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Background Patients treated with neoadjuvant chemotherapy (NACT) for advanced high-grade serous ovarian carcinoma (HGSC) have a higher rate and shorter time to platinum-resistant recurrence compared to patients treated with primary cytoreductive surgery (PCS) and adjuvant chemotherapy. The purpose of this study is to determine the impact of NACT on somatic mutation status in platinum-sensitive and resistant HGSC. Patients with advanced HGSC who had a documented response to platinum-based NACT, a banked blood sample, and a banked tumor sample before and after NACT were identified. Whole exome and/or targeted deep sequencing was performed in matched normal and pre/post-NACT tumor samples from 3 platinum-resistant and 2 platinum-sensitive patients to identify somatic non-synonymous mutations at each time point. Results When comparing exonic non-synonymous mutations in pre-NACT and post-NACT samples from the same patient, an average of 41% (1-68%) of genes were mutated at both time points. There were no trends detected in the mutational burden following exposure to NACT in platinum-resistant vs. platinum-sensitive cases. The majority of mutated genes were unique to each case. We identified several genes that were commonly mutated in pre-NACT samples specific to platinum-resistant (CSPG4, SLC35G5, TUBA3D) or sensitive (CYP2D6, NUTM1, DNAH5) cases. Four mutated genes emerged exclusively in the platinum-resistant cases (ADGRV1, MUC17, MUC20, PAK2) following NACT. Conclusions Patients with advanced HGSC present with significant intra-tumor heterogeneity. NACT significantly impacts the somatic mutation status irrespective of the time to recurrence. The mutated genes detected in chemo-naive pre-NACT tumor samples from either resistant or sensitive cases could potentially have a role in the prediction of chemotherapy response in patients scheduled to receive NACT; larger studies are required to further validate these genes.

Up to 20% of high-grade serous ovarian carcinomas (HGSOC) are hereditary; however, historical uptake of genetic testing is low. We used a unique combination of approaches to identify women in Ontario, Canada, with a first-degree relative (FDR) who died from HGSOC without prior genetic testing, and offer them multi-gene panel testing.From May 2015-Sept 2019, genetic counseling and testing was provided to eligible participants. Two recruitment strategies were employed, including self-identification in response to an outreach campaign and direct targeting of FDRs of deceased HGSOC patients treated at our institution. The rate of pathogenic variants (PV) in established/potential ovarian cancer risk genes and the benefits/challenges of each approach were assessed.A total of 564 women enrolled in response to our outreach campaign (n = 473) or direct recruitment (n = 91). Mean age at consent was 52 years and 96% did not meet provincial testing criteria. Genetic results were provided to 528 individuals from 458 families. The rate of PVs in ovarian cancer risk genes was highest when FDRs were diagnosed with HGSOC60 years (9.4% vs. 3.9% ≥ 60y, p = 0.0160). Participants in the outreach vs. direct recruitment cohort had a similar rate of PVs; however, uptake of genetic testing (97% vs. 89%; p = 0.0036) and study completion (95% vs. 87%; p = 0.0062) rates were higher in the former. Eleven participants with pathogenic variants have completed risk-reducing gynecologic surgery, with one stage I HGSOC and two breast cancers identified.Overall PV rates in this large cohort were lower than expected; however, we provide evidence that genetic testing criteria in Ontario should include individuals with a deceased FDR diagnosed with HGSOC60 years of age.

Key Points Question Can virtual care (VC) be rapidly implemented across a tertiary center during the coronavirus disease 2019 (COVID-19) pandemic, and what are service capacity and quality outcomes? Findings This cohort study of 22 085 VC visits at a single cancer center suggests feasibility of an agile service design process for implementation of VC at scale. This approach preserved outpatient caseloads and maintained care quality in all 6 care-quality domains of care quality laid out by the Institute of Medicine while rendering high patient and practitioner satisfaction. Meaning These data support the value proposition of VC to safeguard system capacity, while minimizing the disruption to patient care during a pandemic., Importance The coronavirus disease 2019 (COVID-19) pandemic has burdened health care resources and disrupted care of patients with cancer. Virtual care (VC) represents a potential solution. However, few quantitative data support its rapid implementation and positive associations with service capacity and quality. Objective To examine the outcomes of a cancer center–wide virtual care program in response to the COVID-19 pandemic. Design, Setting, and Participants This cohort study applied a hospitalwide agile service design to map gaps and develop a customized digital solution to enable at-scale VC across a publicly funded comprehensive cancer center. Data were collected from a high-volume cancer center in Ontario, Canada, from March 23 to May 22, 2020. Main Outcomes and Measures Outcome measures were care delivery volumes, quality of care, patient and practitioner experiences, and cost savings to patients. Results The VC solution was developed and launched 12 days after the declaration of the COVID-19 pandemic. A total of 22 085 VC visits (mean, 514 visits per day) were conducted, comprising 68.4% (range, 18.8%-100%) of daily visits compared with 0.8% before launch (P, This cohort study of a publicly funded comprehensive cancer center examines the outcomes of a cancer center–wide virtual care program in response to the COVID-19 pandemic.

