bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2301: Intraperitoneal CAR-T infusion results in durable protection against peritoneal metastases and control of systemic tumor growth
Authors: Cunetta Marissa , Point Gary , Liu Zhi , Guha Prajna , Darpolor Josephine , Lima Matthew , Espat N J. , Hanna Nader , Boutros Cherif , Junghans Richard P. , Katz Steven
Published: July 2016

Colorectal carcinomatosis due to peritoneal metastases (PM) is a significant cause of morbidity and mortality. Present treatments fail to cure the majority of patients with PM. We are developing a chimeric antigen receptor T cell (CAR-T) regional infusion immunotherapy platform for PM. We treated mice with established CEA+ MC38 PMs with anti-CEA CAR-Ts either via regional intraperitoneal (IP) or systemic tail vein (TV) infusion. When compared to TV infusion, IP delivery resulted in significantly improved responses (37-fold reduction in tumor growth, p<0.01). To measure the durability of protection from IP tumor growth, we re-challenged mice with IP tumor following successful treatment with IP CAR-Ts. Mice who had received initial IP CAR-T treatment were protected from tumor growth following re-challenge for 20 days compared to control animals (1390-fold reduction in tumor growth, p = 0.016). Assessment of IP tumor 10 and 28 days following tumor re-challenge revealed a high proportion of intratumoral CAR-Ts (Day 10- 70.0%, Day 28- 46.6%; p = 0.10) and the proportion of CAR-Ts with an effector memory phenotype (CD44+CD62L-CCR7-, Day 10- 19.3%, Day 28- 60.0%; p<0.01). In separate experiments, we studied the effect of IP CAR-T infusions on tumor growth at distant sites. We established both IP and subcutaneous (SQ) flank tumors in mice prior to CAR-T infusions. Following treatment, IP CAR-T infusion resulted in a significant decrease in SQ tumor growth on day 8 compared to control (5.7-fold, p = 0.03), and a trend toward improvement compared to TV (4.9-fold, p = 0.224). While CAR-Ts were not detected in whole blood, SQ tumor, or draining lymph nodes following IP CAR-T infusion, serum IFNγ levels were significantly elevated on day 4 compared to control mice (p = 0.04), and returned to baseline by day 7. Our findings demonstrate that regional IP CAR-T delivery provides durable protection against IP tumor growth in association with CAR-T effector memory programming, in addition to a systemic anti-tumor effect.

Citation Format: Marissa Cunetta, Gary Point, Zhi Liu, Prajna Guha, Josephine Darpolor, Matthew Lima, N J. Espat, Nader Hanna, Cherif Boutros, Richard P. Junghans, Steven Katz. Intraperitoneal CAR-T infusion results in durable protection against peritoneal metastases and control of systemic tumor growth. [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 2301.
Abstract 2304: Attenuated oncolytic virus HSV1716 increases in vivo expansion of GD2- targeting CAR T cells in murine solid tumor models
Authors: Haile Samuel T. , Conner Joe , Mackall Crystal
Published: July 2016

Neuroblastoma, osteosarcoma, and rhabdomyosarcoma are among the most prevalent childhood solid tumors. Each of these tumor types as well as melanomas exhibit increased levels of the tumor associated carbohydrate, GD2 on their cell surface making them ideal targets for chimeric antigen receptor (CAR) T cell-directed therapies. Despite the ability of GD2 CAR T cells to target tumor cells in vitro, there is great interest in improving tumor clearance in vivo, especially for solid tumors where current outcomes remain poor. We hypothesize that the immunosuppressive milieu present within the solid tumor microenvironment serves as a major factor limiting the effectiveness of GD2 CAR T cells. Additionally, we propose that administration of oncolytic viruses could induce inflammation within the tumor microenvironment that may enhance, rather than inhibit, the effectiveness of immune based therapies. GD2 CAR T cells composed of the 14G2a single chain variable fragment linked to the cytoplasmic signaling domains of CD28 and CD3 zeta (GD2-28z) were expressed in murine lymphocytes and evaluated for the ability to target and lyse GD2-expressing tumor cells. Additionally GD2-28z T cells were evaluated for their ability to secrete the proinflammatory cytokines IFNγ and IL-2. In order to determine the oncolytic ability of attenuated HSV1716, tumor cells were cultured in the presence or absence of HSV1716 and relative cell survival was measured. We observed specific lysis of GD2-expressing tumor cells when co-cultured with GD2-28z, but not mock T cells. Furthermore, GD2-28z T cells secrete IFNγ and IL-2 following co-culture with GD2-expressing tumor cells. Interestingly, melanoma cell lines were not susceptible to oncolytic lysis while rhabdomycosarcoma (RMS) cell lines were susceptible. The combination of GD2-28z and HSV1716 enhanced CAR expansion in both melanoma and RMS models. Given that the melanoma cells in our model are not susceptible to oncolytic lysis yet we observe an increased T cell expansion when used in combination with HSV1716, this supports our hypothesis that HSV1716 is inducing inflammation, which may then triggering T cell expansion.

