bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 482: Atypical expression of βV-tubulin in secretory cells of the fallopian tube as a biomarker for early dysplasia
Authors: Mathew Deepti , Wang Yanhua , Van Arsdale Anne , Horwitz Susan Band , McDaid Hayley
Published: July 2016

Class V βeta tubulin isotype (βV-tubulin), was recently found to have a tissue-specific expression pattern in epithelial tissues with secretory function, and deregulated expression in certain tumors. The overall aim of this study was to define the localization of βV-tubulin in the fallopian tube and its expression in pre-malignant dysplasia. Fallopian tube epithelium (FTE) was obtained from patients undergoing salpingectomy for various reasons including familial history that comprised BRCA mutant cases. Additionally, βV-tubulin expression was also examined in serous ovarian neoplasms. Immunohistochemistry, using a novel antibody developed in our lab against human βV-tubulin, was used to evaluate expression in paraffin-embedded sections of the distal fallopian tube (n = 55) and tumors (n = 13), from prospectively selected cases, categorized according to reasons for salpingectomy. Staining with standard markers, Pax-8 and acetylated α-tubulin was done to demarcate the secretory and ciliated cells of the FTE, respectively. Pilot data indicate that in the FTE, βV-tubulin is present in secretory cells and essentially never in ciliated cells. Morphologically “normal” FTE had only rare, scattered βV-tubulin positive cells; whereas, percentage positivity was increased in cases with serous ovarian neoplasms, and BRCA-wildtype familial breast cancer. The highest expression was observed in FTE from patients with BRCA-mutant breast cancers. Four distinct types of FTE atypia were observed in patients with known BRCA mutations. βV-tubulin was highly expressed in serous ovarian neoplasms, with the extent and intensity of staining elevated in high-grade serous carcinomas, compared to serous borderline tumors. In summary, βV-tubulin was localized to secretory cells of the distal FTE and its expression varied according to the clinical diagnosis. The frequency of these cells, and thus expression of βV-tubulin was dramatically enriched in tissue obtained from BRCA mutant cases. BRCA mutants also exhibited pronounced histologic atypia indicative of early dysplasia. Furthermore, elevated expression of βV-tubulin correlated with poor differentiation status in serous ovarian neoplasms.

Citation Format: Deepti Mathew, Yanhua Wang, Anne Van Arsdale, Susan Band Horwitz, Hayley McDaid. Atypical expression of βV-tubulin in secretory cells of the fallopian tube as a biomarker for early dysplasia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 482.
Abstract 4870: A novel potential target for cancer immunotherapy
Authors: Campagnoli Susanna , Parri Matteo , Grandi Alberto , De Camilli Elisa , Viale Giuseppe , Grifantini Renata Maria , Pileri Piero
Published: July 2016

Suppression of the host's immune system plays a major role in cancer progression. The tumor microenvironment (TME) not only plays a pivotal role during cancer progression and metastasis but also has profound effects on therapeutic efficacy. Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances and provided a new weapon against cancer. This therapy has elicited durable clinical responses and, in a fraction of patients, long-term remissions. Several immune checkpoint inhibitors have been approved by the Food and Drug Administration (FDA), whose therapeutic effect is due to blocking the activity of proteins expressed in T lymphocytes (CTLA4, PD-1) and the interaction with surface protein expressed on tumor cells (PD-L1 and PD-L2).

In our recent research activities we have identified 89 tumor-associated proteins by systematic immunohistochemistry analysis of tissue microarray (TMA) representing breast, colon, lung ovary and prostate cancer by using a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted proteins only marginally characterized. Among them, an interesting protein, which decorated the plasma membrane of breast cancer cells, was selected. This protein has been described to interact with the CD270/BTLA/CD160 pathway that regulates T-cell activation. It has also the capability to interfere with T-cell mediated allo-responses. Our characterization data showed that the protein is localized on the surface of several cancer cell lines. Moreover, it has the ability to stimulate cytokine production and secretion in PBMC from healthy donors. Several monoclonal antibodies have been generated in our lab against this protein and selected for the ability to bind the surface of cancer cells. Moreover, the ability of these monoclonal antibodies to interfere with the immune cells activity and to interfere with tumor growth in animal models is under evaluation. Results will be provided during the meeting.

The currently approved therapeutic targets regimens for triple negative breast cancer and ovarian cancer have limited efficacy. Therefore, the identification of other tumor-associated proteins targetable by specific mAbs is essential to improve patient survival.

