bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5641: Effects of G1T48, a novel orally bioavailable selective estrogen receptor degrader (SERD), and the CDK4/6 inhibitor, G1T38, on tumor growth in animal models of endocrine resistant breast cancer
Authors: Wardell Suzanne E. , Yllanes Alexander P. , Baker Jennifer G. , Baldi Robert M. , Krebs Taylor K. , Sorrentino Jessica , Bisi John , Strum Jay , Norris John D.
Published: July 2017

Background: The combination of targeting the CDK4/6 and ER signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here we report the preclinical characterization of G1T48, a novel, orally bioavailable, non-steroidal small molecule inhibitor of ERα, which is a potent, selective antagonist that down regulates ERα in vitro and in vivo in ER-positive models of breast cancer.

Methods: Breast cancer cells expressing clinically relevant ESR1 mutations (ER-Y537S, ER-D538G) were threated with G1T38, G1T48, and mechanistically distinct SERMs/SERDs and cellular proliferation was assessed by measuring DNA content (Hoechst dye). Ovariectomized nu/nu mice bearing xenograft tumors of clinically relevant tamoxifen (TamR) and aromatase (long term estrogen deprived) resistant ER+ breast cancer were treated with G1T38 and G1T48, alone or in combination, with clinically relevant comparators. Time to progression and tumor volume were assessed over a 4 week dosing period.

Results: G1T38 and G1T48 significantly inhibited cellular proliferation of MCF7 breast cancer cells bearing endocrine resistant ER mutations, ER-Y537S and ER-D538G. G1T48 treatment led to dramatic reductions in ER protein levels. Importantly, tumor growth inhibition was observed in mouse models of sensitive and resistant human breast cancer when G1T48 was dosed as a single agent or in combination with G1T38, a potent, selective CDK4/6 inhibitor.

Conclusions: G1T38, a novel CDK4/6 inhibitor, and G1T48, a novel SERD, either alone or in combination, demonstrated highly potent inhibition of tumor growth in animal models of tamoxifen and aromatase resistance. G1T48 also demonstrated activity in models of endocrine resistance mediated by ER mutation. G1T48 is currently completing IND enabling studies.

Citation Format: Suzanne E. Wardell, Alexander P. Yllanes, Jennifer G. Baker, Robert M. Baldi, Taylor K. Krebs, Jessica Sorrentino, John Bisi, Jay Strum, John D. Norris. Effects of G1T48, a novel orally bioavailable selective estrogen receptor degrader (SERD), and the CDK4/6 inhibitor, G1T38, on tumor growth in animal models of endocrine resistant breast 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 5641. doi:10.1158/1538-7445.AM2017-5641
Abstract 5642: Quantitative comparison of hydrocortisone and prednisone in the postoperative hormone therapy of hypercortisolism
Authors: Tang Kunlong , Huang Yuting , Dong Shangwen , Gao Peng
Published: July 2017

Tumors and several types of malignancies including lung cancer, pancreatic cancer, pituitary tumor, and most often adrenal adenoma, may cause hypercortisolism, which requires glucorticoid (GC) replacement following adrenalectomy and/or surgical resection of the tumor. Based on our previous report of a simplified GC therapy scheme and the perioperative observation, we further investigated its efficacy and safety up to 6 months post-surgery in this retrospective cohort study. All patients received no GC before surgery. The i.v. doses of hydrocortisone (HC) were 100 mg during surgery, 100 mg bid on day 0 and day 1, followed by 100 mg qd on the morning of day 2 post-surgery. Patients were grouped by oral reception of either HC or prednisone since day 2. The doses and the withdrawal schedule are shown in Table. We found in adrenal adenoma patients, the adrenocorticotropic hormone (ACTH) levels were normal, and sufficient to stimulate the recovery of the dystrophic adrenal cortex, thus exogenous supplemental ACTH might not be necessary. By 6 months post-surgery, prednisone and HC exhibited similar efficacy in correcting hypertension, hyperglycemia, and hypokalemia (p>0.05). Most patients lost weight. Both groups reported significant improvement in a subjective evaluation questionnaire. HC showed advantages over prednisone in improving liver function (p=0.035), but also caused significant leg edema (p=0.034). Both groups developed adrenal insufficiency (AI) during GC withdrawal, with no significant difference regarding the incidence rate or severity. Most AI symptoms were relieved by resuming the prior oral doses, while the severe cases were treated in hospital. No particular variable was identified as risk factor for AI. The withdrawal process may last longer time for HC than prednisone. HC may be prioritized for patients with hyperglycemia or abnormal liver function, while prednisone may reduce the incidence of leg edema.

hydrocortisoneprednisonepathology2220adrenal adenoma14/2216/20Cushing's Disease8/224/20oral glucorticoid schemedose on day 240 mg, tid10 mg, tidwithdrawal20 mg/week5 mg/weekaverage length (month)6.64.9correction of symptomshypertension10/1512/19hyperglycemia6/117/10hypokalemia12/1211/11abnormal liver panel7/82/7 *weight loss20/2217/20adrenal insufficiency10/227/20severe case1/101/7leg edema6/220/20 *

Note: This abstract was not presented at the meeting.