Citation Format: Samuel T. Haile, Joe Conner, Crystal Mackall. Attenuated oncolytic virus HSV1716 increases in vivo expansion of GD2- targeting CAR T cells in murine solid tumor models. [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 2304.
Abstract 2310: With a little help from CD4 T cells in adoptive T-cell transfer
Authors: Inderberg Else M. , Wälchli Sébastien , Myhre Marit R. , Lislerud Kari , Kvalheim Gunnar , Gaudernack Gustav
Published: July 2016

Adoptive transfer of genetically engineered T cells offers great opportunities in cancer immunotherapy, however until recently, HLA-Class II-restricted TCRs have been largely ignored.

We have identified and cloned HLA class II-restricted CD4+ T cells isolated from patients vaccinated with long peptides derived from antigens such as hTERT, survivin and frameshift mutated TGFβRII. Several of these cancer patients demonstrated outstanding clinical responses to peptide- or dendritic cell based cancer vaccines with >10-year survival. In these patients strong T-cell responses against peptides other than those used for vaccination were detected, suggesting epitope spreading.

Responses against certain peptides associated with clinical benefit were identified and CD4+ T-cell clones recognizing such peptides were isolated. In depth studies of their specificities lead to the selection of HLA-DR and -DQ restricted T-cells having high functional avidity for the HLA-peptide complexes. We studied tumor cell and/or protein recognition and found one telomerase-specific CD4+ T-cell clone recognizing a melanoma cell line with the corresponding HLA allele.

The TCR sequences of the interesting clones were identified. The ribosome skipping 2A sequence technique was used to express these TCRs in an mRNA expression vector or retrovirus. When expanded T cells were electroporated with these TCRs, both redirected CD8+ and CD4+ T cells produced TNF-α, IFN-γ and the degranulation marker (CD107a) following co-incubation with specific peptide-loaded targets.

We have demonstrated that choosing highly functional CD4+ T-cell clones specific for tumor-associated or -specific antigens from patients with clinical responses after immunotherapy treatment is a successful method for identifying highly functional HLA class II restricted TCRs for adoptive T-cell transfer.

The use of HLA class II-restricted TCRs may be of therapeutic value both in haematopoietic malignancies and in melanoma where tumor cells often express HLA class II. In addition, combining the redirection of T cells with both HLA class I- and class II-restricted TCRs may provide a more powerful therapeutic effect in adoptive T cell therapy.

Citation Format: Else M. Inderberg, Sébastien Wälchli, Marit R. Myhre, Kari Lislerud, Gunnar Kvalheim, Gustav Gaudernack. With a little help from CD4 T cells in adoptive T-cell transfer. [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 2310.
Abstract 2312: TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer
Authors: Byrd Tiara , Fousek Kristen , Pignata Antonella , Szot Christopher , Bielamowicz Kevin , Seaman Steven , Landi Daniel , Rainusso Nino , Sorensen Poul , Koch Joachim , Wels Winfried , Fletcher Bradley , Hegde Meenakshi , St Croix Brad , Ahmed Nabil
Published: July 2016

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no approved targeted therapies. Tumor endothelial marker 8 (TEM8), initially identified as a marker of tumor endothelial cells in colorectal cancer and other solid tumors has recently been shown to be upregulated in TNBC and breast cancer stem cells (BCSCs). We investigated whether TEM8 specific chimeric antigen receptor (CAR) T cells recognize and kill both tumor endothelial cells as well as TNBC tumor cells. TEM8 specific CAR molecules were generated using single chain variable fragment derived from the monoclonal antibody, L2. L2 CAR T cells selectively recognized TEM8, secreted immunostimulatory cytokines and effectively killed both TEM8 positive TNBC and tumor endothelial cell lines. Moreover, L2 CAR T cells targeted breast cancer stem cells significantly reducing the number of mammospheres relative to non-transduced T cells. In vivo, adoptive transfer of L2 CAR T cells induced regression of established vascularized TNBC xenografts. Hence, TEM8 may serve as an attractive target for immunotherapy of TNBC.

Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Kevin Bielamowicz, Steven Seaman, Daniel Landi, Nino Rainusso, Poul Sorensen, Joachim Koch, Winfried Wels, Bradley Fletcher, Meenakshi Hegde, Brad St Croix, Nabil Ahmed. TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative 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 2312.
Abstract 2316: GPC3-specific chimeric antigen receptor T cell in combination with Sorafenib as a novel therapeutic treatment for hepatocellular carcinoma
Authors: Trinh Thu Le , Wu Qunfeng , Chang Lung-Ji , Ho Mitchell , Liu Chen
Published: July 2016