Citation Format: Susanna Campagnoli, Matteo Parri, Alberto Grandi, Elisa De Camilli, Giuseppe Viale, Renata Maria Grifantini, Piero Pileri. A novel potential target for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4870.
Abstract 4881: CD300f-regulated efferocytosis by dendritic cells and macrophages controls the initiation and resolution of experimental colitis
Authors: Lee Ha-Na , Tian Linjie , Davis Jacquice , Quinones Mariam , Belkaid Yasmine , Krzewski Konrad , Coligan John E.
Published: July 2016

Phagocytic clearance of apoptotic cells (efferocytosis) is critical for resolution of inflammation and prevention of chronic inflammatory disorders, including inflammatory bowel diseases, and inflammation-associated cancer. CD300f (CLM-1), a cell surface receptor that recognizes phosphatidylserine exposed on apoptotic cells, has been shown to promote efferocytosis by macrophages. In the present study, we found that CD300f plays a critical role in controlling the severity and duration of dextran sulfate sodium (DSS)-induced colitis, and CD300f-expressing macrophages and dendritic cells (DC) were identified as key players controlling disease pathogenesis. Compared to Cd300f+/+ mice, Cd300f-/- mice showed increased susceptibility to DSS-induced colitis and impaired resolution of inflammation. In Cd300f-/- mice, macrophage-mediated efferocytosis was reduced, resulting in apoptotic cell accumulation in the gut mucosa. In contrast, Cd300f-/- DC engulfed apoptotic cells more efficiently than Cd300f+/+ DC. CD300f-deficient DC transfer exacerbated DSS-induced inflammation and delayed its resolution, whereas CD300f-expressing macrophage transfer attenuated DSS-induced inflammation. Hyperactive efferocytosis by CD300f-deficient gut DC resulted in excessive tumor necrosis factor-á (TNF-á) secretion, which induced secondary interferon-ã (IFN-ã) over-production, both of which impaired the resolution of colitis. TNF-á neutralization resulted in decreased levels of gut pro-inflammatory cytokines and significant disease amelioration. Collectively, these results suggest that CD300f is important for preventing chronic inflammation by enhancing macrophage efferocytosis and inhibiting TNF-á production induced by DC efferocytosis. From these findings, it is conceivable that increasing macrophage-mediated efferocytosis and suppressing DC-mediated efferocytosis through upregulation of CD300f expression would provide a new therapeutic strategy for inflammatory bowel disease patients.

Citation Format: Ha-Na Lee, Linjie Tian, Jacquice Davis, Mariam Quinones, Yasmine Belkaid, Konrad Krzewski, John E. Coligan. CD300f-regulated efferocytosis by dendritic cells and macrophages controls the initiation and resolution of experimental colitis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4881.
Abstract 4882: Identification of potential immune targets in controlling endometrioid endometrial carcinoma metastatic progression
Authors: Billaud Jean-Noel , Tugendreich Stuart , Toburen Debra
Published: July 2016

Endometrial adenocarcinoma is a common cause of gynecological cancer death in Europe and North America. The most dominant subtype, Endometrioid Endometrial Cancer (EEC) accounts for >80% of this cancer and is estrogen-dependent. At diagnosis, 75% of women have the disease confined to the uterus, which is considered Stage One. Five-year survival for Stage One patients is 80%, however, about 15-20% develop metastasis.

Total RNA extracted from tissues obtained after surgical resection from three women at stage one EEC was subjected to RNA-sequencing. The publicly available dataset (SRP045645) was downloaded directly from the Sequence Read Archive and the FASTQ files were processed with Biomedical Genomics Workbench for secondary analysis including mapping, quantification and differential expression analysis. Through streamlined integration the data was uploaded to Ingenuity Pathway Analysis (IPA) for biological interpretation.

We show how in silico solutions developed by QIAGEN Bioinformatics enable us to analyze the biological parameters involved in EEC metastatic progression from an early stage in the three patients diagnosed at Stage One. By comparing these patients’ RNA-seq results, we determined that key Canonical Pathways and other biological processes differentiated the three patients from one other. In particular, the predicted transcriptional program allowed us to visualize key upstream drivers. Three of these transcriptional drivers are immune related molecules (two cytokines: CXCL14 and GDF15 and one growth factor FGF3). They have been predicted to be potential master regulators of a Causal Network that drives increase of EEC, metastasis, epithelial-to-mesenchymal-transition (EMT), and cellular invasion in one of the three patients. Based on these results we propose these three immune molecules as possible therapeutic targets to counteract the metastatic processes in EEC.