Citation Format: Kunlong Tang, Yuting Huang, Shangwen Dong, Peng Gao. Quantitative comparison of hydrocortisone and prednisone in the postoperative hormone therapy of hypercortisolism [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 5642. doi:10.1158/1538-7445.AM2017-5642
Abstract 5643: Physical and functional interactions of exon-skipping variants of androgen receptor with the full-length counterpart
Authors: Uo Takuma , Sprenger Cynthia C. , Sun Shihua , Bradley Robert K. , Nelson Peter S. , Plymate Stephen R.
Published: July 2017

The androgen receptor (AR) is arguably the critical driver of not only early but also advanced prostate cancer. The recent comprehensive splicing landscapes revealed that metastatic castration resistant prostate cancer harbors multiple forms of AR variants (AR-Vs), many of which have diverse patterns of inclusion/exclusion of exons (E4-8) corresponding to the ligand-binding domain. We had used AR-negative M12 prostate cancer cell line and recently reported characteristics of AR-Vs with respect to their subcellular localization and transcriptional activation on luciferase reporter driven by probasin-based promoter (AR2PB-luc)(1). While ARv5es is predominantly nuclear and constitutively active like well-documented ARv567es, many others are castration -induced variants are inactive. As many AR-Vs and the full-length AR (AR-FL) are co-expressed in the same cell, it is important to determine how these inactive AR-Vs interact with active AR including AR-FL. To this end, we examined two inactive AR-Vs namely v7es and v56es in relation to AR-FL. ARv7es, which skips exon 7, is predominantly localized in cytoplasm. ARv7es physically interacted with AR-FL. Moreover, nuclear accumulation of ARv7es was promoted by the addition of dihydrotestosterone (DHT) only in the presence of AR-FL, suggesting that ARv7es and AR-FL heterodimerize in the nuclear compartment. However, DHT-dependent transactivation of AR-FL was not affected by the presence of ARv7es. More specifically, in M12 cells, AR2PB-luc was transactivated by AR-FL to the similar extents either in the presence or absence of ARv7es. In addition, in doxycycline (Dox)-inducible ARv7es stable LNCaP cell line (LNCaP/ARv7es), the presence of ARv7es did not affect DHT-induced expression of endogenous PSA and NKX3.1 transcripts as well as transactivation of AR2PB-luc. In the similar setting, when we examined ARv56es which also physically interacted with AR-FL, Dox-induced expression of ARv56es in LNCaP/ARv56es was sufficient to induce expression of endogenous PSA and NKX3.1 transcripts. Further, DHT-induced expression of these transcripts was enhanced by ARv56es in LNCaP/ARv56es. Accordingly, we propose that AR-Vs display differential modes of action on AR-FL as exemplified by ARv7es as the bystander and v56es as the effector. 1. Oncogene (PMID: 27694897)

Citation Format: Takuma Uo, Cynthia C. Sprenger, Shihua Sun, Robert K. Bradley, Peter S. Nelson, Stephen R. Plymate. Physical and functional interactions of exon-skipping variants of androgen receptor with the full-length counterpart [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 5643. doi:10.1158/1538-7445.AM2017-5643
Abstract 5646: Germline activating mutations in the proto-oncogene GCM2 in primary hyperparathyroidism
Authors: Guan Bin , Welch James M. , Sapp Julie C. , Ling Hua , Vemulapalli Meghana , Li Yulong , Johnston Jennifer J. , Kebebew Electron , Biesecker Leslie G. , Simonds William F. , Marx Stephen J. , Agarwal Sunita K.
Published: July 2017

Primary hyperparathyroidism (PHPT) is a common endocrine disease caused by parathyroid tumors (adenoma or hyperplasia). The hypersecreting parathyroid tumor elevates serum parathyroid hormone level, which causes hypercalcemia and the classical symptoms of PHPT, including kidney stones, osteoporosis, and neuromuscular symptoms associated with hypercalcemia, such as muscle weakness, drowsiness, and depression. Familial PHPT occurs in an isolated nonsyndromal form, termed familial isolated hyperparathyroidism (FIHP), or as part of a syndrome, such as multiple endocrine neoplasia type 1 or hyperparathyroidism-jaw tumor syndrome. We performed exome sequencing on germline DNA of eight index cases from eight unrelated kindreds with FIHP. Rare variants were identified (minor allele frequency <1%), and selected variants were assessed for co-segregation in affected family members by Sanger sequencing. Candidate genes were then screened in an additional 32 kindreds with FIHP. The functional consequence of variants in one FIHP candidate gene (GCM2, a parathyroid tissue-specific transcription factor) was analyzed in a cell culture model. In eight kindreds with FIHP, we identified three rare GCM2 missense variants. Functional characterization of GCM2 variants and deletion-analyses revealed a small C-terminal Conserved Inhibitory Domain (CCID). Two of the three rare variants were recurrent, located in the CCID that enhanced the transcriptional activity of GCM2, and found in seven of 40 (18%) kindreds with FIHP. Functional assays for transcriptional activity demonstrated that these two rare variants acted as gain-of-function mutations, suggesting that GCM2 is a parathyroid proto-oncogene. We further investigated the ethnicity of individuals with GCM2 CCID activating variants in PHPT patient samples and in genome survey datasets. Our results demonstrate that germline activating mutations in the GCM2 CCID can cause FIHP, and specific activating mutations expose a large population at risk for PHPT. Our findings of the first gene with mutations specific for FIHP are expected to have an important impact on clinical practice, including treatment decisions and genetic testing of individuals with PHPT and family members.

