. To identify clinically actionable subtypes in metastatic PDAC (mPDAC), our team analyzes prospectively collected WGTA data from patients enrolled in the PanGen trial (NCT02869802), in parallel with analysis of a set of patient-derived organoids (PDOs). In a cohort of 69 sequenced tumors, four cases (5.8%) had an amplification of chromosome 7q22, which included copy gains of transcriptional co-factor TRRAP, drug metabolizing cytochrome P450 genes CYP3A4 and CYP3A5, and SMURF1. SMURF1 is a ubiquitin-protein ligase that regulates TGFβ receptor signaling, in part via its interaction with SMAD7, and has been implicated in the epithelial-to-mesenchymal program and tumor invasiveness in PDAC. The proteasome inhibitor (PI) bortezomib has been shown to attenuate SMURF1 levels. We investigated whether SMURF1 signaling axis is associated with sensitivity to PIs in mPDAC, by analyzing the cytotoxic effects of the PIs bortezomib, carfilzomib and ixazomib in PDOs. Eight PDOs were treated with PIs at concentrations between 0.1pM-1mM. The presence of live and dead cells was quantified using the IN Cell Analyzer, and cell toxicity was analyzed using GRtoxic metrics (grcalculator.org). The PIs bind catalytic subunits of the 26S proteasome, primarily proteasome subunit beta-5, encoded by PSMB5. The cytotoxic activity of all three PIs negatively correlated with tumor biopsy PSMB5 expression (p75th percentile) tumor SMURF1 expression showed shorter overall survival (OS) compared to the rest of the cohort (median OS 9 vs. 14 months, respectively; p=0.004). Immunohistochemistry analysis of a tumor tissue microarray comprising 175 resected PDAC cases detected an association of concomitant low SMURF1 and high SMAD7 protein levels with adjuvant therapy response (median OS: 40 vs. 12 months with no adjuvant treatment, p=0.002), suggesting that attenuation of SMURF1 in the presence of SMAD7 may improve chemotherapy response in early disease stages. In summary, we present data indicating increased sensitivity to PIs in a subset of tumors with low PSMB5 and high SMURF1 expression, and highlight the translational utility of the investigation of genomic and clinically annotated pre-clinical models in PDAC predictive biomarker discovery. Citation Format: Andrew Metcalfe, Joanna M. Karasinska, James T. Topham, Steve E. Kalloger, Hassan Ali, Dawn Ashforth, Marco A. Marra, Janessa Laskin, Patricia A. Tang, Rachel Goodwin, Oliver F. Bathe, Daniel J. Renouf, David F. Schaeffer. Targeting SMURF1 with low-dose proteasome inhibitors in pancreatic cancer organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B053.

Pancreatic ductal adenocarcinoma (PDAC) can be stratified into distinct transcriptome subtypes, with the ‘basal-like’ or ‘squamous’ subtype being associated with worse prognosis, compared to the ‘classical’ subtype. Our group recently demonstrated that PDAC tumors have unique metabolic transcriptome profiles, and that genes involved in glycolysis and cholesterol synthesis pathways are positively correlated with basal-like and classical gene expression patterns, respectively. The mitochondrial pyruvate complex (MPC) mediates the transport of pyruvate into the mitochondria which attenuates the effect of glycolysis on tumor progression. The mitochondrial pyruvate carrier 1 (MPC1) gene, which encodes one of two subunits of MPC, is deleted in over 60% of metastatic PDAC and PDAC glycolytic tumors have lowest levels of MPC1 expression. Using PDAC tissue microarrays, we also found that reduced MPC1 protein expression correlates with reduced survival in patients. We hypothesized that targeting MPC1 will alter metabolic reprogramming and may modulate tumor aggressiveness and therapeutic vulnerability in PDAC tumor cells. Genomically and clinically annotated patient-derived tumor organoids (PDOs) were generated from metastatic biopsies from patients enrolled in the PanGen study (NCT02869802). PDOs from both basal and classical tumors were used in the study. In order to investigate glycolysis in PDOs, we adapted the Seahorse Glycolytic Stress Test. Glycolysis, glycolytic capacity and reserve were analyzed in PDOs under basal and treated conditions. To alter MPC1 activity, PDOs were treated for 48 hours with 5uM of UK-5099, an MPC1 inhibitor, or 2.5-5uM SRT1720. SRT1720 is an activator of sirtuin 1 (SIRT1) and the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC1-α), which regulates the expression of MPC1. An unpaired t-test with an alpha of 0.05 was used for all statistical analysis. Glycolysis analysis revealed distinct glycolytic profiles in PDOs with differences in glycolytic capacity and reserves trending with different tumor subtypes. Treatment with UK-5099 resulted in an increase in both glycolytic rate and reserve in PDOs from basal and classical tumors. Treatment with SRT1720 resulted in significantly reduced glycolytic rate and capacity. These data suggest that PDAC PDOs exhibit distinct metabolic profiles and that targeting MPC1 can modulate glycolysis in PDOs. Our ongoing efforts aim to further characterize the subtype-specific effect of MPC1 modulators on glycolysis and chemotherapy response in PDAC PDOs. Citation Format: Hassan A. Ali, Andrew Metcalfe, James T. Topham, Cassia S. Warren, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf. Targeting the mitochondrial pyruvate complex to alter metabolic programming in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-021.