Citation Format: Jean-Noel Billaud, Stuart Tugendreich, Debra Toburen. Identification of potential immune targets in controlling endometrioid endometrial carcinoma metastatic progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4882.
Abstract 4885: Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity
Authors: Lu Hailing , Hewitt Jessica , Meulen. Jan ter
Published: July 2016

The tumor microenvironment (TME) plays a critical role in controlling the balance between tumor progression and immune surveillance. Increased infiltration of T cells, especially CD8 T cells has been associated with a good prognosis. We hypothesized that G100, a novel synthetic TLR4 agonist, can modulate TME when directly injected into the tumor and trigger both a local and systemic effective immune response. Balb/c mice with implanted syngeneic A20 lymphoma received intratumoral (IT) injection of G100 (10 ìg) or control PBS three times a week. This treatment significantly inhibited tumor growth and resulted in complete tumor regression in approximately 60% of treated mice. To investigate the effects of G100 on TME, tumors were collected after three IT G100 injections for gene expression analysis by Nanostring and immune phenotyping analysis by FACS. Out of the 770 immune response genes included in the mouse PanCancer Immune Profiling panel, 295 genes were significantly upregulated in G100 treated tumors. The upregulated genes include DC function-related genes (CD40, CD83, CD86, and Ly96) and T cell and NK cell function genes and multiple chemokines and cytokines (IL1b, IL12A, IL18, IL6, IFNã, FcãR4, ICOS, GZMB, CCL3, CCL5, CCL7, CXCL1, CXCL2, CXCL11, CCR5, CCR6, and CCR7). T cell exhaustion markers (CTLA4 and LAG3) and CD274 (PD-L1) were also induced. G100-induced inflammation is also reflected at the cellular level as shown by increased T cells and NK cells in tumor via FACS analysis. To determine the immune cells that mediated tumor rejection, mice were selectively depleted of CD4 or CD8 T cells during G100 treatment. Results showed that the anti-tumor effect of G100 is dependent on CD8 T cells. G100-induced tumor protection was durable as mice surviving the first tumor challenge rejected a secondary tumor challenge without additional G100 treatment. Altogether, our results showed that IT G100 induces a proinflammatory cytokine and chemokine milieu that changes a “cold” tumor to a “hot” tumor, which facilitates the development of a CD8 T cell-dependent potent and durable anti-tumor effect. The induction of PD-L1 also suggests the potential synergy between G100 and checkpoint blockade therapy. These preclinical data support an on-going clinical trial of IT G100 in patients with follicular non-Hodgkin's lymphoma (NCT02501473), alone and in combination with anti-PD-1 therapy (pembrolizumab).

Citation Format: Hailing Lu, Jessica Hewitt, Jan ter Meulen. Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4885.
Abstract 489: NGS analysis of the single CTC or DTC isolated and subtyped by the integrated subtraction enrichment and immunostaining-FISH
Authors: Lin Peter
Published: July 2016

Majority of the circulating or disseminated tumor cell (CTC or DTC) detection methodologies rely on existence of epithelial markers (such as EpCAM and/or cytokeratins) on tumor cells. However, highly heterogeneous expression or even absence of EpCAM, and down-regulation of both EpCAM and cytokeratins in CTCs/DTCs during epithelial-mesenchymal transition (EMT) has been reported elsewhere, resulting in significant false negative detection of those uncatchable and invisible CTCs/DTCs. A novel strategy integrating subtraction enrichment which efficiently enriches non-hematopoietic tumor cells shed from various types of solid tumor into biofluid including peripheral blood, bone marrow, ascites, pleural effusion, and cerebrospinal fluid (CSF), with immunostaining-FISH (iFISH), which enables in situ phenotypic immunofluorescent staining of tumor biomarker and chromosomal FISH on the identical tumor cell, has shown its unique advantage in terms of detecting CTCs/DTCs with higher sensitivity and specificity. Diversified CTCs/DTCs could be identified and subtyped upon specific chromosome ploidy and tumor biomarker expression, and each subtype of CTCs/DTCs respectively correlate with therapeutic drug sensitivity or resistance, tumor metastasis or relapse. Whole genome amplification and subsequent next generation sequencing (NGS) analysis performed on the single subtyped breast, pancreatic, gastric and NSCLC cancer CTC or DTC targeted by iFISH, and collected by means of a microscopic cell manipulator is able to reveal ploidy of all the chromosomes and status of either unknown genes or a series of known oncogenes including HER2, Kras, BRAF, etc. It is anticipated that SE-iFISH could guide and promote more significant single cell based genomic and functional analyses of CTCs and/or DTCs.

Citation Format: Peter Lin. NGS analysis of the single CTC or DTC isolated and subtyped by the integrated subtraction enrichment and immunostaining-FISH. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 489.
Abstract 4890: Preclinical analysis of combination therapy using radiation and cellular immunotherapy
Authors: Wada Satoshi
Published: July 2016

Purpose:

To optimize the combination of ionizing radiation and cellular immunotherapy using a preclinical autochthonous model of prostate cancer.

