bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5912: Tumor micoenvironment modulates RTK signaling
Authors: Thakar Dhruv , Low-Nam Shalini T. , Groves Jay T. , Weaver Valerie M.
Published: July 2017

Receptor tyrosine kinases (RTKs) and their associated signaling molecules are mutated or dysregulated in many aggressive cancers such as non-small cell lung carcinoma (NSCLC), breast cancer, and colorectal cancer. Despite therapeutic interventions such as tyrosine kinase inhibitors (TKI) that target RTKs, majority of patients develop resistance even after initial tumor regression. Although clinical studies suggest sequential treatments with TKI, chemotherapy, and radiation can eliminate most cancer cells, resistance inevitably emerges (Sequist, et al., Sci Trans Med, 2011). We demonstrated that alterations in the extracellular matrix (ECM) correlate with cancer progression and that RTK signaling is subject to spatiotemporal regulation (Paszek, et al., Cancer Cell, 2005.; Salaita, et al., Science, 2010). Thus, geometric and mechanical properties of the ECM may directly modulate RTK signaling. Here, we use silicone gel substrates that exhibit tunable stiffness in the physiological range (100s Pa - 100s kPa) to measure RTK-adaptor protein interactions in mammary epithelial cells (MECs; MCF10A) (Ou, et al. Integ. Biol., 2016). These gels are compatible with total internal reflection fluorescence (TIRF) imaging and enable single molecule detection of activated RTK phosphotyrosine residues via recruitment of Grb2 tagged with the fluorescent protein mEos3.2. We demonstrate a spectrum of Grb2 behaviors that are determined by the stiffness of the substrate. This indicates that the same receptor is capable of different signaling activities that are dictated by material properties. These new insights create opportunities to target the tumor microenvironment to address RTK inhibitor resistance and metastasis in cancer patients and to develop effective therapeutic strategies for better prognosis and prolonged patient survival.

Citation Format: Dhruv Thakar, Shalini T. Low-Nam, Jay T. Groves, Valerie M. Weaver. Tumor micoenvironment modulates RTK signaling [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 5912. doi:10.1158/1538-7445.AM2017-5912
Abstract 5913: The impact of daily exercise on tumor perfusion
Authors: Wiggins Jennifer M. , Lepler Sharon , Pampo Christine , Rice Lori , Lee Jennifer A. , Siemann Dietmar
Published: July 2017

Aberrant blood vessel networks in solid tumors lead to impaired tissue perfusion and areas of hypoxia (pO2 < 10 mmHg). Tumor hypoxia is associated with aggressive progression, dissemination, and therapeutic resistance. It is found in 40% of breast cancers, and can constitute a major obstacle to anticancer therapy. Exercise is associated with improvements in cardiovascular and respiratory function, aerobic capacity and overall health. In cancer patients undergoing therapy, exercise has been shown to decrease treatment related side effects and general fatigue. The goal of the present investigation was to determine whether aerobic exercise could be applied to improve tumor perfusion and oxygenation in a breast cancer model. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of a single and daily bouts of moderate intensity treadmill running were studied in mice bearing the syngeneic murine mammary carcinoma 4T1. The exercise intensity was determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during exercise. Initial studies examined the effect of a single moderate bout of exercise (18 m/min) in mice bearing ~500 mm3 orthotopic tumors. In subsequent experiments mice were exercised at 18 m/min, 5 days a week, for 8 weeks prior to orthotopic injection of tumor cells and continued exercise (5 days a week) for 2 weeks during tumor growth. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of each of the exercise regimen, tumors were harvested, sectioned, stained, and tile mapped to assess physiological changes by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion. Tumor hypoxia was determined using the 2-nitroimidazole (EF5). Blood vessels were stained using the endothelial cell marker CD-31. All markers were quantified using Photoshop and ImageJ NIH software. Our results indicate that in the 4T1 breast cancer model moderate intensity exercise did not significantly alter tumor growth, oxygenation or blood vessel number. However, daily bouts of exercise did significantly increase the number of open tumor vessels indicating improved tumor perfusion. These results suggest that a daily exercise regimen may have the potential to improve drug delivery to mammary tumors

Citation Format: Jennifer M. Wiggins, Sharon Lepler, Christine Pampo, Lori Rice, Jennifer A. Lee, Dietmar Siemann. The impact of daily exercise on tumor perfusion [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 5913. doi:10.1158/1538-7445.AM2017-5913
Abstract 592: Distinct cellular mechanisms mediate anti-CTLA-4 and anti-PD-1 checkpoint blockade
Authors: Wei Spencer C. , Levine Jacob H. , Pe'er Dana , Allison James P.
Published: July 2017

Checkpoint blockade is able to achieve durable responses in a subset of patients, however the biological variables that distinguish responders from non-responders are not well understood. Furthermore, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4 and anti-PD-1 induced tumor rejection. Given that PD-1 and CTLA-4 attenuate T cell activity through distinct mechanisms that are separated spatially and temporally, we hypothesized that responses to anti-CTLA-4 and anti-PD-1 are driven by distinct mechanisms. To address this hypothesis we utilized mass cytometry to comprehensively profile the effect of checkpoint blockade on tumor immune infiltrates in murine tumor models. This approach allows for the interrogation of greater than 40 analytes at single cell resolution. We demonstrate that high dimensional mass cytometry analysis enables unsupervised identification of biologically relevant tumor infiltrating immune populations with high sensitivity and specificity.

