bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A65: Nodal contributes to cisplatin resistance in ovarian cancer cells.
Authors: Bilyk Olena , Postovit Lynne-Marie
Published: May 2016

The capacity of ovarian tumors to grow and propagate is dependent on a small subset of tumor cells, termed cancer stem-like cells, which contribute to drug resistance, and metastasis. Nodal, an embryonic morphogen has been found to sustain stem cell pluripotency and cellular plasticity, but in cancer cells its expression promotes cancer stem cell renewal, tumor growth, invasion, angiogenesis and metastasis. The role of Nodal in the development of recurrent chemoresistant ovarian cancer (OC) has not been previously investigated. The aim of this study was to investigate whether Nodal can modulate resistance of OC cell line A2780s to cisplatin.

Methods. Human OC cell line A2780s was cultured in DMEM/F12 medium supplemented with 10% FBS and treated with cisplatin. Cytotoxity of cisplatin was determined by MTT assay (Roche) and clonogenic assay. Nodal expression after cisplatin treatment was determined by digital PCR and immunofluorescence staining. To increase Nodal signaling, we used a Nodal expression vector (versus an empty pcDNA3.3 vector; pcDNATM3.3-TOPO cloning kit; Invitrogen). We also employed recombinant human Nodal (rhNodal; R&D). Transfection was performed with Lipofectamine (Invitrogen) as per manufacture instructions. Cell cycle analysis after cisplatin treatment and expression of cancer stem cell marker CD133 were determined using flow cytometry analysis (FACS). In vitro sphere limiting diluting assay was applied to measure ovarian cancer stem cell (OCSC) self-renewal.

Results. The expression of Nodal increased significantly in A2780s cells after 24h cisplatin treatment (20,000 copies/1ug RNA versus 800 copies/1ug RNA in untreated cells) and retained for 96h after cisplatin withdrawal (120,000 copies/1ug RNA). Overexpression of Nodal rendered A2780s cells more resistant to cisplatin (IC50 2.9ug/ml) compared to control cells (IC50 1.3ug/ml). Treatment of A2780s cells with rhNodal (100ng for 24h) during cisplatin treatment increased the ability of ovarian cancer cells to form spheres. FACS analysis revealed that cisplatin along with rhNodal treatment increased the population of CD133 positive cancer stem cells in A2780s cell line (5.85% versus 0,65% in untreated cells and 1.75% after cisplatin treatment alone). Treatment of A2780s cells with rhNodal prevented cell arrest in S phase after cisplatin treatment (19.4% of cells in S phase after rhNodal+cisplatin versus 63.6% - after cisplatin alone).

Conclusison. Our findings demonstrate that Nodal may contribute to cisplatin resistance in OC cells and tumor initiation capacity after drug therapy, and may hold promise as a therapeutic target to prevent chemoresistant recurrence.

Citation Format: Olena Bilyk, Lynne-Marie Postovit. Nodal contributes to cisplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A65.
Abstract B13: Discovery of novel subtype-specific ovarian cancer biomarkers via integrated tissue proteomics.
Authors: Leung Felix , Bernardini Marcus Q. , Clarke Blaise , Rouzbahman Marjan , Diamandis Eleftherios P. , Kulasingam Vathany
Published: May 2016

Introduction and Objectives: It is evident that ovarian cancer (OvCa) is not a single disease but is made up of several distinct subtypes, including serous, endometrioid, clear cell and mucinous. The gold-standard biomarker CA125 performs well in serous but not in the other histotypes. We hypothesize that a more focused discovery effort (on non-serous OvCa biomarkers and/or markers that can complement serum CA125) may bring about a sensitivity that is acceptable for all histotypes and be suitable for early diagnosis of OvCa.

Methods: Tissues from patients diagnosed with endometrioid (EC), clear cell (CC), and mucinous ovarian carcinoma (MC), as well as from their appropriate controls (endometriosis and healthy endometrium for EC and CC; mucinous cystadenoma for MC) were subjected to proteomic analysis using a label-free, offline 2-dimensional liquid chromatography tandem mass spectrometry-based approach. Discovery candidates were then filtered using an in-house developed algorithm combining publicly-available resources with our own warehouse of transcriptomic and proteomic sets.

Results and Discussion: Over 8000 unique proteins were identified in this proteomic exercise; specifically, approximately 1500 protein unique to MC, 1100 unique to CC, and 3000 unique to EC were identified when comparing the appropriate cases and controls. A curated list of 60 high-potential candidates was generated after using a range of bioinformatics tools to ensure criteria based on factors including (but not limited to) tissue specificity, cellular localization and transcriptional upregulation were met. These 60 candidates represent putative subtype-specific markers which will be further analyzed and validated in serum cohorts.

