bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5593: Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies
Authors: Willingham Stephen , Hotson Andrew , Ho Po , Choy Carmen , Walter Kim , Yusko Erik , Benzeno Sharon , Laport Ginna , Miller Richard , McCaffery Ian
Published: July 2017

Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index <0.9) were observed in both anti-PD-1 native and refractory patients and correlated to patient responses. CD73 expression, CD8+ infiltration, TREG distribution, and gene expression signatures are currently being evaluated in serial tumor biopsy specimens and will be presented. In total, this shows that CPI-444 is well tolerated in cancer patients, exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of anti-tumor immunity and clinical activity.

Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [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 5593. doi:10.1158/1538-7445.AM2017-5593
Abstract 560: A pan-inflammatory and precancerous disease analysis reveals key biological characteristics in chronic inflammatory diseases with high oncogenic risk
Authors: Zhang Chi , Yao Fang , Cao Sha , Sheng Tao , Liang Sen , Du Wei , Xu Ying
Published: July 2017

It has long been observed that cancer development tends to be associated with chronic inflammation. Recent epidemiology studies have suggested chronic inflammation may be causally linked to a larger number of sporadic cancers. In this work, we have conducted a Pan-inflammatory and precancerous diseases analysis by comparing the 173 transcriptomics data sets consisting 4073 disease and 2669 normal human tissue samples of 10 cancer prone and 8 cancer independent inflammatory diseases, 11 precancerous diseases or benign tumors and 20 cancer types, aiming to identify fundamental differences between cancer-prone chronic inflammations and cancer-independent chronic inflammations and what biological characteristics cause cancer initiation or increased oncogenic risk. By applying differential gene expression, co-expression network, our in-house immune cell deconvolution and machine learning based hypoxia/oxidative stress prediction methods on the collected data, we have discovered the cancer prone chronic inflammatory diseases consistently have: (1) elevated relative proportion of CD4+ T, Macrophage, and Neutrophil cells, (2) decreased CD8 + T, adipocyte, monocyte and B cells, (3) highly disorder tissue repair phase I - IV related pathways, (4) increased oxidative stress level associated with Macrophage and Neutrophil proportions, (5) elevated iron ion metabolism, (6) over expressed extracellular glycosaminoglycan metabolism and (6) suppressed mitochondrial activity. These analyses results strongly suggest that there is a necessary pathway for the majority or all chronic

inflammatory diseases to become cancerous, namely consecutively dysregulated tissue repair process, increased reactive oxygen species producing immune cells, damage of extracellular matrix, Fenton reaction and production of hydroxyl radical and dysregulated mitochondrial activity. We believe these new insights could provide highly useful information in guiding future studies of inflammation associated cancers.

Citation Format: Chi Zhang, Fang Yao, Sha Cao, Tao Sheng, Sen Liang, Wei Du, Ying Xu. A pan-inflammatory and precancerous disease analysis reveals key biological characteristics in chronic inflammatory diseases with high oncogenic risk [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 560. doi:10.1158/1538-7445.AM2017-560
Abstract 5605: Frequencies of HLA-Class I and II alleles between German patients with renal cell carcinoma and healthy controls can be different
Authors: Göbel Steffen , Kehlen Astrid , Bluemke Karen , Altermann Wolfgang , Schlaf Gerald , Fischer Kersten , Fornara Paolo , Wullich Bernd , Wach Sven , Taubert Helge W.
Published: July 2017

The human leukocyte antigen (HLA) system is a major part of the human immune system and has an impact on tumor initiation, tumor progression and immunosurveillance. Renal cell carcinoma tumors are considered to be immunogenic. Therefore, we studied the allele frequencies of four gene loci (HLA-A, -B, -C and HLA-DR) in a cohort of 106 German renal cell carcinoma (RCC) patients and in 201 healthy controls. HLA-A-C were determined using serological methods, whereas HLA-C12, C14, C16, C18 and HLA-DR were characterized through the use of standard molecular biological methods. The presence of HLA-A and HLA-B alleles did not differ significantly between RCC patients and healthy controls. However, the occurrence of the HLA-C*12 allele was significantly increased in German RCC patients compared with healthy controls (P < 0.005; RR=2.3; Fisher’s exact test), whereas the occurrence of the HLA-DRB1*04 allele was significantly reduced in RCC patients compared with healthy controls (P < 0.05; RR=0.7; Fisher’s exact test). But the presence of allele HLA-C*12 was not significantly associated with 10 years overall survival. We suggest that the frequency of HLA alleles can affect development of RCC and could add knowledge as predictive marker for future immunotherapies.