Citation Format: Bin Guan, James M. Welch, Julie C. Sapp, Hua Ling, Meghana Vemulapalli, Yulong Li, Jennifer J. Johnston, Electron Kebebew, Leslie G. Biesecker, William F. Simonds, Stephen J. Marx, Sunita K. Agarwal. Germline activating mutations in the proto-oncogene GCM2 in primary hyperparathyroidism [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 5646. doi:10.1158/1538-7445.AM2017-5646
Abstract 5650: Enhancement of CAR-T cell activity via PD-1 knockdown by self-deliverable RNAi
Authors: Wolfson Alexey , Eliseev Alexey , Shmushkovich Taisia , Betancur-Boissel Monica , Betancur-Boissel Monica , Scholler Nathalie
Published: July 2017

Background: A growing body of evidence indicates that the blockade of PD-1 and other immunosuppressive receptors can increase the efficacy of adoptively transferred cells in immunotherapy for cancer. The expression of PD-1 can be suppressed by ex vivo treatment of transferred lymphocytes by self-deliverable RNAi (sd-rxRNA®) compounds. sd-rxRNAs are based on proprietary chemical modifications of siRNA compounds making them stable to nuclease degradation and capable of penetration into target cells without the use of transfection reagents or physical methods.

Aim: To determine whether the reduction of PD-1 by ex vivo treatment of mesothelin-targeting CAR-T cells with sd-rxRNA will improve the efficacy of meso-CAR-T cells in a mouse model of ovarian cancer.

Methods: Second-generation human CAR-T cells expressing P4 antibody fragments targeting mesothelin (meso-CAR-T) were treated ex vivo with PD-78, a self-deliverable siRNA silencing the PD-1 gene. The meso-CAR-T cells were then injected into tumors in a xenograft mouse model of human ovarian cancer (A1847 cell line expressing mesothelin and PD-L1) in NSG mice. The tumor volumes were monitored over the course of one month. After the animals were sacrificed, human CD45-containing cells extracted from the tumor were analyzed for PD-1 expression by flow cytometry.

Results: We observed a statistically significant reduction in tumor growth in the group of mice injected with meso-CAR-T cells pretreated ex vivo with PD-78. The post-mortem tumor analysis by flow cytometry showed that the expression of PD-1 in meso-CAR-T cells was significantly suppressed for the full duration of the experiment (one month).

Conclusion: Our results suggest that the silencing of immunosuppressive genes by ex vivo treatment with sd-rxRNA may improve efficacy of CAR-T cells in the treatment of solid tumors. A similar approach may be applied to other types of adoptive cell transfer.

Citation Format: Alexey Wolfson, Alexey Eliseev, Taisia Shmushkovich, Monica Betancur-Boissel, Monica Betancur-Boissel, Nathalie Scholler. Enhancement of CAR-T cell activity via PD-1 knockdown by self-deliverable RNAi [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 5650. doi:10.1158/1538-7445.AM2017-5650
Abstract 5651: A LAG-3/PD-L1 bispecific antibody inhibits tumor growth in two syngeneic colon carcinoma models
Authors: Kraman Matthew , Kmiecik Katarzyna , Zimarino Carlo , Everett Katy , Faroudi Mustapha , Wydro Mateusz , Doody Jacqueline
Published: July 2017

Combining immunotherapeutic antibodies in cancer treatment has shown benefits over single agents. An alternative to combining two antibodies is the development of bispecific antibodies that not only bring two biologies together but may result in novel mechanisms of action that are impossible to attain with combinations. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily expressed on activated T cells, NK cells, pDCs, B cells, γδ T cells and participates in immune suppression. Programmed Cell Death receptor (PD-1) binds to its ligand PD-L1, expressed not only on activated immune cells to inhibit cellular immune responses but also on tumor cells. Expression of both these surface molecules therefore leads to T cell exhaustion allowing the tumor to escape immune surveillance. A mAb² TM (bispecific antibody) was engineered which binds murine LAG-3 and PD-L1 simultaneously and with nanomolar affinities. The anti-LAG-3/PD-L1 mAb² inhibits LAG-3 binding to MHCII and PD-L1 binding to PD-1 and CD80, resulting in T cell activation in vitro. This translates into in vivo efficacy, where the mAb² decreased tumor burden in the MC38 colon carcinoma tumor model. At the end of the study tumor-free animals were more numerous in the LAG-3/PD-L1 bispecific group than in the group given a combination of individual anti-LAG-3 and PD-L1 antibodies. The results were recapitulated in the CT26 murine colon cancer model, where the mAb² showed an increase of antitumor activity as compared to the antibodies given in combination. Thus, the preclinical data supports developing an anti-human LAG-3/PD-L1 mAb² for the treatment of cancer patients.