Conclusions: The identification and validation of markers specific to the non-serous subtypes of OvCa remains an unmet clinical need. With our list of putative subtype-specific markers, we aim to develop a novel biomarker panel able to detect all OvCa histotypes with greater sensitivity and specificity than any existing clinical tools.

Citation Format: Felix Leung, Marcus Q. Bernardini, Blaise Clarke, Marjan Rouzbahman, Eleftherios P. Diamandis, Vathany Kulasingam. Discovery of novel subtype-specific ovarian cancer biomarkers via integrated tissue proteomics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B13.
Abstract B32: Serum calcium and serum albumin can discriminate malignant from benign pelvic masses.
Authors: Schwartz Gary G. , Kelly Michael G. , Winkler Stuart S. , Lentz Samuel S. , Berliner Steve H. , Swain Melissa F. , Skinner Halcyon G.
Published: May 2016

Background: Biomarkers that aid in the differential diagnosis of malignant pelvic masses from benign ones prior to surgery are needed in order to triage women with malignant masses to appropriate specialist care. Because high albumin-adjusted serum calcium predicted ovarian cancer among women without evidence of disease, we hypothesized that it might predict cancer among women with pelvic masses that were evident radiographically.

Methods: We studied a cohort of 514 women with pelvic masses who underwent resection at Wake Forest University Baptist Medical Center from July 2009 through June 2013. We divided patients into a “training” set, to identify associations in the data, and a “testing” set, to confirm them. Data were obtained from medical records. A best fit model was selected using the Akaike Information Criterion.

Results: Albumin-adjusted serum calcium was significantly higher in women with malignant vs. benign masses (P= 0.0004). High normocalcemia, i.e., an albumin-adjusted serum calcium ≥ 10 mg/dL, occurred in 53% of women with malignant tumors vs. 12% of benign tumors. High normocalcemia was associated with a ~14-fold increased risk of malignancy. The best fit model (Overall) included albumin, calcium, and non-linear terms. Overall achieved an AUC of 0.83 with a sensitivity of 72% and specificity of 83%, a PPV of 71% and a NPV of 85%.

Conclusions: A model using serum calcium and serum albumin to predict malignancy in women with pelvic masses has high sensitivity and is economical.

Impact: Our model can help triage women with ovarian cancer to appropriate surgical care.

Citation Format: Gary G. Schwartz, Michael G. Kelly, Stuart S. Winkler, Samuel S. Lentz, Steve H. Berliner, Melissa F. Swain, Halcyon G. Skinner. Serum calcium and serum albumin can discriminate malignant from benign pelvic masses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B32.
Abstract B36: In vivo laser-based imaging of the human fallopian tube for future cancer detection.
Authors: Swisher Elizabeth M. , Melville C. David , Johnston Richard S. , Agnew Kathy , Chiang Seine , Seibel Eric J.
Published: May 2016

Inherited mutations in BRCA1 and BRCA2 cause a 20-50% lifetime risk of ovarian, fallopian tubel, or peritoneal carcinoma. Clinical recommendations for these high-risk women include the prophylactic removal of the ovaries and fallopian tubes by age 40 after child-bearing. Recent findings suggest that many presumed ovarian or peritoneal carcinomas arise in fallopian tube epithelium. Although survival rate is >90% when ovarian cancer is detected early (Stage_I), 70% of women have advanced disease (Stage_III/IV) at presentation when survival is less than 30%. Over the years, effective early detection of ovarian cancer has remained elusive, possibly because screening techniques have mistakenly focused on the ovary as the origin of ovarian carcinoma. Unlike ovaries, the fallopian tubes are amenable to direct visual imaging without invasive surgery, using access through the uterine cervix. To develop future screening protocols, we investigated using our 1.2-mm diameter, forward-viewing, scanning fiber endoscope (SFE) to image luminal surfaces of the fallopian tube before laparoscopic surgical removal. Three anesthetized human subjects participated in our protocol development which eventually led to 70-80% of the length of fallopian tubes being imaged in scanning reflectance, using red (632nm), green (532nm), and blue (442nm) laser light. A hysteroscope with saline uterine distention was used to locate the tubal ostia. To facilitate passage of the SFE through the interstitial portion of the fallopian tube, an introducer catheter was inserted 1-cm through each ostia. During insertion, saline was flushed to reduce friction and provide clearer viewing. This is likely the first high-resolution intraluminal visualization of fallopian tubes

Citation Format: Elizabeth M. Swisher, C. David Melville, Richard S. Johnston, Kathy Agnew, Seine Chiang, Eric J. Seibel. In vivo laser-based imaging of the human fallopian tube for future cancer detection. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B36.
Abstract B40: Prediagnosis leukocyte telomere length and risk of ovarian cancer.
Authors: Yang Meng , Prescott Jennifer , Poole Elizabeth M. , Rice Megan S. , Idahl Annika , Lundin Eva , Vivo Immaculata De , Tworoger Shelley S.
Published: May 2016

Introduction: Ovarian cancer is characterized by substantial genomic instability. Telomeres, which protect the physical integrity of chromosomes, are shortened by each cell division, and evidence supports that longer telomeres may reduce genomic instability. While retrospective studies generally support an inverse association of telomere length and ovarian cancer risk, the measures of telomere length after diagnosis may be influenced by treatment. Therefore, we examined the relationship between leukocyte telomere length (LTL) assessed prior to diagnosis and risk of ovarian cancer in three prospective studies.

