bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5541: Clinical impact of hypothyroidism and PD-L1 SNPs in patients having non-small cell lung cancer treated with nivolumab
Authors: Funazo Tomoko , Ozasa Hiroaki , Nomizo Takashi , Tsuji Takahiro , Yasuda Yuto , Yoshida Hironori , Sakamori Yuichi , Nagai Hiroki , Hirai Toyohiro , Kim Young Hak
Published: August 2018

Although Nivolumab is one of the immune-checkpoint inhibitors that improves the prognosis of lung cancer, it is known to cause immune-related adverse events (irAEs). It has recently been reported that patients with irAEs have a longer progression-free survival (PFS) than those without irAEs. However, there are few detailed reports on irAEs in patients with NSCLC. We previously reported the association between single nucleotide polymorphisms (SNPs) of programmed death ligand 1 (PD-L1) and PFS for nivolumab treatment. Therefore, we hypothesized that the SNPs of programed death 1 (PD-1) and PD-L1 were associated with irAEs and PFS.

Between January 2016 and March 2017, 95 consecutive patients with advanced NSCLC were treated with nivolumab and 80 of these patients participated in this study. Patients who did not provide informed consent, patients without follow-up blood tests, patients with double cancer history, and patients diagnosed with disease progression within 15 days were excluded. The prevalence of genotypes between patients with adverse events and those without adverse events was evaluated. PFS was calculated in 59 out of 80 patients excluding liver metastasis at baseline imaging.

The response rate was 15% and the median PFS was 85 days in this cohort. For all adverse events, hypothyroidism, which is defined as a low free T4 level, was associated with SNPs of PD-L1: rs1411262 and rs822339. Moreover, patients with hypothyroidism had a significantly longer PFS than those without hypothyroidism (67 days vs N.R.; P= 0.0055). The T/T genotype of rs1411262 and the A/A genotype of rs822339 were significantly associated with longer PFS than the C/T and C/C genotypes of rs1411262, and the A/G and G/G genotypes of rs822339, respectively (82 versus 175 days; P = 0.0468).

In conclusion, the T allele of rs1411262 and the A allele of rs822339 were significantly associated with hypothyroidism and longer PFS. SNPs of PD-L1 may be associated with functions of the PD-1 and PD-L1 pathway.

Hypothyroidism and SNPs of PD-L1hypothyroidism (-)hypothyroidism (+)P value(n=7)(n=73)rs14112620190.0386CC336CT418TTrs822339AA0190.0386AG336GG418

Citation Format: Tomoko Funazo, Hiroaki Ozasa, Takashi Nomizo, Takahiro Tsuji, Yuto Yasuda, Hironori Yoshida, Yuichi Sakamori, Hiroki Nagai, Toyohiro Hirai, Young Hak Kim. Clinical impact of hypothyroidism and PD-L1 SNPs in patients having non-small cell lung cancer treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5541.
Abstract 5543: Low ATM expression is associated with improved progression-free and overall survival in advanced gastric cancer patients treated with platinum-based chemotherapy
Authors: Bhangoo Munveer S. , Luu Hubert Y. , Kim Seung Tae , Lee Jeeyun , Chao Joseph , Klempner Samuel J.
Published: August 2018

Introduction: Platinum-based combination chemotherapy regimens represent standard approaches for locally advanced and metastatic gastric adenocarcinoma. Ataxia-teleangiectasia mutated protein (ATM) plays a critical role in repairing DNA damage to double-stand breaks. Tumors deficient in ATM (defined as pathologic loss measured by IHC) are hypothesized to demonstrate increased susceptibility platinum chemotherapy.

Methods: We retrospectively reviewed patients from a single center (Samsung Medical Center, Seoul, South Korea) with advanced, unresectable gastric cancer (stage 3b or higher) receiving capecitabine/oxaliplatin (XELOX) from 2013-2017. Clinicopathologic data, treatment response, progression-free survival (PFS), and overall survival (OS) were analyzed according to level of ATM expression by immunohistochemistry (IHC). Low-expression of ATM was defined as greater than 50% loss by IHC. Two-tailed, unpaired t-tests and Fisher's exact tests were used to compare outcomes between patients with preserved ATM versus low ATM expression. Kaplan-Meier survival analysis was used to compare disease-free survival and overall survival; the log-rank test was used to determine statistical significance.