Citation Format: Steffen Göbel, Astrid Kehlen, Karen Bluemke, Wolfgang Altermann, Gerald Schlaf, Kersten Fischer, Paolo Fornara, Bernd Wullich, Sven Wach, Helge W. Taubert. Frequencies of HLA-Class I and II alleles between German patients with renal cell carcinoma and healthy controls can be different [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 5605. doi:10.1158/1538-7445.AM2017-5605
Abstract 5624: Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry
Authors: Thayer Matt , Wong Alden , Draper David , Saims Dan , Wise Scott C.
Published: July 2017

The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4, anti-PD-1 and anti-PD-L1, has altered the paradigm of cancer treatment. Subsequently, the growing interest in the development of new single agent and combination therapies with immune-modulatory effects has generated a need for more powerful immunophenotyping techniques capable of in-depth cell characterization and proliferation assessment. To this end, using the CT26 syngeneic murine colorectal cancer model we have developed an 8 color flow cytometry antibody panel that focuses on the identification of lymphocyte subsets and analysis of proliferation within them utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer (Thermo Fisher Scientific). Splenocytes from tumor-naïve and CT26 tumor-bearing C57BL/6 mice were stained with CFSE (carboxyfluorescein succinimidyl ester), an amine-reactive fluorescent dye which enables generational tracking for up to seven rounds of cell division, allowing for proliferation to be quantified. Splenocytes from these mice were then stimulated with anti-CD3, anti-CD28, and IL-2. After stimulation for four days, cells were stained with antibodies against CD45, CD3, CD19, CD49b, CD335, CD4, CD8, and FoxP3. We were able to identify differential proliferative capacity between naïve and tumor-bearing mice in CD4+ T-cell, CD8+ T-cell, regulatory T-cell, and B-cell populations. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of potentially responsive immune compartments, as well as characterizing proliferation, will facilitate identification and development of potential combination therapies by expanding the depth of our ability to provide mechanistic descriptions of drug function and efficacy. Following this proof-of-concept work, we will also present data from tumor-bearing mice treated with immune checkpoint inhibitors in order to identify potential differences in proliferation after treatment with immune-modulating agents.

Citation Format: Matt Thayer, Alden Wong, David Draper, Dan Saims, Scott C. Wise. Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry [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 5624. doi:10.1158/1538-7445.AM2017-5624
Abstract 5644: The emergence of resistance to tamoxifen in relation to menopausal status, HER-2 status, status of steroid receptor and lymph nodes metastases status
Authors: Maksimovic Sinisa
Published: July 2017

Background: The study used data from medical and counselling of patients who were diagnosed with hormonedependent breast cancer. Aim: The objective of the paper is to identify within a group of patients diagnosed with hormone sensitive breast cancer and those who have received adjuvant tamoxifen, and then to isolate the patients with whom the therapeutic effect of tamoxifen stopped.

Methods: The study analyzed 153 patients in the period from 2005 to 2011, at the Public Health Institution Hospital, Sveti Vračevi" in Bijeljina. Resistance to tamoxifen was developed by 60 patients (39.2%) and 93 patients (60.8%) did not develop resistance to it.

Results: More common emergence of resistance is in the premenopausal group of patients (p<0.001). Statistically significant difference in frequency of resistance to tamoxifen was observed in the group of patients with ER-/PrR+ status of steroid receptors (p<0.001). In relation to HER-2 status of diagnosed cancer, a statistically significant difference in frequency of resistance emergence during tamoxifen therapy in patients with HER2-positive status (p<0.001) was observed. We found that there is a statistically significant difference between patients with metastatic in lymph nodes compared to patients who had no metastases in lymph nodes (X2=39.494; p<0.001).

Conclusions: The analysis of menopausal status of patients, status of ER/PgR receptors status, HER-2 status of diagnosed cancer and status of lymph nodes trying to sort out the parameters on the basis of which a group of patients who can be expected to develop resistance to tamoxifen could be differentiated.

Note: This abstract was not presented at the meeting.

Citation Format: Sinisa Maksimovic. The emergence of resistance to tamoxifen in relation to menopausal status, HER-2 status, status of steroid receptor and lymph nodes metastases status [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 5644. doi:10.1158/1538-7445.AM2017-5644
Abstract 5655: Inhibition of CCR2 potentiates checkpoint inhibitor immunotherapy in murine model of pancreatic cancer
Authors: Janson Christine , Jung Heiyoun , Ertl Linda , Liu Shirley , Dang Ton , Zeng Yibin , Krasinski Antoni , McMahon Jeff , Zhang Penglie , Charo Israel , Singh Rajinder , Schall Thomas J.
Published: July 2017