Methods: We used buffy coat samples collected from healthy participants in the Nurses' Health Study (NHS), NHSII, and the Northern Sweden Health and Disease Study (NSHDS). Women who later developed ovarian cancer were matched to one or two controls on age, menopausal status, and date of blood collection. LTL was assessed using quantitative PCR-based assays in 5 batches with coefficients of variation of 10-15%. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using conditional logistic regression, based on study-specific quartiles in controls, for each study separately. We used fixed-effects models for meta-analysis. Multivariate models adjusted for oral contraceptive use, tubal ligation, family history of ovarian cancer, parity, smoking status, and body mass index.

Results: In total, there were 487 cases and 810 controls across the three studies. The mean age at blood collection ranged from 45 (NHSII) to 57 (NHS) years. In unadjusted and multivariate models, we observed a suggestion of an inverse association between LTL and ovarian cancer risk in each study, although none of the trend tests were statistically significant. In a meta-analysis of the multivariate adjusted models, women in the longest versus shortest quartile of LTL had a non-significant 26% lower risk of ovarian cancer (OR=0.74; 95%CI=0.49-1.12; p-trend=0.33).

Conclusion: In this first prospective study of telomere length and ovarian cancer risk, we observed that longer leukocyte telomere length was suggestively associated with lower ovarian cancer risk. Given that serous tumors are more likely to exhibit genomic instability, we are currently evaluating the association for this subtype as well as conducting pooled analyses with common quartile cut points across studies.

Citation Format: Meng Yang, Jennifer Prescott, Elizabeth M. Poole, Megan S. Rice, Annika Idahl, Eva Lundin, Immaculata De Vivo, Shelley S. Tworoger. Prediagnosis leukocyte telomere length and risk of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B40.
Abstract B58: Functional analysis of the cell adhesion molecule Nectin-4 in the unique tumor microenvironment of ovarian cancer.
Authors: Boylan Kristin L.M. , Buchanan Petra C. , Shukla Dip , DeRycke Melissa , Walcheck Bruce , Skubitz. Amy P.N.
Published: May 2016

A unique aspect of ovarian cancer progression is the collection of ascites fluid in the abdominal cavity, which contains free-floating tumor cells in the form of single cells and multicellular aggregates (spheroids). In previous studies, we showed that the cell adhesion molecule Nectin-4 is overexpressed in ovarian cancer tumors and that the soluble fragment of Nectin-4 (sN4) is detected in the ascites fluid and sera of ovarian cancer patients. The objectives of this study were to determine how Nectin-4 is cleaved from the surface of ovarian cancer cells and the functional role that Nectin-4 plays in the aggregation, invasion, and proliferation of ovarian cancer cells. Functional assays were conducted using human ovarian cancer cell lines that differed in their levels of expression of Nectin-4. First, NIH:OVCAR5 cells, which express moderate levels of Nectin-4, were either: (a) transfected with shRNA targeting Nectin-4 (resulting in almost complete knockdown of Nectin-4 expression) or (b) transfected with full length Nectin-4 to overexpress Nectin-4. Second, MA148 cells, which do not endogenously express Nectin-4 were transfected with a full length Nectin-4 cDNA. Cells that expressed high levels of Nectin-4 formed multicellular aggregates and proliferated more rapidly than cells in which Nectin-4 levels had been knocked down by shRNA. Nectin-4 knock down cells also migrated more slowly in a wound healing assay than cells transfected with a control shRNA. Cells that overexpressed Nectin-4 took a longer time to disaggregate and invade monolayers of mesothelial cells. In addition, cells that overexpressed Nectin-4 shed sN4 following 3 hours of stimulation with phorbol myristate acetate. A broad spectrum inhibitor of matrix metalloproteases and A Disintegrin And Metalloproteases (ADAMs) completely inhibited Nectin-4 shedding. Our results suggest that Nectin-4 promotes cell-cell adhesion (important in the formation of multicellular aggregates), yet it can be cleaved from the surface of cells to allow cells to disaggregate (important in invasion). Understanding the role of Nectin-4 in the multifaceted ovarian cancer tumor microenvironment is critical in order to facilitate its development as a novel therapeutic target for ovarian cancer.