Using this approach we analyzed immune infiltrates of MC38 and B16BL6 murine tumors in mice treated with anti-CTLA-4, anti-PD-1, or control antibodies. In both tumor models we identify 15 distinct T cell populations with 0.5% or greater frequency. The T cell populations identified in MC38 and B16BL6 tumors were highly congruent. Notably, some but not all of these T cell populations were responsive to checkpoint blockade. A subset of tumor infiltrating CD8 T cell populations expanded following both anti-CTLA-4 and anti-PD-1. Conversely, a subset of regulatory T cell populations contracted following both anti-CTLA-4 and anti-PD-1. Interestingly, we observed expansion of a Th1-like CD4 effector T cell population only in response to anti-CTLA-4 treatment. Thus, we find that anti-PD-1 predominantly engages subsets of tumor infiltrating CD8 T cells whereas anti-CTLA-4 engages both the CD4 and CD8 effector compartments.

Our findings indicate that anti-CTLA-4 and anti-PD-1 utilize distinct cellular mechanisms to induce tumor rejection. These findings highlight the importance of expanding our mechanistic understanding of immunotherapeutic approaches for the rational design of combinatorial therapeutic approaches. Furthermore, these results demonstrate that mass cytometry analysis can be utilized to identify biologically relevant tumor infiltrating T cell populations.

We acknowledge the MDACC core facility NCI Support Grant P30CA16672.

Citation Format: Spencer C. Wei, Jacob H. Levine, Dana Pe'er, James P. Allison. Distinct cellular mechanisms mediate anti-CTLA-4 and anti-PD-1 checkpoint blockade [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 592. doi:10.1158/1538-7445.AM2017-592
Abstract 5925: Mapping the molecular communication within the tumor microenvironment of pancreatic cancer as well as between tumor and peritumoral tissue
Authors: Alhamdani Mohamed S. , Bauer Andrea S. , Hoheisel Jörg D.
Published: July 2017

We are analyzing in detail the molecular communication between the different cell types in the microenvironment of pancreatic ductal adenocarcinomas (PDAC). Also, its regulation is being investigated, looking at aspects such as the kind and mixture of molecules needed for triggering a particular regulative function, and unraveling the inter- and intracellular processes utilized to transmit a molecular message.

Although a rare tumor entity, adding up to only about 3% of all cancer cases in the Western world, PDAC is the fourth most frequent cause of cancer-related death; mortality is nearly identical to incidence. There is no real remedy apart from surgery, which can be applied to only 10 to 20% of patients. Even then, basically all patients will experience tumor recurrence.

Our study is based on a large basic set of data at the levels of DNA, RNA and protein that was generated from some 1000 pancreatic tumor samples [1], supplemented with information from studies that aim at an elucidation of functional molecules and comparative analyses in other tumors. This has already led to the creation of basic functional knowledge about metastasis [e.g., 2], the establishment of means for diagnosis [3, 4], an accurate disease prognosis [1], the characterization of communication processes [5] and the identification of new therapeutic avenues [6].

At the conference, we will report about the status of mapping particularly the protein-mediated communication between the different cell types in the tumor microenvironment. Also, we will show results about the effect that PDAC tissue has on the wider peritumoral environment and vice versa. The peritumoral tissue is exhibiting a field defect that does not seem to be dependent on DNA-methylation. A comparison of PDAC and cystic pancreatic tumors revealed that differences in communicating with the peritumoral tissue could actually be more important to the known pathological difference between the two cancer entities than the molecular variations in the tumor cells.

1. Bauer et al. (2017), work submitted for publication

2. Botla et al. (2016) Cancer Res. 76, 4149-4159

3. Keller et al. (2014) BMC Med. 12, 224

4. Moskalev et al. (2015) Oncotarget 6, 4418-4427

5. Dror et al. (2016) Nature Cell Biol. 18, 1006-1017

6. Jandaghi et al. (2016) Gastroenterology, in press (doi: 10.1053/j.gastro.2016.08.040)

www.dkfz.de/funct_genome/

Note: This abstract was not presented at the meeting.