Pancreatic cancer is an aggressive malignancy with a 5 year survival rate of less than five percent. The predominant immune cells infiltrating the tumor microenvironment are monocytes/macrophages, which are reported to support tumor growth by suppressing host immune responses to the tumor. Recruitment of monocytes to various tissues, including tumors, is dependent upon activation of the chemokine receptor CCR2 by one or more of the chemokines CCL2, CCL8 and CCL13. In preclinical and clinical studies, inhibition of CCR2 in pancreatic cancer has shown to decrease tumor progression by blocking recruitment and accumulation of monocytes/macrophages in the tumor microenvironment. Analysis of human pancreatic tumors revealed elevation of both CCL2 and CSF1, which recruit monocytes, as well as the monocyte marker CD14, in advanced pancreatic cancers. Current immunotherapy using checkpoint inhibitors are effective in some tumors, but lack efficacy in immune insensitive cancers, including pancreatic cancer. Here, we report that the inhibition of CCR2 using a small molecule antagonist potentiates anti-PD-1 immunotherapy in a syngeneic, orthotropic mouse model of pancreatic cancer. Our data reveal that blocking CCR2 decreases tumor burden by blocking monocyte infiltration and creating a microenvironment more favorable for CD8 T cells activity, and provide a mechanistic rationale for investigating the combination of a CCR2 antagonist and an immune checkpoint inhibitor in pancreatic cancer.

Citation Format: Christine Janson, Heiyoun Jung, Linda Ertl, Shirley Liu, Ton Dang, Yibin Zeng, Antoni Krasinski, Jeff McMahon, Penglie Zhang, Israel Charo, Rajinder Singh, Thomas J. Schall. Inhibition of CCR2 potentiates checkpoint inhibitor immunotherapy in murine model of pancreatic 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 5655. doi:10.1158/1538-7445.AM2017-5655
Abstract 5657: Objective measurement and significance of VISTA (PD-1H) expression in non-small cell lung cancer (NSCLC)
Authors: Villarroel-Espindola Franz , Datar Ila J. , Velcheti Vamsidhar , Rimm David L. , Herbst Roy S. , Schalper Kurt A.
Published: July 2017

Introduction: VISTA (PD-1H) is a member of the B7 family of immune co-regulatory molecules and has been proposed as a novel anti-cancer immunotherapy target. The intra- and extracellular domains of VISTA show homology to PD-1 and PD-L1, respectively, suggesting a role in anti-tumor immune evasion. The expression of VISTA, its association with PD-1 axis components and biological role in human NSCLC are unknown.

Methods: Using multiplex quantitative immunofluorescence (QIF), we simultaneously measured the levels of VISTA (clone D1L2G, CST), PD-L1 (clone 405.9A11, CST) and PD-1 (clone EH33, CST) protein in 732 stage I-IV NSCLCs from 3 retrospective collections represented in tissue microarray format (cohort #1 [n=297, Yale], cohort #2 [n=329, Greece]; and cohort #3 [n=106, Yale]). To evaluate the tumor tissue distribution, VISTA was also selectively measured in cytokeratin+ tumor cells, CD3+ T-cells, CD4+ T-helper cells, CD8+ cytotoxic T-cells and CD20+ B-lymphocytes. Associations between the marker levels, clinico-pathological-molecular variables and survival were studied.

Results: VISTA protein was detected in all NSCLCs, showed a membranous staining pattern and was localized predominantly in the tumor cells in 27.4% of cases; and in the stromal compartment in 98.5%. Although VISTA was detected in all major tumor infiltrating lymphocyte (TIL) subsets, the signal was higher in CD20+ B-cells than in CD3+ T-lymphocytes (P<0.05); and in CD4+ helper than in CD8+ cytotoxic T-cells (P<0.001). Using the median score as cut point, elevated VISTA in tumor and/or stromal cells was significantly associated with high PD-L1, PD-1 protein expression and increased CD8+TILs in the cohorts (P<0.001 - P<0.05). No consistent association between VISTA levels and age, gender, smoking status, specific NSCLC histology, stage, EGFR/KRAS mutations and overall survival was found.

Conclusion: VISTA is expressed in the majority of NSCLCs and shows differential distribution in tumor/stromal cells and TIL subsets, suggesting a complex function as a ligand and receptor. Elevated expression of VISTA in NSCLC is associated with increased PD-1 axis targets and cytotoxic T-cell density, indicating its possible modulation by pro-inflammatory signals. Our results support VISTA as a candidate target for anti-cancer immunotherapy in NSCLC alone or in combination with PD-1 axis blockers.

Citation Format: Franz Villarroel-Espindola, Ila J. Datar, Vamsidhar Velcheti, David L. Rimm, Roy S. Herbst, Kurt A. Schalper. Objective measurement and significance of VISTA (PD-1H) expression in non-small cell lung cancer (NSCLC) [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 5657. doi:10.1158/1538-7445.AM2017-5657
Abstract 5681: Utility of a targeted NSCLC genomic test for cfDNA variants in the community setting
Authors: Hahn Westen , Thurston Scott , Jackson Leisa , Weaver Amanda , Tschida Cherie , Sage Brent , Alexander Kristin , Pelletier Tunee , Charrington Nia , Cooper Samantha , Marr Dianna , Bowling Paul M. , Mellert Hestia , Pestano Gary
Published: July 2017