Citation Format: Matthew Kraman, Katarzyna Kmiecik, Carlo Zimarino, Katy Everett, Mustapha Faroudi, Mateusz Wydro, Jacqueline Doody. A LAG-3/PD-L1 bispecific antibody inhibits tumor growth in two syngeneic colon carcinoma models [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 5651. doi:10.1158/1538-7445.AM2017-5651
Abstract 5654: Lymphodepletion induces T cell homeostatic proliferation and augments antitumor effects of PD-1/PD-L1 blockade therapy
Authors: Arita Masashi , Watanabe Satoshi , Miho Takahashi , Sato Miyuki , Ohtsubo Aya , Ichikawa Kosuke , Kondo Rie , Abe Tetsuya , Tanaka Junta , Koya Toshiyuki , Kikuchi Toshiaki
Published: July 2017

Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has demonstrated to enhance antitumor immunity. Recent evidence has shown that anti-PD-1/PD-L1 therapy restores the function of exhausted effector T cells and augments antitumor immune responses. The expression of PD-L1 has been best studied as a biomarker for the anti-PD-1/PD-L1 therapy. Because PD-L1 expression on tumor cells is induced by IFN-γ which is mainly produced by effector T cells, induction of antitumor effector T cells in cancer patients could be an important for the anti-PD-1/PD-L1 therapy. Indeed, previous studies have suggested that the increase of tumor-infiltrating lymphocytes is the predictive biomarker for PD-1/PD-L1 blockade therapy. We and others have demonstrated that lymphodepletive therapies, such as chemotherapy and radiotherapy, induce tumor-specific effector T cells from naïve T cells. Naïve T cells rapidly proliferate and elicit memory-like functions after lymphodepletion (homeostatic proliferation). These findings indicate that lymphodepletive regimens could augment the antitumor efficacies of the anti-PD-1/PD-L1 therapy. In the current study, we transferred naïve T cells into mice after whole-body irradiation or chemotherapy. These mice were inoculated s.c. with PD-L1+ B16F10 melanoma cells or PD-L1- MCA205 fibrosarcoma and then were treated with anti-PD-1 mAbs. Lymphodepletion by whole body irradiation, but not cytotoxic agents following the transfer of naïve T cells significantly enhanced antitumor immunity of anti-PD-1 mAbs. This augmentation required both of CD4+ and CD8+ T cells, but not NK cells. Further experiments revealed that immune cells infiltrating MCA205 expressed PD-L1. Transfer of naïve T cells from IFN-γ knockout mice abrogated the enhancement of antitumor effects of anti-PD-1 mAb therapy by lymphodepletion. These results suggest that lymphodepletion induces homeostatic proliferation of T cells and augments antitumor effects of PD-1/PD-L1 blockade therapy.

Citation Format: Masashi Arita, Satoshi Watanabe, Takahashi Miho, Miyuki Sato, Aya Ohtsubo, Kosuke Ichikawa, Rie Kondo, Tetsuya Abe, Junta Tanaka, Toshiyuki Koya, Toshiaki Kikuchi. Lymphodepletion induces T cell homeostatic proliferation and augments antitumor effects of PD-1/PD-L1 blockade therapy [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 5654. doi:10.1158/1538-7445.AM2017-5654
Abstract 566: A pathway based drug selection for cancer precision medicine
Authors: Vasudevaraja Varshini , Cheng Lijun , Inavolu Sai Mounika , Radovich Milan
Published: July 2017

Background and Purpose:

Precision medicine tailors the right therapy to the right patient based on his/her tumor molecular profiles. In clinic and ongoing clinical trials, drug-targeted selection schemes depends more or less on drug targets’ mutations or expression status. However, there is a lack of systematic pathway based method to connect individual genomics information with properly drug target and treatment in precision medicine. The paper aims to integrate various knowledge-bases, including cancer drugs, drug-targets and gene-gene regulatory pathway, to construct a drug target and properly drug selection algorithm for individual patient in precision medicine.

Materials and Methods:

In the drug selection algorithm, drug targets and properly drugs recommendation are based on the patient specific molecular profile data, including copy number variation, mutation, and gene expression. Our target and drug selections are based on whether these drug targets act as hub genes that either regulate or control the signaling pathways for many other genes in the biological pathways. Only those patients whose molecular profiles do not show targets, the usual chemotherapy treatment will be recommended. This novel algorithm is applied to individual Pancreatic Adenocarcinoma (PAAD) for drug target and drug selection. All of patients’ genomic data obtained from the Cancer Genome Atlas (TCGA). The pathway information is from Pathway Commons and FDA approved cancer drugs and their targets is from DrugBank. The Cancer Cell Line Encyclopedia (CCLE) 46 pancreases cancer cell line are used to validate the algorithm result reliability.

Results:

Our algorithm identifies targets, such as ERBB2, CDK2, SRC, CDK9, SMAD2, CDK4, HDAC1, PPP1CA, AKR1B1, EGFR, IGF1R, AKT and MEK, for pancreatic adenocarcinoma cancer patients. In which, they cover the clinic first line effective drug targets, such as Gemcitabine for the Akt-mTOR signaling pathway (AKT) and CDK4/6 inhibitor and Erlotinib for HER2 Kinase Family (ERBB2, EGFR) activation inhibitor. In addition, new inhibitors include Src inhibitors (dasatinib, saracatinib and bosutinib), TGF beta inhibitor for target SMAD2 (galunisertib) and IGF-1R/insulin receptor inhibitors (ceritinib, brigatinib) are recommended accordingly and validated in pancreatic cancer cells.

