bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4013: Transcriptionally mediated effects of radiation on the expression of immune susceptibility markers in murine and human melanoma
Authors: Werner Lauryn R. , Gressett Monica , Riegert Maureen , Huang Shyhmin , Kern Joseph G. , Erbe Amy , Harari Paul M. , Sondel Paul M. , Morris Zachary S.
Published: July 2016

Radiation therapy (RT) may enhance tumor susceptibility to immune response. We and others have observed a cooperative interaction between RT and various immunotherapies, which appears dependent on the relative sequencing of each therapy. We reported a synergistic interaction between RT and the antibody-dependent cell-mediated cytotoxicity (ADCC) response to tumor-specific monoclonal antibodies (mAb) in murine melanoma. We reported enhanced cooperative effect when immunotherapy is administered 6-10 days after RT compared to administration on days 1-5 or 11-15. We hypothesized that this might reflect a delayed, transcriptionally-mediated effect of RT on the expression of tumor markers of immune susceptibility. In this study, we investigated the effect of RT on a variety of immune susceptibility markers at both transcriptional and post-translational levels to explore mechanisms behind the observed RT-induced changes.

We used flow cytometry to examine the time course of phenotypic changes in immune susceptibility markers in B78 murine melanoma cells following in vitro RT. We observed a time- and RT dose-dependent increase in the expression of specific death receptors (Fas, DR5), as well as T cell co-stimulatory/co-repressor ligands (PD-L1, CD80). The timing of these changes correlated with tumor susceptibility to ADCC immune response in vivo. All protein expression changes observed by flow cytometry were found to occur over a similar time course by quantitative polymerase chain reaction (qPCR), suggesting that RT-induced protein expression changes were mediated by changes in transcriptional activity. Using high throughput qPCR, we observed a similar time course of transcriptional effects in additional markers of tumor cell immune susceptibility, including Fas, MHC I, CD40, and others. We compared the effect of RT on these markers in human and mouse melanoma cell lines and observed a comparable time course for transcript-level changes across species.

This study sheds light on the mechanistic basis of time sensitivity in the interaction of RT with ADCC in vivo and suggests opportunities to enhance anti-tumor immune response by combining RT and immunotherapeutic agents. Such findings bear relevance for research investigating the potential role for RT in driving and optimizing the response to various cancer immunotherapies.

Citation Format: Lauryn R. Werner, Monica Gressett, Maureen Riegert, Shyhmin Huang, Joseph G. Kern, Amy Erbe, Paul M. Harari, Paul M. Sondel, Zachary S. Morris. Transcriptionally mediated effects of radiation on the expression of immune susceptibility markers in murine and human melanoma. [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 4013.
Abstract 4014: Combination of high-dose irradiation and local interleukin-12 treatment enhance tumor killing and have less toxicities than either treatment alone
Authors: Hong Ji-Hong , Yu Ching-Fang , Chen Fang-Hsin , Chiang Chi-Shiun
Published: July 2016

Immunotherapy such as anti-PD-1/PD-L1 is effective in several types of tumors, but in general the response rate is less than 30%. Systemic administration of cytokine such as IL-12 could create anti-tumor immunity but has unacceptable side effects. This study is to test our hypothesis that combination of high-dose local radiotherapy (RT) and local production of IL-12 inside tumors within short period could have potent anti-tumor effects but low systemic toxicity. TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment, or irradiated with 25 Gy, or intra-tumorally injected with Ad-sc-IL12 virus (1×10sup8 pfu), or treated with both modalities at the tumor size of 4mm. RT alone and Ad-sc-IL12 alone could not shrink tumors and only caused tumor growth delay around 7 and 12 days, respectively. The combination of RT with Ad-sc-IL12 virus could shrink the tumors to the sizes of < 2mm and further extended the tumor growth delay more than 20 days. The percentage of lymphocytes and IFN-γproduction were evaluated by flow cytometry and ELISA. The percentages of CD4 cells in tumors were too low to be reliably measured in all groups, but the percentages of CD8 cells were increased in Ad-sc-IL12 virus-treated groups and even much higher in those combined with RT. For mice treated with Ad-sc-IL12 virus injection, the serum IL-12 levels were around 4.5 times increase at day7, but those combined with with RT had significantly lower levels than those treated with virus alone. The serum IFN-γ(Th1 response) levels were around 12 times higher in Ad-sc-IL12 groups at day7 as compared to control or RT only group, but had no significant differences between those treated with combined modality and with Ad-sc-IL12 virus alone. Liver damage is an index for IL-12 toxicity and the levels of GOT and GTP were also lower in those treated IL-12 combined with RT than those treated with IL-12 alone. This study supported our hypothesis and showed that combination of local IL-12 production and local RT are effective in increasing CD8 cells, exerting Th1 response, and enhancing tumor growth delay. The most important is that this combination has less systemic toxicity than local IL-12 production alone. This study provides evidence to develop new strategy for producing a strong anti-tumor and low toxicity for radioresistant tumors by combining RT and IL-12 immunotherapy.