FDA-approved targeted therapy for advanced hepatocellular carcinoma (HCC) is limited with the only option of Sorafenib treatment with severe side effects. Immunotherapy based on adoptive transfer of tumor-specific T cells have demonstrated a promising clinical outcome for solid tumors. In our study, we developed T cells expressing the fourth generation of GPC3-specific chimeric antigen receptor (α-GPC3 CAR) that efficiently eliminated GPC3-positive HCC cells (Huh7 and HepG2) without killing GPC3-negative primary hepatocytes. This CAR was constructed using a lentiviral vector containing CD28, CD27 and 41BB costimulatory domains, a CD3ζ signaling domain fused to a FKBP-iCasp9 apoptosis inducing gene, and a GPC3-specific sequence derived from HN3, a single-domain VH human monoclonal antibody (gift from Mitchell Ho, NCI). After 16h of incubation at the E (α-GPC3 CAR T cells) : T (Huh7 or HepG2 cells) ratio of 1:1, α-GPC3 CAR T cells effectively lysed Huh7 (39%) and HepG2 (77%) but not primary hepatocytes (9%), while the control CAR T cells (α-CD30 CAR) could not initiate a specific lysis on those cells. At the E:T ration of 3:1, the specific killing increased to 78% (Huh7) and 99% (HepG2) while remaining low with primary hepatocytes (11%). Especially at the very low E:T ratio of 1:10, combination with Sorafenib priming (24h, IC10 concentration) prior to CAR T incubation boosted the specific lysis up to 25% (p = 0.0416). These results indicate that combination of GPC3-specificCAR T cells transfer and Sorafenib regimen could be a promising therapeutic option for the treatment of HCC, with the doses of both CAR T and Sorafenib reduced up to 10 times, hence lower side effects while preserving the efficacy of tumor-specific cytotoxicity.

Citation Format: Thu Le Trinh, Qunfeng Wu, Lung-Ji Chang, Mitchell Ho, Chen Liu. GPC3-specific chimeric antigen receptor T cell in combination with Sorafenib as a novel therapeutic treatment for hepatocellular 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 2316.
Abstract 2331: HDAC6, new role as master regulator of PD-L1 and immune-related pathways
Authors: Knox Tessa , Lienlaf Maritza , Perez Patricio , Pabon Mibel , Lee Calvin , Cheng Fengdong , Sahakian Eva , Powers John , Deng Susan , Keiran Smalley , Kozikowski Alan , Pinilla Javier , Sarnaik Amod , Seto Ed , Weber Jeffrey , Sotomayor Eduardo , Villagra Alejandro
Published: July 2016

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immunobiology is still not completely understood, and the reported immunological outcomes when using HDACi are heterogeneous. Our group recently reported that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modulates the expression of immuno-regulatory proteins, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. We primarily focused in PD-L1, which is an important negative regulator of T-cell function and often over-expressed in cancer cells. In a mechanistic point of view, we have found that the pharmacological inhibition and genetic abrogation of HDAC6 inactivates the STAT3 pathway, impairs the nuclear translocation and the recruitment of STAT3 to the PD-L1 promoter and subsequently down-regulates the expression of PD-L1. Moreover, the in vivo abrogation of HDAC6 reduces tumor growth in melanoma models, effect that is enhanced in the presence of the immune check-point blocking antibodies anti-PD-1 and anti-CTLA4. These results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immunomodulatory agents in cancer.

Citation Format: Tessa Knox, Maritza Lienlaf, Patricio Perez, Mibel Pabon, Calvin Lee, Fengdong Cheng, Eva Sahakian, John Powers, Susan Deng, Smalley Keiran, Alan Kozikowski, Javier Pinilla, Amod Sarnaik, Ed Seto, Jeffrey Weber, Eduardo Sotomayor, Alejandro Villagra. HDAC6, new role as master regulator of PD-L1 and immune-related pathways. [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 2331.
Abstract 2332: Patient-derived tumor xenografts in humanized NSG-SGM3 mice: a new immuno-oncology platform
Authors: Yao Li-Chin , Cheng Mingshan , Wang Minan , Banchereau Jacques , Palucka Karolina , Shultz Leonard , Bult Carol , Keck James G.
Published: July 2016

Humanized mice engrafted with tumors enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently found that humanized NOD-scid IL2Rγnull (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of check-point inhibitors. Next generation NSG strains include triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSG-SGM3). Here we provide a direct comparison of check-point inhibitors evaluation in NSG and NSG-SGM3 mice engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from the same donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune system in the blood was faster and more robust in NSG-SGM3 compared to NSG recipients throughout the course of the study (18 weeks). Human CD45+ cells reached 25% of total blood cells at week 4 in hu-NSG-SGM3 mice and at week 9 in hu-NSG mice. A majority of blood hCD45+ cells in hu-NSG-SGM3 at week 4 were CD33+ myeloid cells. Circulating hCD3+ T cells reached 10% at week 9 and included regulatory T cells (Tregs), consistent with earlier studies. Hu-NSG mice displayed comparable hCD3+ T cells in the blood only at 12-15 weeks and did not contain Tregs. PDX tumors were then engrafted into partially HLA-matched hu-NSG-SGM3 mice at 9 weeks post engraftment. Two PDX models previously shown to respond to anti-PD1 therapy in hu-NSG mice, BR1126 and LG1306, were used. Treatment with the anti-PD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth in both models. Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies.