Results: During the study period, 138 patients underwent treatment with XELOX for a diagnosis of advanced gastric cancer. Median (IQR) follow-up time was 11.8 mo. (6.8-17.3). Between patients with ATMintact or ATMlow, there were no significant differences in mean age, sex, final pathologic diagnosis, or mean number of cycles of chemotherapy received. Kaplan-Meier analysis demonstrated that patients with ATM low were associated with a longer PFS (ATMlow 14.9 mo versus ATMintact 6.8 mo.) (HR 0.24, [0.18-0.34, 95% CI], p<0.0001), and longer OS (ATMlow 30.0 mo. versus ATMintact 14.7) (HR 0.23, [0.17-0.33, 95% CI], p<0.0001). There was no difference in response rates between the two groups.

Conclusions: This retrospective, case-control study in patients with advanced gastric cancer shows improved survival in patients with ATMlow tumors compared with ATMintact tumors after treatment with platinum-based chemotherapy. Findings are suggestive and require prospective validation in cohorts of patients with ATMlow/ATMintact tumors.

Citation Format: Munveer S. Bhangoo, Hubert Y. Luu, Seung Tae Kim, Jeeyun Lee, Joseph Chao, Samuel J. Klempner. Low ATM expression is associated with improved progression-free and overall survival in advanced gastric cancer patients treated with platinum-based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5543.
Abstract 5546: eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response
Authors: Sharma Rajesh K. , Goel Vikas K. , Staunton Jocelyn , Barrera Maria , Parra Ana , Sung Eric , Chiang Gary G. , Webster Kevin R.
Published: August 2018

An effective and durable T-cell response is a cornerstone of current immunotherapies. We show that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes a regulatory program that promotes multiple steps in the cancer immunity cycle including expansion of memory T cells and prevention of T-cell exhaustion. Using OT-I and OT-II transgenic systems, we show that eFT508 shifts the distribution of T cells towards a CD62LhighCD44high central memory (CM) phenotype in both CD4 and CD8 T cells upon activation with SIINFEKL peptide in vitro without adverse effects on T-cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where eFT508 treatment also enriches the CM T-cell pool in a SIINFEKL vaccine-induced OT-I adoptive T-cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T-cell response upon rechallenge. In addition, the CM bias elicited by eFT508 remains dominant when combined with agonists of co-stimulatory molecules, such as 4-1BB, OX-40 and GITR, or checkpoint inhibitors, such as PD-1, PD-L1 and CTLA-4, suggesting that eFT508 can affect the rate of T-cell differentiation in these combinations. eFT508 treatment also reduces the expression of exhaustion markers such as PD-1, LAG3 and TIM3, leading to increased cytotoxic T-cell function. eFT508 is currently under evaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solid tumors and patients with advanced lymphoma. In addition, a phase 2 study evaluating eFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed or refractory CRC patients is ongoing. The preclinical studies presented here provide further evidence that eFT508 may combine well with additional immunotherapies beyond checkpoint blockade.

Citation Format: Rajesh K. Sharma, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Ana Parra, Eric Sung, Gary G. Chiang, Kevin R. Webster. eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5546.
Abstract 556: Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants
Authors: Flores Brianna , Hull Elizabeth E.
Published: August 2018

The tumor suppressor p53 protein is mutated in over 50% of cancers. The most frequent mutations in p53, termed “hotspot” mutations, occur in the DNA binding domain of the p53 protein. These mutations not only remove normal p53 function but also convey additional oncogenic properties greater than loss of tumor suppressor activities associated with loss of wild-type (WT) p53 activities. With newly acquired oncogenic properties, mutations in the DNA binding domain of p53 are classified as gain of function (GOF) mutations. GOF p53 mutants can be subdivided into conformational mutations (e.g. R175H, H193Y) involving residues necessary for maintenance of structure and contact mutations (e.g. R248Q, R273H) involving resides necessary for DNA interaction. Cancer cells bearing a GOF p53 mutation increase metastasis with application of histone deacetylase inhibitors (HDACi). This uncharacterized pro-oncogenic GOF p53 property is of clinical relevance as HDACi are FDA approved for hematological cancers but have failed in clinical trials for solid tumors. To explore this hypothesis, metastatic capacity and proliferation rates of common GOF p53 mutations were assessed with and without HDACi treatment and in the presence or absence of specific inhibitors of target GOF p53. The p53-null, H1299 non-small cell lung carcinoma line was transfected with GOF p53 mutants and WT p53 using a tetracycline inducible expression system. Inducible expression of stable subclones was varified by immunofluorescence and western blot. Proliferation rate was measured by incorporation of EdU and metastatic capacity was assessed by measuring MMP activity and migration assays with and without HDACi treatment. Specific GOF p53 mutations possess a subset of the oncogenic properties of the entire GOF p53 class. Data suggest that the GOF p53 mutants display differing rates of proliferation with the R175H mutation exhibiting the highest rate. Application of NSC319726 significantly decreases the proliferation rate and appears to successfully refold the R175H mutant to a WT conformation. The R248Q mutation exhibited morphological change suggestive of the epithelial to mesenchymal transition associated with increased metastatic capacity. Preliminary data suggest that HDACi affect among these cell lines in conjunction with NSC 319726 and SCH 529074 treatment. In conclusion, HDACi treatment may be contraindicated while treatment with HDACi may have great benefit in tumors which are WT or null for p53 protein expression.