Conclusion:

This novel algorithm might act as a better source for off-label drug selection and further cell line validations may help in providing a better treatment strategy in precision medicine.

Note: This abstract was not presented at the meeting.

Citation Format: Varshini Vasudevaraja, Lijun Cheng, Sai Mounika Inavolu, Milan Radovich. A pathway based drug selection for cancer precision medicine [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 566. doi:10.1158/1538-7445.AM2017-566
Abstract 5668: CCL2/CCR2-driven monocyte recruitment to the tumor following radiotherapy influences the outcome of the treatment of head and neck cancer
Authors: Mondini Michele , Loyher Pierre L. , Berthelot Kevin , Clémenson Céline , Boissonnas Alexandre , Deutsch Eric
Published: July 2017

Local radiotherapy is the major treatment to control locally advanced head and neck cancer. Evidence supporting the importance of immune response in the response to radiation therapy is growing and the possibility to potentiate the effects of radiotherapy by modulating the function of the immune system offers new exciting therapeutic perspectives. Preclinical findings suggest that immunomodulatory agents may synergize with radiotherapy, increasing local response rates and inducing sustained systemic responses. The role of monocytes/macrophages in the response to radiotherapy is under extensive investigation. Among the many cytokines secreted by tumor cells, CCL2 is involved in inflammatory monocyte recruitment from bone marrow to the inflammatory site. We have recently developed a model of human papillomavirus (HPV)-related head and neck cancer using immunocompetent mice and validated its usefulness to evaluate the tumor response to radiotherapy. Using the same model, based on the injection of TC1/luciferase cells in the inner lip of C57Bl/6 mice, we show that local radiotherapy induces an increase of CCL2 levels in the bloodstream and intratumorally accompanied by a neo-infiltration of monocytes in the tumor that subsequently differentiate into tumor-associated macrophages (TAM). When TC1/Luc cells were engrafted in CCL2-/- or CCR2-/- mice, the tumor response to radiotherapy and survival were improved. This was accompanied by an impaired infiltration of inflammatory monocytes, and a subsequent decrease of TAM levels at later time points. In vitro, exposure to ionizing radiation increased the secretion of CCL2 by TC1/Luc cells, and such increase was significantly enhanced when tumor cells were co-cultured with irradiated RAW 264.7 macrophage-derived cells. This data suggests interplay between tumor and TAM that can amplify the effects of radiotherapy on the immune system. We propose that reducing radiation-induced monocyte recruitment may yield improved results in the treatment of HPV-related head and neck squamous cell carcinomas by radiotherapy. Immunologically-augmented radiotherapy could allow reduction of the delivered radiation dose, thus minimizing the risk of treatment sequelae while maintaining optimal tumor control.

Citation Format: Michele Mondini, Pierre L. Loyher, Kevin Berthelot, Céline Clémenson, Alexandre Boissonnas, Eric Deutsch. CCL2/CCR2-driven monocyte recruitment to the tumor following radiotherapy influences the outcome of the treatment of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5668. doi:10.1158/1538-7445.AM2017-5668
Abstract 5669: Targeting post-surgical breast cancer metastasis through combining natural killer T cell immunotherapy and low dose chemotherapy
Authors: Gebremeskel Simon , Tanner Kaitlyn , Lobert Lynnea , Johnston Brent
Published: July 2017

Breast cancer is the most common cancer in Canadian women and the second leading cause of cancer deaths. Given that most mammary tumors are surgically resectable and over 90% of breast cancer-associated deaths are due to metastasis, new therapeutic strategies targeting metastasis are required. Natural killer T (NKT) cells are a rare population of immune cells that have been shown to limit primary tumor growth and target distant metastatic disease in various animal models. We have shown that NKT cell activation improves survival in a model of post-surgical metastatic breast cancer. We are now expanding this work to determine whether NKT cell activation can be combined with chemotherapies to improve outcomes. In our model, 4T1 mammary carcinoma cells were injected into the mammary fatpad of syngeneic BALB/c mice. Tumors were resected at day 12, and mice were treated with cyclophosphamide or gemcitabine. On day 17, NKT cells were activated by transfer of dendritic cells loaded with the glycolipid antigen α-GalCer. We also examined whether gemcitabine or mafosphamide (active component of cyclophosphamide) would induce immunogenic cell death of 4T1 cells in culture. Chemotherapeutics did not affect NKT cell activation as measured by serum IFNγ levels. Treatment with cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cells alone reduced metastasis and prolonged survival. Combined treatments significantly enhanced survival. NKT cell activation decreased the frequency and immunosuppressive function of myeloid derived suppressor cells (MDSCs). Treatments resulted in enhanced tumor specific immunity as surviving mice exhibited slower tumor growth following secondary tumor challenge. Gemcitabine and mafosphamide also increase the immunogenicity of cancer cells in vitro by increasing the exposure/release of MHCI, MHCII, CD1d, Calreticulin, HMGB1 and ATP. NKT cell activation therapy can successfully be combined with low doses of gemcitabine or cyclophosphamide to enhance protection against tumor metastasis and recurrence. This work provides a clear rationale for combining chemotherapy with NKT cell immunotherapy to target metastatic disease in the clinical setting.