Variability in the immunogenic landscape of metastatic cancer lesions has revealed insight into detection of potential immunotherapy-responsive patients, though underlying mechanisms driving such variation are not fully understood. Endogenous retrovirus (ERV)-containing transcripts have recently emerged as a potential source of tumor-associated antigen that are orthogonal to somatic mutation-derived neoantigens. To characterize the intersection between ERV levels and predicted immunogenicity in metastatic cancer, we comprehensively profiled the transcript abundance of 702,533 ERV loci in 199 metastatic tumors from breast, colorectal, and pancreatic cancer patients. In all three cancer types, overall ERV transcript load was associated with upregulation of genes involved in innate antiviral response pathways as well as genes involved in both adaptive and innate immune signaling. In colorectal and pancreatic tumors, samples with concomitant increases in ERV load and antiviral response gene expression, termed viral mimicry tumors, showed high expression of the DNA demethylation gene TET2, a gene previously described to promote transcription of ERV-containing transcripts. Collectively, these data are compatible with the notion that an increased level of ERV-containing transcripts may account for increased immunogenicity in a subset of metastatic tumors and support the relationship between DNA demethylation and ERV load in colorectal and pancreatic tumors. Citation Format: James T. Topham, Emma Titmuss, Erin Pleasance, Laura M. Williamson, Joanna M. Karasinska, Luka Culibrk, Michael K.C. Lee, Steve E. Kalloger, Shehara Mendis, Richard A. Moore, Andrew J. Mungall, Janessa Laskin, Jonathan M. Loree, Dixie L. Mager, Marco A. Marra, Steven J.M. Jones, David F. Schaeffer, Daniel J. Renouf. Endogenous retrovirus transcript levels are associated with immunogenic signatures in multiple metastatic cancer types [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B56.

Reprogramming of metabolic pathways allows cancer cells to survive and thrive in the tumor microenvironment. Glycolysis-inducing factors including oncogenic KRAS mutations, loss of function in TP53 and hypoxia are prevalent in PDAC. Cholesterol and its metabolites support tumor cell growth and the mevalonate pathway, which uses glycolysis products for de novo cholesterol synthesis, has been found to be upregulated in cancer. However, whether intertumoral heterogeneity in these metabolic networks influences outcome in pancreatic cancer has not been well established. We profiled the expression of glycolytic and cholesterogenic genes in 325 resected and metastatic pancreatic ductal adenocarcinoma (PDAC) tumors and identified four distinct subgroups: quiescent, glycolytic, cholesterogenic, and mixed. Glycolytic tumors were associated with the shortest median survival in resectable and metastatic disease settings. Patients with cholesterogenic tumors had the longest median survival. KRAS and MYC amplified tumors had higher expression of glycolytic genes than tumors with normal or lost copies of these oncogenes. The mitochondrial uptake of pyruvate, the end product of glycolysis, facilitates the generation of acetyl-CoA for cholesterol synthesis. PDAC tumors with a glycolytic gene signature had the lowest expression of mitochondrial pyruvate carriers MPC1 and MPC2. Glycolytic and cholesterogenic gene expression correlated with the expression of reported prognostic PDAC subtype classifier genes. Our results indicate that PDAC tumors have unique metabolic profiles that influence disease outcome and provide functional correlate to previously identified subtypes. The findings also raise the possibility of a shift in balance between the glycolytic and cholesterogenic pathways as a factor in PDAC progression and a potential target for therapy. Citation Format: Joanna M. Karasinska, James T. Topham, Steve E. Kalloger, Gun Ho Jang, Robert E. Denroche, Luka Culibrk, Laura M. Williamson, Hui-li Wong, Michael K.C. Lee, Grainne M. O'Kane, Richard A. Moore, Andrew J. Mungall, Malcolm J. Moore, Cassia Warren, Andrew Metcalfe, Faiyaz Notta, Jennifer J. Knox, Steven Gallinger, Janessa Laskin, Marco A. Marra, Steven J.M. Jones, Daniel J. Renouf, David F. Schaeffer. Gene expression along the glycolysis-cholesterol synthesis axis and outcome in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A24.