Citation Format: Kristin L.M. Boylan, Petra C. Buchanan, Dip Shukla, Melissa DeRycke, Bruce Walcheck, Amy P.N. Skubitz. Functional analysis of the cell adhesion molecule Nectin-4 in the unique tumor microenvironment of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B58.
Abstract IA13: Myc, metabolism, and cancer.
Authors: Dang. Chi Van
Published: May 2016

The MYC oncogene is highly amplified in many human cancers, and importantly is frequently amplified in ovarian cancer. It encodes a helix-loop-helix transcription factor, Myc, that dimerizes with a partner, Max, to bind the DNA consensus E-box sequence and broadly affects transcription by alleviating RNA polymerase pausing. Myc activates hundreds to thousands of genes that involve ribosome biogenesis, metabolism, and cell cycle control. Its deregulated expression in cancer cells causes a constitutive drive for cell growth, thereby rendering MYC-overexpressing cells addicted to specific metabolic pathways that may be dependent on the mutational spectrum of the cancer cell. For example, loss of p53 in combination with deregulated MYC, relieve cells from apoptotic pressure and unleash MYC's full potential as an oncogenic transcription factor. The dependence of MYC-overexpressing cells on nutrients provide a synthetic lethal approach to target metabolism for therapy. Further, deregulated MYC disrupt the circadian clock and metabolism in favor of cell growth-related metabolism. Hence, timing of metabolic therapies for MYC-induced cancers such as ovarian cancer may provide a new therapeutic window for emerging metabolic therapies.

Note: This abstract was not presented at the conference.

Citation Format: Chi Van Dang. Myc, metabolism, and cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA13.
Abstract IA17: Molecular characterization of mucinous ovarian carcinoma.
Authors: Levine Douglas A. , Schlappe Brooke
Published: May 2016

Mucinous ovarian carcinoma (MOC) is a rare, chemoresistant tumor that shares pathologic features with tumors of the gastrointestinal and pancreaticobiliary tracts and remains clinically, morphologically, and epidemiologically distinct from other more common ovarian cancer histologic subtypes. MOC accounts for 1-8% of all ovarian, typically affecting younger women and has a poor prognosis when disease has spread beyond the ovary. Unlike other epithelial ovarian cancers, increased risk of MOC is associated with cigarette smoking in a dose-dependent fashion. Oral contraceptive use that has been shown to be protective in serous ovarian cancer does not extend to MOCs. Despite clinical, pathologic, and epidemiologic differences from other epithelial ovarian cancers, MOC treatment continues to use standards defined for high grade serous ovarian cancer including comprehensive surgical staging followed by platinum and taxane combination chemotherapy for disease spread beyond the ovary. Most mucinous adenocarcinomas that involve the ovary are now recognized to be metastatic disease from an extraovarian site, most commonly gastrointestinal and pancreatic. In advanced stage cases, defining clear diagnostic criteria that reliably differentiates primary versus malignant tumors remains a challenge. Despite a similar site of origin, the molecular profile of MOC has few similarities to high-grade serous ovarian carcinoma, which is characterized by ubiquitous TP53 mutations, frequent BRCA1/2 mutations, and few KRAS mutations. The mutational landscape of MOC is characterized by frequent KRAS, TP53, ARID1A, and PIK3CA mutations as well as frequent CDKN2A homozygous deletions. Co-mutations of KRAS and TP53 are common. New data describe the molecular features of MOC using an integrative and comprehensive approach including next-generation sequencing, copy number analysis and immunohistochemistry that may lead to expanded treatment options for women with this rare disease. Data suggest a shared molecular pattern with pancreatic adenocarcinoma that may guide future therapeutics.

Citation Format: Douglas A. Levine, Brooke Schlappe. Molecular characterization of mucinous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA17.
Abstract PR03: Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth.
Authors: Huang Ruea-Yea , McGray AJ , Odunsi Kunle
Published: May 2016

We have previously shown that tumor-associated or infiltrating T cells (TALs or TILs) often co-express immune inhibitory receptors lymphocyte activation gene-3 (LAG-3) and programmed cell death 1 (PD-1) in human and murine ovarian tumor microenvironment (TME). Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed ovarian tumor growth. The relative contribution of other immune inhibitory receptors to immune suppression in the ovarian TME, and whether they co-regulate each other are currently unknown. To address this, we analyzed the expression of multiple immune checkpoint proteins and their cognate ligands in ovarian tumor-bearing wild-type (WT), LAG3KO and PD1KO mice. We found that PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and CD160 were elevated in TILs from the WT mice. Interestingly, compared with TILs of WT mice, the levels of LAG-3, TIM-3, and CTLA-4 were further significantly elevated in TILs from the tumor-bearing PD1KO mice; while the levels of PD-1, CTLA-4, and CD160 were further increased in the tumor-bearing LAG3KO mice. These results suggest that PD-1 and LAG-3 regulate the expression of multiple inhibitory receptors in the ovarian TME. Furthermore, combinatorial blockade with anti-LAG3 and anti-CTLA4 antibodies in the tumor-bearing PD1KO mice resulted in 30% tumor-rejection as compared to 10% by anti-CTLA4 alone or only delayed tumor growth by anti-LAG3. These data provide a strong basis for rational clinical testing of combinatorial blockade of the PD-1, LAG-3 and CTLA-4 immune inhibitory pathways for ovarian cancer immunotherapy.