Citation Format: Simon Gebremeskel, Kaitlyn Tanner, Lynnea Lobert, Brent Johnston. Targeting post-surgical breast cancer metastasis through combining natural killer T cell immunotherapy and low dose chemotherapy [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 5669. doi:10.1158/1538-7445.AM2017-5669
Abstract 5677: Performance of a robust and sensitive liquid biopsy workflow for mutation detection in plasma
Authors: Choi Jonathan , Choi Cindy , Dinis Jorge , Li Jingchuan , Chien Sean , Min Bosun , Munoz Abraham , Jin Janet , Pati Amrita , Feng Liang , Wu Johnny , Singh Ashla , Casey Fergal
Published: July 2017

The discovery of circulating tumor DNA (ctDNA) and the development of highly sensitive technologies have made the utility of liquid biopsies in clinical practice more feasible. Potential applications for liquid biopsies include early cancer detection, tumor genotyping in the absence of tissue biopsies, disease monitoring, and identification of drug resistance markers. However, there is a multitude of technical challenges to accurately detect ctDNA with high sensitivity and reproducibility. Various methods have been established by research centers and companies, each with its advantages and limitations. We have developed a sensitive NGS-based liquid biopsy assay, called the AVENIO ctDNA Analysis Kits (research use only), which includes three panels targeting biomarkers for solid tumors. The AVENIO ctDNA Analysis Kits include cfDNA extraction from up to 5ml plasma input, optimized reagents and workflow for NGS library preparation and target enrichment. It is accompanied with an intuitive software and data analysis package to easily obtain results. The assay sensitively detects 4 cancer relevant mutation classes - single nucleotide variations (SNVs), insertions/deletions, copy number variations and structural rearrangements.

We conducted a study with 200+ samples of plasma-derived cfDNA, cell line blends containing relevant cancer mutations, and clinical samples, with which we demonstrate the analytical sensitivity of the three distinct AVENIO gene panels (covering 17, 77 and 197 genes). We demonstrate >95% sensitivity for SNVs at 0.5% mutant allele frequency; >95% sensitivity for 1% indels; >95% sensitivity for 1% structural variants. We also demonstrate assay robustness through multiple replicates, day-to-day, and operator-to-operator reproducibility.

In conclusion, the AVENIO ctDNA Analysis Kits for solid tumors encompasses a comprehensive assay and analysis workflow aimed to provide researchers with robust, reliable, highly sensitive and ease to use liquid biopsy assays to conduct the necessary studies to demonstrate the utility of circulating biomarkers in routine clinical testing.

Citation Format: Jonathan Choi, Cindy Choi, Jorge Dinis, Jingchuan Li, Sean Chien, Bosun Min, Abraham Munoz, Janet Jin, Amrita Pati, Liang Feng, Johnny Wu, Ashla Singh, Fergal Casey. Performance of a robust and sensitive liquid biopsy workflow for mutation detection in plasma [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 5677. doi:10.1158/1538-7445.AM2017-5677
Abstract 5683: Evaluation of the utilization of blood collection tubes for cell-free DNA research
Authors: Endo Fumitaka , Iwaya Takeshi , Chiba Takehiro , Yaegashi Mizunori , Sato Kei , Kume Kohei , Arisue Atsuhiro , Nishinari Yutaka , Kawagishi Ryoko , Segawa Takenori , Nishizuka Satoshi , Sasaki Akira
Published: July 2017

Many studies have been conducted investigating cell-free DNA (cfDNA) for disease diagnosis. To prevent cellular DNA release into the plasma, plasma separation within 2 hours of blood collection is required, when utilizing the most commonly used type of tube (BD Vacutainer® CPTTM; CPT). These temporal restrictions render operations requiring plasma separation a laborious task. It has been demonstrated that Cell-Free DNA BCT® tubes (Streck Inc; BCTs) prevented nucleated cell disruption. The use of BCTs may enable us to separate plasma without temporal restrictions. The aim of this study was to evaluate changes in DNA levels in plasma after preservation using two types of collection tubes. Samples were drawn from 7 healthy donors into CPTs and BCTs, and stored at room temperature. Plasma was separated immediately (d0), and on days 3, 6, 9, 12, and 14 after blood collection. The concentrations of DNA in the extracted plasma samples were measured. Blood stored in CPTs showed increases in plasma DNA concentrations over time. The mean DNA concentrations at each time-point were 0.39 (d0), 2.96 (d3), 40.4 (d6), 84.9 (d9), 91.4 (d12), and 94.9 ng/μl (d14). However, plasma DNA levels remained stable in BCTs from 6 days. There were significant differences between the DNA concentrations in BCTs on d0 (0.74 [0.43-1.44] ng/µl) and d14 (1.78 [0.63-4.20] ng/µl) (p = 0.0378). Although significant differences were only observed between d0 and d14 in BCTs, the amount of plasma DNA gradually increased after d6. Furthermore, obvious color changes in the plasma were observed, and the boundary between plasma and PBMCs became unclear over time. These results indicate that BCTs may yield stable cfDNA samples for up to 6 days after blood collection. BCTs may be a novel collection tube for cfDNA research, including liquid biopsies in cancer patients.