Citation Format: Brianna Flores, Elizabeth E. Hull. Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 556.
Abstract 5563: Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins
Authors: Ongaro Tiziano , Bigatti Martina , Sladojevich Filippo , Donckele Etienne , Villa Alessandra Micaela , Neri Dario
Published: August 2018

Cytokines are able to mediate a potent anticancer activity but have significant side effects that prevent dose escalation to therapeutically-active regimens. Targeting the cytokines to the site of disease helps to improve their therapeutic index while decreasing associated toxicities. However, peak concentrations in blood are still associated with side-effects. We have developed a novel technology (termed “CORK Technology”), which consists in masking the undesired activity of a cytokine in circulation. The “cork” can be either an antibody fragment or a small molecule, which specifically binds to the cytokine thus preventing the interaction with its receptor. Once the complex accumulates in the tumor, the cork dissociate from the cytokine allowing it to exert its function. Capitalizing on the fact that the kinetic dissociation constant koff of the complex is identical in different species, this technology can be easily translated from mouse to man with comparable results. In this poster we describe the experimental implementation of CORK Technology to improve the therapeutic index of the clinical stage L19-IL2 (L19 is an antibody fragment specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis). Two classes of IL2 inhibitors were discovered and characterized: (i) a fully human scFv fragment, and (ii) a small organic ligand, featuring a modified methylindole moiety. Both types of agents reduced IL2 activity in vitro, were able to dissociate from the cognate cytokine in a kinetically-tuned process and allowed the administration of the L19-IL2 fusion protein at higher doses, both in normal and in tumor-bearing mice. The technology is applicable to other cytokine payloads and facilitates the development of targeted cytokine products with “activity on demand” (i.e., with increased activity at the tumor site, promoting an activation and proliferation of tumor-resident lymphocytes). Albeit still in its infancy, this elegant approach provides promising results and a strong rationale to further investigate this therapeutic power of the CORK Technology.

Citation Format: Tiziano Ongaro, Martina Bigatti, Filippo Sladojevich, Etienne Donckele, Alessandra Micaela Villa, Dario Neri. Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5563.
Abstract 557: Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia
Authors: Shimada Akira , Kanzaki Hirotaka , Ishida Hisashi , Matsubara Takehiro , Aoe Michinori
Published: August 2018

Introduction: Not only somatic mutations, but also epigenetic modifications in acute myeloid leukemia (AML) are estimated to have a pivotal role in leukemogenesis and it could be a therapeutic target. Especially, aberrant methylation of the promotor region of tumor suppressor genes (TSGs) were the causative events in leukemogenesis, however, there was only limited information in pediatric myelodysplastic syndrome (MDS) and AML. Materials and Methods: We analyzed the aberrant methylation of promotor region of 25 TSGs in pediatric 7 low grade MDS, 4 advanced MDS and 22 AML patients by FAB classification by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) method (10 Male, 12 female, 0-14 years old, median 6 years ols). Simultaneously, detection of SNVs, In/del, and copy number variants were also analyzed by target sequence of more than 150 leukemia related genes. Especially, specific chimeras were analyzed in each patient. Results: Total 12/22(54.5%) patients had more than one methylated TSGs (number of methylated TSGs was 0-5 in each patient, median 1). More than 20% of patients had methylated TSGs including CDKN2B, CADM1, CDH13 and ESR1. Especially, there was a specific methylation pattern of TSGs, for example, all (4/4) t(8;21) patients had CDH13 methylation, 3/4 (75%) patients with inv(16) had ESR1 mutation and normal karyotype did not. Normal control samples, complete remission samples, and low grade MDS samples were not completely methylated. All advanced MDS patients had 1 or 2 methylated TSGs. Simultaneously, several leukemogenic genes were mutated such as GATA1, IKZF1, KRAS, KIT, FLT3, SH2B3, PIK3CD, PU.1, SMARC1, ALK, etc. Several genes were deleted such as CTCF, RB1, SRP72, ATM, NF1, U2AF1 etc. Discussion: In adult MDS, the increased number of methylated TSGs correlated to the development of AML and the poor prognosis. In this study, a few TSGs were methylated as the adult study (Hess CJ., Leuk Lymphoma 2008), furthermore, many genes related to epigenetic modification such as ASXL1, TET2 and EZH2 were not mutated in this study. Recently, anti-methylation drug were used in adult MDS/AML, larger study will be needed for adjusted use of epigenetic modifier in pediatric MDS/AML.