Citation Format: Ji-Hong Hong, Ching-Fang Yu, Fang-Hsin Chen, Chi-Shiun Chiang. Combination of high-dose irradiation and local interleukin-12 treatment enhance tumor killing and have less toxicities than either treatment alone. [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 4014.
Abstract 4023: Combination therapy with immune checkpoint inhibitor and CCK-receptor blockade increases survival of pancreatic cancer
Authors: Smith Jill P. , Wang Shangzi , Reham Ajina , Jablonski Sandra A. , Weiner Louis M.
Published: July 2016

Introduction: Advanced pancreatic ductal adenocarcinoma (PDAC) has typically been resistant to chemotherapy and immune therapy; therefore, novel strategies are needed to enhance therapeutic response. Cholecystokinin receptors (CCKRs) are present on pancreatic cancer epithelial cells, fibroblasts of the microenvironment, and lymphocytes. We hypothesized that CCK receptor blockade would improve response to immune checkpoint blockade and survival by promoting influx of tumor infiltrating lymphocytes (TILs) and reducing fibrosis.

Methods: Subcutaneous tumors were established using Panc02 murine pancreatic cancer cells (1.0- or 2.0 x10E6) in a syngeneic immune competent mouse model. Three separate experiments were performed with 40 mice each. Mice (C57BL/6) were divided into 4 groups (N = 10 mice each) and treated with PBS (control), a CCKR antagonist (L364,718 or proglumide), an immune checkpoint blockade antibody (PD-1 or CTLA-4; Im-Ab), or the combination of CCKR antagonist and immune checkpoint blockade antibody (combination). Tumor growth and animal survival were evaluated. Tumors were subjected to immunohistochemical staining for TILs, trichrome staining, and flow cytometry.

Results: On day 50 after inoculation of 10E6 cells all control mice had died while 42%, 42% and 71% of L364,718, CTLA-4 mice, or combination-treated mice, respectively were alive (p = 0.02). By day 90 all mice died except one in the combination-treated group. In the experiment where mice were injected with 2×10E6 Panc02 cells, survival was also increased in mice receiving a proglumide and PD-1 antibody combination (p = 0.0009). CD8+TILs increased by 3-,6.3-,and 7.5-fold in CCKR antagonist, Im-Ab, and combination therapy, respectively compared to controls (p<0.0001). CD4+ TILs also significantly increased (p = 0.001) in the tumors of mice treated with the combination. Trichrome stain analysis revealed 50% less fibrosis in mice treated with CCKR antagonist compared to PBS controls (p = 0.017).

Conclusions: The combination of CCKR antagonism and immune checkpoint blockade antibodies significantly improve survival in a murine model of PDAC. The mechanism is associated with decreased fibrosis in the microenvironment and an accompanying influx of CD8+ and CD4+ TILs.

Supported by NCI CA50633, CA51008, and CA194745

Citation Format: Jill P. Smith, Shangzi Wang, Ajina Reham, Sandra A. Jablonski, Louis M. Weiner. Combination therapy with immune checkpoint inhibitor and CCK-receptor blockade increases survival of pancreatic 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 4023.
Abstract 4025: Impact of Kras mutant subtypes on PD-L1 expression in lung adenocarcinoma
Authors: Ilie Marius , Fazzalari Laetitia , Guibert Nicolas , Yazbeck Nathalie , Signetti Laurie , Hofman Véronique , Long Elodie , Zahaf Katia , Fayada Julien , Lespinet Virginie , Bordone Olivier , Tanga Virginie , Washetine Kevin , Vénissac Nicolas , Mouroux Jérôme , Marquette Charles Hugo , Brest Patrick , Hofman Paul
Published: July 2016

Clinical responses to immune checkpoint blockade by anti-PD-1/PD-L1 monoclonal antibodies in non-small-cell lung cancer (NSCLC) may be associated with PD-L1 expression. This study was undertaken to determine the expression profile of PD-L1 in patients with Kras-mutant lung adenocarcinoma (LUAD) and to investigate the activation of Kras codon subtypes as a mechanism of PD-L1 regulation.

Immunohistochemistry analysis of PD-L1 expression (SP142 clone) on 117 LUAD (KrasWT, n = 51; KrasG12D, n = 25; KrasG12V, n = 14; KrasG12C, n = 27), showed significantly greater expression of PD-L1 in both tumor and immune cells compartments in Kras mutant LUAD compared with KrasWT wild-type tumors (37% vs. 18%; P = 0.005). PD-L1+ tumors had greater CD66b+ neutrophil infiltrate and lower CD8+ T-cell infiltrate than PD-L1− tumors, notably in KrasG12D tumors.