Citation Format: Mohamed S. Alhamdani, Andrea S. Bauer, Jörg D. Hoheisel. Mapping the molecular communication within the tumor microenvironment of pancreatic cancer as well as between tumor and peritumoral tissue [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 5925. doi:10.1158/1538-7445.AM2017-5925
Abstract 5927: Eribulin modulates tumor microenvironment through vascular remodeling for antitumor effect in multiple mouse xenograft models
Authors: Ito Ken , Hamamichi Shusei , Abe Takanori , Akagi Tsuyoshi , Shirota Hiroshi , Kawano Satoshi , Asano Makoto , Asano Osamu , Yokoi Akira , Matsui Junji , Umeda Izumi O. , Fujii Hirofumi
Published: July 2017

Eribulin mesilate (Eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been reported to remodel tumor vasculature (i.e., improvement of tumor vessels and perfusion) in human breast cancer xenograft models; however, the role of this vascular remodeling in anti-tumor effects is not fully understood. Here, we investigated the effects of eribulin-induced vascular remodeling on anti-tumor activities in multiple human cancer xenograft models. Microvessel densities (MVDs) were evaluated by immunohistochemistry (CD31 staining), and anti-tumor effects were examined in 10 human cancer xenograft models treated with eribulin. Eribulin treatment significantly increased MVDs compared to the corresponding control groups in 7 out of 10 models with a correlation between enhanced MVD levels and anti-tumor effects (R2=0.55). Because of observed increases in MVDs, we next utilized 111In-labeled PEGylated liposomes to examine if eribulin treatment would result in increased tumoral accumulation levels of such liposomes, and indeed, we found that eribulin, unlike vinorelbine another tubulin inhibitor, enhanced them. Since eribulin treatment increased accumulation of 111In-labeled PEGylated liposomes, we postulated that this treatment might enhance anti-tumor effect of Doxil (a liposomal anti-cancer agent). As expected, eribulin enhanced anti-tumor activity of Doxil in the PE-H1650 xenograft model. Then, we evaluated whether eribulin facilitated the recruitment of immune cells into the tumor. Infiltrating CD11b-positive immune cells were significantly increased in the multiple eribulin-treated xenografted tumors, and natural killer (NK) cell depletion reduced anti-tumor effects of eribulin. Collectively, these findings suggested a contribution of the immune cells for anti-tumor activities of eribulin. Taken together, our results obtained from multiple human cancer xenograft models suggested that vascular remodeling induced by eribulin therapy would act as a microenvironment modulator, and contributed to show anti-tumor effects of eribulin.

Citation Format: Ken Ito, Shusei Hamamichi, Takanori Abe, Tsuyoshi Akagi, Hiroshi Shirota, Satoshi Kawano, Makoto Asano, Osamu Asano, Akira Yokoi, Junji Matsui, Izumi O. Umeda, Hirofumi Fujii. Eribulin modulates tumor microenvironment through vascular remodeling for antitumor effect in multiple mouse xenograft 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 5927. doi:10.1158/1538-7445.AM2017-5927
Abstract 5937: Obesity-induced destabilization of p16INK4A promotes the procarcinogenic effects of breast stromal adipocytes through miR-141/miR-146b-5p-dependent inhibition of leptin
Authors: Alkhalaf Huda H. , Aboussekhra Abdelilah
Published: July 2017

Obesity, a disease linked to increased number of adipocytes in various tissues, is increasingly recognized as a risk factor for the development of breast cancer. However, the molecular basis of adipocyte-related breast carcinogenesis remains elusive. In this study, we established and characterized normal stromal adipocytes from post-plastic surgery of breast tissues from obese and lean women. We have shown that adipocytes from obese women express low level of the tumor suppressor p16 protein, have higher secretion of various adipokines and higher invasion and migration capabilities as compared to adipocytes from lean women. In addition, down-regulation of p16INK4A by shRNA in adipocytes from lean women increased the expression/secretion levels of various adipokines including leptin, activated breast adipocytes and enhanced their migration/invasion abilities. Consequently, media conditioned with p16-deficient adipocytes or adipocytes from obese women promote epithelial-to-mesenchymal-transition in normal breast luminal cells in a leptin-dependent manner and induced tumor growth in vivo. Indeed, p16INK4A suppresses Leptin at the mRNA level through miR-141 and miR-146b-5p, which inhibits Leptin expression through a specific sequence at the Leptin 3’UTR. These results show the existence of active adipocytes in the mammary gland of obese women in absence of breast cancer cells and provide the first indication that p16INK4A through targeting miR-141 and miR-146b-5p suppresses Leptin and its procarcinogenic effects. This indicates that p16 pathway has non-cell-autonomous tumor suppressor function in breast stromal adipocytes.

Note: This abstract was not presented at the meeting.

Citation Format: Huda H. Alkhalaf, Abdelilah Aboussekhra. Obesity-induced destabilization of p16INK4A promotes the procarcinogenic effects of breast stromal adipocytes through miR-141/miR-146b-5p-dependent inhibition of leptin [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 5937. doi:10.1158/1538-7445.AM2017-5937
Abstract 5938: In vivo targeting of laminin-411-β1 integrin-Notch signaling pathway using nanobioconjugate alters glioma microenvironment for effective treatment
Authors: Ljubimova Julia Y.
Published: July 2017

Introduction: Brain gliomas have been extensively characterized by genomic and molecular marker analysis. These studies have underscored glioblastoma multiforme (GBM) heterogeneity, which may underlie failure of even the newest drugs to improve patient survival. For this reason, we turned to genetic modulation of glioma microenvironment using a novel nanobioconjugate.