Time to treatment can have significant impact on progression of cancer, and treatment decisions rely heavily upon genomic and proteomic testing. However, multiple factors can delay the availability of molecular diagnostic test results, including the use of tissue-based testing. Almost one third of patients with non-small cell lung cancer (NSCLC) are either not candidates for biopsies, or have insufficient tissue samples from their initial biopsy. Additionally, most physicians in the community setting want to initiate treatment quickly but do not have access to on-site molecular testing at their practice. As a result, physicians may delay their decision to commence treatment with specific targeted therapies or treat before the mutation status is known. The goal of this study was to assess the utility of a blood-based cell-free (cf) nucleic acid Laboratory Developed Test (LDT) in the community setting. The tests utilize Droplet Digital™ PCR (ddPCR) technology to detect the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750), the EGFR resistance mutation (T790M), the KRAS mutations G12C, G12V and G12D, and the BRAF V600E mutation. Patient samples for testing were collected and shipped at ambient room temperature using a Biodesix® whole blood sample collection kit and processed at the Biodesix laboratory. Metrics were reviewed from the real-time measurement of established molecular diagnostic markers in the plasma of patients with NSCLC. For this study we analyzed results from greater than 4,000 patient cases (~24,000 individual variants). Greater than 70% of test orders were received from physician practices that self-identified as community based. Test mutation status results were reported within 72 hours of sample receipt from the physician’s office (95%). The percentage of tests requested that were positive for each variant category were 9% for EGFR sensitizing, 9% for EGFR resistance, 11% for KRAS, and 1% for BRAF. Notably, we generated test results successfully for 98% of tests submitted. We have developed fast, highly robust and sensitive blood-based assays to expedite time to treatment and expand the laboratory testing options for patients with NSCLC.

Citation Format: Westen Hahn, Scott Thurston, Leisa Jackson, Amanda Weaver, Cherie Tschida, Brent Sage, Kristin Alexander, Tunee Pelletier, Nia Charrington, Samantha Cooper, Dianna Marr, Paul M. Bowling, Hestia Mellert, Gary Pestano. Utility of a targeted NSCLC genomic test for cfDNA variants in the community setting [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 5681. doi:10.1158/1538-7445.AM2017-5681
Abstract 5691: ESR1 methylation in plasma cfDNA of patients with high-grade serous ovarian cancer
Authors: Giannopoulou Lydia , Mastoraki Sophia , Strati Areti , Chebouti Isaam , Kasimir-Bauer Sabine , Lianidou Evi S.
Published: July 2017

Introduction: ESR1 methylation is frequently observed in many types of solid malignancies, but few studies attempted to investigate ESR1 methylation status in ovarian cancer so far. We examined for the first time ESR1 methylation in plasma samples of high-grade serous (HGSC) ovarian cancer patients, using a novel real-time MSP assay.

Materials and methods: We first developed and validated a highly specific and sensitive real-time MSP assay for ESR1 methylation. A commercially available 100% methylated standard was used as positive control in all experiments. We then evaluated ESR1 methylation in plasma (2mL) of 59 pre-surgery, mainly advanced stage HGSC patients. A control group of 51 plasma samples from healthy women was recruited for the evaluation of specificity of our assay. All cfDNA samples isolated from plasma were subjected to sodium bisulfite (SB) treatment and were then analyzed by real-time MSP for ESR1 methylation. For the normalization of the results, we used ACTB as a reference gene. The real-time PCR assay for ACTB was designed to amplify specifically SB treated DNA.

Results: The real-time MSP assay is highly sensitive, as it detects down to 0.1% of ESR1 methylation in the presence of 99.9% non-methylated sequences. ESR1 methylation was detected in 18/59 (30.5%) high-grade serous ovarian patients, but only in 1/51 (2.0%) of healthy women. All results were obtained after normalization with ACTB as a reference gene.

Conclusion: We report for the first time that ESR1 methylation can be detected in cfDNA of high-grade serous ovarian cancer patients, but not in healthy women. Further investigation is required to determine the clinical significance of ESR1 methylation status in cfDNA of HGSC patients. We also aim to evaluate our findings in a larger group of patients.

Citation Format: Lydia Giannopoulou, Sophia Mastoraki, Areti Strati, Isaam Chebouti, Sabine Kasimir-Bauer, Evi S. Lianidou. ESR1 methylation in plasma cfDNA of patients with high-grade serous ovarian 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 5691. doi:10.1158/1538-7445.AM2017-5691
Abstract 5718: Outcome prediction in colorectal cancer using digitized tumor samples and machine learning
Authors: Bychkov Dmitrii , Turkki Riku , Haglund Caj , Linder Nina , Lundin Johan
Published: July 2017