To determine the effect of Kras codon subtypes on PD-L1 expression, stable cell lines were generated by transfection of KrasG12D, KrasG12V, KrasG12C and KrasWT plasmids into Beas2B bronchial cells. At basal level, mutant Kras led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, notably in KrasG12C and KrasG12V cells, suggesting transcriptional regulation. Moreover, there was differential activation of NF-kB, ERK and Pi3k/Akt pathways between Kras mutant subtypes. In addition, PD-L1 was upregulated 3-fold by stimulation with IFNγ, independently of the Kras codon subtypes. Instead, hypoxia significantly increased PD-L1 expression in KrasG12C and KrasG12D cells. Co-culture experiments with human PBMCs from healthy patients were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 expression by tumor cells induced by Kras mutations led to decreased PBMCs proliferation and increased apoptosis.

PD-L1 is expressed in 37% of Kras mutant LUAD, suggesting PD-L1 as a therapeutic target in this subset. According to the Kras mutation subtype, potential drugs targeting the NF-kB, ERK or Pi3k/Akt pathways may increase the antitumor adaptive immune responses.

Citation Format: Marius Ilie, Laetitia Fazzalari, Nicolas Guibert, Nathalie Yazbeck, Laurie Signetti, Véronique Hofman, Elodie Long, Katia Zahaf, Julien Fayada, Virginie Lespinet, Olivier Bordone, Virginie Tanga, Kevin Washetine, Nicolas Vénissac, Jérôme Mouroux, Charles Hugo Marquette, Patrick Brest, Paul Hofman. Impact of Kras mutant subtypes on PD-L1 expression in lung adenocarcinoma. [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 4025.
Abstract 403: Performance assessment of highly sensitive NGS assay for TP53
Authors: Fredebohm Johannes , Mehnert Daniel , Löber Ann-Kathrin , Shimizu Hiroyuki , Holtrup Frank , Diehl Frank
Published: July 2016

We have designed a highly sensitive assay based on the Safe-Sequencing technology(1) to detect de novo mutations in the TP53 gene. A custom panel was designed to cover 95% of all reported mutations in TP53. To demonstrate assay performance, we assessed LoD, LoB, reproducibility, and repeatability and evaluated concordance with BEAMing(2). A customized data analysis pipeline for ultra-deep sequencing runs was developed that includes extensive quality control and advanced mutation calling. This highly sensitive NGS-based workflow can be applied to monitor recurrent or minimal residual disease in cancer patients after surgery or chemotherapy.

References:

(1) Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. 2011 Jun 7; 108(23): 9530-9535

(2) Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods. 2006 Jul;3(7):551-9.

Citation Format: Johannes Fredebohm, Daniel Mehnert, Ann-Kathrin Löber, Hiroyuki Shimizu, Frank Holtrup, Frank Diehl. Performance assessment of highly sensitive NGS assay for TP53. [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 403.
Abstract 4036: Prognostic value of infiltrating immune cell profiling in lung adenocarcinoma
Authors: Kurebayashi Yutaka , Emoto Katsura , Hayashi Yuichiro , Kamiyama Ikuo , Ohtsuka Takashi , Asamura Hisao , Sakamoto Michiie
Published: July 2016

Neoplastic cancer cells and cancer stroma (including infiltrating immune cells) determine the biology and prognosis of cancer. Various types of adaptive and innate immune cells are known to infiltrate the cancer stroma. However, the patterns and spatial distribution of immune cell infiltration as well as their association with tumor histology remain poorly understood. To address these issues, we comprehensively analyzed the infiltrating immune cells present in lung adenocarcinoma. The principal types of both adaptive and innate infiltrating immune cells were immunohistochemically evaluated in the predominant histological components of 111 lung adenocarcinomas. The same analysis was also carried out on 143 samples of histological subtypes making up more than 20% of tumors. As a result, plasma cells and B cells with interfollicular distribution were almost exclusively observed in invasive histological subtypes, while increased number of mast cells were observed in noninvasive histological subtypes. Cluster analysis revealed four distinct immunosubtypes: those characterized by high CD8/CD3 ratios with low stromal cellularity (CD8 subtype), those with increased mast cell numbers (mast cell subtype), those with increased macrophage/DC infiltration with variable amounts of T cell infiltration (mϕ/DC subtype), and those with increased plasma cell infiltration in addition to increased macrophage/DC infiltration and variable increases in other infiltrating immune cells (T cells, Tregs, pDCs, and neutrophils) (plasma cell subtype). These immunosubtypes correlated with histological subtypes. Univariate and multivariate analyses identified the plasma cell subtype as an independent negative prognostic factor. Furthermore, we found that these plasma cells may be one of the major producers of immunosuppressive cytokine IL-35 in cancer stroma. Consequently, comprehensive immunohistochemistry-based profiling of infiltrating immune cells has potential as a powerful method for analyzing the cancer stroma of various cancer tissues.