Methods/Results: Clinical material from 107 GBM patients was analyzed, and tumors with overexpression of “malignant” tumor vascular basement membrane laminin-411 were found to have higher recurrence rate and shorter patient survival. In 92% of human GBM samples, overexpression of laminin-411 was associated with high expression of cancer stem cell (CSC) markers. To examine the interaction mechanisms between GBM cells and their extracellular matrix microenvironment, blood-brain barrier (BBB) passing nanobioconjugates based on poly(β-L-malic acid) (PMLA) were synthesized that specifically inhibit α4 and β1 chains of trimeric laminin-411. In vitro. Normal brain endothelial cells and astrocytes had higher expression of β1 integrin, Notch-1, and Notch ligands when seeded on “malignant” laminin-411 as compared with “normal” laminin-421. All these markers were downregulated in two GBM cell lines treated with antisense oligonucleotides against laminin α4 and β1 chains, suggesting regulation of Notch pathway by laminin-411 through integrin β1. In vivo. In GBM xenograft mouse models increased expression of laminin-411 correlated with overexpression of integrin β1 and Notch signaling pathway members. In two mouse models with intracranial human LN229 and U87MG GBMs, treatment with PMLA-based nanobioconjugate against tumor microenvironment protein laminin-411 led to significantly increased animal survival, associated with marked suppression of laminin-411- β1 integrin-Dll4-Notch axis and CSC markers CD133, Nestin, and c-Myc.

Conclusion: BBB crossing and brain tumor-targeted nanodrug therapy using laminin-411 suppression provided a unique tool to study mechanistic interactions between tumor microenvironment and signaling pathways, and showed promise for efficient GBM treatment affecting both the bulk of tumor cells and CSC.

Support: NIH grants U01 CA151815, R01 CA136841, R01 CA188743, R01 CA209921

Citation Format: Julia Y. Ljubimova. In vivo targeting of laminin-411-β1 integrin-Notch signaling pathway using nanobioconjugate alters glioma microenvironment for effective treatment [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 5938. doi:10.1158/1538-7445.AM2017-5938
Abstract 5939: Dissemination of breast cancer
Authors: Kakkad Samata , Hanna Mikayla , Jacob Desmond , Krishnamachary Balaji , Bhujwalla Zaver M.
Published: July 2017

Breast cancer is the second leading cause of death in US women. Despite advances in early detection, diagnosis and treatment of breast cancer, metastasis and tumor recurrence persist leading to continued morbidity and mortality. Several breast cancer studies have highlighted the role of cyclooxygenase-2 (COX-2) and its interaction with the tumor microenvironment (TME) in breast cancer progression. The TME consists of stromal cells that include fibroblasts, macrophages, the extracellular matrix (ECM), and several secreted factors that contribute to aggressiveness of the cancer and resistance to therapy. Increased density of collagen 1 (Col1) fibers, a major structural component of the ECM in solid tumors, and their alignment in tumors frequently result in increased invasion and metastasis of cancer cells. To understand the role of COX-2 at the lung metastatic site, and its relationship with Col1 fibers, COX-2 overexpressing triple negative SUM-149 human breast cancer cells (SUM-149-COX-2) or empty vector SUM-149 cells (EV) were injected through tail vein in SCID mice to compare Col1 fiber patterns in metastatic lesions. We isolated the lungs, processed the tissue, and imaged for Col1 fiber by second harmonic generation microscopy on H&E stained sections. The number of cancer associated fibroblasts (CAFs) were detected in adjacent sections by immunostaining for α-SMA. COX-2 overexpression did not increase the size of the cancer cell emboli in blood vessels or the size of pulmonary metastasis formed in the lungs. However, COX-2 overexpression did significantly increase the number of pulmonary metastases. Significantly higher Col1 fiber volume was observed in emboli compared to pulmonary metastases independent of COX-2 overexpression. Interestingly, the Col1 fibers were significantly more aligned in the COX-2 overexpressing pulmonary metastases compared to COX-2 overexpressing and EV emboli. The COX-2 overexpressing emboli had significantly higher amounts of CAFs detected by α-SMA expression compared to the EV emboli and the EV and COX-2 overexpressing pulmonary metastases. Pulmonary metastases from COX-2 overexpressing cells were detected further away from the nearest blood vessel compared to the metastases from EV cells suggesting that COX-2 may facilitate the formation of distant metastases. These data provide new insights into the role of COX-2, Col1 fibers, and CAFs in enabling emboli to extravasate to form pulmonary metastatic foci. These insights are important as COX-2 upregulation during treatment may potentiate metastasis. This work was supported by NIH R01 CA82337.

Citation Format: Samata Kakkad, Mikayla Hanna, Desmond Jacob, Balaji Krishnamachary, Zaver M. Bhujwalla. Dissemination of 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 5939. doi:10.1158/1538-7445.AM2017-5939
Abstract 6: New chk1 inhibitor development & the feasibility of use of chk1 inhibitor with antimetabolite for NSCLC
Authors: Chung Injae
Published: July 2017