Additional prognostic stratification of colorectal cancer patients is needed to improve management of patients. Visual microscopic assessment of tumor samples remains the standard method for disease subtyping. However, visual analysis of samples is subjective and weakly reproducible due to inter- and intra-observer variations. Recent progress within machine learning, especially its novel branch called deep learning, enables accurate evaluation of complex patterns observed in microscopic tissue images. Here, we combined deep learning techniques to evaluate a set of digitized formalin fixed paraffin embedded hematoxylin-eosin stained tumor tissue microarray (TMA) samples from 420 randomly selected patients with colorectal cancer. For each patient a set of clinicopathological characteristics including histological grade, Dukes stage and age at diagnosis are available as well as outcome data. Using convolutional neural networks and Long Short-Term Memory networks we validated the predictive power of the colorectal TMAs with regards to patient outcome. Univariate Cox proportional hazard regression analysis demonstrated that the prognostic accuracy of the deep learning algorithm on TMAs (hazard ratio 2.3; CI 95% 1.79-3.03) outperforms visual histological grading performed by a certified pathologist on a whole slide level (hazard ratio 1.65; CI 95% 1.30-2.15). In multivariate Cox proportional hazard regression, the deep learning based model was a prognostic factor, independent of histological grade, Dukes stage and age at diagnosis (Wald p-value < 0.001). Thus, we demonstrate that novel deep learning models can serve as digital prognostic biomarkers in colorectal cancer.

Citation Format: Dmitrii Bychkov, Riku Turkki, Caj Haglund, Nina Linder, Johan Lundin. Outcome prediction in colorectal cancer using digitized tumor samples and machine learning [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 5718. doi:10.1158/1538-7445.AM2017-5718
Abstract 5725: Search for a combination of risk stratification markers for recurrence after curative resection of gastric cancer
Authors: Oshima Takashi , Miyagi Yohei , Kimura Yayoi , Sakamaki Kentaro , Yamanaka Shoji , Oue Naohide , Aoyama Toru , Sato Tsutomu , Cho Haruhiko , Shiozawa Manabu , Yoshikawa Takaki , Rino Yasushi , Yasui Wataru , Imada Toshio , Masuda Munetaka
Published: July 2017

Objective: Adjuvant chemotherapy after curative resection is standard treatment for stage II/III gastric cancer. Personalized therapy based on biomarker analysis is expected to further improve outcomes. We analyzed frozen specimens of gastric cancer to investigate markers for stratification of recurrence risk and report our findings.

Methods: We studied 778 patients with stage II/III gastric cancer in whom ≥5 years had elapsed since curative resection. We first measured the expression levels of 127 genes in gastric cancer tissue by quantitative polymerase-chain-reaction (PCR) assay in 255 patients (training set, 145; validation set, 110) to identify poor prognostic factors. Next, we studied patients who received 1 year of adjuvant chemotherapy after curative resection of T4a, Nx, M0, stage III gastric cancer and performed an exploratory proteomic analysis to compare the expressions of 1756 proteins between 12 patients who died of recurrence within 3 years and 12 patients who survived with no recurrence for at least 5 years. Proteins with a max fold change of >2 and significant differences (P<0.001) were identified. Immunostaining of tissue microarray sections from another cohort of 499 patients was performed to confirm our results and to identify combinations of markers for risk stratification.

Results: On quantitative PCR assay, SPARC, HER2, CXCR4, INHBA, VSNL1, MMP11, CCR7, MRP1, CEACAM7, p53, EZH2, PDGFRB, and VCAM1 were identified as candidate markers. On exploratory proteomic analysis, SPARC, CLC, PRG2, and PRG3 were identified. A tissue microarray was constructed for the 16 candidate markers to evaluate combinations of markers. The combination of SPARC, EZH2, HER2, and p53 could be used for risk stratification (5-year survival rate, 68% vs. 33%; P=0.0111).

Conclusions: The combination of SPARC, EZH2, HER2, and p53 can be used as a marker to stratify the risk of recurrence after curative resection of stage II/III gastric cancer.

Citation Format: Takashi Oshima, Yohei Miyagi, Yayoi Kimura, Kentaro Sakamaki, Shoji Yamanaka, Naohide Oue, Toru Aoyama, Tsutomu Sato, Haruhiko Cho, Manabu Shiozawa, Takaki Yoshikawa, Yasushi Rino, Wataru Yasui, Toshio Imada, Munetaka Masuda. Search for a combination of risk stratification markers for recurrence after curative resection of gastric 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 5725. doi:10.1158/1538-7445.AM2017-5725
Abstract 5743: Characterization of mitochondrial DNA mutations and tumor evolution in ulcerative colitis-associated colorectal cancer
Authors: Baker Kathryn Terese , Nachmanson Daniela , Kennedy Scott R. , Brentnall Teresa A. , Risques Rosa Ana
Published: July 2017