Citation Format: Fumitaka Endo, Takeshi Iwaya, Takehiro Chiba, Mizunori Yaegashi, Kei Sato, Kohei Kume, Atsuhiro Arisue, Yutaka Nishinari, Ryoko Kawagishi, Takenori Segawa, Satoshi Nishizuka, Akira Sasaki. Evaluation of the utilization of blood collection tubes for cell-free DNA research [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 5683. doi:10.1158/1538-7445.AM2017-5683
Abstract 569: Dexamethasone inhibits T-cell proliferation through a CTLA-4 mediated pathway
Authors: Hutchinson Marsha-Kay N. , Giles Amber J. , Sonnemann Heather M. , Reid Caitlin M. , Park Deric M. , Gilbert Mark
Published: July 2017

BACKGROUND: The use of corticosteroids for therapeutic benefit has to be weighed against the risks of adverse consequences associated with these drugs. Brain tumor patients in particular, are routinely prescribed dexamethasone (a glucocorticoid) to reduce edema associated with their lesion. Checkpoint blockade, a type of immune therapy, is currently being investigated as a potential treatment for brain tumors. However, glucocorticoid signaling has been shown to attenuate the immune response through several mechanisms including the repression of transcription of genes controlling pro-inflammatory cytokines and chemokines.

HYPOTHESIS: Here, we propose that dexamethasone’s ability to upregulate inhibitory T-cell molecules such as CTLA-4 and PD-1 might be an additional immunosuppressive mechanism.

METHODS: Healthy donor T cells were tested for response to dexamethasone. T cell proliferation, cell cycle analysis, apoptosis, and protein expression were assessed with flow cytometry. Protein expression was also measured with Western blots. Transcriptional changes were assessed with qPCR. A monoclonal antibody, ipilimumab, was used to block CTLA-4 binding.

RESULTS: Unexpectedly, dexamethasone did not elicit a direct lymphotoxic effect on T cells as measured by absolute cell number. However, we found that dexamethasone significantly reduced T cell entry into the cell cycle, but did not impact cells already undergoing mitosis. Checkpoint molecules CTLA-4 and PD-1 were increased with dexamethasone treatment when cells are stimulated. Blockade of CTLA-4 with Ipilimumab resulted in a substantial reversal of cell cycle entry inhibition that was induced by dexamethasone.

CONCLUSIONS: These results suggest that dexamethasone impairs T cell expansion by inhibiting cell cycle entry. Upregulated CTLA-4 expression contributes to cell cycle entry blockade which is reversed by inhibiting CTLA-4 with ipilimumab. These findings indicate that administration of ipilimumab before dexamethasone diminishes the negative proliferative effect on anti-tumor T cells suggesting that when needed, corticosteroids can be used after immune checkpoint blockade has been established.

Citation Format: Marsha-Kay N. Hutchinson, Amber J. Giles, Heather M. Sonnemann, Caitlin M. Reid, Deric M. Park, Mark Gilbert. Dexamethasone inhibits T-cell proliferation through a CTLA-4 mediated pathway [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 569. doi:10.1158/1538-7445.AM2017-569
Abstract 5690: Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA)
Authors: Artieri Carlo G. , Beauchamp Kyle A. , Vysotskaia Valentina S. , Welker Noah C. , Evans Eric A. , Chu Clement , Tezcan Haluk , Haque Imran S.
Published: July 2017

The evaluation of cfDNA allows novel approaches to noninvasive detection of actionable alterations, resistance mechanisms, and tumor monitoring in patients with cancer. Importantly, tumor-specific DNA fragments represent a small minority of the cfDNA and can be obscured by false positive (FP) variants introduced by chemical damage and sequencer error. To address this, we improved key processes in the design of NGS libraries, including a new molecular barcoding approach, that maximize molecular recovery while eliminating spurious variants. We engineered a set of Illumina sequencing chemistry compatible adaptors incorporating unique molecular identifiers (barcodes) enabling reconstruction of the sequence of both strands of the original DNA molecule. These barcodes incorporate a number of key design improvements as compared to published methodologies, which enhance sequencer cluster density, thereby increasing library diversity and molecular recovery. Our new design identified both chemical and sequencer errors, reducing incorrect base calls to rates below 5e-7. We validated our methodology for use in cfDNA using both dilution experiments and patient blood samples with known oncogenic alterations via a custom capture panel targeting actionable genomic alterations in a 55kb region. By identifying the molecular origin of each read, we found that the sensitivity of detection obtained from barcoded libraries followed ideal binomial sampling expectations. We obtained an average molecular depth of 1,000 molecules per site from the plasma extracted from a single blood collection tube, which corresponded to an 80% sensitivity of detection of known oncogenic single-nucleotide and indel mutations at 0.15% mutant allele frequency (MAF) in cfDNA with no FP calls. Furthermore, we successfully detected known gene-fusions at 0.5%, and amplifications (>10 copies) down to 1% MAF. We designed and validated a custom-engineered error-correcting sequencing adapters, ideal for broad range of applications requiring high accuracy detection of ultra-low frequency alterations.

Note: This abstract was not presented at the meeting.