Citation Format: Akira Shimada, Hirotaka Kanzaki, Hisashi Ishida, Takehiro Matsubara, Michinori Aoe. Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 557.
Abstract 560: Identification of a novel MUTYH mutation in non-small cell lung cancer
Authors: Fong Alvin Hong-Wai , Lau Eunice Yuen-Ting , Cheung William Ka-Chun , Cho William Chi-Shing
Published: August 2018

Introduction: Lung cancer is the leading cause of death from cancer in Hong Kong and worldwide. Nearly 80-85% of lung cancers are diagnosed with non-small cell lung cancer (NSCLC). Treatment strategies can be refined according to the distinct difference of NSCLC molecular profiling among different ethnic populations. Moreover, oncogenic driver mutations are closely linked to NSCLC in non-smokers and young people. Therefore, the urge of finding pathogenic mutations is inevitable. This study aims to discover novel mutations in NSCLC patients in Hong Kong by next-generation sequencing and characterize the clinical significance of these mutations. Methods: In order to identify mutations, DNA was purified from 6 pairs of lung tumor-normal tissues from 2 stage I adenocarcinoma (LUAD), 2 stage III LUAD and 2 stage I squamous cell carcinoma (LUSC) patients. Samples were sequenced by targeted sequencing with the TOMA OS-Seq 130 cancer gene panel. Sequence alignment and variant calling were performed by combining TOMA Stratus and in-house pipelines. Results: The mean aligned reads exceeded 14 million and the mean coverage of target region was over 1000X. MUTYH deletion was found in 66.7% (4/6) of the patients. Data from The Cancer Genome Atlas (TCGA) revealed that MUTYH deletion is significantly associated with poorer disease-free survival (DFS) (p=0.0063) and overall survival (p=0.022) in LUAD patients. TCGA data also showed that MUTYH copy number correlates with gene expression (r=0.473) and LUAD patients with MUTYH mRNA overexpression have better DFS (p=0.0205). However, such correlations were not observed in LUSC patients. MUTYH is a DNA glycosylase which involves in the base excision repair and it repairs oxidative DNA damage by preventing G:C to T:A transversion. Numerous studies indicated that biallelic mutations in MUTYH cause MUTYH-associated polyposis and increase the lifetime risk of colorectal cancer. MUTYH mutations could be associated with increased risk of endometrial and breast cancer. Conclusion: To elucidate the clinical significance of MUTYH in LUAD, an independent cohort will be sequenced to validate the primary result. Immunohistochemistry, RT-qPCR and functional assays will be performed to examine the protein expression, the gene expression and the functional role. All these findings suggested that MUTYH deletion may play a crucial role in LUAD and potentially lead to the development of biomarker and therapeutic target.

Citation Format: Alvin Hong-Wai Fong, Eunice Yuen-Ting Lau, William Ka-Chun Cheung, William Chi-Shing Cho. Identification of a novel MUTYH mutation in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 560.
Abstract 5651: Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging
Authors: Natan Michael
Published: August 2018

With very high spatial and biological complexity, tumor micro-environments present a significant

characterization challenge, which is only heightened by the need to image entire sections of cancer

tissue on many different samples. A variety of techniques offer very high levels of multiplexing, but

always fail on either speed requirements (incredibly long times required to image a whole slide) or on

compatibility issues (expensive, hard-to-operate CapEx not suitable for high-throughput CLIA operation).

Ultivue has developed a platform reagent technology (InSituPlex™) that simultaneously enables whole-

slide higher multiplexing by immunofluorescence AND complete compatibility with existing

instrumentation.

The InSituPlex technology comprises five key foundational elements. First, short unique DNA barcodes

are covalently attached to each primary antibody (Ab). These conjugates are purified and functionally

characterized in tissue for optimal staining specificity. Importantly, all of this work is done in advance by

Ultivue. Second, tissue samples are stained in a single step. Third, all the DNA sequences attached to

targets (via primary Ab binding) are simultaneously amplified, allowing for detection of less abundant

markers e.g. PD-1, PDL-1 etc. Fourth, complementary DNA sequences each with different, covalently-

attached fluorescent labels (“imager strands”) are simultaneously hybridized to the amplified DNA on

the targets. All these wet chemistry steps are carried out in walkaway mode using commercially

available auto-stainers. The samples are then imaged using a standard, whole-slide multi-color

fluorescence imaging system. Finally, in an optional fifth step, the imager strands are gently removed

via an enzymatic dehybridzation- with complete retention of antigenicity- to enable higher multiplexing

via additional rounds of hybridization/imaging.