Objectives To identify differentially expressed genes between relapsed and non-relapsed clinical stage I testicular germ cell tumours (TGCTs). Materials and methods We reviewed patients with clinical stage I non-seminoma and seminoma from an institutional database (2000-2012) who were managed by active surveillance. Patients with non-relapsed non-seminoma and non-relapsed seminoma were defined as being relapse-free after 2 and 3 years of surveillance, respectively. RNA extraction and gene expression analysis was performed on archival primary tumour samples and gene-set enrichment analysis (GSEA) was conducted in order to identify differentiating biological pathways. Results A total of 57 patients (relapsed non-seminoma, n = 12; relapsed seminoma, n =15; non-relapsed non-seminoma, n = 15; non-relapsed seminoma, n = 15) were identified, with a median (range) relapse time of 5.6 (2.5-18.1) and 19.3 (4.7-65.3) months in the relapsed non-seminoma and relapsed seminoma cohorts, respectively. A total of 1 039 differentially expressed genes were identified that separated relapsed and non-relapsed groups. In patients with relapse, GSEA revealed enrichment in pathways associated with differentiation, such as skeletal development (i.e. FGFR1, BMP4, GLI2, SPARC, COL2A1), tissue (i.e. BMP4, SPARC, COL13A1) and bone remodelling (i.e. CARTPT, GLI2, MGP). A discriminative gene expression profile between relapsed and non-relapsed cases was discovered when combining non-seminoma and seminoma samples using 10- and 30-probe signatures; however, this profile was not observed in the seminoma and non-seminoma cohorts individually. Conclusion A discriminating signature for relapsed disease was identified for clinical stage I TGCT that we were not able to identify by histology alone. Further validation is required to determine if this signature provides independent prognostic information to standard pathological risk factors.

BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, are essential for BCR-ABL1+, but not WT, pre-B-cell proliferation. The mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.

e14122 Background: Massive volumes of patient health information (PHI) are required to realize the anticipated benefits of artificial intelligence in future clinical medicine. To maintain public trust in medical research however, consent policies must evolve to reflect contemporary patient preferences. Methods: From January-December 2019, patients attending clinics at a large academic cancer centre were invited to complete a 27-item iPad survey on consent preferences. Survey items focused on: (a) broad vs. specific consent; (b) opt-in vs. opt-out approaches for research contact; (c) comfort sharing with different recipients; (d) perceptions on commercialization; and (e) options to track information use and study results. Demographic questions addressed cancer type, treatment stage, age, gender, ethnicity, education level, and household income. Results: A total of 222 participants were included in the analysis (112 males, 108 females; 2 rather not say); 83% were comfortable sharing PHI with researchers at their own hospital. While 56% of patients preferred broad consent, 38% preferred to be contacted with study details and asked to consent every time (specific consent); 6% prefer not to share at all. Younger patients ( < 49 years) most often chose specific consent (50%); significantly more than those 75+ years (24%; p < .05). Younger patients ( < 49 years) were also significantly more uncomfortable than older patients (50+ years) sharing even within their own hospital (13% uncomfortable vs. 1% uncomfortable; p < .05). A significant majority of patients (63%, p = .0001) preferred to be asked for permission before being entered into a contact pool vs. automatic entry with opportunity to opt-out. The majority of patients were uncomfortable sharing PHI with commercial enterprises (51% uncomfortable, 27% comfortable, 22% neutral). A significant majority expressed the desire to track who is using their PHI (61%, p < .0001), and be notified regarding study results (70%, p < .0001). Conclusions: While most patients were willing to share their PHI with researchers at their own hospital, many preferred a transparent and reciprocal consent process. These data also suggest a generational shift, wherein younger patients preferred more informed consent options. Modernizing consent policies to reflect increased patient interest in the exercise of their autonomy is crucial in fostering sustained public engagement in medical research.

HNSCC is the 6th most common malignancy in the world. The prognosis is favorable during early stages; however, the disease is rarely diagnosed early due to the lack of symptoms. Most HNSCC patients present with metastatic disease for which the survival rates remain low. Hypoxia serves as a bad prognostic factor in HNSCC correlating with worse survival and resistance to radiotherapy. We aimed to discover novel targets in metastatic HNSCC utilizing a unique collection of matched sets of cell lines derived from primary tumors and their respective metastatic sites. We carried out expression profiling across the lines to reveal potential common changes in gene expression between the cells derived from primary and metastatic sites in each patient as well as between normoxia and hypoxia. This analysis revealed significant changes in gene expression between the described conditions. Interestingly, beta defensin 2 was one of the genes that came up as overexpressed in metastasis as well as in cells cultured in hypoxia. Beta defensins are small cationic peptides that belong to the innate immune system and exhibit antimicrobial and antiviral activities. Few studies reported their abnormal expression patterns in various cancers including HNSCC. While various novel anticancer therapies are currently being developed, early diagnosis remains one of the biggest challenges in cancer research. A discovery of soluble serum factors that can reliably detect the presence of primary or metastatic disease would serve as an extremely valuable tool in defining diagnosis and prognosis, predicting the response to treatment, and monitoring disease progression. We hypothesized the beta defensin 2 can serve as a serum biomarker of hypoxia and metastasis in HNSCC patients. Utilizing a commercial ELISA kit developed to detect and quantify levels of beta-defensin in human sera, we demonstrated that media collected from cell lines derived from metastases contained higher levels of beta-defensin compared to the cell lines derived from the primary tumors. Moreover, sera from HNSCC patients showed higher levels of beta defensin compared to the normal controls. As a next step, we tested sera samples from 40 HNSCC patients, of whom 20 had lymph node metastasis and 20 did not. While these data are still under analysis, preliminary results suggest that higher concentrations of beta-defensin correlate with the presence of lymph node metastasis in HNSCC patients. Utilizing two large chemical libraries that together contain about 4,000 FDA-approved drugs, we performed high-throughput screening of the HNSCC lines described above in order to discover new drugs targeting head and neck cancer, including drugs that target selectively metastatic cells compared to their primary tumor counterparts. Interestingly, many of the metastasis-specific drugs were antibiotics. Together with the findings described above, these data clearly suggest a connection between patient microbiome and the metastatic process in HNSCC. Citation Format: Maria Kondratyev, Aleksandra Pesic, Carl Virtanen, Marianne Koritzinsky, Brad Wouters. Antibacterial defense and metastatic progression—lessons from head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B10.