Citation Format: Yutaka Kurebayashi, Katsura Emoto, Yuichiro Hayashi, Ikuo Kamiyama, Takashi Ohtsuka, Hisao Asamura, Michiie Sakamoto. Prognostic value of infiltrating immune cell profiling in lung adenocarcinoma. [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 4036.
Abstract 4038: Developmentof a murine tumor immunophenotyping platform to support drug discovery anddevelopment in immuno-oncology
Authors: Belmontes Brian , Matyas Stephanie , O’Brien Sarah , Tan Hong , Ganley Kenneth , Merriam Kimberly , Rottman Jim , Egen Jackson , Beltran Pedro , Moody Gordon
Published: July 2016

Recent clinical data highlights the importance of immune cell localization, phenotype, and gene signature as it correlates to a productive anti-tumor response. Preclinically, multiple syngeneic mouse models have been used to study the effects of immunomodulation and define anti-tumor responses to transplanted “self” tumors. However, while the literature describes distinct aspects of many of these models, there is no comprehensive dataset comparing and contrasting their tumor-immune microenvironments across models. These data are critical for better understanding the role that various immune populations play in the anti-tumor response and interpreting observed changes in tumor clearance following treatment with immunomodulatory agents. We have therefore established a platform that 1) quantitates the types of immune cells within murine tumor models, and 2) describes the location of these cells within the tumor. In parallel to the immunophenotyping efforts we have benchmarked tumor models based on their response to antibodies against T cell checkpoint pathways.

We sought to use this immunophenotyping platform to identify specific immune modulation that occurs in syngeneic tumors post depletion of macrophages via CSF1R blockade. Tumor-associated macrophages (TAMs) are believed to help promote tumor survival through suppression of the adaptive immune response and the secretion of growth factors that promote tumor growth and angiogenesis. Therefore depletion of TAMs should lead to T-cell recruitment and bolster the antitumor T-cell response. Here we show that treatment of CT-26 and RENCA syngeneic tumors with a CSF1R antagonist leads to depletion of MHCII+ and F4/80+ expressing cells. Future experiments will seek to understand if CSF1R blockade improves the response to T-cell checkpoint immunotherapies. In summary, the development of a murine tumor immunophenotyping platform has allowed insight and evaluation of immune cells in the tumor microenvironment that can ultimately be leveraged to understand the synergistic effects of immunotherapeutics.

Citation Format: Brian Belmontes, Stephanie Matyas, Sarah O’Brien, Hong Tan, Kenneth Ganley, Kimberly Merriam, Jim Rottman, Jackson Egen, Pedro Beltran, Gordon Moody. Developmentof a murine tumor immunophenotyping platform to support drug discovery anddevelopment in immuno-oncology. [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 4038.
Abstract 4039: Mimotope variance analysis: A novel immunoprofiling method to monitor progression of cervical cancer
Authors: Enerly Espen , Nygård Mari , Orumaa Madleen , Pihlak Arno , Pihelgas Susan , Langseth Hilde , Neuman Toomas , Palm Kaia
Published: July 2016

The purpose of the study was to identify and describe cervical cancer specific changes in humoral immune response by using a novel technology platform Mimotope Variation Analysis (MVA) developed by Protobios.

The Janus Serum Bank is one of the world's oldest and largest population-based research biobanks established in 1973. The cohort is annually linked to the Cancer Registry of Norway using personal identification numbers. More than 1000 women have developed cervical cancer after donating sera. We identified sets of successive sera samples from ten patients with invasive cervical cancer, ten patients with pre-invasive cervical neoplasia and twenty cancer-free individuals (matched for age, gender, length of sample storage +3 months). For each subject we identified 4-10 samples donated to Janus over a period of up to 18 years, in total 213 samples. We applied MVA which combines phage display technology and high-throughput sequencing analysis to generate quantitative serologic profiles of millions of 12-mer peptide antigens called mimotopes from 2 μl of blood serum per analysis.

Hierarchical clustering analysis of the top 5000 mimotopes from each sample resulted in individual-specific immunoprofiles. Multiple samples from the same person clustered together. Moreover, clustering reflected the time the sera was drawn suggesting that part of the individual immunoprofile is stabile over time.

In conclusion, preliminary results suggests that Mimotope Variance Analysis generates individual-specific immunological profiles. This profile most likely reflect prior exposure environmental pathogens. Further, we aim to decode the differences in the immunological profiles between cancer patients and cancer-free subjects.