DNA damaging anticancer agents activate checkpoints that stop the cell cycle to prevent additional DNA replication & mitosis until damaged region has been repaired. One of the important regulatory proteins activated in DNA damage response pathway is Checkpoint kinase 1 (Chk1). Therefore, Chk1 inhibitors have emerged as promising new therapeutics for cancer. In order to find novel Chk1 inhibitor, we have conducted a structure based virtual screening. Several small molecules have shown 75 - 100 % inhibition of Chk1 in the binding affinity assay. Combined with the results of kinase assay with ATP & mechanism based cell assay, hit candidates has been led. Lung cancer is the leading cause of cancer related deaths in Korea. About 85 % of lung cancer is non-small cell lung carcinoma (NSCLC) worldwide. Chemotherapy for NSCLC is often regarded ineffective. To test the feasibility of Chk1 inhibitor to sensitize antimetabolite in NSCLC, growth inhibition induced by gemcitabine plus MK8776 was assessed in A549 & H1129 cells. These cells are insensitive to the MK8776 when treated as a single agent (IC50 >10 μM). In contrast, MK8776 reduced the concentration of gemcitabine required to inhibit H1299 or A540 cell growth by almost 3-30 fold or 6-15 fold, respectively, depending on the concentration used. These results would imply the potential clinical utility of Chk1 inhibitor as an adjuvant for antimetabolite such as gemcitabine for NSCLC. Further studies to evaluate the effectiveness of Chk1 inhibitor including new molecules would be warranted in additional preclinical and clinical settings. [The chemical library used in this study was kindly provided by Korea Chemical Bank & KIST. This study was supported by a grant (2015R1D1A1A01057014) from the National Research Foundation of Korea.]

Citation Format: Injae Chung. New chk1 inhibitor development & the feasibility of use of chk1 inhibitor with antimetabolite for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 6. doi:10.1158/1538-7445.AM2017-6
Abstract 60: Reducing toxicity of antibody-drug conjugates through modulation of pharmacokinetics
Authors: Simmons Jessica , Zapata Francisco , Neff-Laford Haley , Hunter Joshua , Cochran Julia , Burke Patrick , Lyon Robert P.
Published: July 2017

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications. Research on ADCs has revealed that the physicochemical properties of the drug-linker component can exert a significant impact on the disposition of the ADCs, particularly at higher levels of drug loading. We have recently reported (Nature Biotechnology 33, 733-735 (2015); Molecular Cancer Therapeutics, manuscript accepted) that these properties can be modulated through the judicious incorporation of small, discrete PEG chains of varying lengths into a monomethyl auristatin E (MMAE) drug-linker. Homogeneous DAR 8 ADCs prepared with these drug-linkers using native cysteine conjugation display a continuum of pharmacokinetic behaviors that mirror the length of the incorporated PEG chain. We selected four of these drug-linkers that span the range of observed pharmacokinetics and used them as model compounds to evaluate the impact of ADC clearance on the concentration profile of released MMAE in normal tissues and consequent toxicology in the Sprague-Dawley rat. Faster clearing ADCs (prepared with drug-linkers containing very short or no PEG modifier) produced higher tissue MMAE Cmax values at early post-dose time points relative to slower clearing ADCs that incorporate longer PEG chains. This finding indicates that MMAE concentrations in tissues are proportional to the rate at which the ADC is catabolized, a process which converts the conjugated payload into free drug. Faster clearing ADCs also exhibited diminished tolerability, with greater histologic depletion of bone marrow and more dramatic decreases and/or delayed recovery in select peripheral hematology parameters. These results provide a strategy for reducing the non-antigen-mediated toxicity of ADCs through modulation of pharmacokinetics.

Citation Format: Jessica Simmons, Francisco Zapata, Haley Neff-Laford, Joshua Hunter, Julia Cochran, Patrick Burke, Robert P. Lyon. Reducing toxicity of antibody-drug conjugates through modulation of pharmacokinetics [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 60. doi:10.1158/1538-7445.AM2017-60
Abstract 603: Evaluation of CD19 targeted T cells in relapsed or refractory ALL patients unable to afford allogenic bone marrow transplant in India
Authors: Dwivedi Alka , Kumar Sushant , Fry Terry , Narula Gaurav , Purwar Rahul
Published: July 2017

CAR-T cell therapy has demonstrated remarkable success in long-term remission of relapsed or refractory B-ALL. Unlike western countries, most of the relapsed/refractory B-ALL patients in India are unable to afford allogeneic stem cell transplant (allo-SCT), and simply choose palliation. To assess the potential use of CAR-T cells as a first line therapy, it is critical to evaluate CAR-T cell efficacy in patients unable to undergo allo-SCT. As a first step, a novel anti-CD19 CAR was designed and CD19-vector was produced using lentiviral mediated gene delivery system. To generate CD19+CAR T cells, T cells from healthy subjects were transduced and expanded either with rIL-2 or rIL-15. Almost 20-50% transduced T cells express CD19-CAR on their surface as analysed by flow cytometry. Next, efficacy of CD19-CAR-T cells was examined by cytotoxicity assay using CD19+ malignant B cell lines (Raji cells) and autologous B cells. CAR-T cells were able to kill majority of CD19+B cells effectively (100% killing). In addition, CAR-T cells secreted high levels of IFNΓ and IL-2. The CAR-T cells expanded in presence of rIL-15 showed fewer Treg compared to CAR-T cells expanded using rIL-2, however, there were no differences in numbers of T stem cells (TSC) and central memory T cells (TCM). In addition to data on healthy volunteers reflected above, results of above tests in relapsed/ refractory B-ALL patients is ongoing and being collated for inclusion in the final analysis. In conclusion, our data demonstrate the successful development of an indigenous CAR-T cell platform for subsequent use in clinical trials of CD19+B cell malignancies.