Background: AML is driven by a small subpopulation of leukemia stem cells (LSCs), which possess stem-cell properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- cell fractions from 78 AML patients. The LSC17 score was strongly associated with survival in 4 independent cohorts of AML patients treated with curative intent (n = 908), and accurately predicted initial response. Patients with high LSC17 scores had poor outcomes with standard treatment strategies. The LSC17 score remained highly significant in multivariate analyses, independent of commonly used prognostic factors. A critical advantage of the LSC17 test over cytogenetic analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a powerful tool for upfront risk stratification. To date, no RNA-based, stem cell-derived score has been transitioned into a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Study design and methods: The study consists of 2 phases. Phase 1 aims to validate the assay in a CLIA certified laboratory setting. Phase 2 aims to determine prospectively the feasibility and prognostic power of LSC17 score testing in newly diagnosed AML patients in the real-world setting. Clinical endpoints include primary induction failure rate, relapse free survival and overall survival. All patients with a suspected diagnosis of de novo or secondary AML, who are deemed fit and appropriate by their treating physician to undergo intensive induction chemotherapy, are considered for this study. Patients who received any prior anti-leukemia treatments (except hydroxyurea) and patients with a confirmed diagnosis of acute promyelocytic leukemia are excluded. Current participating centres include Princess Margaret Cancer Centre (Toronto), Juravinski Cancer Centre (Hamilton), and The Ottawa Hospital Cancer Centre (Ottawa). Pre-study sample size analysis suggests that 150 patients will be required to demonstrate a hazard ratio for death of 2.3 between patients with a high and low LSC17 score (α = 0.05, power = 0.8). The survival for the high and low LSC17 score groups will be compared using the Cox proportional hazards model. Traditional risk stratification will also be tested within a Cox proportional hazards model. Phase 1 of the study has been completed and several key quality control measures have been created. Initial derivation and validation of the LSC17 score was performed using standard chemistry on the NanoString platform; for CLIA lab validation, the assay was transitioned to Elements© chemistry, which does not require custom codeset manufacture by NanoString. The original AML reference cohort was retested using Elements© chemistry to derive an absolute median threshold for prospective LSC17 score determination in individual patients. The lab validation process compared and found no difference in LSC17 scores between samples processed by Ficoll or collected in Paxgene for ease of processing. A standardised quality assurance (QA) process was completed to identify optimal sample requirements as well as specimen storage conditions, score stability during sample storage and turnaround time for testing. An algorithm has been created using the laboratory information system to allow standardised and rapid calculation of the LSC17 score from NanoString nCounter output data. The LSC17 score can be tested on peripheral blood or bone marrow, although bone marrow samples are preferred for patients with very low peripheral blast counts. Samples are ideally stored in RNA Paxgene tubes for RNA stability and to maximize RNA yield. The prospective phase of the study (Phase 2) opened in April 2018 and as of June 2019, 233 patients have been enrolled, of which 120 received induction chemotherapy. 54 patients were excluded due to an alternative diagnosis or failed QA. The remaining patients had non-intensive therapy based on patient choice. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a 49-gene AML panel are performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. The study continues to recruit and is open to collaborations in other centres. Disclosures Ng: Celgene: Research Funding. Leber:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sabloff:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTX: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Canada: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Wang:NanoString: Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation.

We conducted a case–control exome-wide association study to discover germline variants in coding regions that affect risk for pancreatic cancer, combining data from 5 studies. We analyzed exome and genome sequencing data from 437 patients with pancreatic cancer (cases) and 1922 individuals not known to have cancer (controls). In the primary analysis, BRCA2 had the strongest enrichment for rare inactivating variants (17/437 cases vs 3/1922 controls) (P=3.27×10−6; exome-wide statistical significance threshold P

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture–based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient–derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patient's tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.