Citation Format: Li-Chin Yao, Mingshan Cheng, Minan Wang, Jacques Banchereau, Karolina Palucka, Leonard Shultz, Carol Bult, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: a new immuno-oncology platform. [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 2332.
Abstract 2333: Possible prediction of tumor specific checkpoint inhibitor response based on TCGA somatic mutation load
Authors: Colli Leandro Machado , Machiela Mitchell J. , Myers Timothy , Jessop Lea , Yu Kai , Chanock Stephen J.
Published: July 2016

Immune checkpoint inhibitor therapy has been shown to be effective in a subset of patients with melanoma, non-small cell lung cancer (NSCLC) and kidney cancer. Recent studies have suggested that the number of non-synonymous mutations (NsM) can be used to select melanoma and NSCLC patients most likely to benefit from immune checkpoint inhibitor treatment. It is hypothesized that NsMs generate novel epitopes and gene products which can be detected by the immune system. The aim of this study is to apply prior information on NsM count and immune checkpoint inhibitor treatment to a range of tumor subtypes in the TCGA database to evaluate the proportion cases that could be possible responders. In our analysis of published studies of melanoma and NSCLC, the receiver operator characteristic (ROC) curve suggests a cutoff of 192 NsM would have a maximum combination of sensitivity (74%) and specificity (59.3%) for potential clinical benefit for patients. We conducted an analysis of 7,757 samples from 26 different TCGA tumor types and observed that approximately 16.2% of TCGA samples harbor more than 192 NsM, and thus could be in the category associated with a higher likelihood of response to immune checkpoint inhibitors. Based on NsM count, bladder, colon, gastric, and endometrial cancers each contained more than 30% of tumors with a high NsM and thus, could be possible candidates for checkpoint immune therapy. Although our model estimates clinical response to immune checkpoint treatment from NsM count is preliminary and needs future validation, information on NsM count can be useful for selecting tumor types most likely to benefit in clinical trials of immune checkpoint inhibitor treatment.

Citation Format: Leandro Machado Colli, Mitchell J. Machiela, Timothy Myers, Lea Jessop, Kai Yu, Stephen J. Chanock. Possible prediction of tumor specific checkpoint inhibitor response based on TCGA somatic mutation load. [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 2333.
Abstract 2343: The immune modulatory roles of IAP inhibitor, LCL161, and its connection to immune-checkpoint molecules
Authors: Pinzon-Ortiz Maria , Hastings William , Longmire Tyler , Shaw Pamela , Rong Xianhui , Murakami Masato , Lee Benjamin H. , Dranoff Glenn , MacIsaac Kenzie , Cao Z. Alexander
Published: July 2016

Tumor immunotherapy is a unique therapeutic modality in our fight against human cancers. The recent success of immune-checkpoint therapies highlights the value and potential of this approach. Previous studies have demonstrated positive immune-modulatory activities of the IAP inhibitor, NVP-LCL161. In this study, we sought to further explore the immune-modulatory activities of NVP-LCL161 in connection with immune-checkpoint molecules.

In PBMC stimulation assays, NVP-LCL161 potently induced IFN and enhanced proliferation, while down-modulating IL-10 production. Additionally, the role of NVP-LCL161 on immune-checkpoint modulation was revealed in CyTOF analysis of in vitro stimulated PBMCs. NVP-LCL161 treatment led to increases in TIM-3 expression on multiple T cell subsets, and on cells of the myeloid lineage. These observations illustrate potential modulation of specific immune-checkpoint molecule by NVP-LCL161, thus suggesting possible synergy between TIM-3 immunecheckpoint blockade and NVP-LCL161. NVP-LCL161 was further combined in vivo with anti-PD1 antibody in the murine syngeneic tumor model, MC38. At multiple dosing schedules, synergy was observed with this combination, which appeared to be independent of single agent efficacy. Expression profiling of MC38 tumor tissues by a customized Nanostring panel revealed elevation of gene signatures in T cells, dendritic cells, cytokines and chemokines, amongst others.

In summary, the preclinical data support the hypothesis that NVP-LCL161 is an active immune modulator. Its combination activity with immune_checkpoint inhibitors would warrant further exploration in both preclinical and clinical setting.

Citation Format: Maria Pinzon-Ortiz, William Hastings, Tyler Longmire, Pamela Shaw, Xianhui Rong, Masato Murakami, Benjamin H. Lee, Glenn Dranoff, Kenzie MacIsaac, Z. Alexander Cao. The immune modulatory roles of IAP inhibitor, LCL161, and its connection to immune-checkpoint molecules. [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 2343.
Abstract 234A: Aberrant sodium current contributes to constitutive mTOR activity in malignant melanoma cells
Authors: Gallant Benjamin , Lizza Nicole , Garrity Ryan , Madigan Audrey , Jost Zachary , Gonzalez Alfredo , Justiniano Jeanine , Xie An , Wan Yinsheng
Published: July 2016