The InSituPlex reagent technology offers many advantages for cancer research over current approaches:

• the ability to detect 5, 10, or more targets simultaneously via whole-slide imaging on an each

and every sample, with no loss in antigenicity;

• the ability to leverage existing auto-stainers and existing fluorescent microscopes with

Ultivue's reagent kits to carry out high-throughput analyses of multiple cancer

biomarkers; and

• the ability to design custom biomarker panels that leverage Ultivue's unique barcoding

capability and infrastructure.

This poster will elaborate on each step of the technology, and provide multiple examples of data

acquired using different biomarker panels and different cancer tissue types.

Citation Format: Michael Natan. Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5651.
Abstract 5657: Use of a novel immunofluorescence multiplexing technology, InSituPlex™, for the simultaneous detection of immune cells in multiple cancer types
Authors: Downing Sean , Patel Katir , Buell Jamie , Hebert Courtney
Published: August 2018

Introduction: The field of immuno-oncology has enthusiastically adopted multiplexed

immunohistochemistry (IHC) techniques to establish the spatial relationships between various

immune cells in tumor biology. Multiplexing enables researchers to gain a deeper understanding

and insight into the tumor microenvironment. Unfortunately, many of the multiplexing

technologies currently utilized in the immuno-oncology field face a number of challenges. These

challenges include complicated and difficult assay optimizations for high-level multiplexing and

the need of specialized imaging instrumentation. In addition, harsh stripping methods and

irreversible staining techniques compromise the integrity of precious tissue samples that ultimately

can no longer be used for additional forms of testing. InSituPlex™ is a new method of multiplexed

IHC that relies on the detection of a unique DNA barcode conjugated to primary antibodies. This

technology removes the need to perform multiple, harsh stripping methods to perform multiplexed

IHC and allows for higher order of multiplexing in less time than currently available multiplexing

techniques, all the while maintaining tissue integrity for use in downstream analyses.

Methods: InSituPlex was used to perform a multiplexed IHC assay on 5 samples from 4 tumor

types (breast, lung, colon, and melanoma). The assay consisted of the following markers: CD4,

CD8, CD20, CD68, PD1, Ki67, FoxP3, and a tumor marker; pan-cytokeratin (CK) for breast,

colon, and lung carcinomas and Sox10 for melanoma. Staining was performed using the Leica

BOND RX autostainer, imaging was performed on various platforms including the Leica VERSA

whole slide scanner, and image analysis was performed using HALO from Indica Labs.

Data: All markers were detected utilizing the InSituPlex technology across all tumor types. Data

indicates a range of phenotypes for infiltrating immune cells in tumor types from highly inflamed

to “immune deserts”. The panel provided for analysis of many phenotypic subtypes, (e.g.,

CD4+/PD1+, CD8+/Ki67+, and Ki67+/CK+), cell densities, proliferative indexes, and spatial

analysis (e.g., CD8+ to tumor cells).

Conclusion: InSituPlex technology is proficient in maintaining tissue integrity and detecting a

range of immune cell phenotypes in multiple tumor types. InSituPlex can be implemented by any

lab possessing a standard fluorescent microscope and does not rely on the purchase of, nor access

to, more expensive platforms.

Citation Format: Sean Downing, Katir Patel, Jamie Buell, Courtney Hebert. Use of a novel immunofluorescence multiplexing technology, InSituPlex™, for the simultaneous detection of immune cells in multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5657.
Abstract 5663: Proteomic analysis of exosomes isolated from ccRCC cell lines
Authors: Mizutani Kosuke , Kawakami Kyojiro , Fujita Yasunori , Horie Kengo , Ito Masafumi , Deguchi Takashi
Published: August 2018

Purposes of the study: Clear renal cell carcinoma (ccRCC) is one of the most lethal malignant tumors. Molecular targeted therapies have been used to improve outcomes for metastatic or advanced ccRCC. However, currently approved therapies have limited success and a cure remains elusive. Since ccRCC could be curable if diagnosed at the early stage, novel diagnostic approach is needed to identify circulating markers that may help diagnose early and evaluate prognosis. Exosomes are membrane vesicles with a diameter of 40-150 nm that are secreted by various types of cells including cancer cells. Exosomes contain a variety of molecular constituents of their origin such as proteins and nucleic acids. Over the years, exosome has been suggested to be a novel diagnostic marker. The purpose of this study is to find candidates of ccRCC markers in exosomes. Experimental procedures: Exosomes were collected from cell culture supernatant of RPTEC human renal proximal tubule epithelial cells and ccRCC cell lines (Caki-1, KMRC-1, and OS-RC-2) by ultracentrifugation. Isolation of exosome was confirmed by western blot analysis for CD9. Exosome fraction was analyzed by iTRAQ-based quantitative proteomics. Results: A total of 52 proteins were detected and 3 proteins were found to be upregulated by more than 1.2 fold in exosomes isolated from ccRCC cells compared with those from RPTEC cells. Twelve proteins were found to be downregulated by less than 0.8 fold in exosomes from ccRCC cells compared with those from RPTEC cells. The expression profiles of several proteins were verified by western blot analysis. Conclusions: While major challenges still remain, exosome studies in ccRCC could provide a wealth of information to find novel diagnostic and prognostic markers.