Citation Format: Espen Enerly, Mari Nygård, Madleen Orumaa, Arno Pihlak, Susan Pihelgas, Hilde Langseth, Toomas Neuman, Kaia Palm. Mimotope variance analysis: A novel immunoprofiling method to monitor progression of cervical 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 4039.
Abstract 4048: Stem cell properties of normal human keratinocytes influence transformation responses to HPV16
Authors: Woappi Yvon , Creek Kim E. , Pirisi Lucia
Published: July 2016

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. While HPV infection is very common, cervical cancer is a rare outcome. Why some women develop cervical cancer and others do not is not well understood, but likely involves host cell-specific factors and a woman's ability to get rid of HPV-infected cells. We have previously reported marked variability of epidermal growth factor receptor (EGFR) expression in normal human epidermal tissue from different individuals and demonstrated that increased immortalization by HPV16 E6 and E7 oncogenes correlated with lower basal levels of EGFR. We now report a significantly greater transformation response in stem cell-like keratinocytes after transfection with the full length HPV16 genome. Primary normal human keratinocyte strains (NHKc) from 59 individuals were cultured in 3-D anchorage-free suspension to assess spheroid forming ability and to determine resistance to anchorage-independent cell death. Only about 40% of NHKc strains survived and formed spheroids in 3-D culture and grew when plated back in monolayers as spheroid-derived NHKc (SD-NHKc). Fluorescence-activated cell sorting analysis found that SD-NHKc were enriched in cells expressing known keratinocyte stem cell markers: low levels of EGFR and high levels of integrin α6, while spheroid-non forming strains (SNF-NHKc) had dramatically reduced numbers of EGFRlo/ITGα6hi cells. Isolation and cultivation of EGFRlo/ITGα6hi keratinocytes isolated from primary monolayer NHKc cultures revealed this cell population to behave like SD-NHKc with respect to anchorage-independent survival, proliferative potential, cell morphology, and clonogenicity. Upon transfection with HPV16 DNA, most NHKc strains acquired anchorage-independence and exhibited increased expression of EGFR. Importantly, EGFRlo/ITGα6hi keratinocytes and SD-NHKc displayed exuberant growth responses after transfection with HPV16 DNA and significantly higher transformation efficiency by HPV16. In contrast, SNF-NHKc grew more sluggishly and were relatively resistant to HPV16-mediated immortalization. Our study indicates that stem cell-like keratinocytes are preferentially transformed by HPV16 DNA and that stemness is a phenotypic determinant of susceptibility to HPV16-mediated transformation in primary human keratinocytes.

Citation Format: Yvon Woappi, Kim E. Creek, Lucia Pirisi. Stem cell properties of normal human keratinocytes influence transformation responses to HPV16. [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 4048.
Abstract 4049: Conserved region 2 of Human Papilloma Virus 18 protein E7 is required for E7 binding to centromere protein C
Authors: Tokuda Aoi , Takahashi Shoko , Yoshimoto Masafumi , Yaginuma Yuji
Published: July 2016

The molecular genetics of cervical cancer have not been thoroughly elucidated, despite the fact that cervical cancer is responsible for the deaths of over 250,000 women annually worldwide. Infection with human papillomaviruses (HPVs) is linked to the pathogenesis of a variety of human diseases, including genital warts, laryngeal papillomas, and most notably, cervical cancer. Recently HPV infection was reported to be associated with head and neck cancers.High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. HPV infection leads to aneuploidy, a numerical chromosomal aberration caused by dysregulation of chromosomal segregation. We found that high-risk HPV18 and HPV58 E7 proteins bind to centromere protein C (CENP-C), a component of the kinetochore that is essential for proper chromosomal segregation. Low-risk HPV4, HPV6, and HPV11 E7s do not bind to CENP-C. The PxDLLCxE sequence in conserved region 2 (CR2) of HPV18 E7 is required for E7 binding to CENP-C. Our results indicate that differences in the ability of high- and low-risk HPV E7s to bind to CENP-C reflect the different oncogenic potentials of high- and low-risk type HPVs.We demonstrated that the E7 proteins from low-risk HPVs do not bind to CENP-C, and that the PxDLLCxE sequence in conserved region 2 of the HPV18 E7 protein is required for E7 binding to CENP-C. CENP-C is a component of the inner kinetochore, and as such, it plays an essential role in proper chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Therefore, we hypothesize that differences in the capability of various E7s from low- and high-risk type HPVs to bind to CENP-C reflect the different oncogenic potential of high- and low-risk type HPVs.

Citation Format: Aoi Tokuda, Shoko Takahashi, Masafumi Yoshimoto, Yuji Yaginuma. Conserved region 2 of Human Papilloma Virus 18 protein E7 is required for E7 binding to centromere protein C. [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 4049.
Abstract 4067: Cell phone use is associated with an inflammatory cytokine profile of parotid gland saliva
Authors: Gomez Ricardo S. , Siqueira Elisa C. , Souza Fabrício Tinôco A. , Ferreira Efigênia F. , Souza Renan P. , Friedman Eitan , Gomez Marcus V. , Gomes Carolina C.
Published: July 2016