Background: Advanced pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality. There has been a rising incidence of early-onset pancreatic cancer (EOPC; ≤55 years). Reported treatment and survival outcomes in EOPC remain limited and have only been reported in the pre-FOLFIRINOX era. We characterized the genomic and transcriptomic landscapes of EOPC, while also leveraging provincial health data to investigate survival outcomes in advanced EOPC in a separate dataset. Methods: We generated a comprehensive and integrative dataset utilizing RNA-seq data and matched clinical metadata for 402 PDAC patients across 5 distinct studies and 4 sequencing centers, encompassing both resectable (ICGC, TCGA) and advanced (Personalized OncoGenomics and COMPASS) disease. 345 (85.8%) and 371 (92.3%) of samples had SNV/indel and CNV data available, respectively. Patients were stratified into EOPC (n=96), average-onset pancreatic cancer (AOPC, ≥70 years; n=121), and intermediate (>55 and Results: CDKN2A SNV/indels were identified in 22% and 26% of intermediate and AOPC patients, and in only 7% of EOPC patients (p Conclusions: Using an extensive PDAC sequencing dataset, we highlight a novel association between CDKN2A SNV/indel frequency and EOPC. Transcriptome-based analysis identified significant associations between age of onset and expression of synaptic signal transduction pathways. Collectively, these data indicate potential age-specific differences in the mutational and developmental trajectories of PDAC and generate novel hypotheses for further study of EOPC. Citation Format: Erica S. Tsang, James T. Topham, Joanna M. Karasinska, Michael K.C. Lee, Shehara Mendis, Luka Culibrk, Robert Denroche, Gun Ho Jang, Steve E. Kalloger, Richard A. Moore, Andrew J. Mungall, Janessa Laskin, Grainne M. O'Kane, Jennifer J. Knox, Steven Gallinger, Steven J. Jones, Marco A. Marra, Jonathan M. Loree, David F. Schaeffer, Daniel J. Renouf. Early-onset pancreatic ductal adenocarcinomas are characterized by a distinct mutational landscape [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B57.