Citation Format: Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Kengo Horie, Masafumi Ito, Takashi Deguchi. Proteomic analysis of exosomes isolated from ccRCC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5663.
Abstract 5668: The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction
Authors: Boohaker Rebecca J. , Sambandam Vijaya , Suto Mark , Xu Bo
Published: August 2018

Tumor cells exploit immune checkpoints by expressing checkpoint ligands such as PD-L1, effectively masking them from immune destruction. Programmed cell death- 1 (PD-1), plays a major role in tumor immune escape. The interaction of PD-1/PD-L1 inhibits cytotoxic T lymphocyte (CTL) proliferation, induces apoptosis of tumor-specific T cells, and promotes the resistance of tumor cells to CTL attack. Monoclonal antibodies to these proteins have been developed and approved for use in the clinic. Theoretically, peptide mimics, which have the benefit of lower half-life and significantly smaller size, are able to penetrate into solid tumors and tissues better than antibodies, and can function as inhibitors for the PD1/PD-L1 interaction. Here we report for the first time the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 immune checkpoint as a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We established the binding constant for the interaction and PL120131 reduces association of PD-L1 in a dose dependent manner. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity. Understanding the mechanism(s) of action of

this peptide has yielded information on the pathways that are drivers of immune evasion in a tumor microenvironment, which may improve and synergize with other forms of immunotherapy, thus increasing efficacy of cancer treatment. Outcomes from this study have provided a path forward to further development of small molecule inhibitors to the PD-1/PD-L1 interaction, and outlines a strategy for the design and development of small molecules to additional cell-surface checkpoint proteins.

Citation Format: Rebecca J. Boohaker, Vijaya Sambandam, Mark Suto, Bo Xu. The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5668.
Abstract 5672: Facile generation of single-cell transcriptome and immune repertoire freshly isolated from clinical tumor specimens
Authors: Zhu Junjie , Salahudeen Ameen A. , Giangarra Valeria , Montesclaros Luz , Sapida Jerald , Sharifi Osman , Lee Josephine , Zheng Grace X. , Wagh Dhananjay , Coller John , Sabatti Chiara , Kuo Calvin J.
Published: August 2018

Immunotherapies including cell-based therapies generate deep and durable responses in patients with chemotherapy-refractory cancers. However, in solid tumors, particularly those with stereotypic driver mutations and resultant neoantigens, the clonality and cell states of tumor-infiltrating lymphocytes (TILs) remain poorly understood. Here, we use a droplet-based 5' single-cell RNA-sequencing (scRNA-seq) to simultaneously profile transcriptome and immune repertoire of the same cells, enabling the phenotypic characterization (including cytotoxic and helper T, B, T regulatory and exhausted T cells) of each clonotype. We performed scRNA-seq on unsorted and CD45+-FACS sorted cells from fresh clinical samples of multiple tissue types, including lung, liver and kidney, and obtained on average tens of thousands of cells per sample. We demonstrated that high-quality single-cell suspension can be rapidly and reliably generated from clinical samples. We devised different diversity metrics to appropriately classify the immune repertoire within samples, and cross samples of different tissues. We observed distinct cell type compositions and, more importantly, context-specific T-cell clonal expansion patterns, suggesting the activation of different molecular programs in these tumors. In addition, comparison of non-small cell lung cancer (NSCLC) tumors varying by histology type and patient smoking status showed differences among these phenotypes, suggesting a potential link between immune microenvironments and cancer etiology. In summary, we provide a proof of concept for rapid generation of large number of single-cell transcriptomes of TILs paired with their corresponding TCR cDNA sequence in fresh tumor samples across different tissue types. Further analysis with this methodology on larger clinical cohorts will provide robust correlative prognostic markers of clinical phenotypes of immunotherapy responses. Our analysis strategy of TCR sequences among large clinical cohorts across multiple tumor types will facilitate cell-based therapeutic efforts, including CAR T cell or autologous T cell therapies.