This abstract is also presented as Poster A51.

Citation Format: Ruea-Yea Huang, AJ McGray, Kunle Odunsi. Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR03.
Abstract 01: Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS)
Authors: Tilak Anup , Sompallae Ramakrishna , Stence Aaron , Guseva Natalya , Bossler Aaron
Published: May 2016

Background: AML is a clinically and genetically heterogeneous clonal disorder. Approximately 50% are characterized by recurrent clonal chromosome aberrations which have contributed to the classification of disease and are recognized as important prognostic factors. AML patients who lack these recurrent structural abnormalities have been grouped as “intermediate cytogenetic risk” and are being further subcategorized by the sequence alterations that are being identified. A more complete understanding of the genetic changes that are relevant to the pathogenesis of AML will improve the classification of risk and ultimately better selection of therapy. Our hypothesis is that mutational profiling and analysis of patient outcomes will help better define the risk subgroups of patients and predict prognosis in patients with AML.

Methods: Archived DNA from 37 patients with various AML diagnoses were obtained with IRB approval (IRB#201502763). A panel of 30 commonly mutated genes in AML were designed using the Ion Ampliseq Designer (Ion Torrent) and targeted for “hotspot” or complete coding sequence (CDS). Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) technology. Library clonal amplification was performed by emulsion PCR using Ion PGM™ Template OT2 Reagents Kit200. Simultaneous sequencing of clonally amplified DNA targets was performed by Ion-Torrent Personalized Genome Machine (IT-PGM) using 318v2 chip. Genomic variants were identified by variant caller3.4 and further filtered and analysed through a custom-made clinically validated pipeline. Novel mutations were confirmed by either single nucleotide primer extension or Sanger sequencing.

Results: The assay successfully identified previously identified point mutations in FLT-3 and CEBPA genes. Additional variants were identified in 81% (30/37) of specimens; no mutations were found in 19% (7/37) specimens. The genes most frequently mutated were DNMT3A (23%), TET2 (17%), IDH2 (14%) and TP53 (14%). U2AF1 (11%) and RUNX1 (8%) were exclusively mutated in specimens with non-recurrent cytogenetic abnormalities. The identified variants will be used to risk stratify patients based on clinical outcome.

Conclusion: This targeted multi-gene panel successfully identified several variants along with previously known mutations in archived specimens.

Citation Format: Anup Tilak, Ramakrishna Sompallae, Aaron Stence, Natalya Guseva, Aaron Bossler. Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 01.
Abstract 09: Laminin binding integrin copy number variations in distinct types of epithelial cancers
Authors: Pond Erika , Harryman William L. , Cress Anne E.
Published: May 2016

Background: The laminin binding integrin (LBI) family are adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. Our objective was to investigate whether copy number variation (CNV) of a five gene signature (ITGB4, ITGA3, LAMB3, PLEC and SYNE3) comprised of essential genes for laminin adhesion would correlate with the reported clinical outcome in human epithelial tumors.

Methods: We investigated the relative alteration frequency of the CNV of integrin B4 (ITGB4), integrin A3 (ITGA3), laminin beta 3 chain (LAMB3), plectin (PLEC) and nesprin 3 (SYNE3) as a five gene signature independent of the epithelial cancer type using the publically available and published data. Provisional data was excluded from the analysis. We rank ordered the results using a 20% alteration frequency cut off and limited the analysis to studies containing at least 100 samples. The Kaplan Meier survival curves were analyzed to determine any alterations in patient survival in terms of months.

Results: Eight different cancer types representing 1,985 samples, contained at least a 20% alteration frequency of the five gene signature. The frequency of alteration ranged from 32.2% to 22.9% with the rank order (highest to lowest) as skin cutaneous melanoma, ovarian serous adenocarcinoma, stomach adenocarcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma and uterine corpus endometrial carcinoma. Within the gene signature, PLEC was the most commonly altered followed by LAMB3, ITGB4, ITGA3 and SYNE3 across all eight cancer types. Although specific mutations occurred in PLEC, amplification was the most common feature. Within a cancer type, there was little overlap of the individual amplified genes with the specific case, suggesting different specific amplicons may alter the LBI adhesion structures. Of the eight cancer types, a significant alteration in survival was indicated in bladder urothelial carcinoma (p=0.052714) with a median survival change of 19.02 to 24.28 months.