Background: Secreted Protein, Acid, Cysteine-Rich (SPARC) has been classified as a marker of poor prognosis and has been postulated as a therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The clinical trial findings have been discordant with Phase I/II trials declaring that patients treated with nab-paclitaxel and gemcitabine whose tumors express SPARC in the stromal component have a significantly improved response. Unfortunately, these results were not validated in a subsequent Phase III trial. The focus of examining SPARC expression in a particular component and using nab-paclitaxel, which binds to SPARC, sets the stage for partial clinical efficacy due to component expression heterogeneity. The goal of this study is to develop an integrated component based approach to the quantification of SPARC in PDAC to reveal discrete prognostic and predictive groups in a cohort of resected patients treated with an adjuvant pyrimidine analog or subjected to post-surgical observation. Methods: Tissue-microarray based immunohistochemical quantification of SPARC expression was performed in the epithelial and stromal histological components of 246 resected PDACs from the Vancouver Coastal Health Region collected between 1989-2013. The semi-quantitative H-Score methodology was used where the percent of cells staining for SPARC is multiplied by the subjective assessment of its intensity (1-3) resulting in a range of scores between 0 - 300. These scores were subjected to unsupervised hierarchical clustering using Ward’s algorithm. The number of clusters was determined through an a priori decision that no cluster could have a resultant N < 30. Comparisons of component specific H-Scores across the resultant clusters were performed with the non-parametric Steel-Dwass test for multiple comparisons. Univariable disease specific survival (DSS) analysis was performed with the Kaplan-Meier method to examine the cluster specific survival profiles with regard to prognostic and predictive effects. The proportional hazards model was used to perform multivariable DSS to determine if the resultant prognostic groups were statistically independent when other known prognostic variables were considered. Results: The distribution of H-Scores spanned the entire range of possible values for both the epithelial and stromal components with medians [IQRs] of 120 [120] and 270 [120] respectively. The two-variable hierarchical clustering procedure yielded six clusters ranging in size from 31 to 51 cases. Kaplan-Meier curves showed no statistically significant differences between any of the clusters. However, the cluster exhibiting the best prognosis (N=36) also had significantly lower H-Scores for the stromal component compared to the remaining 5 clusters (p Conclusion: This study illustrates that there is enhanced value in a combinatorial approach to the examination of SPARC in the stromal and epithelial components of PDAC where we have confirmed its status as a negative prognostic marker. While this cohort cannot address the issue of nab-paclitaxel in PDAC, we have noted that heterogeneity of SPARC expression at the component level can yield discrete predictive groups with regard to response to adjuvant pyrimidine analogs. Citation Format: Steve E. Kalloger, Joanna M. Karasinska, HuiLi Wong, Daniel J. Renouf, David F. Schaeffer.{Authors}. Novel assessment of SPARC expression by hierarchical clustering in pancreatic ductal adenocarcinoma shows distinct prognostic and predictive groups. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B86.

Background: Mutant KRAS stimulates glucose uptake and lactate production in pancreatic ductal adenocarcinoma (PDAC), contributing to metabolic pathway reprogramming and tumor progression. A prognostic effect for glucose transporter GLUT1 and lactate transporter MCT4 expression in PDAC has been demonstrated but it is not known if the expression of markers of glycolytic and lactate metabolism pathways is predictive of treatment response. We aimed to validate the prognostic and assess the predictive effects of GLUT1 and MCT4 protein levels in resectable PDAC. Methods: Immunohistochemical analysis for GLUT1 and MCT4 was performed on a tissue microarray (TMA) comprising 261 resected PDAC tumors with associated clinical outcome data. The expression of GLUT1 and MCT4 in the epithelial compartment of PDAC was quantified and patient samples were scored as low (negative and weak staining) and high (moderate and strong staining) expression groups. Univariable disease-specific survival (DSS) was assessed using the Kaplan-Meier method. Results: 70% (182) of the patients included in the TMA had high GLUT1 staining and 58% had high MCT4 staining. GLUT1high patients had reduced median DSS compared to GLUT1low patients (1.34 vs. 2.05 years, p=0.0136). Median DSS was also reduced in the MCT4high group (1.33 vs. 1.91 years in MCT4low, p=0.0153). There was a significant co-occurrence of high GLUT1 with high MCT4 expression (70%, p Conclusions: GLUT1 and MCT4 expression are poor prognostic markers in PDAC and co-expression of these biomarkers enhances this effect. Neither GLUT1 nor MCT4 were found to be of predictive relevance when considered individually. Patients with low tumor GLUT1 and MCT4 expression had the best outcomes with chemotherapy with a pyrimidine analog. Hence, immunohistochemical analysis of GLUT1 and MCT4 in resected PDAC defines patient subgroups with different survival prognosis and predicted sensitivity to pyrimidine analog chemotherapy. The combined effect of GLUT1 and MCT4 expression on PDAC outcome suggests that therapeutic agents which can alter the glycolytic/lactate pathway may have potential for increasing sensitivity to treatment. Citation Format: Joanna M. Karasinska, Steve E. Kalloger, Hui-li Wong, Taixiang Wang, Daniel J. Renouf, David F. Schaeffer.{Authors}. Co-expression of GLUT1 and MCT4 is a poor prognostic marker and predicts response to adjuvant chemotherapy in PDAC. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B87.