Methods and Materials:

Transgenic mice expressing a model antigen under a prostate-specific promoter were treated using a platform that integrates cone-beam CT imaging with 3-dimensional conformal therapy. Using this technology we investigated the immunologic and therapeutic effects of combining ionizing radiation with granulocyte/macrophage colonystimulating factor-secreting cellular immunotherapy for prostate cancer in mice bearing autochthonous prostate tumors.

Results:

The combination of ionizing radiation and immunotherapy resulted in a significant decrease in pathologic tumor grade and gross tumor bulk that was not evident with either single-modality therapy. Furthermore, combinatorial therapy resulted in improved overall survival in a preventive metastasis model and in the setting of established micrometastases. Mechanistically, combined therapy resulted in an increase of the ratio of effector-to regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes.

Conclusions:

Our preclinical model establishes a potential role for the use of combined radiation-immunotherapy in locally advanced prostate cancer, which warrants further exploration

in a clinical setting.

Citation Format: Satoshi Wada. Preclinical analysis of combination therapy using radiation and cellular immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4890.
Abstract 4901: Short-course enzalutamide reveals immune activating properties in patients with biochemically recurrent prostate cancer
Authors: Donahue Renee N. , Madan Ravi A. , Richards Jacob , Grenga Italia , Lepone Lauren M. , Heery Christopher R. , Gulley James L. , Schlom Jeffrey
Published: July 2016

Background: Enazalutamide (enz) is an androgen receptor signaling molecule approved by the FDA for the treatment of patients with metastatic castration-resistant prostate cancer; however, trials are evaluating this agent in earlier stages of disease. Here, we have investigated the effect of enz on peripheral immunity in patients with biochemically recurrent prostate cancer enrolled in a clinical trial of enz with/without a therapeutic vaccine (NCT01875250).

Methods: Patients from the arm receiving enz alone (160 mg daily for 84 days, without ADT) were assessed in this study. Peripheral blood mononuclear cells (PBMCs) collected from 12 patients pre and post enz (days 14, 28, 84, and 100) were analyzed by flow cytometry to identify 123 immune cell subsets, including 9 standard subsets (CD4+ and CD8+ T cells, T-regulatory cells (Treg), B cells, conventional and plasmacytoid dendritic cells (cDC, pDC), natural killer cells (NK), natural killer T cells (NKT), and myeloid derived suppressor cell (MDSC)), and 114 subsets relating to maturation and function. PBMCs were also assessed for T-cell receptor excision circles (TREC) to identify recent thymic emigrants, and by microarray to determine changes in global gene expression.

Results: Treatment with enz induced several notable alterations in peripheral immune cells, suggesting that it has potential immune activating properties. These changes occurred early following treatment, and included an increase of NK cells, decreased frequencies of MDSCs with a suppressive phenotype (e.g. PDL1+ MDSC, gMDSC, and CD16+ MDSC), and decreased frequencies of both CD4+ and CD8+ T-lymphocytes expressing the immune inhibitory checkpoint molecule CTLA4. Additionally, treatment with enz increased TREC levels by >75% in 7 out of 12 patients compared to pre-therapy levels (p = 0.012); naïve CD4+ and CD8+ T-lymphocytes were also elevated by >25% in those patients demonstrating the greatest increase in TREC. Gene expression analysis of PBMCs corroborated these findings, showing that enz increased activation of interferon-gamma signaling and related immune activating pathways.

Conclusions. These findings show that short-course enz has immune activating properties in cancer patients, and support the combination of enz with immunotherapy.

Citation Format: Renee N. Donahue, Ravi A. Madan, Jacob Richards, Italia Grenga, Lauren M. Lepone, Christopher R. Heery, James L. Gulley, Jeffrey Schlom. Short-course enzalutamide reveals immune activating properties in patients with biochemically recurrent prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4901.
Abstract 4906: The selective class I HDAC inhibitor entinostat enhances the antitumor effect of PD-1 inhibition in a syngeneic orthotopic murine model of renal cell carcinoma
Authors: Orillion Ashley R. , Shen Li , Adelaiye-Ogala Remi , Elbanna May , Chintala Sreenivasulu , Arisa Sreevani , Pili Roberto
Published: July 2016

Background: Recent advances in immunotherapy have highlighted the antitumor effects of immune checkpoint inhibition. Novel anti-PD-1/PD-L1 immunotherapies have been shown to effectively overcome tumor avoidance of immune surveillance in several tumor types including renal cell carcinoma. Our group has recently shown that the selective class I HDAC inhibitor entinostat is effective in suppressing regulatory T cells and enhancing immunotherapies in murine renal and prostate models, RENCA and Myc-Cap respectively. In this study we have evaluated the combination of entinostat with an anti-PD-1 antibody in the RENCA renal cell carcinoma model.