Citation Format: Alka Dwivedi, Sushant Kumar, Terry Fry, Gaurav Narula, Rahul Purwar. Evaluation of CD19 targeted T cells in relapsed or refractory ALL patients unable to afford allogenic bone marrow transplant in India [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 603. doi:10.1158/1538-7445.AM2017-603
Abstract 611: Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGFβ1 (TGFβ TRAP)
Authors: David Justin M. , Schlom Jeffrey , Lan Yan , Palena Claudia
Published: July 2017

Background: Evasion of antitumor immunity is widely regarded as a hallmark of cancer. Tumor cells can avoid immune destruction by upregulating the immune checkpoint molecule PD-L1, and antibodies targeting the PD-1/PD-L1 axis have demonstrated clinical efficacy in several tumor types. Antitumor immunity can also be suppressed by TGFβ, a pleiotropic cytokine that reduces innate and adaptive antitumor immune responses. Additionally, TGFβ impacts tumor cells by activating a differentiation program called epithelial-mesenchymal transition (EMT), in which tumor cells lose epithelial features and gain mesenchymal characteristics. The gain of mesenchymal traits is associated with metastasis, stemness, and resistance to multiple therapeutic modalities, including chemotherapy and radiation. The objective of this work is to investigate the therapeutic potential of M7824 in non-small cell lung cancer (NSCLC). M7824 is a first-in-class bi-functional immunotherapeutic agent designed to target two negative regulatory pathways in immunosuppression. It is a novel fusion protein comprised of the extracellular domain of human TGFβRII covalently linked to the C-terminus of the Fc domain of the fully human anti-PD-L1 IgG1 antibody avelumab.

Methodology: In vitro assays were conducted using NSCLC cell lines treated with TGFβ1 alone or in combination with avelumab or M7824 to assess the ability of M7824 to revert features of tumor cell EMT, including molecular marker expression, cell proliferation, and response to various chemotherapies. The ability of M7824 to mediate antibody-directed cellular cytotoxicity (ADCC) of TGFβ1-treated cells in vitro was also assessed.

Results: Treatment with TGFβ1 induced mesenchymal marker expression, reduced cell proliferation, and enhanced resistance to docetaxel, paclitaxel, and gemcitabine. Treatment with M7824, but not avelumab, was able to both prevent and revert these effects of TGFβ1. M7824 also exhibited the ability to mediate ADCC of TGFβ1-treated cells in vitro.

Conclusions: M7824 effectively antagonizes TGFβ1-mediated tumor cell mesenchymalization, including resistance to chemotherapy. These results suggest that M7824 has the potential to relieve immunosuppression and block treatment-resistance induced by TGFβ1 in patients with NSCLC.

Citation Format: Justin M. David, Jeffrey Schlom, Yan Lan, Claudia Palena. Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGF⓵ (TGFββ TRAP) [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 611. doi:10.1158/1538-7445.AM2017-611
Abstract 618: Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T cells
Authors: Li Peng , Du Xin , Xin Yun , Weng Jianyu , Lai Peilong
Published: July 2017

Chimeric antigen receptor (CAR) T cell immunotherapies have shown unprecedented success in treating leukemia but lack efficacy in solid tumors. Here, we generated 1928zT2 and m28zT2, targeting CD19 and mesothelin, respectively, by introducing the Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 2 (TLR2) to 1928z and m28z. T cells expressing 1928zT2 or m28zT2 showed enhanced effector function, expansion and persistency against CD19+ leukemic or mesothelin+ lung cancer cells in vitro and in vivo. In a patient with relapsed B cell acute lymphoblastic leukemia, a single dose of 5×104/kg 1928zT2 T cells resulted in robust expansion and leukemia eradication and led to complete remission. Hence, our results demonstrate that TLR2 signaling is a contributing component to CAR T cells for both leukemia and solid tumors and is capable of increasing the efficacy of CAR T cells and facilitating low dose clinical usage.

Citation Format: Peng Li, Xin Du, Yun Xin, Jianyu Weng, Peilong Lai. Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T cells [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 618. doi:10.1158/1538-7445.AM2017-618
Abstract 636: Comprehensive analysis of T cells responsive to neoantigens derived from tumor-specific genetic mutations
Authors: Sasada Tetsuro , Ohtake Junya , Wada Satoshi , Yada Erika , Yoshida Shintaro
Published: July 2017