Background The clinical utility of molecular profiling of tumor tissue to guide treatment of patients with advanced solid tumors is unknown. Our objectives were to evaluate the frequency of genomic alterations, clinical “actionability” of somatic variants, enrollment in mutation-targeted or other clinical trials, and outcome of molecular profiling for advanced solid tumor patients at the Princess Margaret Cancer Centre (PM). Methods Patients with advanced solid tumors aged ≥18 years, good performance status, and archival tumor tissue available were prospectively consented. DNA from archival formalin-fixed paraffin-embedded tumor tissue was tested using a MALDI-TOF MS hotspot panel or a targeted next generation sequencing (NGS) panel. Somatic variants were classified according to clinical actionability and an annotated report included in the electronic medical record. Oncologists were provided with summary tables of their patients’ molecular profiling results and available mutation-specific clinical trials. Enrolment in genotype-matched versus genotype-unmatched clinical trials following release of profiling results and response by RECIST v1.1 criteria were evaluated. Results From March 2012 to July 2014, 1893 patients were enrolled and 1640 tested. After a median follow-up of 18 months, 245 patients (15 %) who were tested were subsequently treated on 277 therapeutic clinical trials, including 84 patients (5 %) on 89 genotype-matched trials. The overall response rate was higher in patients treated on genotype-matched trials (19 %) compared with genotype-unmatched trials (9 %; p

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient. Citation Format: Maria Kondratyev, Aleksandra Pesic, Azin Sayad, Troy Ketela, Natalie Stickle, Carl Virtanen, Jason Moffat, Laurie Ailles, Marianne Koritzinsky, Brad Wouters. Cell surface targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3054.

Background The prognosis for esophageal cancer is poor but may be improved by neoadjuvant therapy. A complete pathologic response (pCR) is associated with improved survival. We conducted a study to profile the expression of microRNAs (miRNAs) in esophageal cancer before and after induction therapy. Our aims were to identify those miRNAs that are differentially regulated after induction therapy and attempt to describe a miRNA pattern that could predict pCR. Methods Total RNA was extracted from pretreatment and posttreatment specimens from 25 patients who underwent trimodal therapy using concurrent irinotecan/cisplatin and radiotherapy followed by surgical treatment. miRNAs were labeled and hybridized to the Illumina miRNA BeadChip microarray (Illumina, Inc, San Diego, CA). Expression data was quantified using BeadStudio software (Illumina), using a cutoff for significant gene differences of p less than 0.05 with a 2-fold difference in expression. Survival analysis was performed using SPSS, version 18 (SPSS, Inc, Chicago, IL). Results Using pretreatment biopsy specimens, 71 miRNAs were significantly different between pCR and non-pCR groups. Of these, 5 miRNAs were greater than 2-fold differentially regulated, including miR-296, recently shown to be of prognostic significance in esophageal carcinoma. After induction therapy, 568 miRNAs were found to be significantly upregulated or downregulated, 111 of which had a 2-fold difference. Patients with high levels of miR-135b or miR-145 in the posttreatment biopsy specimens had significantly shorter median disease-free survival (DFS) than did those with low levels (11.5 versus 5.1 months; p = 0.04; 11.5 versus 2.8 months; p = 0.03). Conclusions miRNA expression profiling of pretreatment biopsy specimens revealed 5 miRNAs differentially expressed in patients with pCR compared with patients without pCR. We have also identified 111 miRNAs significantly upregulated or downregulated after induction therapy, some of which may be predictive of outcome. Further study of these miRNAs may elucidate a novel understanding of mechanisms of resistance to chemotherapy or radiotherapy.

// James B. Greenaway 1 , Carl Virtanen 2 , Kata Osz 1 , Tamas Revay 1 , Daniel Hardy 3 , Trevor Shepherd 4 , Gabriel DiMattia 4 , Jim Petrik 5 1 Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada 2 Princess Margaret Genomics Centre, University Health Network, Toronto, ON, M5G 1L7, Canada 3 Department of Ob/Gyn and Physiology and Pharmacology, Children’s Health Research Institute, Western University, London, ON, N6A 5C1, Canada 4 Department of Ob/Gyn and Oncology, Anatomy and Cell Biology, London Regional Cancer Program, Western University, London, ON, N6A 4L6, Canada 5 Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada Correspondence to: Jim Petrik, email: jpetrik@uoguelph.ca Keywords: ovarian cancer, mevalonate pathway, simvastatin, p53 Received: January 12, 2016 Accepted: June 04, 2016 Published: June 17, 2016 ABSTRACT Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo , daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.