Background: In the absence of defined tumor molecular subtypes and validated predictive markers, PDAC has been largely treated as a single disease. Recent studies of molecular subtyping in PDAC [1-4] reveal a complex mutational landscape with data suggesting the presence of genomic and gene expression signatures that may have prognostic and therapeutic significance. However, most of these PDAC datasets consisted of resected tumors, cell lines or xenografts and lack data on metastatic tumors. The aim of this study is to evaluate gene signatures using whole genome sequencing (WGS) and transcriptome (RNA-Seq) data from metastatic biopsy samples in patients with advanced PDAC. Methods: Patients with incurable advanced cancers undergo fresh tumor biopsies for indepth WGS and RNA sequencing as part of an ongoing prospective study (NCT02155621). DNA and RNA extraction and library construction were performed according to standard protocols. Paired-end reads were generated on an Illumina HiSeq2500 sequencer. RNA-Seq expression values were converted into centile expression ranks against the TCGA PDAC dataset. Centile distributions for genes in published signatures were compared by pairwise Wilcoxon-Mann-Whitney tests using one-sided p=0.1 as the significance cutoff. Survival analysis was performed using the Kaplan-Meier method. Results: Molecular data is available for 12 patients with metastatic PDAC; median age 63 years, 6 males, 8 with de novo metastatic disease (67%). 10 tumor samples (83%) were obtained from liver biopsies; average tumor content was 41% (range 24-51%). The average number of structural variants per sample was 125 (range 40-271). Rearrangement-based subtypes [3] were distributed as follows: stable (n=3), locally rearranged (n=1), scattered (n=7) and unstable (n=1). 1 patient harbored a germline BRCA1 185delAG founder mutation but had a stable genotype. Gene expression analysis for classical and basal subtypes similar to those recently described [4] identified 3 and 7 patients with classical and basal expression patterns respectively. Gene signatures were undetermined for 2 patients, where no significant difference in expression of classical or basal signature genes was noted. At median follow-up of 16.7 months, 8 patients had died. Median overall survival was 19.1 vs 7 months in patients with classical and basal subtypes respectively (p=0.078). Conclusion: Despite small patient numbers, gene expression analysis demonstrated the presence of distinct signatures in metastatic PDAC, with a trend towards worse outcomes for patients with a basal expression subtype. Future challenges include prospective validation in larger cohorts, standardization of RNA data acquisition and analysis, and better definition of prognostic and predictive signatures that may be of clinical utility in metastatic PDAC. References 1. Collison E, et al. Nat Med. 2011 [PMID: 21460848] 2. Biankin A, et al. Nature. 2012 [PMID: 23103869] 3. Waddell N, et al. Nature. 2015 [PMID: 25719666] 4. Moffitt R, et al. Nat Genet. 2015 [PMID: 26343385] Citation Format: Hui-li Wong, Joanna M. Karasinska, Martin Jones, Peter Eirew, Kasmintan Schrader, Howard Lim, Yaoqing Shen, Steven Jones, Stephen Yip, Janessa Laskin, Marco Marra, David F. Schaeffer, Daniel J. Renouf.{Authors}. Gene expression analysis demonstrates prognostic subtypes in metastatic pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B81.

Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient's personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient's PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G>A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications. Citation Format: Kasmintan A. Schrader, Carolyn Chu’ng, Eric Zhao, Hui-li Wong, Yaoqing Shen, Martin Jones, Tom Thomson, Howard Lim, Sean Young, Carol Cremin, Robert Holt, Peter Eirew, Joanna Karasinska, Jacquie Schein, Yongjun Zhao, Andy Mungall, Richard Moore, Yussanne Ma, Alexandra Fok, Robyn Roscoe, Stephen Yip, Gillian Mitchell, Aly Karsan, Steven Jones, David Schaeffer, Janessa Laskin, Marco Marra, Daniel Renouf. Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis. [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 5226.