Transformation from disciplined melanocytes to untamed melanoma cells remains an enigma. Our previous studies have demonstrated that melanoma cells are more resistant to oxidative stress and melanoma cells exhibit constitutive mTOR activity. Surprisingly, further studies have failed to present the expression and activity of EGFR using conventional anti-EGFR antibodies. We hypothesized that constitutive mTOR activity in melanoma cells may be due to mutated EGFR variants and membrane channel activities. Using patch clamping technique, we have shown that melanoma cells (WM 266-4) but not human skin melanocytes exhibit Na+ current which is blocked by TTX. Interestingly, mTOR inhibitor, rapamycin, blocks Na+ current. Western blot and confocal microscopy data further revealed that Na+/Ca2+ exchanger blocker KB-R7943 (KBR), and L-type Ca2+ channel blocker Nifedipine (NIF) inhibits mTOR activity in melanoma cells in a dose dependent manner. To further characterize Na+ channels, we used commercially available channel antibodies. Western blot analysis data showed that melanoma cells but not human melanocytes express Na+ v1.5 and Na+ v1.6 and NCX3. Functional studies also indicated that KBR and NIF inhibit melanoma cell proliferation and migration. Taken all together, our data suggest that aberrant Na+ current contributes to constitutive mTOR activity in melanoma cells and channel blockers may be potential for the treatment of melanoma.

Citation Format: Benjamin Gallant, Nicole Lizza, Ryan Garrity, Audrey Madigan, Zachary Jost, Alfredo Gonzalez, Jeanine Justiniano, An Xie, Yinsheng Wan. Aberrant sodium current contributes to constitutive mTOR activity in malignant melanoma cells. [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 234A.
Abstract 2356: Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for in situ cancer immunotherapy
Authors: Cremel Magali , Guerin Nathalie , Barthe Quitterie , Bourgeaux Vanessa , Berlier Willy , Horand Françoise , Godfrin Yann
Published: July 2016

Introduction: In current vaccine therapy, efficient delivery of tumor antigens (TA) to antigen-presenting cells (APC) represents a major step toward the development of strong TA-specific immune response including cytotoxic T lymphocyte (CTL) induction. Our innovative approach is to use the property of erythrocytes to be naturally phagocytosed by APC when senescent. Indeed, TA can be encapsulated into erythrocytes that will be used as carrier to specifically deliver the TA to APC, which will ensure their degradation and presentation to T cells.

Materials and Methods: The proof of concept was established with Ovalbumin (OVA), tyrosinase-related protein 2 (TRP2 (melanoma TA) and prostate specific antigen (PSA, prostate TA). These TA were encapsulated into erythrocytes by a hypotonic lysis process. To stimulate the erythrophagocytosis by APC, the membranes of erythrocytes were coated with antibodies. Then, erythrocytes containing TA were intravenously injected to mice concomitantly with Poly(I:C) adjuvant. The CTL response was analyzed by IFNγ ELISPOT and in vivo lysis of TA-expressing target cells injected to immunized mice. Humoral response was assessed by the determination of TA-specific immunoglobulin titer in mouse serum.

Results: All TA were efficiently encapsulated in erythrocytes in a dose-dependent manner. A strong T-cell response was induced after 2 injections of TA encapsulated in erythrocytes: 2μg of OVA and 20μg of TRP2 induced the in vivo lysis of 97% and 96% of TA-target cells, respectively. By comparison, injections of free antigen induced less than 5% of lysis. Furthermore, a significant number of TA-specific IFNγ-secreting cells was generated (1585 and 286 cells / 106 cells for TRP2 and PSA, respectively) compared to the free TA (< 80 cells / 106 cells for both TA). In addition, we established a dose-dependent PSA-specific humoral response. Finally, a significant delay of tumor growth was obtained in mice more than twenty days after the subcutaneous implantation of EG7-OVA or B16F10 (melanoma) tumor cell lines, compared to injections with free antigen or control vehicle.

Conclusion: The use of erythrocytes as TA carrier to specifically deliver antigen to APC and induce efficient immune response against tumor can be a very promising strategy in cancer immunotherapy.

Citation Format: Magali Cremel, Nathalie Guerin, Quitterie Barthe, Vanessa Bourgeaux, Willy Berlier, Françoise Horand, Yann Godfrin. Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for in situ 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 2356.
Abstract 2364: Spontaneous and vaccine-induced clearance of Mus Musculus Papillomavirus1 (MusPV1) infection
Authors: Jiang Rosie T. , Wang Joshua W. , Peng Shiwen , Hung Chien-Fu , Roden. Richard B.S
Published: July 2016