The role of mitochondrial DNA (mtDNA) mutations in cancer is highly controversial. While specific mtDNA mutations have been associated with certain cancers, it has been recently reported that mtDNA mutations are negatively selected in most tumors. We aimed to clarify the role of mtDNA mutations in cancer progression by: 1) capitalizing on the multi-step sequence of tumorigenesis in ulcerative colitis (UC), a premalignant disease ideal for the study of tumor progression, and 2) employing a highly accurate, ultra-deep sequencing method to fully characterize the mtDNA mutational profile at different stages of tumor evolution. UC is an inflammatory bowel disease characterized by chronic colonic inflammation that increases the risk of colorectal cancer (CRC). UC CRC evolves through a stepwise process that involves the accumulation of molecular alterations in otherwise histologically normal epithelium, low-grade dysplasia (LGD), high-grade dysplasia (HGD), and cancer. Our lab has previously identified mitochondrial alterations as a facet of dysplastic progression in UC. Here we have worked to fully characterize the spectrum and frequency of mtDNA mutations in UC tumorigenesis by applying a novel, highly sensitive NGS technology, Duplex Sequencing (DS), that can accurately detect mutant allele frequencies (MAF) as low as 0.01%. For two patients that developed cancer, we analyzed 15-20 colon biopsies obtained at colectomy, representing all stages of tumorigenesis. The median duplex sequencing depth was 4,000x, which allowed unprecedented resolution to detect subclonal mutations (MAF 1-99%) and rare mutations (MAF<1%). Several subclonal mutations were present in all biopsies, but with variable MAF, thus enabling construction of phylogenetic trees via PHYLIP analysis. Rare mutations were abundant in all biopsies; on average one in every 10,000 nucleotides sequenced was mutated. Most rare mutations were G to A transitions, indicative of replicative errors, but G to T and C to A transversions, the oxidative damage mutational signature, were prevalent in some biopsies. We conclude, therefore, that most mtDNA mutations are caused by proliferation with a contribution by inflammatory processes, and that this may have greater implications for their role in other cancer types.

Citation Format: Kathryn Terese Baker, Daniela Nachmanson, Scott R. Kennedy, Teresa A. Brentnall, Rosa Ana Risques. Characterization of mitochondrial DNA mutations and tumor evolution in ulcerative colitis-associated colorectal 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 5743. doi:10.1158/1538-7445.AM2017-5743
Abstract 5749: VCAN promotes clear cell renal cell carcinoma tumor progression and metastasis, and predicts poor prognosis
Authors: Mitsui Yozo , Kato Taku , Maekawa Shigekatsu , Hashimoto Yutaka , Shiina Marisa , Sumida Mitsuho Imai , Wong Ryan Kenji , Yamamura Soichiro , Shahryari Varahram , Majid Shahana , Saini Sharanjot , Deng Guoren , Dahiya Rajvir , Nakajima Koichi , Tanaka Yuichiro
Published: July 2017

Introduction and Objectives: Although versican (VCAN) is known to promote tumor progression and enhance metastasis of several types of cancers, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Interestingly, a recent report showed that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC cases. In the present study, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC.

Methods: VCAN expression was analyzed in 3 RCC and normal kidney cell lines, as well as 84 matched ccRCC and normal renal tissues. We also performed various functional analyses of growth and progression properties using VCAN-depleted ccRCC cells. Microarray analysis was then employed to investigate the target genes of the pathway involved in ccRCC tumorigenesis and development.

Results: There are 4 isoforms of VCAN containing the N-terminal globular (G1 domain) and C-terminal globular (G3 domain) domains, each of which was found to be over-expressed in the ccRCC samples as compared to the controls. Higher VCAN expression was significantly correlated with metastasis (p<0.001) and worse 5-year overall survival after a radical nephrectomy (p=0.014). In vitro, VCAN knockdown by siRNA in Caki-2 and 786-O cells significantly decreased cell proliferation and increased apoptosis, and was also found to be associated with alteration of several TNF signaling-related genes, such as TNF-α, BID, and BAK. Furthermore, VCAN depletion markedly decreased cell migration and invasion associated with reduced MMP7 and CXCR4 levels.

Conclusion: Our results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis, showing it to be an attractive novel target for diagnostic, prognostic, and therapeutic strategies for affected patients.

Note: This abstract was not presented at the meeting.

Citation Format: Yozo Mitsui, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Mitsuho Imai Sumida, Ryan Kenji Wong, Soichiro Yamamura, Varahram Shahryari, Shahana Majid, Sharanjot Saini, Guoren Deng, Rajvir Dahiya, Koichi Nakajima, Yuichiro Tanaka. VCAN promotes clear cell renal cell carcinoma tumor progression and metastasis, and predicts poor prognosis [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 5749. doi:10.1158/1538-7445.AM2017-5749
Abstract 5779: Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies
Authors: Sablatura Lindsey K. , Harrington Daniel A. , Farach-Carson Mary C.
Published: July 2017