Citation Format: Junjie Zhu, Ameen A. Salahudeen, Valeria Giangarra, Luz Montesclaros, Jerald Sapida, Osman Sharifi, Josephine Lee, Grace X. Zheng, Dhananjay Wagh, John Coller, Chiara Sabatti, Calvin J. Kuo. Facile generation of single-cell transcriptome and immune repertoire freshly isolated from clinical tumor specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5672.
Abstract 5688: Humanized double knock-in mouse models for evaluating in vivo efficacy of the combination therapy of immune-oncology drugs
Authors: Gan Tian , Jin James , Guo Chaoshe , Shen Yuelei , Guo Yanan
Published: August 2018

Over the past years, monoclonal antibodies have been successfully utilized in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA-4, PD-1, PD-L1 and OX40 et.al,. Although significant benefits including complete regression and long-term survival were reported among the majority of participants, not all patients respond. In order to optimize the efficacy and minimize toxicities of anticancer immunotherapies, immune checkpoint antibodies are being tested in combination with other conventional therapeutics (radiation, chemotherapy and targeted therapies) and newer immunotherapies (cancer vaccines, oncolytic viruses). However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially for the combination therapy of two IO antibodies. Therefore, we generated humanized double knock-in mouse to evaluate the in vivo efficacy of combination therapy. For example, humanized double Knock-in B-hPD-1/hOX40 mice pairing with mouse cancer cells, demonstrated a synergistic effect of using hPD-1 and hOX40 antibodies. Additionally, more double knock-in mice targeting immune checkpoint molecules were developed, such as B-hPD-1/hPD-L1, B-hPD-1/hCTLA-4, B-hPD-1/hLAG3, B-hPD-1/hTIM3, B-hPD-1/hTIGIT, B-hPD-1/hBTLA, et al,. Humanized double knock-in mice, represent useful in vivo efficacy models for evaluation of the combination effects of various IO drugs.

Citation Format: Tian Gan, James Jin, Chaoshe Guo, Yuelei Shen, Yanan Guo. Humanized double knock-in mouse models for evaluating in vivo efficacy of the combination therapy of immune-oncology drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5688.
Abstract 5694: Evaluation of the in vivo efficacy of immune-oncology drugs targeting stimulatory immune checkpoint molecules using humanized knock-in mice
Authors: Xia Tongmei , Guo Chaoshe , Shen Yuelei , Guo Yanan
Published: August 2018

Cancer immunotherapy is one of the most promising research areas in the field of cancer therapy. Many pharmaceutical and biotech companies in the world are devoting great effort to develop cancer immunity-related treatment antibodies. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step. In most cases, a human monoclonal antibody does not have mouse cross-reactivity. Mouse surrogate antibodies were often used in immune-competent syngeneic mouse models to evaluate in vivo efficacy of IO drugs. However, the efficacy of a surrogate antibody cannot fully represent the human drug in the clinical scenario. Therefore, we generated humanized knock-in mice to evaluate the in vivo efficacy of human IO antibodies. For example, human OX40 knock-in (B-hOX40) mice were generated with a chimeric OX40 receptor, which is recognizable by stimulatory human OX40 antibodies. Additionally, more knock-in mice targeting stimulatory immune checkpoint molecules were developed and validated, such as B-hCD137, B-hGITR, B-hCD27, B-hCD40, B-hCD28, B-hCD3 et al,. All these mouse models response well to the corresponding human IO antibodies, proving that they are powerful tools for in vivo efficacy evaluation of human stimulatory immune checkpoint antibodies.

Citation Format: Tongmei Xia, Chaoshe Guo, Yuelei Shen, Yanan Guo. Evaluation of the in vivo efficacy of immune-oncology drugs targeting stimulatory immune checkpoint molecules using humanized knock-in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5694.
Abstract 5695: Use of certain antihistamines among breast cancer patients confers survival benefit
Authors: Olsson Hakan L.
Published: August 2018

H1-antihistamines have not been widely studied in relation to cancer therapy, despite being among the most common medications used globally, and having become increasingly sophisticated, cheap and safe.

In a nation-wide study of the entire Swedish population using the Drug Prescription Registry, Cancer Registry and Cause of Death Registry- the first of its kind - we investigated the effects of the six major antihistamines (cetirizine, clemastine, desloratadine, ebastine, fexofenadine and loratadine) on breast cancer-related (as well as all-cause) mortality. Our study includes 61,627 Swedish women with breast cancer diagnosed between July 1st 2005 and December 31st 2013. 14,711 used at least one of the six major antihistamines. Both peri- and post-diagnostic antihistamine use was analyzed, as well as the use among different subgroups of patients, divided according to age, stage and estrogen receptor status, was analyzed.