Methods: 32 BALB/c female mice were implanted with the syngenic, orthotopic, renal cell carcinoma mouse model, RENCA - luciferase tagged - at day -8. Treatment (8 mice /group) with anti-mouse-PD-1 (aPD-1; 10mg/kg twice a week, I.P.), entinostat (5mg/kg 5 days a week), or combination of the two was begun at day 1. Bioluminescence imaging was performed at days -1, 9 and 19 to assess the orthotopic tumor growth. End point tumor weights were taken to assess the effect of combination treatment.

Results: Analysis of tumor growth showed a reduction of bioluminescence across the three time points in the combination group as compared to the vehicle and single agent treatments. Additionally, end point analysis of tumor weights revealed an overall reduction in the size of the tumors in the entinostat/anti-mPD-1 combination group (88% inhibition) as compared to the vehicle (p = 0.0002), aPD-1 alone (25% inhibition)(p = 0.0181), and entinostat alone (63% inhibition)(p = 0.0481) groups. Examination of the status of the infiltrating immune cells of the tumor microenvironment via flow cytometry, qRT-PCR, immunohistochemistry, and/or immunofluorescence analysis is ongoing.

Conclusions: Our preliminary results suggest that the immunomodulatory activity of the selective class I HDAC inhibitor entinostat may enhance the antitumor effect of PD-1/PD-L1 inhibition and provide the rationale for the clinical testing of this novel combination in patients with RCC.

Citation Format: Ashley R. Orillion, Li Shen, Remi Adelaiye-Ogala, May Elbanna, Sreenivasulu Chintala, Sreevani Arisa, Roberto Pili. The selective class I HDAC inhibitor entinostat enhances the antitumor effect of PD-1 inhibition in a syngeneic orthotopic murine model of renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4906.
Abstract 4911: SOCS-1 inhibits tumor growth by enhancing T cell mediated antitumor immunity related to PD-L1
Authors: Nakagawa Satoshi , Serada Satoshi , Matsuzaki Satoko , Ueda Yutaka , Yoshino Kiyoshi , Fujimoto Minoru , Kimura Tadashi , Naka Tetsuji
Published: July 2016

[Background]

In many solid cancers, constitutive activation of JAK/ signal transducers and activators of transcription (STAT) signaling pathway is associated with poor prognosis. Constitutive JAK/STAT signaling enhances the expression of cyclins and anti-apoptosis proteins, leads to the increased tumor cell proliferation and survival. Furthermore, one of the immune checkpoint molecules programmed cell death 1 ligand 1 (PD-L1), which is involved in the suppression of T cell mediated antitumor immunity, is induced by STATs. Suppressor of cytokine signaling (SOCS)-1 is a negative feedback molecule, which is induced by JAK/STAT signaling and inhibits the JAK activity, resulted in the suppression of JAK/STAT signaling. This study aimed to reveal the antitumor effects by SOCS-1 in vitro and in vivo.

[METHODS]

Five human OC cell lines (OVCAR-3, SKOV-3, RMG-1, A2780, ES-2) and 4 murine cancer cell lines (B16F10, LLC, 4T1, CT26) were assessed. SOCS-1 or LacZ were overexpressed by adenoviral vector. Anti-proliferative effect was assessed by WST-8 assay. Female BALB/c mice were injected with CT26 for subcutaneous xenograft experiments. AdSOCS-1 or AdLacZ was intra-tumorally administered every other day and PD-L1 expression in tumor cells and activation levels of tumor infiltrated T cells were analyzed by flow cytometry.

[RESULT]

Overexpression of SOCS-1 inhibited proliferation of all cancer cell lines. Three OC (OVCAR-3, SKOV-3, ES2) and all murine cancer constitutively expressed PD-L1. Expression levels of PD-L1 in OVCAR-3 and CT26 cells were downregulated about 30% compared with control by overexpressed SOCS-1 in vitro. In CT26 allografted model, SOCS-1 treatment significantly inhibited tumor growth (62.2±5.2%, P<0.01)and suppressed expression of PD-L1 on CT26 cells in the tumor (65.2±8.4%, P<0.05). Granzyme B and CD107a expression of intratumorally infiltrated CD8 T cells were increased more than 50% (P<0.05) in the AdSOCS-1 injected group compared with AdLacZ injected group. PD-L1 Fc fusion protein significantly inhibited antitumor effect of SOCS-1.

[Conclusion]

SOCS-1 mediated inhibition of JAK/STAT signaling shows not only direct antitumor effect against tumor cells but also enhances T cell mediated anti-tumor immunity in vivo by downregulating the expression of PD-L1 on tumor cells and preventing the interaction of PD-1/ PD-L1.