Purpose: To assess inflammation-related gene expression in nonmalignant fallopian tube epithelium (FTE) from BRCA1/2 mutation carriers and control patients obtained during the luteal and follicular phase, and to determine the impact of BRCA1 and disabled homolog 2 (DAB2) on NF-κB–mediated proinflammatory signaling. Experimental Design: A list of inflammation-related and NF-κB–responsive genes was compiled through gene set enrichment and PubMed database search, corresponding probes identified, and unpaired t tests conducted to identify differentially expressed genes in previously profiled FTE samples. ES2 and A549 cells were cotransfected with DAB2- or BRCA1-targeting siRNA and an NF-κB–responsive luciferase reporter, treated with TNF-α and luciferase activity determined. To determine whether DAB2 or BRCA1 alters mRNA expression of NF-κB target genes, cells were transfected with siRNA, treated with TNF-α, and harvested for total RNA extraction and quantitative real-time PCR. Results: A subset of BRCA1-mutated luteal phase samples previously found to group with adnexal high–grade serous carcinomas (HGSCs) differentially expressed 124 inflammation–associated probesets relative to remaining FTE samples. These samples also differentially expressed 264 probes relative to other luteal phase samples exposed to the same postovulatory environment. Both BRCA1- and DAB2–targeting siRNA increased TNF-α-induced NF-κB activity and mRNA expression of NF-κB–dependent target gene SOD2 relative to nontargeting siRNA, suggesting that both proteins repress proinflammatory signaling. Conclusions: These data provide evidence of elevated proinflammatory signaling in a subset of BRCA1-mutated luteal phase FTE, consistent with an altered response to ovulation-associated cytokines. Furthermore, both BRCA1 and DAB2 affect NF-κB activity, indicating a novel link between BRCA mutation status, ovulation, and predisposition to HGSC. Clin Cancer Res; 18(16); 4334–44. ©2012 AACR.

Allan Siu 1 , Carl Virtanen 2 and Jan Jongstra 1 1 Genetics and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Canada and Department of Immunology, University of Toronto, Toronto, Canada 2 Microarray Centre, University Health Network, Toronto, Canada. Received: December 17, 2011; Accepted: December 20, 2011; Published: December 21, 2011; Keywords: M-110, PIM-1, MIG6, EGFR, Gefitinib, prostate cancer Correspondence: Jan Jongstra, email: // // Abstract The PIM family of oncogenic serine/threonine kinases regulates tumour cell proliferation. To identify proliferative signaling pathways that are regulated by PIM kinases we analyzed gene expression differences in DU-145 and PC3 prostate cancer derived cells induced by treatment with the recently developed highly selective PIM kinase inhibitor M-110. This identified 97 genes the expression of which is affected by M-110 in both cell lines. We then focused on the M-110 induced up regulation of the MIG6 gene that encodes a negative regulator of EGFR signaling. Here we show that M-110 and the structurally unrelated PIM kinase inhibitor SGI-1776 up regulate MIG6 in DU-145 and PC3 cells. Knockdown of PIM-1 but not of PIM-2 or PIM-3 also up regulates MIG6 expression, which identifies MIG6 as a PIM-1 regulated gene. In agreement with the role of MIG6 protein as a negative regulator of EGFR signaling we found that M-110 treatment inhibits EGF induced EGFR activation and the activation of the downstream ERK MAPkinase pathway. The biological significance of these findings are demonstrated by the fact that co-treatment of DU-145 or PC3 cells with the EGFR tyrosine kinase inhibitor Gefitinib and M-110 or SGI-1776 has synergistic inhibitory effects on cell proliferation. These experiments define a novel biological function of PIM-1 as a co-regulator of EGFR signaling and suggest that PIM inhibitors may be used in combination therapies to increase the efficacy of EGFR tyrosine kinase inhibitors.

We previously reported that BRCA1/2-mutated fallopian tube epithelium (FTE) collected during the luteal phase exhibits gene expression profiles more closely resembling that of high-grade serous carcinoma (HGSC) specimens than FTE collected during the follicular phase or from control patients. Since the luteal phase is characterised by high levels of progesterone, we determined whether the expression of progesterone receptor (PR) and PR-responsive genes was altered in FTE obtained from BRCA mutation carriers during the luteal phase of the menstrual cycle. RT-qPCR confirmed a decreased expression of PR mRNA in FTE during the luteal phase relative to follicular phase, in both BRCA1/2 mutation carriers and control patients. Immunohistochemistry using isoform-specific antibodies confirmed a low level of both PR-A and PR-B in HGSC and a lower level of staining in FTE samples obtained during the luteal phase compared with the follicular phase. No significant difference in PR-A or PR-B staining was found based on patient BRCA mutation status. Analysis of our previously reported gene expression profiles based upon known PR-A- and PR-B-specific target genes did not partition samples by BRCA mutation status, indicating that overall FTE PR response is not altered in BRCA mutation carriers. HGSC samples grouped separately from other samples, consistent with the observed loss of PR expression. These findings indicate no overall difference in PR signalling in FTE as a function of BRCA mutation status. Thus, the molecular similarity of BRCA1/2-mutated luteal phase FTE and HGSC likely results from an altered response to luteal phase factors other than progesterone.

Head and neck carcinoma (HNSCC) is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. The idea behind this selective targeting is based on discovering molecular pathways that became essential in metastatic cells, and then exploiting this vulnerability through targeted agents. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA profiled gene expression in the HNSCC cell lines cultured under normoxia and hypoxia using Illumina microarrays. These analyses led to the discovery of genes that are differentially essential in metastatic cells as well as in cells exposed to hypoxic conditions. Utilizing CRISPR technology, we assembled a library of guide RNAs targeting the discovered hits and are currently validating the top identified genes using both in vitro and in vivo systems. To validate hits that are essential in metastatic cells in vivo, we engineered selected “matched” pairs of HNSCC cell lines to express CAS9 protein upon induction with doxycycline. The cells are then transduced with the library of guides and injected subcutaneously into mice; tumors from control and doxycycline treated animals are compared. The difference in genes that are essential for growth of tumors seeded by primary tumor derived and metastasis derived HNSCC cell lines is then assessed in a quantitative manner. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Jason Mofat, Carl Virtanen, Natalie Stickle, Reider Grenman, Brad Wouters. Novel targets in metastatic HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 407.