There is controversy on the effects of the non-ionizing radiation emitted by cell phones on cellular processes and the impact of such radiation exposure on health. Conflicting results were reported on the association between cell phone use and parotid tumor development. Chronic inflammation is associated with an increased risk for cancer, such as seen in ulcerative colitis. To evaluate the effect that cell phone use has on the parotid gland, cytokine expression profile was determined in the saliva produced by the parotid glands in healthy volunteers, and correlated with self-reported cell phone use and laterality. The following parameters were determined, in 83 Brazilian individuals in saliva produced by the parotid glands comparing the saliva from the gland exposed to cell phone radiation (ipsilateral) to that from the contralateral parotid: salivary flow, total protein concentration, IL-1β, IL-6, IL-10, IFN-γ and TNF-α by ELISA. After multiple testing correction, decreased IL-10 and increased IL-1β salivary levels in the ipsilateral side compared with the contralateral side (p<0.05) were detected. Subjects who used cell phones for more than 10 years presented higher differences between IL-10 levels in ipsilateral versus contralateral parotids (p = 0.0012). No difference was observed in any of the tested parameters in correlation with cell phone monthly usage in minutes. We conclude that exposure of parotid glands to cell phones can alter salivary IL-10 and IL-1β levels. These findings suggest a pro-inflammatory profile on parotid naturally exposed to cell phones, which can be associated not only to the radiation emitted by the device but with the warming caused by radiation and the battery of the cell during use.

Supported by: FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil

Citation Format: Ricardo S. Gomez, Elisa C. Siqueira, Fabrício Tinôco A. Souza, Efigênia F. Ferreira, Renan P. Souza, Eitan Friedman, Marcus V. Gomez, Carolina C. Gomes. Cell phone use is associated with an inflammatory cytokine profile of parotid gland saliva. [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 4067.
Abstract 4069: The carcinogenic effects of electronic cigarettes in oral cancer
Authors: Korrapati Avinaash , Yu Vicky , Saad Maarouf A. , Rahimy Mehran , Xuan Yinan , Zou Angela , Krishnan Aswini , Brumund Kevin , Ongkeko Weg M.
Published: July 2016

Alcohol consumption, tobacco use, and human papillomavirus infection have been well-established as the primary risk factors for head and neck squamous cell carcinoma (HNSCC). However, the surge in popularity of e-cigarettes (e-cigs) has prompted speculation of e-cig use potentially emerging as a new risk factor. With previous research on the safety of e-cigs still collectively inconclusive, a comprehensive study of the carcinogenicity of these devices remains urgently necessary. We therefore investigated the potential genotoxic, cytotoxic, and invasive and migratory effects of e-cig vapor exposure on human epithelial cells. A panel of normal epithelial and head and neck cancer cell lines was treated with 0.5%, 1.0%, and 2.0% by volume e-cig vapor from two popular e-cig brands, over periods ranging from 24 hours to 4 weeks. Neutral comet assay and immunostaining for γ-H2AX foci revealed significant induction of DNA double-stranded breaks (up to 3-fold increase, p < 0.05) in cell lines incubated with both nicotinized and nicotine-free e-cig vapor. To evaluate the cytotoxicity of e-cigarettes, trypan blue exclusion and clonogenic assays were performed following short-term e-vapor exposure. Our results indicate that in epithelial cells, short-term treatment induces up to a 5-fold increase in cell death without nicotine, and up to a 10-fold increase with nicotine as compared to untreated controls (p < 0.001). We subsequently assessed the effects of e-cigarettes on HNSCC progression and metastatic potential through exposure of established HNSCC cell lines to e-cigarette vapor. Wound healing assays revealed increased migration of HNSCC cells following e-cigarette treatment, with significant upregulation of key EMT-promoting genes observed via qRT-PCR. We will next examine the effects of long-term (6-9 months) e-cig vapor exposure in vivo using mouse models. Mouse oral epithelium will be harvested for histologic analysis and sequenced to determine the mutagenic potential of long-term e-cig vapor exposure. Nevertheless, our present findings based on short-term e-vapor exposure alone already pose alarming implications for the carcinogenic effects of e-cigarette use.

Citation Format: Avinaash Korrapati, Vicky Yu, Maarouf A. Saad, Mehran Rahimy, Yinan Xuan, Angela Zou, Aswini Krishnan, Kevin Brumund, Weg M. Ongkeko. The carcinogenic effects of electronic cigarettes in oral 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 4069.
Abstract 4072: Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins
Authors: Tate Karen , Tran Lucy , Baker Melanie , Evans Kamilah , Rouse Mechelle , Chung Seyung , Nguyen Duy , Kim Juri , Smith-Joe Enijah , Vadgama Jay , Li Mengtao , Lin Yu-Ling , Patillo Roland , McGhee Eva
Published: July 2016