Citation Format: Satoshi Nakagawa, Satoshi Serada, Satoko Matsuzaki, Yutaka Ueda, Kiyoshi Yoshino, Minoru Fujimoto, Tadashi Kimura, Tetsuji Naka. SOCS-1 inhibits tumor growth by enhancing T cell mediated antitumor immunity related to PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4911.
Abstract 4937: Atm expression predicts ABT-888 and CPT-11 sensitivity in gastric cancer cells by mediating p53 independent regulation of cell cycle and apoptosis
Authors: Vijayasubhash Vinod , Tan Shihui , Yan Fui Leng , Liem Natalia , Yong Wei Peng
Published: July 2016

Background: Identification of synthetically lethal cellular targets and synergistic drug combinations are important in cancer chemo therapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signalling and cell cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its potential implication as a predictive biomarker for cancer chemotherapy remains unexplored. The present study evaluated ATM induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, ABT-888 and CPT-11 respectively.

Methods: ATM expression in a panel of 10 gastric cancer cells were detected by immunohistochemistry and the IC50 against ABT-888 and CPT-11 was determined by MTS cell proliferation assay. The synergistic effect of ABT-888 and CPT-11 in gastric cancer cells was determined by cell cycle and DNA damage assays. ATM expression was knocked down using siRNA and its effect in P53 induced cell cycle regulation and apoptosis was determined by immunoblot analysis.

Results and Conclusion: ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy, however showed a higher synergistic effect towards ABT-888 and CPT-11 combinational therapy that predisposed cells to enhanced drug sensitivity. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell cycle progression and apoptosis in a P53 independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cell to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM dependent regulation of cellular processes.

Citation Format: Vinod Vijayasubhash, Shihui Tan, Fui Leng Yan, Natalia Liem, Wei Peng Yong. Atm expression predicts ABT-888 and CPT-11 sensitivity in gastric cancer cells by mediating p53 independent regulation of cell cycle and apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4937.
Abstract 4952: Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer
Authors: Pértega-Gomes Nelma , Tyekucheva Svitlana , Penney Kathryn , Zadra Giorgia , Mucci Lorelei A. , Loda Massimo
Published: July 2016

Introduction: AKT and MYC are amongst the most prevalent oncogenes in prostate cancer (PCa). MYC cooperates with activated AKT in pre-clinical, genetically engineered in vivo models accelerating disease progression and increasing resistance to therapy. However, the clinical relevance of phosphoAKT/MYC co-expression in human prostate tumors remains unknown.

Experimental Procedures: A prospective cohort (Physicians’ Health Study and Health Professionals Follow-up Study) of 578 men with PCa with a mean 13-year follow up was used. Protein expression of MYC and phosphoAKT (Serine 473) was characterized by immunohistochemistry on tissue microarrays. pAKT and MYC expression was analyzed using the Cri Vectra/InForm multispectral analysis platform. The InForm software package was trained to segment PCa regions only (i.e., no stromal regions were included in the analysis). We used quantile normalization to adjust for the potential batch effects of protein expression staining across each of the tissue microarrays using the limma package R. The continuous score for pAKT and MYC were divided into quartiles and assigned a value of 1, 2, 3 or 4 for low (1), moderate (2 or 3) and high staining (4). Spearman correlations between pAKT, MYC and the clinico-pathological data were calculated.

Results: High pAKT and High MYC were co-expressed in 10% of patients. (Quartile 4). Also 10% were negative or weakly positive for both pAKT and MYC (Quartile 1). Only 3% of the High pAKT patients had weak or no expression of MYC and 4% of High_MYC patients had Low pAKT expression. The majority of patients were distributed in the Quartiles 2 and 3 (73%). Importantly, only High pAKT and High pMYC co-expressors were associated with tumor stage (pT3 or pT4) (p = 0.03), high Gleason Score (p = 0.021) and presence of metastatic disease (p = 0.011). GEP analysis showed that the same patients exhibited up-regulation of genes involved in cell invasion/migration capacity and stromal remodeling.

Conclusions: Co-expression of AKT and MYC is associated poor clinical outcome in PCa patients. Gene expression profiling suggest that concomitant activation of AKT and MYC results in the activation of pathways associated with an invasive phenotype.