Objective. Low-grade serous carcinoma (LGSC) is a chemoresistant ovarian neoplasm thought to potentially arise in a background of low malignant potential tumors (LMP), which are typically non-aggressive. However, LMP with micropapillary features (LMP-MP) have more aggressive clinical behavior and may represent an intermediate in progression to LGSC. The objective of this study was to obtain and compare gene expression profiles of LMP, LMP-MP and LGSC to determine if LMP-MP more closely resembles LGSC, and to identify genes involved in LGSC carcinogenesis. Methods. Epithelial cells from LMP ( n =17), LMP-MP ( n =9) and LGSC ( n =11) were isolated by laser capture microdissection. RNA was extracted, reverse transcribed to cDNA, amplified and hybridized to Affymetrix U133 Plus2 genechip arrays. Gene expression data were checked for quality, filtered and significantly altered genes between subgroups were identified. Differential expression of selected genes was verified by RT-qPCR and immunohistochemistry. Results. Gene expression analysis identified differential expression between LMP and LMP-MP, LMP and LGSC but not LMP-MP and LGSC. Integration of differentially expressed genes into the protein interaction database CytoScape highlighted gene products in the MAPK pathway as differentially regulated between LMP and LGSC. Four genes were selected and validated by RT-qPCR performed on microarray samples ( n =15) and immunohistochemistry on a representative microarray ( n =57). Conclusion. The gene expression profile of LMP-MP is similar to LGSC and distinct from LMP, reflecting their more aggressive clinical behavior. Candidate genes in the MAPK pathway were highlighted which may play a role in LGSC carcinogenesis and indicate potential therapeutic targets.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that “fixing” the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Carl Virtanen, Laurie Ailles, Soroush Samadian, Mikhail Bashkurov, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2017-3020

Aberrant activation of the Notch signaling system and over-expression of the Notch ligand JAG1 are associated with poor outcome in breast cancer. The plasminogen activator system, which includes urokinase-type plasminogen activator (uPA), has been validated as a marker of recurrence, high metastasis risk and death in breast malignancy. By using microarray profiling of breast cancer cell lines that had undergone siRNA-mediated abrogation of Notch signaling we uncovered a link between activated Notch and uPA expression. An association between elevated expression of the Notch ligand JAG1, uPA and the basal breast cancer subtype was confirmed in breast cancer cell lines. The association between JAG1 and uPA expression persisted in a survey of primary carcinomas of the breast. We found that Notch knockdown reduced transcription of uPA and phenocopied uPA knockdown in breast cancer cells. Through mutational analysis we identified a CBF-1 binding site in the uPA promoter that is required for direct transcriptional regulation by Notch. These data suggest that JAG1-induced Notch activation results in breast cancer progression through up-regulation of the plasminogen activator system and directly links these two poor prognosis pathways. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-12.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Stephano Marastoni, Troy Ketela, Jason Moffat, Carl Virtanen, Azin Sayad, Mikhail Bashkurov, Alessandro Dati, Laurie Ailles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer. [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 3794.

493 Background: Genomic signatures may compliment pathological features in identifying appropriate patients who may benefit from adjuvant therapy in Stage I (SI) TGCT. This study aimed to identify a gene expression pattern to differentiate between relapsed (R) and non-relapsed (NR) SI TGCT. Methods: Patients with SI non-seminoma (NS) and seminoma (S) were identified from an institutional database from 2000 to 2012. All patients were managed with active surveillance. NR-NS and NR-S patients were defined as having no evidence of relapse after 2 and 3 years of surveillance respectively. Following pathology review, RNA extraction and gene expression analysis was performed on archived paraffin embedded tumor and normal testicular tissue using Illumina Whole Genome DASL Human HT-12 V4 BeadChip. Hierarchical clustering analysis, ANOVA and t-tests were used to evaluate candidate genes and expression patterns that could differentiate NR and R samples. Results: 57 patients (12 R-NS, 15 R-S, 15 NR-NS, 15 NR-S) were identified with median relapse time of 5.6 (2.5-18.1) and 19.3 (4.7-65.3) months in NS and S cohorts respectively. 3 additional normal testis samples were included. Poor prognostic factors were more frequent in R versus NR cases (NS: vascular invasion [5/12 vs 0/15]; S: median size [4cm vs 2.8cm]). Unsupervised hierarchical clustering of 22822 probes randomly separated S from NS, indicating no batch effect. One-way ANOVA revealed 4525 significantly varying probes (p < 0.05) however, no statistically significant gene expression profile differentiated the 4 cohorts. A discriminative gene expression profile between R and NR cases was discovered when combining NS and S samples using 10 (p = 0.03) and 30 (p = 0.03) probe signatures with a 10 fold cross-validation. However, this profile was not observed in the S and NS cohorts individually. Conclusions: A discriminating signature for R and NR was identified for SI testis tumors, but not separately for NS and S. Biological relevance of these signatures is to be determined. Further studies are required to corroborate this profile in NS and S. If validated, these expression patterns could help identify patients beyond standard pathological risk algorithms for optimal management.