Human papilloma virus (HPV) high risk (hr) types cause several cancers, including cervical, vaginal, penile, and oropharyngeal, whereas low risk types cause benign diseases such as skin warts, anogenital warts and recurrent respiratory papillomatosis. Although hrHPV infection is one of the most common sexually transmitted diseases most infections remain asymptomatic and are cleared while a sub-population develops persistent HPV-related pathologies including cancer. Further, immune-compromised populations, notably organ transplant and HIV-positive patients, are particularly vulnerable to HPV-related pathologies. Here, we describe the first establishment of mouse papillomavirus-1 (MusPV1) disease in outbred and immune competent SKH1-Elite mice (Crl:SKH1-Hrhr). Challenge of SKH1 mice with MusPV1 resulted in three clinical outcomes: 1) persistent (>2 months) papillomas (∼15%), 2) transient papillomas that spontaneously regress typically within 2 months (∼10%), 3) no visible papillomas and viral clearance (∼75%). SKH1 mice with persisitent papillomas were treated using a potentially preventive and therapeutic DNA vaccine composed of human calreticulin (hCRT) linked to MusPV1 early proteins, mE6 and mE7 and the late protein mL2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations (15 μg in PBS) via in vivo electroporation were administered weekly and immune responses were measured, including mE6/mE7/mL2 serum antibody response and T-cell epitope mapping studies. Previously persistent papillomas disappeared within 2 months after final vaccination. Clearance of MusPV1 was confirmed by loss of mE6/mE7 transcript staining using RNA-CISH on formalin-fixed, paraffin-embedded tail sections. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice. Interestingly, mL2 antibody responses were faster and stronger in asymptomatic challenged mice and in those that spontaneously cleared their papillomas after challenge as compared to mice with persistent papillomas. In sum, MusPV1 challenge of outbred, immune-competent SKH1 mice represents a promising model to further study potential immunotherapies for HPV-related disease and the relationship between spontaneous clearance of papillomavirus and host genetics.

Citation Format: Rosie T. Jiang, Joshua W. Wang, Shiwen Peng, Chien-Fu Hung, Richard B.S Roden. Spontaneous and vaccine-induced clearance of Mus Musculus Papillomavirus1 (MusPV1) infection. [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 2364.
Abstract 237: Enrichment of AXL-high/ MITF-low melanoma cells in the presence of MAPK inhibitors in vitro
Authors: Dutton-Regester Ken , Garraway Levi
Published: July 2016

BRAF and MEK inhibitors have become a standard of care for patients with metastatic BRAF mutant (V600) melanoma. Despite their success, 10- 20% of patients exhibit ‘intrinsic’ resistance and fail to respond to treatment. Recently, distinct transcriptional profiles have been associated with sensitivity to these drugs with intrinsically resistant melanomas exhibiting an AXL-high/ MITF-low phenotype (1). Using melanoma cell lines from the Cancer Cell Line Encyclopedia with matched gene expression data, we confirmed that AXL-high/ MITF-low cell lines had increased resistance to both BRAF (Dabrafenib) and MEK (Trametinib) inhibitors, either singly or in combination. Using flow cytometry with an AXL antibody, we observed that AXL-high/ MITF-low resistant cell lines frequently exhibited a high percentage of AXL+ cells (≥90%) while AXL-low/ MITF-high sensitive cell lines showed the opposite (≤5%). We hypothesized that the small percentage of AXL+ cells in the sensitive cell lines may be responsible for mechanisms of adaptive or acquired resistance (a feature that is frequently observed with the use of BRAF and MEK inhibitors in vitro and in the clinic). To explore this, we cultured sensitive cell lines in vitro for 5 days in the presence of combined Dabrafenib and Trametinib (DT) and performed flow cytometry to determine if there was a change in the percentage of AXL+ cells. All sensitive cell lines exhibited an enrichment of AXL+ cells with increasing concentrations of DT (maximum increase compared to DMSO control across individual cell lines ranged from 10% to 85%). It is unknown whether this phenomenon could be explained by a clonal selection or ‘plastic’ epigenetic reprogramming process and as such, we are currently performing experiments to determine this. Looking to the future, identifying potential dependencies of AXL-low/ MITF-high melanomas will deepen our understanding of the biology supporting this resistant state and provide a platform for the design of future clinical intervention strategies in this subset of patients.

1) Konieczkowski et al. 2014. Cancer Discovery. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. 4(7):816-27.

Citation Format: Ken Dutton-Regester, Levi Garraway. Enrichment of AXL-high/ MITF-low melanoma cells in the presence of MAPK inhibitors in vitro. [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 237.
Abstract 2375: Multiclonal invasion in breast cancer identified by single cell DNA sequencing
Authors: Casasent Anna K. , Long Annalyssa N. , Schalck Aislyn , Sei Emi , Gao Ruli , Davis Alexander , Wang Yong , Edgerton Mary E. , Navin Nicholas E.
Published: July 2016