Citation Format: Nelma Pértega-Gomes, Svitlana Tyekucheva, Kathryn Penney, Giorgia Zadra, Lorelei A. Mucci, Massimo Loda. Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4952.
Abstract 4977: Immunogenic chemotherapy synergize PD-1 blockade by enhancing dendritic cells infiltration in triple-negative breast cancer (TNBC)
Authors: Yuan Xiangliang , Xiao Yi , Kuo Wenling , Yu Dihua
Published: July 2016

Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) has achieved noteworthy benefit in multiple cancers. However, their efficacy remains unsatisfactory in triple-negative breast cancer (TNBC). Breast cancer is not highly mutagenic and immune checkpoints blockade therapies for TNBC is not as effective as for melanoma or lung cancer with high mutation rates. Combination of different modalities offers an opportunity to enhance their effect. We studied the synergistic effect of cancer chemotherapy and immunotherapy in TNBC mouse model. Here we report that the combination of Doxisome (liposomal encapsulated formulation of Doxorubicin) and anti-PD-1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing 4T1-triple negative breast cancer. Moreover, the combination of Doxisome and anti-PD-1 was able to overcome low dose Doxisome resistance in mouse models of TNBC. The success of anticancer chemotherapy is linked to a durable immune response of targeting tumor. We present evidence that the synergistic therapeutic activity of Doxisome with anti-PD1 blockade results in enhanced T cell anti-tumor immune responses. Moreover, the synergetic effect is due to increased dendritic cells (DCs) infiltration in tumor microenvironment, which internalize tumor antigens and subsequently activate tumor-reactive T cells. In TNBC mouse model, by combining anti-PD1 checkpoint blockade while shifting the immune response towards CD4+ Th1 with Doxisome, immune tolerance to TNBC tumor can be overcome. Enhancing antigen presenting cells (APCs, e.g. DCs) infiltration by immunogenic chemotherapy and T cell activation by anti-PD1 blockade increase therapy response in TNBC. These results provide rationale for several newly planned clinical trials combining immunogenic chemotherapy with PD-1 or PD-L1 blockade in breast cancer and exploration of such a combination in others solid tumors.

Citation Format: Xiangliang Yuan, Yi Xiao, Wenling Kuo, Dihua Yu. Immunogenic chemotherapy synergize PD-1 blockade by enhancing dendritic cells infiltration in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4977.
Abstract 501: PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer
Authors: Zavridou Martha , Strati Areti , Malamos Nikos , Georgoulias Vasilis , Lianidou Evi S.
Published: July 2016

Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis.

Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors.

Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer.

Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples.

Citation Format: Martha Zavridou, Areti Strati, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 501.
Abstract 5050: The significance of the expression of E-cadherin and CD44 in patients with unresectable metastatic colorectal cancer
Authors: Iseki Yasuhito , Shibutani Masatsune , Maeda Kiyoshi , Nagahara Hisashi , Tamura Tatsuro , Ohira Go , Yamazoe Sadaaki , Sakurai Katsunobu , Kimura Kenjiro , Toyokawa Takahiro , Amano Ryosuke , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Ohira Masaichi , Hirakawa Kosei
Published: July 2016

The loss of the adhesion molecules is reported to correlate with tumor invasion and metastasis in patients with various cancers. E-cadherin is an important molecule for cell-to-cell adhesion. While, CD44 is important for cell-to-extra cellular matrix adhesion. The aim of the present study is to evaluate the significance of the expression of E-cadherin and CD44 in patients with the unresectable metastatic colorectal cancer who are undergoing palliative chemotherapy. Formalin-fixed, paraffin-embedded samples were obtained from 49 patients who underwent primary tumor resection and who were receiving palliative chemotherapy for unresectable metastatic colorectal cancer. We evaluated the expression of E-cadherin and CD44 by immunohistochemistry. The expression of E-cadherin was not significantly associated with progression-free survival (PFS) or overall survival (OS). Although, the expression of CD44 did not correlate with PFS, it tended to correlate with the OS (p = 0.0699). According to the combination of CD44 and E-cadherin, both low expression group showed a significant poor PFS (p = 0.0101) and OS (p = 0.0009). Objective response rate and disease control rate were significantly decreased in both low expression group (p = 0.0076 and p = 0.0294, respectively). A univariate analysis to determine the variables that were associated with PFS indicated that the both low expression of E-cadherin and CD44 (p = 0.0474) and gender (p = 0.0330) were significantly associated with decreased survival. A multivariate analysis showed that the both low expression of E-cadherin and CD44 was an independent risk factor for decreased PFS (hazard ratio [HR]: 8.276, 95% confidence interval [CI]: 1.383-43.311; p = 0.0227). According to OS, the both low expression of E-cadherin and CD44 expression was revealed a prognostic factor by univariate and multivariate analysis(HR:15.118, 95%CI: 2.645-77.490; p = 0.0039). This study suggested that the combined low expression of both E-cadherin and CD44 was a strong independent predictor of decreased chemotherapeutic outcome and survival in patients with unresectable metastatic colorectal cancer.