Human Papillomavirus (HPV) high-risk types 16 and 18 are directly associated with approximately 90% incidence of invasive cervical carcinoma. Epithelial cells infected with HPV become transformed and acquire genomic instability. As a result of this transformation, E6 and E7 oncogenes are perpetually expressed. E6 degrades the tumor suppressor p53; E7 inhibits tumor suppressor pRB, leading to disproportionate target cell growth and proliferation. While there have been studies exploring genomic instability induced by HPV oncoproteins E6/E7, the full scope of the genomic damage has not been clearly characterized. Three HPV positive cervical cancer cell lines, HeLa (HPV18), SiHa (HPV16) and CaSki (HPV16), were studied using Spectral Karyotyping (SKY) and Giemsa banding (G-banding). Our results using SKY and G-banding analysis showed HeLa cells exhibited frequent translocations on chromosomes 4 and 11, and deletions on chromosomes 11 and 20. The SiHa cells exhibited translocations primarily on chromosomes 9 and 20, with deletions on chromosome 10. CaSki revealed translocations on chromosomes 10 and 4, with deletions on chromosome 21. All three cell lines gained copies on chromosome 5. The aforementioned aneuploidy is demonstrative of the induced genomic instability acquired from HPV infection, which include tumor suppressor genes; p53 and pRB disruptions caused by E6 and E7 oncoproteins. HeLa cells showed extensive genomic instability on chromosome 11q22-23, where the ATM gene is located. Acquired genomic instability to the ATM chromosome region may further increase the rate of cervical cancer.

Citation Format: Karen Tate, Lucy Tran, Melanie Baker, Kamilah Evans, Mechelle Rouse, Seyung Chung, Duy Nguyen, Juri Kim, Enijah Smith-Joe, Jay Vadgama, Mengtao Li, Yu-Ling Lin, Roland Patillo, Eva McGhee. Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins. [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 4072.
Abstract 4074: Macrophages promote the progression of premalignant mammary gland lesions through activation of the Axl signaling cascade
Authors: Carron Emily C. , Machado Heather L. , Homra Samuel
Published: July 2016

Infiltrating inflammatory cells, including tumor-associated macrophages (TAMs), have been shown to promote breast cancer cell invasion and have been correlated with metastasis and poor prognosis. While it is well-established that macrophages are recruited to the invasive fronts of established tumors to exert pro-tumor signals, their role in premalignancy remains poorly understood. Using a novel p53−/− syngeneic transplantable model of premalignancy, we show that inflammatory cells, including macrophages, are indeed recruited to early lesions prior to invasion. Microarray analysis was performed on two different pre-invasive lines, termed PN1a, (high tumor-forming potential) and PN1b (low tumor-forming potential). Interestingly, several pro-inflammatory chemokines, macrophages markers, and the pro-inflammatory cytokine growth arrest specific 6 (Gas6) were increased in PN1a lesions as compared to PN1bs. In a 3-D co-culture system, macrophages were recruited to PN1a acini and induced invasion, while PN1b cells remained non-invasive, supporting our in vivo data. Depletion of macrophages in vivo with clodronate containing liposomes significantly delayed progression of PN1a lesions to invasive cancer, indicating a critical role for macrophages in early progression. Gas6 and its receptor tyrosine kinase, Axl, were highly expressed in PN1a preinvasive lesions, but decline in invasive tumors, suggesting that this pathway is a key driver of the transition from pre-invasive to invasive cancer. Further studies using our 3-D co-culture system with Gas6−/− macrophages demonstrated that macrophages promote the formation of non-polar disorganized structures by activating Axl. Finally, transplantation of PN1a cells into Gas6 +/− mice resulted in a delay in tumor formation, indicating that stromal-derived Gas6 (and potentially macrophage derived) may promote the progression of early stage lesions through the paracrine activation of Axl. As a plethora of Axl inhibitors have been developed and are currently in clinical trial, these studies have critical implications for the prevention and treatment of invasive breast cancer.

Citation Format: Emily C. Carron, Heather L. Machado, Samuel Homra. Macrophages promote the progression of premalignant mammary gland lesions through activation of the Axl signaling cascade. [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 4074.
Abstract 4087: Mammary fibroblasts exert divergent effects on the survival of invasive breast cancer cells
Authors: Hagel Kimberly , Weigel Kelsey , Boyd Meaghan , McCormack Luke , Champion Matthew , Schafer Zachary
Published: July 2016