Ductal carcinoma in situ (DCIS) is the most common form of early stage breast cancer and is frequently detected by mammography. Only 10% of low-grade and 30% of high-grade DCIS patients will later present with an invasive carcinoma, making it difficult to determine which patients to treat aggressively. Although DCIS is often considered a precursor to invasive breast carcinoma, a major question in the field is whether invasive subpopulations evolve directly from the in situ populations or independently. We hypothesize that early tumor cells evolve over an extended period of time in the ducts, generating multiple subpopulations that migrate out of the ducts together. To test this hypothesis, we developed an approach that combines laser-capture microdissection (LCM) with single cell sequencing to perform DNA copy number profiling while preserving the topographic location of cells. We applied this method to 10 high-grade breast cancer patients with defined in situ, invasive, and normal regions. Using these data, we delineated the clonal substructure and location of the in situ and invasive subpopulations. We found that most high-grade tumors are composed of 1-4 major clonal subpopulations that are intermixed in both the in situ and invasive regions. These subpopulations share a common evolutionary lineage or cell of origin. We also performed deep-exome sequencing (100X) of matched microdissected regions of in situ, invasive and adjacent normal tissue from these breast cancer patients. In each tumor we identified a large number of shared somatic mutations between the in situ and invasive regions, further supporting our direct genomic lineage hypothesis. We also identified a few mutations confined to the invasive tumor regions that may play an important role in the invasive phenotype. These data support a multiclonal invasion model in which distinct clones evolve in the ducts and subsequently migrate into the adjacent tissues to establish invasive tumors. This model has important implications for the diagnosis and therapeutic treatment of DCIS breast cancer patients since multiple clones may need to be targeted to inhibit invasion.

Citation Format: Anna K. Casasent, Annalyssa N. Long, Aislyn Schalck, Emi Sei, Ruli Gao, Alexander Davis, Yong Wang, Mary E. Edgerton, Nicholas E. Navin. Multiclonal invasion in breast cancer identified by single cell DNA sequencing. [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 2375.
Abstract 2386: Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition
Authors: Ragoussis Ioannis , Savage Paul , Wang Yu-Chang , Revil Timothee , Badescu Dunarel , Saleh Sadiq , Iacucci Ernesto , Bertos Nicolas , Monast Anie , Omeroglou Attila , Zuo Dongmei , Park Morag
Published: July 2016

Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor, a finding replicated in several independent experiments. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. One population was characterized by high MYC and the other high EGFR/KRT14. These findings were validated using immunohistochemistry on PDX and primary tumor material. Further functional experiments showed that that EGFR subpopulation has stem cell character istics and forms metastasis in the animal model. Microfluidic whole genome amplification followed by whole exome capture of 81

single cells, along with exome sequencing of the germline, primary and post treatment tumor and whole PDX showed that BRCA1 and TP53 are mutated in all single cells, as well as a number of sub-clonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 known cancer driver genes. Careful comparison of the exome sequencing data allowed the association of driver gene mutation prevalence with tumor progression. These findings are important in our understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance.

Citation Format: Ioannis Ragoussis, Paul Savage, Yu-Chang Wang, Timothee Revil, Dunarel Badescu, Sadiq Saleh, Ernesto Iacucci, Nicolas Bertos, Anie Monast, Attila Omeroglou, Dongmei Zuo, Morag Park. Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition. [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 2386.
Abstract 2396: Heterogeneity of colorectal cancers and multiple liver metastases by mutation and copy number profiling
Authors: Alagaratnam Sharmini , Høst Brunsell Tuva , Aske Danielsen Stine , Sveen Anita , Eknæs Mette , Hektoen Merete , Røsok Bård , Bjørnbeth Bjørn Atle , Nesbakken Arild , Lothe Ragnhild
Published: July 2016

To investigate genetic and genomic heterogeneity in liver metastases of colorectal cancer, multiple tumor foci in the liver per patient were identified on the basis of radiological images and samples from these systematically biobanked. For a total of 67 patients, 258 metastatic tumor samples were collected (minimum of 2 and maximum of 9 samples per patient, median of 4 samples per patient). Additionally, for n = 14 patients, tumor samples from the corresponding primary colorectal cancer were also available. DNA isolated from these samples was subjected to mutation profiling by Sanger sequencing for the genes BRAF, KRAS, PIK3CA and TP53, as well as allele-specific copy number profiling by CytoscanHD SNP array. For the four genes investigated for mutations, 39/67 (58%) of the patients were homogeneous in their mutation profile, while 21/67 (32%) were heterogeneous (one or more samples had mutations not detected in all samples). For 7/67 patients (10%), no mutations were detected in any of these four genes. For 9/14 patients with matching primary tumors, there was complete concordance between the primary tumor and all liver metastases profiled for mutations, while the remaining 5/14 patients showed discordance in mutation profile between the primary tumor and at least one of the liver metastases profiled. High resolution copy number profiles of this cohort detected many of the aberrations previously described in primary colorectal cancer, specifically gain of chromosomes 7, 8q, 13q and 20q, and loss of chromosomes 5q, 8p, 15q, 17p and 18q. The extent of heterogeneity observed in patient-wise mutation profiles was largely mirrored in the copy number profiles of the same tumors. 16/27 (60%) of the patients for which both mutation and copy number profiles were available showed concordance in the degree of heterogeneity per patient. In summary, intra-individual heterogeneity varied from patient to patient, with results from the genetic and genomic data types largely concurring.