The high attrition rate of potential anti-cancer drugs entering clinical trials predicates a need for more predictive pre-clinical model systems. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment than traditional cell culture methods—including relevant extracellular matrix (ECM) cues and cell-cell interactions—while allowing greater experimental control and higher-throughput studies than animal models. Prevalent cancers, including breast and prostate cancer, metastasize preferentially to bone, where they become incurable. The bone marrow microenvironment is a complex network of interacting bone homeostatic and hematopoietic niches that contribute to osteotropic cancer homing, proliferation, and treatment resistance. We created a 3D in vitro tri-culture model of the bone metastatic microenvironment to serve as a new platform to study the “trialogue” between bone metastatic cancer, bone marrow stroma, and bone marrow endothelial cells. This tool can be used to inform drug discovery and to combat treatment resistance. Within the present study, the utility of peptide-functionalized hyaluronan hydrogels for supporting the self-organization of 3D bone marrow microvascular networks was explored. Gel crosslinking density was varied to examine the effect of matrix porosity on cell migration and organization over time. Tubule length and diameter were quantified and the endothelial structures were assessed for indicators of mature vasculature, including lumen formation, basement membrane production, and the recruitment of supporting stromal cells. Breast/prostate cancer cell growth, survival, and physical association with bone marrow stromal and bone marrow microvascular endothelial cell networks was examined in tri-culture models. Ongoing studies seek to identify and disrupt key cell signaling pathways that mediate cell-cell interaction and promote cancer cell proliferation and survival, providing new tools to combat bone metastatic disease.

Citation Format: Lindsey K. Sablatura, Daniel A. Harrington, Mary C. Farach-Carson. Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies [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 5779. doi:10.1158/1538-7445.AM2017-5779
Abstract 5791: 3D tissue engineering bladder model for cancer invasion study
Authors: Goulet Cassandra Ringuette , Bernard Geneviève , Chabaud Stéphane , Pouliot Frédéric , Bolduc Stéphane
Published: July 2017

INTRODUCTION: Our understanding of the biological processes involved in bladder cancer (BCa) is greatly limited by the models currently available. In fact, the combination of in vitro and in vivo models of BCa has failed to elucidate all the fundamental aspects of the disease. The eighth most commonly diagnosed cancer in Western societies, BCa has become a growing public health concern, and more realistic models are needed to reveal the mechanisms involved in tumor initiation and progression. METHODS: Bladder substitutes have been constructed by tissue engineering with healthy human fibroblasts and urothelial cells, using the self-assembly method. Meanwhile, spheroids have been produced from non-invasive (RT4) and invasive (T24) BCa cell lines expressing DsRed fluorescent protein. The invasive potential of these spheroids was characterized in a type-I collagen gel (2.5mg/mL). Then, the spheroids were implanted on the surface of bladder substitutes, after which their development was followed by fluorescence microscopy. RESULTS: Both of the cancer cell lines used were able to form compact spheroids and grow on bladder equivalents. The invasive behaviour of spheroids varied depending on the nature of the cells used. The non-invasive RT4 cell line was unable to cross the basal lamina whereas the invasive T24 cell line was able to do so. CONCLUSION: The establishment of such a model for studying cancer biology in a physiological environment will help bridge the gap between overly simple cell culture models and more complex transgenic mice models. This new model offers a unique opportunity to study separately the players involved in the development of BCa and thus represents a powerful tool for the mechanistic analysis of this complex pathology.

Citation Format: Cassandra Ringuette Goulet, Geneviève Bernard, Stéphane Chabaud, Frédéric Pouliot, Stéphane Bolduc. 3D tissue engineering bladder model for cancer invasion study [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 5791. doi:10.1158/1538-7445.AM2017-5791
Abstract 5800: Epidermal growth factor-induced ANGPTL4 enhances anoikis resistance and tumor metastasis in head and neck squamous cell carcinoma
Authors: Liaong Yu-Han
Published: July 2017

Epidermal growth factor (EGF) is important for cancer cell proliferation, angiogenesis and metastasis in many types of cancer. However, the mechanisms involved in EGF-induced head and neck squamous cell carcinoma (HNSCC) metastasis remain largely unknown. In this study, we reveal that angiopoietin-like 4 (ANGPTL4) plays an important role in the regulation of EGF-induced cancer metastasis. We showed that EGF-induced ANGPTL4 expression promoted anoikis resistance and cancer cell migration and invasion in head and neck squamous cell carcinoma (HNSCC). In addition, depletion of ANGPTL4 inhibited EGF-induced cancer cell invasion. Autocrine production of EGF-induced ANGPTL4 regulated the expression of MMPs. The induction of MMP-1 gene expression by ANGPTL4-activated integrin β1 signaling occurred through the AP-1 binding site in the MMP-1 gene promoter. Furthermore, down-regulation of MMP-1 impeded EGF- and recombinant ANGPTL4-enhanced HNSCC cell migration and invasion. Depletion of ANGPTL4 significantly blocked EGF-primed anoikis resistance and extravasation. Moreover, depletion of ANGPTL4 significantly blocked EGF-primed metastatic seeding of tumor cells and MMP-1 expression in lungs. However, no effect of ANGPTL4 on tumor growth was observed. These results suggest that EGF-induced expression and autocrine production of ANGPTL4 enhances HNSCC metastasis via the up-regulation of MMP-1 expression. Inhibition of ANGPTL4 expression may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Citation Format: Yu-Han Liaong. Epidermal growth factor-induced ANGPTL4 enhances anoikis resistance and tumor metastasis in head and neck squamous cell carcinoma [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 5800. doi:10.1158/1538-7445.AM2017-5800
Abstract 5844: Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, improved radiosensitivity of human hepatocellular carcinoma
Authors: Chen Fang-Hsin , Yu Ching-Fang , Hong Ji-Hong
Published: July 2017