Citation Format: Yasuhito Iseki, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsuro Tamura, Go Ohira, Sadaaki Yamazoe, Katsunobu Sakurai, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. The significance of the expression of E-cadherin and CD44 in patients with unresectable metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5050.
Abstract 5058: Hitting the brakes on the migratory capacity of tumoral cells: Targeting key regulators of actin dynamics in prostate cancer
Authors: Paez Alejandra V. , Pallavicini Carla , Schuster Federico , Giudice Jimena , Valacco Pia , Labanca Estefania , Anselmino Nicolas , Ortiz Emiliano , Binaghi Maria , Cotignola Javier , Marti Marcelo , Bruno Luciana , Levi Valeria , Navone Nora , Vazquez Elba , Gueron Geraldine
Published: July 2016

Prostate Cancer (PCa) is the second leading cause of cancer in men. PCa cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize other organs. We have previously shown that heme-oxygenase 1 (HO-1) has a strong anti-tumoral effect in prostate cancer (PCa) and regulates the adhesive properties of PCa cells. Innovative high-throughput proteomic platforms are now identifying and quantifying new specific and sensitive biomarkers for PCa detection, stratification and treatment. Towards this end, we undertook an in-depth mass spectrometry-based proteomics study to build the Heme-oxygenase 1 (HO-1) interactome in PCa, in an effort to identify HO-1 molecular partners associated to the integrity of the cellular architecture and assess actin dynamics of PCa cells under HO-1 modulation.

The proteomics analysis of HO-1 interacting proteins yielded several cytoskeletal-associated proteins regulating actin filament dynamics, such as gelsolin, lasp1, SIPA1L1, testin, moesin, tropomodulin and vinculin. The bioinformatics screening across the Oncomine platform revealed that the RNA expression profiles of the cytoskeletal HO-1 interacting proteins, lie within the 10 percent of the most consistently low-expressed genes in prostate adenocarcinoma compared to normal tissue.

Motility changes were assessed on fiber-like 2D migration scenarios displaying a reduced frequency in migration events and in migration speed under hemin exposure, a specific pharmacological inducer of HO-1. A significant higher proportion of filopodia-like protrusions among neighboring cells and cellular contacts were observed when HO-1 was induced; effects reversed under HO-1 silencing. Altogether, our experimental findings demonstrate that HO-1 modulation in PCa induces the remodeling of the actin filament architecture at filopodia, alters the migratory patterns and cellular morphology, showcasing the relevance of the cytoskeletal proteins as potential therapeutic targets against the aggressive and metastatic disease.

Citation Format: Alejandra V. Paez, Carla Pallavicini, Federico Schuster, Jimena Giudice, Pia Valacco, Estefania Labanca, Nicolas Anselmino, Emiliano Ortiz, Maria Binaghi, Javier Cotignola, Marcelo Marti, Luciana Bruno, Valeria Levi, Nora Navone, Elba Vazquez, Geraldine Gueron. Hitting the brakes on the migratory capacity of tumoral cells: Targeting key regulators of actin dynamics in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5058.
Abstract 5061: Evaluating the role of durotactic migration in the tumor microenvironment
Authors: DuChez Brian J. , Yamada Kenneth M.
Published: July 2016

Durotaxis is a mechanism of directional migration in which cells respond to a stiffness gradient in their local microenvironment. While durotaxis has been characterized primarily in cells of mesenchymal origin, its role in cancer biology has not been clearly defined. At a cellular and molecular level, evidence suggests cancer cells sense and respond to the stiffening tumor microenvironment in a manner that promotes malignant and invasive characteristics. Given the gradual stiffening of tumors, it is interesting to speculate how durotaxis might contribute to cell flux to and from primary tumors and metastatic sites. We hypothesize that a durotactic mechanism may, in part, contribute to the dissemination of cancer cells into the stiffened microenvironment associated with primary tumors and also possibly with pre-metastatic sites.

To directly characterize the impact of local stiffness on directional migration, cancer cells were cultured on a hydrogel possessing a stiffness gradient, then imaged by time-lapse microscopy and tracked using custom automated tracking software. Automated cell tracking allows for unbiased, accurate, and high-speed generation of large migratory datasets not feasible with conventional manual tracking and enables more robust analysis of cell populations. Preliminary data reveal that multiple cancer cell lines respond to a stiffness gradient with directed migration towards the stiffer part of the gel. The strength of the directional migratory response to stiffness gradients was found to depend on the magnitude of stiffness a cell encounters. By manipulating stiffness gradients to accurately reflect stiffness encountered by cancer subtypes, we will better understand if a durotactic mechanism has a global or cancer-specific impact on disease progression.

Citation Format: Brian J. DuChez, Kenneth M. Yamada. Evaluating the role of durotactic migration in the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5061.