Neoantigens derived from tumor-specific genetic mutations can be recognized as foreign by the host immune system, and might be suitable as target for cancer immunotherapy possibly due to their higher immunogenicity. In this study, to really know the immunogenicity of tumor-specific neoantigens, we comprehensively investigated T cell responses against neoantigens derived from genetic mutations in gastric cancer. Using next-generation sequencing, 156 missense mutations were identified in tumor cells from two gastric cancer patients. From them, we selected 30 potentially immunogenic amino acid sequences, which were derived from the mutations and predicted to potentially bind to HLA-class I (A*0201, A*0206, or A*2402) by an epitope prediction server, IEBD. We synthesized 30 kinds of 27mer long peptides, in which the mutated sequences were located in the center, and then cultured peripheral blood mononuclear cells (PBMC) from healthy donors in the presence of the synthetic peptides to evaluate whether they could really induce antigen-specific T cell responses. In the analysis with PBMC from 18 healthy donors, 27/30 (90%) synthetic peptides showed an ability to induce antigen-specific T cell responses in at least one donor, assessed by cytokine production assay. Among them, 15 peptides were immunogenic in more than one donor. The antigen-specific T cell responses were detected more frequently in CD4+ T cells (70%) than in CD8+ T cells (43%). The specificity of T cell responses to mutated sequences, but not to the corresponding wild type sequences, were confirmed in 5 of 8 (63%) peptides examined. In addition, antigen-specific T cell responses induced by mutated peptides were shown to be much higher than those induced by the corresponding wild type peptides. These findings clearly demonstrated high immunogenicity and specificity of neoantigens derived from tumor-specific genetic mutations. Further studies would be recommended to develop a novel immunotherapeutic approach, “personalized cancer vaccination”, targeting mutation-derived neoantigens.

Citation Format: Tetsuro Sasada, Junya Ohtake, Satoshi Wada, Erika Yada, Shintaro Yoshida. Comprehensive analysis of T cells responsive to neoantigens derived from tumor-specific genetic mutations [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 636. doi:10.1158/1538-7445.AM2017-636
Abstract 642: Detection of circulating antibodies against KRAS in patients with advanced cancers
Authors: Kheder Ed , Huang Helen J. , Wu Alice , Hong David S. , Piha-Paul Sarina A. , Karp Daniel D. , Fu Siqing , Subbiah Vivek , Tsimberidou Apostolia M. , Naing Aung , Diab Adi , Javle Milind , Kopetz Scott , Sood Anil K. , Kurie Jonathan M. , Meric-Bernstam Funda , Gleeson Martin , Janku Filip
Published: July 2017

Introduction: KRAS gene plays a major role in tumorigenesis, cell proliferation and survival. Yet, there has been no effective targeted therapy against KRAS mutation-mediated tumors. We hypothesized that KRAS can induce humoral-mediated immune response.

Methods: Plasma or serum samples from patients with progressing advanced cancers with or without KRAS mutations were tested for the presence of circulating KRAS antibodies. We used the Maverick Detection System (Genalyte, San Diego, CA), which can perform real-time detection of macromolecules in crude samples using biologically functionalized silicon photonic biosensors lithographically printed on disposable silicon chips.

Results: We collected serum or plasma samples from 213 patients with advanced cancers (KRAS-mutant, N=100; KRAS wild-type, N=113) and 50 (23%) were found to have circulating KRAS antibodies. There was no association between KRAS antibodies and tumor KRAS mutation status (21/100, 21% for KRAS-mutant vs. 29/113, 26% for KRAS wild-type; P=0.52). In addition, there was no difference in detection of KRAS antibodies in colorectal cancer (21/89, 24%) compared to other cancers (29/124, 23%; P=1.00). There was no difference in the median survival in patients with KRAS antibodies compared to patients without KRAS antibodies (9.0 months vs. 10.1 months; P= 0.825). Similarly, there was no difference in the median survival according to the presence of circulating KRAS antibodies in subgroups of patients with tumor KRAS mutations (P= 0.96) and without tumor KRAS mutations (P=0.63). On the contrary, the median survival of patients with tumor KRAS mutation was shorter compared to patients with KRAS wild-type (7.2 months vs. 11.5 months; P<0.001).

Conclusion: Circulating KRAS antibodies can be detected in 23% of patients with advanced cancers. Biological implications of circulating KRAS antibodies remain to be understood.

Citation Format: Ed Kheder, Helen J. Huang, Alice Wu, David S. Hong, Sarina A. Piha-Paul, Daniel D. Karp, Siqing Fu, Vivek Subbiah, Apostolia M. Tsimberidou, Aung Naing, Adi Diab, Milind Javle, Scott Kopetz, Anil K. Sood, Jonathan M. Kurie, Funda Meric-Bernstam, Martin Gleeson, Filip Janku. Detection of circulating antibodies against KRAS in patients with advanced cancers [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 642. doi:10.1158/1538-7445.AM2017-642
Abstract 659: Multiplex cocktails for immunotherapy targets: PD-L1 with tumor specific transcription factors
Authors: Altree-Tacha David , Yuan Wei , Yang George
Published: July 2017

Introduction

Blocking the interaction between the programmed cell death (PD)-1 protein and one of its ligands, PD-L1, has been reported to generate impressive antitumor responses. Therapeutics targeting PD-L1 in several cancers are currently in clinical trials, and the U.S. FDA has granted accelerated approval for Keytruda (pembrolizumab) to treat patients with advanced (metastatic) non-small cell lung cancer (NSCLC). Various pathology scoring and interpretation methods have been put forth to assay PD-L1 expression, and variability of non-PD-L1 staining across tumors may have important prognostic aspects. PD-L1 expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes. PD-L1 expression is also associated with certain subtypes of tumor-associated lymphocytes, macrophages and dendritic cells. In certain cancers, the cross reactivity of PD-L1 can make interpretation and scoring difficult. Thus, a strategy using IHC multiplex stains could help resolve challenging cases by cocktailing PD-L1 with target tissue transcription factor antibodies along with other potential markers.