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Radiotherapy (RT) is one of modalities for HCC therapy in clinics, but the therapeutic effect is unsatisfied due to high radiosensitivity of normal liver tissues that limits the optimal dose. This study is to develop a new theraprutic approach to enhance the efficiency of RT on HCC tumors. Suberoylanilide hydroxamic acid (SAHA), a FDA approved chemotherapeutic drug, is an inhibitor of histone deacetylase (HDACs) and has been reported to enhance radiosensitivity in many cell lines. This study hypothesized that combination of SAHA and RT could improve tumor control on HCC tumors. HCC-834 cell, a primary culture of human HCC, had a dose-dependent response to RT when analyzed by clonogenic assay. SAHA had mild cytotocixity to HCC-834 cell (IC50=1μM). Combination of SAHA and RT reached a synergestic effect to prevent colony formation. In detailed, SAHA treatment did not induce DNA damage on HCC-834 cells when assayed by the expression of γH2AX. RT of 2Gy induced highest γH2AX expression within 10min and quickly returned to baseline after 24hrs. The expression of γH2AX was sustained at 24hrs when treated by SAHA and RT, and gradually recovered after 48hrs. This data indicated that SAHA inhibited the DNA repair process in HCC-834 cell lines after RT. An orthotopic tumor model was developed for further exploring the role of SAHA on HCC tumor radiotherapy. Mice were orally fed with SAHA (100mg/kg) 3hrs and 24hrs before RT of 25Gy. Mice were sacrificed at day10 after RT and maximum tumor area was examined by H&E staining. Either RT (38.81 ± 15.6mm2) or SAHA alone (45.09 ± 4.493mm2) inhibited tumor growth significantly compared with control tumors (99.1 ± 0.732mm2). Combined SAHA with RT had an additive effect on tumor control (28.03±12.25mm2). These results supported our hypothsis and demonstrated that SAHA combined with local RT was effective on inhibiting DNA repair and anti-tumor growth. This study provided a new strategy on HCC tumor therapy that SAHA could act as a radiosensitizer to improve the therapeutic efficiency of RT on HCC tumors.

Citation Format: Fang-Hsin Chen, Ching-Fang Yu, Ji-Hong Hong. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, improved radiosensitivity of human hepatocellular carcinoma [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 5844. doi:10.1158/1538-7445.AM2017-5844
Abstract 5866: t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites
Authors: Magdziarz Patrycja , Feng You , Jiang Dianlu , Parga Elizabeth , Celis Arianna , Denny Erin , Wang Xiaoying , Phillips Martin , Zhou Feimeng , Kane Susan E. , Momand Jamil
Published: July 2017

t-Darpp, one of two protein isoforms encoded by the PPP1R1B gene, is overexpressed in gastric, esophageal, colon, prostate, and breast cancers; and confers resistance to trastuzumab in Her2+ breast cancer cells. To gain insight into the trastuzumab-resistance mechanism of t-Darpp, we studied its structure, oligomerization status, metal-binding properties, and sites of phosphorylation. Circular dichroism spectroscopy analysis showed that recombinant t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil character at 25°C. Upon mild heat treatment (50°C for five min) the secondary structure does not appreciably change. Metal ion-binding analyses through inductively coupled plasma-mass spectrometry and graphite furnace-atomic absorption indicated that t-Darpp co-purifies with calcium, but not other metals commonly found in biological systems. The T75 site, critical for t-Darpp activation of the Akt signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, native blue gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments showed that the majority of t-Darpp (calculated mass = 19 kDa) exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a relatively large Stokes radius of 4.4 nm, suggesting an elongated structure. In summary, this study indicates that t-Darpp is an elongated, mild-heat-stable, and monomeric calcium-binding protein that is capable of being phosphorylated at T75 by CDK1 and CDK5. Blockage of t-Darpp calcium binding capacity or T75 phosphorylation may therefore help sustain Her2+ breast cancer sensitivity to trastuzumab therapy.

Citation Format: Patrycja Magdziarz, You Feng, Dianlu Jiang, Elizabeth Parga, Arianna Celis, Erin Denny, Xiaoying Wang, Martin Phillips, Feimeng Zhou, Susan E. Kane, Jamil Momand. t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites [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 5866. doi:10.1158/1538-7445.AM2017-5866