Citation Format: Sharmini Alagaratnam, Tuva Høst Brunsell, Stine Aske Danielsen, Anita Sveen, Mette Eknæs, Merete Hektoen, Bård Røsok, Bjørn Atle Bjørnbeth, Arild Nesbakken, Ragnhild Lothe. Heterogeneity of colorectal cancers and multiple liver metastases by mutation and copy number profiling. [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 2396.
Abstract 24: Loss of ubiquitin-specific peptidase 18 (USP18) causes cold sensitive mice by destabilizing the critical regulator of thermogenesis: uncoupling protein-1 (UCP-1)
Authors: Liu Xi , Lu Yun , Zheng Lin , Sekula David J. , Freemantle Sarah J. , Dmitrovsky Ethan
Published: July 2016

USP18 is the major ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We recently reported that USP18 null mice spontaneously develop leiomyosarcomas. Intriguingly, these USP18 null mice are also markedly cold sensitive as compared to their wild-type littermates. When briefly exposed to cold stimuli, USP18 null mice significantly (P < 0.05) decreased their body temperature. In contrast, wild-type mice exposed to the same experimental conditions exhibited transient changes in their body temperature. We sought to elucidate the engaged mechanism. Expression profiles of critical thermogenic regulatory proteins were examined by immunoblot analyses of brown fat tissues of USP18 null mice. Strikingly, UCP-1 expression was substantially reduced in these tissues of USP18 null versus wild-type mice. To independently confirm that reduced UCP-1 expression was caused by loss of USP18, stable knock-down of USP18 was independently achieved in a panel of murine cell lines by use of transfected small hairpin RNAs (shRNAs). The obtained results were compared to that of transfected control vectors. USP18 down-regulation by different shRNAs markedly reduced UCP-1 protein as compared to controls. As expected, engineered gain of USP18 expression in the same murine cell lines stabilized UCP-1 protein. We explored if UCP-1 destabilization was due to its complex formation with the ubiquitin-like protein ISG15. This was the expected experimental outcome since USP18 is the distinct deconjugase that removes ISG15 from complexed proteins. Immunoprecipitation assays were performed to establish this direct association. These studies revealed a complex formed between ISG15 and UCP-1 protein. Thus, engineered loss of USP18 in mice led to cold sensitive mice from repression of UCP-1. As a consequence of its complex with ISG15, the thermogenic regulator UCP-1 was destabilized. Taken together, this study extends prior work by showing a previously unrecognized link between USP18 and regulation of thermogenesis. This defines a novel role for the USP18 protease in metabolism.

Citation Format: Xi Liu, Yun Lu, Lin Zheng, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of ubiquitin-specific peptidase 18 (USP18) causes cold sensitive mice by destabilizing the critical regulator of thermogenesis: uncoupling protein-1 (UCP-1). [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 24.
Abstract 2424: Genomic landscape of aggressive tumor subtype in MYCN-non-amplified neuroblastoma
Authors: Ohira-Ichikawa Miki
Published: July 2016

Neuroblastoma is known to exhibit wide ranges of clinical phenotypes, from spontaneous regression to highly resistant to chemotherapy. One of the challenges in the clinic is to develop suitable therapeutic strategies for the intermediate risk-type of patients (stage 3 or 4 without MYCN amplification) whose prognosis sometimes tends to be poor in terms of long-term follow-up (10 years survival rate was 51% in our database, n = 161). To identify key biological pathways involved in the unfavorable phenotypes in this patient group, we have analyzed 63 primary tumors (dead:32, alive:31) without MYCN amplification nor ALK alteration by integrated genomic analyses such as array CGH, whole exome sequencing (SureSelect XT Human All Exon V4, average read depth:157, x20 coverage:95%), RNA sequencing, as well as Methylome profiling (Infinium HumanMethylation450 BeadChip). By using the Karkinos and ANNOVAR software, we found so far 816 non-synonymous somatic single nucleotide variations (SNVs) in coding exons in total (approximately 20 per tumor in average) and number of alterations in each tumor strongly correlated with age at diagnosis (p<0.0001) and patient prognosis (p = 0.0077). Recurrent somatic SNVs (n = 2 to n = 5) were found in 66 genes, 19 of those were involved only in the tumors from the 24 patients with poor outcome. Gene Ontology and pathway analysis (GeneMANIA) using the 66 genes showed that mutations occurred in the features including cytoskeleton, ion channel and extracellular matrix were significantly correlated with patient survival of the 63 MYCN-non-amplified tumors with logrank p-values of 0.0039, 0.0482 and 0.0011, respectively. ATRX mutations were observed in four tumors (6.3%), all of them were occurred in patients with unfavorable phenotypes (3 dead, 1 alive but relapsed twice). By methylome signature, the 63 tumors were subdivided into at least three clusters, which also showed strong correlation with patient prognosis (cluster-1 vs. cluster-2 and -3, p = 0.0368). Unfavorable cluster-2 and -3 exhibited hyper-methylation phenotypes in CpG islands and the former was quite similar to that of MYCN-amplified tumors. Thus, these prognosis-related genomic features could be useful markers for risk classification and help understanding molecular mechanism for aggressive phenotypes of MYCN-non-amplified neuroblastomas.

Citation Format: Miki Ohira-Ichikawa. Genomic landscape of aggressive tumor subtype in MYCN-non-amplified neuroblastoma. [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 2424.