Method and Materials

Formalin-fixed paraffin-embedded tissues and tissue microarrays for various tumor types including lung, bladder and melanoma were processed and cut at 4-5 microns. PD-L1 rabbit monoclonal antibody was cocktailed with the following antibodies: TTF-1 (lung cancer); CD163 (lung cancer); p40 + GATA3 (bladder cancer); and SOX10 (melanoma). Cocktails were detected with double stain detection systems using brown, red and blue chromogens for visualization. Sections were counterstained with a standard hematoxylin or Wiegert’s iron hematoxylin.

Results

PD-L1 and TTF-1 with or without CD163 (macrophage) and PD-L1 + CD163 were used in lung cancers. The nuclear staining of TTF-1 in lung adenocarcinoma (blue chromogen) helped define tumor positive PD-L1 (DAB) positive cells and CD163 (Fast Red) marked macrophages. PD-L1 combined with p40 and GATA3 was deployed for bladder cancers. PD-L1 membranous expression in tumor cells and co-expression of p40 and GATA3 gave robust nuclear staining in bladder tumor cells. Finally, SOX10 nuclear staining was observed in most melanoma cells and could be easily separated from PD-L1 membrane staining.

Conclusion

PD-L1 cocktailed with strategic nuclear or cytoplasmic antibodies can help discriminate tumor cells from non-tumor cells, and may facilitate quantitation or scoring methods for more accurate assessment of this key immunotherapy marker.

Citation Format: David Altree-Tacha, Wei Yuan, George Yang. Multiplex cocktails for immunotherapy targets: PD-L1 with tumor specific transcription factors [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 659. doi:10.1158/1538-7445.AM2017-659
Abstract 674: HPV16 was the preponderant type of HPV infection in cutaneous squamous cell carcinoma
Authors: Shi Gang , Fischer Anne C. , Zenilman Jonathan M. , Gravitt Patti E. , Fischer Max , Sander Inbal B. , Cuda Jonathan D. , Taube Janis M. , Lilo Mohammed , Lee W P Andrew , Tufaro Anthony P.
Published: July 2017

Purpose: Human papillomavirus (HPV) has been well established as a causative factor in mucosal oropharyngeal cancer and anogenital surface malignancies. However, its etiological role in cutaneous squamous cell carcinoma (cSCC) is still debated. The aim of this study is to identify the presence of HPV types in cSCC and identify their associations with cSCC.

Methods: 77 cSCC blocks from 54 patients underwent DNA isolation and a broad spectrum of HPV PCR tests to detect the presence of HPV genotypes by the use of a universal primer pair GP5+/GP6+. The resultant PCR products were cloned to T Vectors and subsequently underwent DNA sequencing and Nucleotide BLAST search to identify the HPV types in cSCC. To ensure the accuracy of the HPV PCR test, 16 blocks from 10 head and neck SCC (oropharyngeal) patients were also included in the HPV presence and type analyses. Cutaneous SCC patients’ demographics, pathology and clinical parameters were compared and analyzed statistically in conjunction with the HPV test results.

Results: HPV DNA was found in 37 of 77 (48.1%) blocks representing 32 of 54 (59.3%) cSCC patients. HPV16 was the preponderant type of HPV Infection, accounting for 28 of 32 (87.5%) and 30 of 37 (81.08%) HPV positive patients and blocks. Other HPV genotypes found included HPV18, 57, 10, 2, accounting for 4 of 32 (12.5%), 2 of 32 (6.25%), 2 of 32 (6.25%), 2 of 32 (6.25%) of the HPV positive patients and 4 of 37 (10.81%), 2 of 37 (5.41%), 2 of 37 (5.41%), 2 of 37 (5.41%) of the HPV positive blocks, respectively. HPV infection was significantly associated with the cSCC tumor size in this cohort of patients. Tumors with a size of ≤20mm were more frequently associated with HPV. Moreover, the average tumor size of the HPV positive group was also significantly smaller than that of the HPV negative group.

Conclusions: HPV infection is very commonly found in cSCC patients and HPV16 is the major type of HPV involved in cSCC oncogenesis, accounting for >80% of HPV infections. HPV is a contributing factor, which appears to be contributing to a smaller size of cSCC, which may improve the prognostic classification of patients to provide a new insight into tumorigenesis.

Citation Format: Gang Shi, Anne C. Fischer, Jonathan M. Zenilman, Patti E. Gravitt, Max Fischer, Inbal B. Sander, Jonathan D. Cuda, Janis M. Taube, Mohammed Lilo, W P Andrew Lee, Anthony P. Tufaro. HPV16 was the preponderant type of HPV infection in cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2017-674