Conclusions: A five gene signature was created representing the essential and genetically verified functional components of laminin binding integrins and their adhesion structures. The CNV signature of the laminin binding integrins may have prognostic and predictive significance depending on the type of epithelial cancer. These may be promising biomarkers but also rational targets for personalized therapy in selected epithelial tumors for preventing metastatic spread. (Supported in part by NIH grants CA 23074 and CA 159406)

Citation Format: Erika Pond, William L. Harryman, Anne E. Cress. Laminin binding integrin copy number variations in distinct types of epithelial cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 09.
Abstract 13: Individualized approach for ovarian cancer: Identification of potential therapeutic targets based on genomic analyses, testing efficiency of treatments, and monitoring
Authors: Harris Faye R. , Halling Geoffrey C. , Becker Marc A. , Haluska Paul , Vasmatzis George , Kovtun Irina V.
Published: May 2016

Ovarian cancer (OCa) is fifth leading cause of death among women. Most of OCa patients diagnosed with high stage invasive disease. Although, majority experience remission after surgical debulking and adjuvant chemotherapy, about 80% of patients relapse and/or develop chemoresistant disease. More efficient diagnostics and therapies are needed to improve outcome in ovarian cancer.

We used an integrated approach to characterize genomic rearrangements in serous OCa inordet to identify potentially targetable alterations and test treatments with corresponding drugs using matching mouse avatar models. Mate-pair next generation sequencing protocol and BIMA2 algorithm were used to identify chromosomal rearrangements, including gene fusions and copy number changes, in primary tumors. Further analysis was then performed to determine alterations that may also be therapeutically targeted. Total of 25 primary tumors were analyzed, out of which four were selected for genomically guided treatments in corresponding mouse avatars. The tumors propagated in the selected mouse lines were also sequenced and showed identical to original patient tumor landscape of rearrangements. In addition, the selected targetable alterations were validated in both patient and avatar tumor DNA. For several of the analyzed cases pre-surgical and postsurgical blood was available for detection of selected alterations in cell free circulating tumor DNA. The analysis revealed presence of selected rearrangements in pre-surgical blood of all tested cases and in a number of those that showed presence of the disease after surgery. In summary, we showed that the whole genome sequencing of DNA rearrangements in conjunction with analyses of therapeutic targets and treatment testing in mouse avatars, developed using primary tumors, is a promising approach to aid treatment decisions for individual ovarian cancer patients.

Note: This abstract was not presented at the conference.

Citation Format: Faye R. Harris, Geoffrey C. Halling, Marc A. Becker, Paul Haluska, Jr., George Vasmatzis, Irina V. Kovtun. Individualized approach for ovarian cancer: Identification of potential therapeutic targets based on genomic analyses, testing efficiency of treatments, and monitoring. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 13.
Abstract IA09: Single cell RNA-sequencing of circulating tumor cells
Authors: Miyamoto David T. , Zheng Yu , Wittner Ben S. , Lee Richard J. , Zhu Huili , Broderick Katherine T. , Desai Rushil , Brannigan Brian W. , Arora Kshitij S. , Dahl Douglas M. , Sequist Lecia V. , Smith Matthew R. , Kapur Ravi , Wu Chin-Lee , Shioda Toshi , Ramaswamy Sridhar , Ting David T. , Toner Mehmet , Maheswaran Shyamala , Haber Daniel A.
Published: May 2016

Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR <0.1), with the majority implicated in growth factor, cell adhesion, and hormone signaling. Gene mutations and alternative splice variants of the Androgen Receptor (AR) gene were rare in primary prostate tumors and CTCs from untreated patients, but were prevalent in patients with castration-resistant prostate cancer. Distinct AR variants, including AR-V7, were present within different cells of individual patients, as well as within individual CTCs. Together, single cell molecular analysis of human CTCs points to multiple mechanisms of drug resistance in advanced prostate cancer, and suggests the role of heterogeneous signaling pathways that cooperate with co-existing abnormalities in AR in mediating disease progression.

Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.
Abstract IA19: Human melanoma neoantigens: Identification and vaccination
Authors: Linette Gerald P.
Published: May 2016

Cancer medicine is in the midst of a dramatic shift towards new therapeutics directed by alterations in the cancer genome. Melanoma genomes harbor somatic mutations that are caused by exposure to mutagens such as ultraviolet light. Tumor missense mutations (MM), translated into amino acid substitutions (AAS), may provide a form of non-self that elicits tumor specific T cell immunity. Indeed, T cell immunity directed against tumor encoded AAS has been reported in humans with cutaneous melanoma, thus implicating MM as a source of patient specific (private) neoantigens. However, a systematic evaluation of these putative neoantigens as validated targets is lacking and it is unknown whether the immune response directed against tumor encoded AAS can be augmented by vaccination. We show that vaccination against melanoma AAS-encoding peptides augments naturally occurring T cell immunity and reveals new HLA class I restricted private neoantigens in patients with advanced melanoma. Neoantigen specific T cells recognized naturally processed and presented antigen but failed to recognize wild type (non-mutated) antigen. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. As determined by TCR beta CDR3 DNA sequencing, vaccination promotes a repertoire of neoantigen-specific T cells that is highly diverse in terms of both TCR beta usage and clonal composition. No evidence of autoimmunity was apparent after vaccination. Our results demonstrate that tumor MM can be targeted as neoantigens and vaccination directed at tumor AAS somatic mutations broadens the antigenic breadth and clonal diversity of anti-tumor immunity. Personalized cancer immunotherapies targeting private somatic tumor alterations may become feasible in the near future.