Citation Format: Carlo G. Artieri, Kyle A. Beauchamp, Valentina S. Vysotskaia, Noah C. Welker, Eric A. Evans, Clement Chu, Haluk Tezcan, Imran S. Haque. Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA) [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 5690. doi:10.1158/1538-7445.AM2017-5690
Abstract 5697: Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads
Authors: Mizutani Kosuke , Kawakami Kyojiro , Fujita Yasunori , Horie Kengo , Kameyama Koji , Ito Masafumi , Deguchi Takashi
Published: July 2017

Purposes of the study: Exosome has been demonstrated to be a useful non-invasive biomarker for several cancers including prostate cancer. For better understanding cancer-derived exosome, their proteomic analysis is necessary. The aim of this study is to establish the method for isolation and analysis of prostate cancer-related exosomes. Experimental procedures: Total exosomes were isolated from the conditioned media of LNCaP cells by ultracentrifugation. Then, exosomes positive for CD9 or prostate specific membrane antigen (PSMA) were isolated by magnetic beads conjugated with anti-CD9 or -PSMA antibody, respectively. Isolated exosomes were subjected to proteomic analysis. The PSMA positive fraction was also collected by anti-PSMA beads from diluted serum of 3 prostate cancer patients and analyzed by proteomics. Results: A total of 126, 139 and 13 proteins were detected in the LNCaP exosome fractions that were isolated by ultracentrifugation, anti-CD9 beads and anti-PSMA beads, respectively. Out of 126 proteins identified in ultracentrifuge isolated exosome, 56 and 6 protein were found in exosomes that were isolated by anti-CD9 beads and anti-PSMA beads, respectively. Seven proteins were commonly detected in exosomes that were isolated by both beads. Fifty, 54 and 48 proteins were identified in the PSMA positive fraction from serum of 3 prostate cancer patients (P1: localized prostate cancer, P2: advanced prostate cancer and P3: castration resistant prostate cancer). Thirty-three proteins were detected in one patient, 22 in two patients and 25 in three patients. Most proteins detected in all patients were serum- or immunoglobulin-related proteins. The numbers of proteins that were detected only in P2 and P3 were 16 and 8, respectively. Six proteins were found in both P2 and P3 patients. Conclusion: In the present study, we analyzed prostate cancer-related exosomes that were isolated by immunoaffinity-based method. Although most proteins were serum- or immunoglobulin-related proteins, proteins that had been reported as a cancer marker were also detected, which could be candidates for exosomal marker of prostate cancer.

Citation Format: Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Kengo Horie, Koji Kameyama, Masafumi Ito, Takashi Deguchi. Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads [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 5697. doi:10.1158/1538-7445.AM2017-5697
Abstract 5698: Next generation sequencing of circulating-free DNA from advanced non small cell lung cancer patients using Gene Reader®
Authors: Casas Clara Mayo de las , Garzón Mónica , Ariza Nuria Jordana , Balada Ariadna , Bertran-Alamillo Jordi , García Beatriz , Villatoro Sergio , Aldeguer Erika , Rodriguez Sonia , Campos Raquel , Ramirez Santiago Viteri , Gonzalez-Cao Maria , Karachaliou Niki , Costa Rafael Rosell , Molina-Vila Miguel Angel
Published: July 2017

Background: Stand alone tests such as PCR-derived techniques, FISH or IHC are usually employed to determine clinically relevant alterations in non-small cell lung cancer (NSCLC). However, they target single genes and proteins. Mutiplex techniques can reduce the turnaround time and quantity of sample in this setting, but require a careful validation.

Methods: A total of 41 cfDNA samples from serum and plasma from advanced NSCLC p were analyzed with the Actionable Insights Tumor Panel, which covers mutations in 15 clinically relevant genes, using the Gene Reader platform (Qiagen). The samples had been previously genotyped for EGFR, KRAS and BRAF mutations by stand alone, PNA-Taqman assays. Paired biopsies were available in 37 cases. The remaining 4 corresponded to p.T790M-positive blood samples of p progressing to EGFR TKIs.

Results: Of the 41 samples taken into the GeneReader workflow, some had a DNA input concentration below specifications, in spite of this limitation, good results were obtained. 14 mutations were fully concordant between tissue, Taqman and GeneReader and the four p.T790M mutations were concordant between Taqman and GeneReader. Five mutations present in tissue were detected by GeneReader and not by Taqman and 11 mutations detected by Taqman were below the 1% detection threshold of GeneReader. Finally, 12 mutations present in tissue were not detected in cfDNA by any of the assays. Concordance between the stand alone tests and the Gene Reader in cfDNA was 64%, raising to 84% if mutations <1% allelic fraction were excluded.

Conclusions: Application of NGS to liquid biopsies is challenging and requires a careful validation. However, once fully validated, NGS will probably become the methodology of choice for cfDNA analysis in NSCLC patients at presentation and at progression.

Citation Format: Clara Mayo de las Casas, Mónica Garzón, Nuria Jordana Ariza, Ariadna Balada, Jordi Bertran-Alamillo, Beatriz García, Sergio Villatoro, Erika Aldeguer, Sonia Rodriguez, Raquel Campos, Santiago Viteri Ramirez, Maria Gonzalez-Cao, Niki Karachaliou, Rafael Rosell Costa, Miguel Angel Molina-Vila. Next generation sequencing of circulating-free DNA from advanced non small cell lung cancer patients using Gene Reader® [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 5698. doi:10.1158/1538-7445.AM2017-5698