Radiotherapy is a widely used cancer treatment. However, understanding how ionizing radiation affects tumor cells and their vasculature, particularly at cellular, subcellular, genetic, and protein levels, has been limited by an inability to visualize the response of these interdependent components within solid tumors over time and in vivo. Here we describe a new preclinical experimental platform combining intravital multimodal optical microscopy for cellular-level longitudinal imaging, a small animal x-ray microirradiator for reproducible spatially-localized millimeter-scale irradiations, and laser-capture microdissection of ex vivo tissues for transcriptomic profiling. Using this platform, we have developed new methods that exploit the power of optically-enabled microscopic imaging techniques to reveal the important role of the tumor microvasculature in radiation response of tumors. Furthermore, we demonstrate the potential of this preclinical platform to study quantitatively - with cellular and sub-cellular details - the spatio-temporal dynamics of the biological response of solid tumors to ionizing radiation in vivo.

Aberrant activation of the Notch receptor signaling pathway and overexpression of the Notch ligand JAG1 are associated with poor outcome in breast cancer. The plasminogen activator system, which includes urokinase-type plasminogen activator (uPA), has been validated as a marker of recurrence, high metastasis risk and death in breast malignancy. By using microarray profiling of breast cancer cell lines that had undergone siRNA-mediated abrogation of Notch signaling we uncovered a link between activated Notch signaling and uPA expression. An association between elevated expression of the Notch ligand JAG1, uPA, and the basal-like breast cancer subtype was confirmed in breast cancer cell lines. The association between JAG1 and uPA expression persisted in a survey of primary carcinomas of the breast. We found that Notch knockdown reduced transcription of uPA and phenocopied uPA knockdown in breast cancer cells. Through mutational analysis we identified a CBF-1 binding site in the uPA promoter that is required for direct transcriptional regulation by Notch. These data suggest that JAG1-induced Notch activation results in breast cancer progression through upregulation of the plasminogen activator system, directly linking these 2 important pathways of poor prognosis. Cancer Res; 71(1); 277–86. ©2010 AACR.

Introduction: The molecular characterization of patients who exhibit a unique response to treatments that are not effective in most patients, so called “exceptional responders”, can shed light on the biological underpinnings of the response and provide rationale for selection of future patients for the same treatment. Here we present the genomic characterization of a 22-year-old female patient who presented with an unclassifiable kidney cancer and diffuse bilateral lung metastases. The primary tumour was resected and platinum plus gemcitabine therapy achieved a complete resolution of the lung metastases. Approximately 2 years later the patient again developed bilateral lung metastases and an additional course of platinum plus gemcitabine once again achieved a complete radiological response. To better understand the genomic contributors to this dramatic and recurrent response, we performed whole exome sequence analysis of the diagnostic formalin-fixed paraffin-embedded tumor tissue and a matched blood sample. Methods and Results: The patient provided informed consent and was profiled through the Princess Margaret Cancer Centre Genomic Investigation of Unusual and Spectacular responders (GENIUS) program. From tumour and matched blood specimens, we isolated coding and untranslated regions followed by deep sequencing (∼250X median coverage) on an Illumina HiSeq 2000. Copy-number analysis using VarScan2 revealed 23 somatic copy-number alterations (12 gains, 11 losses) including loss of 9p and gains of 3q and 8q, consistent with reports on clear cell renal cell carcinomas. Mutational analysis using muTect found 60 somatic, coding mutations, consistent with mutation rates seen in other kidney cancers. Of these variants, only one was a previously reported hotspot mutation, GNAS p.R201H (NM_000516, also known as p.R844H on transcript NM_080425). This mutation causes constitutive activation of the G-protein complex and activates adenylate cyclase to produce cyclic-AMP (cAMP) that can activate oncogenic pathways. However, excess cellular cAMP levels have also been found to promote apoptosis. Furthermore, a germline SNP in GNAS (rs7121), thought to increase GNAS transcript stability, is associated with improved response to gemcitabine plus platinum in non-small-cell lung cancer patients. The current patient was found to be heterozygous for the rs7121 SNP, which may compound the chemosensitivity phenotype observed. Conclusion: We have identified a p.R201H mutation in GNAS that may both drive tumour progression and confer exceptional chemo-sensitivity in a patient with an unclassified kidney cancer. Further analysis is underway in vitro to validate the hypothesis that this mutation confers sensitivity to chemotherapy. Confirmation of this hypothesis may suggest that chemotherapy could be considered for patients harbouring this mutation. Citation Format: Jeff P. Bruce, Arnavaz Danesh, Neil Winegarden, Patrick Yau, Carl Virtanen, Suzanne Kamel-Reid, Michael H. Roehrl, Anthony M. Joshua, Jennifer Knox, Trevor J. Pugh. Exome analysis of an exceptional responder uncovers an activating GNAS mutation that may confer sensitivity to cytotoxic chemotherapy. [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 3898. doi:10.1158/1538-7445.AM2015-3898