Citation Format: Gerald P. Linette. Human melanoma neoantigens: Identification and vaccination. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA19.
Abstract IA22: Reflections on precision medicine
Authors: Sawyers Charles L.
Published: May 2016

The clinical success of molecularly targeted therapies such as imatinib for chronic myeloid leukemia sparked a revolution in cancer drug development as well as global efforts to define the landscape of genomic alterations across all cancers. Today we live in a world with a near complete understanding of all cancer gene mutations, giving us an unprecedented opportunity to leverage this knowledge to improve treatment outcomes, through precision medicine. Despite the many remarkable successes over the past 15 years, the goal of improved therapy for all cancers remains daunting due to the complexity of the genomic landscape and the challenges of overcoming drug resistance. With the rapid spread of next generation sequencing into the clinical arena at various cancer centers and health care institutions throughout the world, we have an opportunity to accelerate our understanding of treatment response and resistance through data sharing across these growing silos of genomic and clinical datasets. In this lecture I will illustrate the power of this approach through specific examples in prostate cancer and discuss interactional efforts to catalyzed data sharing through the Global Alliance for Genomics and Health (GA4GH).

Citation Format: Charles L. Sawyers. Reflections on precision medicine. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA22.
Abstract A018: Onset of dysthyroidism during treatment with immune checkpoint inhibitors is increased in responder patients
Authors: Signorelli Diego , Vecchio Michele Del , Garassino Marina , Intini Alice , Cona Maria Silvia , Magni Michele , Seregni Ettore , Nicola Massimo Di , Braud Filippo De
Published: May 2016

Background: Immune checkpoints inhibitors (ICI) are characterized by a high therapeutic index; among toxicities, immune-related thyroiditis has been reported. The aim of this study is to evaluate either the grade and severity of thyroiditis or its frequency in responder patients.

Material and Methods: We retrospectively evaluated 104 metastatic solid tumor patients treated at our Institute with ICI from 2010. We defined thyroid toxicity according to CTCAE version 4.0 and the disease control rate (DCR: CR, PR, SD) as efficacy endpoint. Correlation between dysthyroidism and DCR was assessed by Fisher's exact test.

Results: Of 104 patients, 32 (30.8%) were treated by anti PD-1, 32 (30.8%) by anti PDL-1, 34 (32.7%) by anti CTLA-4 and 6 (5.7%) by combo (anti PD-1+anti CTLA-4). Population was heterogeneous, ranging from the first to the 8th line of therapy, and affected by the following cancers: 42 (40.4%) lung (38 NSCLC, 4 SCLC), 40 (38.5%) melanoma, 9 (8.6%) RCC, 7 (6.7%) gastric, 2 (1.9%) bladder and one each for colon, salivary glands, thyroid and uterine leiomiosarcoma. Best responses were: 3 CR, 14 PR, 33 SD and 54 PD; 50 (48%) of patients achieved DCR, 18 (37.5%) treated by anti PD-1, 16 (33.3%) by anti PDL-1, 12 (25%) by anti CTLA-2 (4.2%) by combo. Overall, 29 patients developed dysthyroidism (subclinic, G1, G2 hypo/hyperthyroidism): 2/34 (6%) in anti CTLA-4, 10/32 (31%) in anti PD-1, 13/32 (41%) in anti PDL-1, 4/6 (67%) in combo group, respectively; 19/29 (65.5%) of these patients achieved DCR. Of 75 patients who did not develop dysthyroidism, only 29 (38.6%) achieved DCR. The onset of dysthyroidism statistically significant correlated with DCR (65.5% vs 40.0%, p= 0.016); no correlation was detected between toxicity grade (subclinic and G1 from oneside, G2 from the other side) and DCR (p= 1.0).

Conclusions: In responding patients, we found a statistically significant increase of thyroiditis during treatment with ICI. Thyroid function evaluation should be recommended routinely during ICI therapy, in particular with anti PD-1 and anti PDL-1 agents. Finally, the role of thyroiditis in predicting response to ICI must be confirmed in a large and homogeneous series of patients.

Note:This abstract was not presented at the conference.