Cancer development and progression are inherently inefficient processes riddled with anti-tumorigenic obstacles. Anoikis, or apoptosis induced by detachment from the extracellular matrix (ECM), represents one prominent hindrance to cancer progression that transformed cells must overcome in order to survive, particularly during the ECM-bereft metastatic cascade. Far from acting in isolation, we report that one mechanisms of anoikis evasion by invasive breast cancer cells involves the exploitation of carcinoma-associated fibroblasts (CAFs) from the surrounding tumor microenvironment. CAFs secrete insulin-like growth factor-binding proteins (IGFBPs) that facilitate non-canonical activation of integrin-linked kinase (ILK), activation of ERK/MAPK, and ultimately stabilization of the anti-apoptotic protein Mcl-1 to promote cancer cell survival during ECM detachment. Further investigation into the influence of the tumor microenvironment on cancer cell survival revealed that, contrary to the role of CAFs, normal mammary fibroblasts (NMFs) appear to exert anti-tumorigenic effects on ECM attached cancer cells. Utilizing conditioned media from NMFs, we have found that NMFs secrete factors that increase caspase activation in multiple cancer cell lines, suggestive of NMF-mediated cancer cell death. Because cancer progression necessitates the intermingling of cancer cells with normal stromal cells, particularly at early time points in primary tumor and metastases formation, we hypothesize that this NMFs pose yet another obstacle to disease progression that may compliment the process of anoikis by capping the metastatic cascade with environments that are inhospitable to cancer cell survival. Similar to anoikis evasion, our data suggests that cancer cells must evade NMF-induced death stimuli in order to survive, possibly through the accrual of CAFs in the tumor microenvironment to combat or replace the pro-apoptotic NMFs. Using traditional and 3D mammalian cell culture in combination with in vivo studies, we aim to delineate the roles of both CAFs and NMFs on cancer cell survival under conditions of differential ECM availability. A more complete understanding of the nuances dictating cancer cell-fibroblast interactions could yield informative data relevant to chemotherapeutic research and development.

Citation Format: Kimberly Hagel, Kelsey Weigel, Meaghan Boyd, Luke McCormack, Matthew Champion, Zachary Schafer. Mammary fibroblasts exert divergent effects on the survival of invasive breast cancer 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 4087.
Abstract 4089: Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells
Authors: Tuli Neha Yashpal , Bednarczyk Robert B. , Ben Rahoma Ghada M. , Moscatello Augustine , Geliebter Jan , Tiwari Raj K.
Published: July 2016

Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy.

Citation Format: Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory 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 4089.
Abstract 409: Immunofluorescent digital slide technology to evaluate correlation between Glypican-3 expression and response to codrituzumab
Authors: Tanaka Takayoshi , Sugitani Yasuo , Abe Tokiya , Kawaida Miho , Yamazaki Ken , Hashiguchi Akinori , Sakamoto Michiie , Ohtomo Toshihiko
Published: July 2016

Background: Codrituzumab/GC33/RO5137382 is a recombinant humanized monoclonal antibody against Glypican-3 (GPC3) which is highly expressed in HCC cells. As part of phase I clinical trial, GC-002US study, evaluating escalating doses of codrituzumab plus sorafenib in HCC, immunofluorescent staining of GPC3 were applied in addition to GPC3 immunohistochemistry (IHC) with conventional DAB staining. The objectives of this work were to compare GPC3 expression levels by different staining methods and evaluate the correlation between these GPC3 scores and codrituzumab activities.

Methods: Biopsies specimens (n = 34) collected at baseline in the phase I trial were evaluated by GPC3-IHC and immunofluorescent quantification digital slide (IQD) methods. GPC3-IHC were conducted using mouse anti-human GPC3 antibody, mouse GC33 (Ventana Medical Systems) as reported previously (Ikeda M. et al., Cancer Sci. 2014: 105; 455-462) and H scores were derived by two pathologists. For IQD, biopsy specimens were stained with same primary antibody, and were incubated with Qdot 655-conjugated secondary antibody. Fluorescent images of whole slides were obtained with the Nano-Zoomer (Hamamatsu Photonics K. K.). The IQD intensity score (immunofluorescent intensity per pixel) and cell score (immunofluorescent intensity per cell) were calculated respectively using MatLab (MathWorks Inc.).

Results: There was a statistically significant correlation between GPC3 H scores and IQD scores. Among GPC3 scores, only the IQD cell score was correlated with serum GPC3 C-terminus increase by treatment (p = 0.032), whereas in the patients treated with higher dose of GC33 (≥ 10 mg/kg/week) the cytoplasmic H and the IQD intensity score were also correlated with serum GPC3 C-terminus increase by treatment. Interestingly, increasing cut-off values of IQD cell score levels led to lower HR and longer PFS. Best HR with lowest p value was observed at a cut-off value of 66,183 (HR = 0.24, p = 0.038).

Conclusion: This study suggested that higher GPC3 expression on HCC cells quantified by IQD method was superior to other scores to predict codrituzumab activity, though this analysis was conducted only with limited number of subjects assessed. Further assessment and validation in clinical study of codrituzumab would be necessary to make a final conclusion on the clinical application of GPC3 IQD.

Citation Format: Takayoshi Tanaka, Yasuo Sugitani, Tokiya Abe, Miho Kawaida, Ken Yamazaki, Akinori Hashiguchi, Michiie Sakamoto, Toshihiko Ohtomo. Immunofluorescent digital slide technology to evaluate correlation between Glypican-3 expression and response to codrituzumab. [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 409.