Background: High-grade serous ovarian cancer is rarely diagnosed at an early and potentially curable stage, effective early detection and preventative strategies are few. Recently the discovery of occult invasive and intraepithelial tubal carcinomas in BRCA1 mutation carriers, who are at high risk of serous cancer, undergoing prophylactic surgery has focused attention on the fallopian tube epithelium as the cell of origin and has led to the reporting of putative serous cancer precursor lesions. However, little is known of the early molecular events of serous oncogenesis, or why cancers in BRCA 1 mutation carriers are found preferentially in tissues which are responsive to reproductive hormones. We hypothesize that molecular alterations are present in morphologically normal tubal epithelium from BRCA1 heterozygotes, and that these changes may reflect the earliest alterations in serous carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. To identify cancer predisposition candidate genes of high-risk fallopian tube epithelium, we compared gene expression profiles of microdissected tubal epithelium from BRCA1 mutation carriers, control women and patients with HGSC. Methodology: Snap-frozen tissues were selected from the UHN Biobank; control and BRCA cases controlled for age, ovarian cycle status at surgery, and hormone therapy. Cases included 12 BRCA1 mutation carriers and 12 control women, undergoing salpingo-oophorectomy for reasons other than adnexal malignancy or family history, 15 HGSC and 8 contralateral normal tubes. Epithelium was microdissected in all cases using laser capture, RNA extracted and cDNA amplified. The gene expression profiles were generated using Affymetrix Human Genome U133 Plus 2.0 Array. Candidate genes were validated by qPCR and IHC on 2 fallopian tube TMAs. IHC was scored using Spectrum. Statistical analysis was performed using ANOVA (p Results: We focused the microarray analyses on identifying differential expression between BRCA1 heterozygotes and controls. Comparisons between FTE-nonBRCA and FTE-BRCA, resulted in 440 probe sets with more than 2-FC in gene expression. We selected 5 genes to validate by qPCR and IHC, based on gene ontology and known associations to cancer pathways. Conclusions: These results show that histological normal fallopian tube epithelium from BRCA1 heterozygotes have altered gene expression and these differences are most pronounced in the post-ovulatory phase of the ovarian cycle in pre-menopausal women, suggesting that factors associated with the luteal phase are implicated in the increased risk of HGSC in BRCA1 mutation carriers. We also show that genes involved in inflammation pathways are up-regulated in mutation carriers and that these genetic signatures are maintained in HGSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2011-2835

Serous ovarian cancer (SOC) typically presents with advanced disease. Current therapy significantly increases survival, yet nearly all patients recur within five years and die of their disease. The cancer-initiating cell (CIC) hypothesis holds that only a subset of cells have the potential to extensively self-renew and give rise to other tumor cells. As their properties may differ from bulk tumor cells, CIC may be spared by available therapies. Identification and characterization of CIC may lead to more effective therapeutic strategies. Previous reports suggested the presence of ovarian CIC, but these findings require validation in primary human samples. Primary SOC were dissociated and depleted of CD45+ cells. Cell surface (CD133/CD44/CD117/CDCP1/MUC-1/VEGFR2) and functional (ALDH1) markers were examined by flow cytometry (n=105). All markers demonstrated intra-/inter-tumor heterogeneity; however only CD133, VEGFR2 and ALDH marked minority populations in all samples. In contrast to a report that CD44+/CD117+ cells identify ovarian CIC, CD117 was present on only half of SOC samples examined (N=75) and only one quarter had a CD44+/CD117+ population. Limiting dilution analysis of primary SOC injected in the mammary fat pad of NOD/SCID mice (95% take at 106 cells) revealed the CIC frequency in primary tumors (n=13) and metastases (n=6 +5 matched) to be ∼1/40000 (n=13). The CIC frequency was significantly higher (∼1/9000) in primary and recurrent ascites (n=16, p=0.002). Xenografts could be passaged at least 3 times, providing evidence of self-renewal. The CIC frequency remained constant in nearly all xenografts from primary tumors, but increased substantially with passage of recurrences, suggesting greater genetic instability. CD133+ cells from primary tumors (n=2), matched metastases (n=2), ascites (n=6) and passage 1 xenografts (n=6) were enriched for CIC (1/300-1/4000), and all (or the vast majority) of CIC activity resided within the CD133+ fraction. Xenografts from CD133+ cells gave rise to CD133+ and CD133- cells and could be serially passaged at least 1-3x. VEGFR2+ and ALDH1+ cells also were enriched for CIC, to a lower extent than CD133. In contrast, after sorting for CD117/CD44 (n=3), tumors arose from all fractions but CD117+/CD44+ cells. Our data are consistent with a hierarchical model of SOC and indicate that CD133 is a marker for ovarian CIC. Current work is devoted to profiling the CD133+ population and identifying additional markers, using high throughput flow cytometry and a panel of 234 antigens and other methods. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4303.