Citation Format: Diego Signorelli, Michele Del Vecchio, Marina Garassino, Alice Intini, Maria Silvia Cona, Michele Magni, Ettore Seregni, Massimo Di Nicola, Filippo De Braud. Onset of dysthyroidism during treatment with immune checkpoint inhibitors is increased in responder patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A018.
Abstract A043: Safety and efficacy of a genetic vaccine targeting telomerase against various canine cancers
Authors: Impellizeri Joseph A. , Gavazza Alessandra , Lubas George , Greissworth Eliana , Jemiolo David , Ciliberto Gennaro , Aurisicchio Luigi
Published: May 2016

Purpose of the study: we have recently shown that a genetic vaccine targeting dog telomerase (dTERT) and based on Ad (Adenovirus)/DNA-EP (DNA electroporation) technology can induce strong immune response and increase overall survival (OS) of dogs affected by B-cell malignant lymphoma (ML) when combined to COP therapy in a limited number of subjects. In this study, we have conducted a double arm clinical trial with an extended number of ML patients and evaluated the immunotherapy in dogs affected by other tumor types, such as mesothelioma, hemangiosarcoma, melanoma and renal cancer.

Methods: Dogs are vaccinated by a protocol consisting of 2Ad vector injections (Priming phase) and followed by DNA-EP (Boosting phase) by means of a Cliniporator device, currently utilized and approved in Europe for Electrochemotherapy applications and adapted to Electro-Gene-Transfer. The vaccine was combined with Standard of care (SOC) therapies. The immune response was measured by ELISPOT, Intracellular staining for cytokines and ELISA assays.

Results: No adverse effects were observed in any dog patient that could be attributed to treatment. dTERT-specific immune response was induced in almost all treated animals. The OS of vaccine/COP treated ML-dogs was significantly increased over contemporaneous COP-treated animals (>76.1 vs 29.3 weeks, respectively, p<0.0001). The evaluation of the OS for the other tumor types is in progress. The association of the therapeutic effects and vaccine potency with biomarkers such as T regulatory cells (Tregs) and Myeloid derived suppressor cells (MDSC) is also in progress.

Conclusions: Ad/DNA-EP-based cancer vaccine against dTERT in combination with COP chemotherapy is safe and significantly prolongs the survival of ML canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and hold promise for the treatment of dogs affected by other cancer types. Most importantly, our study outlines the relevance of canine patients as relevant preclinical models for human cancer vaccines.

Citation Format: Joseph A. Impellizeri, Alessandra Gavazza, George Lubas, Eliana Greissworth, David Jemiolo, Gennaro Ciliberto, Luigi Aurisicchio. Safety and efficacy of a genetic vaccine targeting telomerase against various canine cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A043.
Abstract A116: A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade
Authors: Chowell Diego , Krishna Sri , Snyder Alexandra , Vladimir Makarov , Chan Timothy A. , Anderson Karen S.
Published: May 2016

Recent studies have shown that CD8+ T cells that recognize neoantigens presented by tumor cells, but the target specificity of these tumor infiltrating T cells are largely unknown. Developing a predictive model that identifies immunogenic neoepitopes presented by major histocompatibility complex (MHC) class I molecules is critical to identify potential vaccine targets and to improve prediction of response to immune checkpoint inhibitors. We hypothesize that the immunogenic heterogeneity of tumors may explain, in part, differences in clinical response that individual patients derive to checkpoint blockade therapy. We have recently shown that the relative hydrophobicity of T cell receptor (TCR) contact residues is a hallmark of immunogenic MHC class I epitopes, and that this may represent a biochemical basis by which T cells discriminate immunogenic epitopes within the background of self-peptides. Here, we extend this analysis using candidate MHC class I tumor neopeptides from patients with melanoma. Somatic missense mutations were identified from recently published data on whole-exome sequencing of tumors from 11 patients who derived a long-term benefit and 14 patients who derived a minimal or no benefit from ipilimumab or tremelimumab. Mutational burden ranged from 1 to 1,304 mutations per tumor. More than 3,200 potential MHC class I neopeptides were identified based on a predicted binding algorithm (binding affinity, ≤500 nM). Our preliminary results indicate significant differences in hydrophobicity at the anchor residues P2 and P9 (P < 0.001 for both), reflecting MHC binding differences. In addition, there is an increase in hydrophobicity at the TCR contact residues P4 and P7 (P < 0.01 for both) in long-term responders. The overall frequency of mutations in neoepitopes is significantly different at the TCR contact residues P4 and P7 between long-term responders and minimal or no responders (P < 0.01), suggesting genetic immunoediting of these tumors. Based on these results, we have developed a probabilistic Bayesian-based model to predict immunogenic neoepitopes. The model is being applied on the overall pool of neopeptides from each individual patient with melanoma. The sets of predicted immunogenic neoepitopes may serve as potential therapeutic vaccine candidates for patients with melanoma.

Citation Format: Diego Chowell, Sri Krishna, Alexandra Snyder, Makarov Vladimir, Timothy A. Chan, Karen S. Anderson. A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A116.