Here, we show a consistently beneficial effect of desloratadine and ebastine use on breast cancer survival (in the peri-diagnostic analysis, for desloratadine, HR=0.79 [95% CI 0.63-0.99], P=0.044; and for ebastine users, HR=0.57 [95% CI 0.32-1.03], P=0.064; relative to non-users) and a consistently negative effect on breast cancer survival of clemastine use (in the peri-diagnostic analysis, HR=1.24 [95% CI 1.05-1.47], P=0.013; relative to non-users). Loratadine use is associated with better survival in the peri-diagnostic analysis (HR=0.78 [95% CI 0.63-0.97], P=0.022) relative to non-use. No significant effects were seen for cetirizine use or fexofenadine use. Post diagnostic use. A woman with breast cancer using desloratadine will lower her relative risk of breast cancer-related death with 15% after one

year's use and 17% after two years' use (both risks relative to a woman of the same age and the same tumor status).

Conclusion. Use of desloratadine and ebastine may may significantly improve survival in breast cancer with few side effects.

Citation Format: Hakan L. Olsson. Use of certain antihistamines among breast cancer patients confers survival benefit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5695.
Abstract 5724: Homologous recombination repair gene mutations for DNA repair and immune oncology drug combinations
Authors: Dry Jonathan R. , Lai Zhongwu , Dougherty Brian , Barrett Carl , Criscione Steven
Published: August 2018

Exciting clinical benefit has been seen from both immune checkpoint inhibitors and PARP inhibitors in several cancer types. Initial reports have suggested the tumor mutational burden (TMB) may be a tumor agnostic marker for predicting immunotherapy (IO) response. Deficiencies in DNA damage repair (DDR) resulting from BRCA1 and BRCA2 mutations can lead to an accumulation of tumor mutations, and sensitivity to PARP inhibitors. We hypothesized that patients with germline or somatic mutations in BRCA1/2 or other homologous recombination repair (HRR) genes, termed “HRRm”, will have higher TMB, higher immune infiltration and activity, and greater benefit from the combination of DDR and IO therapies. We investigated the relationship between DDR deficiencies, TMB, PDL1, transcriptomic immunoscores and drug response across The Cancer Genome Atlas (TCGA) disease cohorts, data from AstraZeneca's clinical studies and a linked NSCLC genomic and EHR dataset from Foundation Medicine and FlatIron Health. HRRm cancer samples show higher TMB in several cancer types. This does trend with higher PDL1 but does not associate with transcriptomic immunoscores. HRRm could be a practical clinical biomarker to select patients with DDR defects likely to benefit from combination of DDR and IO therapies.

Citation Format: Jonathan R. Dry, Zhongwu Lai, Brian Dougherty, Carl Barrett, Steven Criscione. Homologous recombination repair gene mutations for DNA repair and immune oncology drug combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5724.
Abstract 5728: DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer
Authors: Ettayebi Ilias , Mehdipour Parinaz , Singhania Rajat , Chakravarthy Ankur , Medina Tiago , Shen Shu Yi , Ishak Charles , Roulois David , Carvalho Daniel De
Published: August 2018

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Times New Roman'} Colorectal cancer (CRC) is the second leading cause of death from cancer in men and the third in women in Canada. There is accumulating evidence suggesting that CRC is organized in a hierarchical manner, at the apex of which are the Cancer-Initiating Cells (CIC). Furthermore, results from xenograft models and human clinical trials indicate a selective enrichment of CICs in tumours that are resistant to therapy, suggesting that targeting CICs may represent a new paradigm in cancer treatment. We have previously reported the treatment with low dose of the DNA demethylating agent, 5-Aza-2'-deoxycytidine (5-AZA-CdR), can target CICs through the activation of the RIG1-MDA5 viral sensing sensing pathway, leading to an anti-viral response in cancer cells. Although it is now known that 5-AZA-CdR induces a state of “viral mimicry” in these cancer cells, the mechanism by which 5-AZA-CdR can specifically target CICs is not well understood. In this study, we propose a novel intersection between RIG1-MDA5-IRF7 and Wnt/β-catenin pathways. We found that treatment of CIC enriched cancer cells with both 5-AZA-CdR and type I and III interferons reduced canonical Wnt/β-catenin signalling. Furthermore, treatment with type I and III interferons showed a reduced CIC frequency in colorectal cancer cells in vitro. Mechanistically, we found that IRF7 and β-catenin interact in the nucleus, giving rise to a novel, non-canonical pathway that can modulate WNT signalling. These findings may explain a novel pathway by which 5-AZA-CdR can specifically target CIC enriched populations in colorectal cancer, and highlights the importance of innate immune pathways in epigenetic therapy.

Citation Format: Ilias Ettayebi, Parinaz Mehdipour, Rajat Singhania, Ankur Chakravarthy, Tiago Medina, Shu Yi Shen, Charles Ishak, David Roulois, Daniel De Carvalho. DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5728.