bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4516: A new MLH1 rabbit monoclonal antibody for immunohistochemical application
Authors: Cobb Sean , Pan Hongyang , Yang Qiong , Chan Jackie , Wu Julia , Law Jason , Li Aihua
Published: August 2018

Background: MLH1, a DNA mismatch repair gene, plays a critical role in colorectal cancer (CRC) development. Loss of nuclear MLH1 is attributed to hypermethylation of its promoter, occurring in 78% of microsatellite instability (MSI) which composes 15% of all CRC. Immunohistochemical (IHC) analysis of MLH1 in CRC is routinely performed in pathology laboratories, but remains challenging with the current selection of available MLH1 antibodies. Here we report a new MLH1 rabbit monoclonal antibody (RabMAb) developed with RabMAb® technology, designated as clone EP481.

Design: Sera collected from rabbits immunized with recombinant MLH1 protein were screened by ELISA, WB, and IHC. After fusion, antibody from a final hybridoma cell line was further characterized by extensive IHC testing using formalin-fixed paraffin-embedded human normal and tumor tissue microarrays (TMAs) comprised of 99 CRCs. EP481 performance was compared against MLH1 mouse monoclonal antibodies, G168-15, G168-728, and the current benchmark, ES05. Cases without tumor cell staining were considered negative; those also lacking stroma staining were excluded, as it could not be determined if there was germline mutation or poor tissue quality. Agreement was evaluated with Cohen's kappa.

Results: An 85 kDa MLH1 protein was detected by WB in SW480 cell lysate, but absent in the MLH1-negative cell line, HCT116. IHC analysis of EP481 was positive in normal epithelium and surrounding stroma, but negative in 10% of tumors. G168-728 showed nonspecific cytoplasmic staining and deviated from its counterparts. Concordance data (Table 1) demonstrates that EP481 is equivalent with ES05 and incongruous with G168-15.

Conclusion: The RabMAb MLH1 antibody, clone EP481, specifically labels normal cells and is absent in 10% of CRCs. This finding is consistent with both literature and the gold-standard anti-mouse clone, ES05. EP481 is a new alternative for MLH1 IHC and a promising tool to identify MSI in CRC.

Table 1. Concordance staining of MLH1 clone EP481 with clones ES05 and 168-15 on CRC TMA.ES05 +ES05 -Total168-15 +168-15 -TotalEP481 +7417566975EP481 -178268Total75883681583Cohen's kappaRatersCoefficientAgreementEP481/ES0586%ExcellentEP481/168-1545%Moderate

Citation Format: Sean Cobb, Hongyang Pan, Qiong Yang, Jackie Chan, Julia Wu, Jason Law, Aihua Li. A new MLH1 rabbit monoclonal antibody for immunohistochemical application [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 4516.
Abstract 4531: Development of a companion diagnostic assay for the detection of phosphatase and tensin (PTEN) protein loss and treatment with ipatasertib in metastatic castration-resistant prostatic cancer (mCRPC)
Authors: Stephens Crystal , Harnish Erica , Bowermaster Rebecca , Djalilvand Azita , Smith Dustin , Kim Doris , Gendreau Steven , Valle Edmundo Del
Published: August 2018

Background: Ventana Medical Systems, Inc. is developing in collaboration with Genentech, Inc., an immunohistochemical (IHC) companion diagnostic to aid in selecting patients with metastatic castration-resistant prostate cancer (mCRPC) that may benefit from ipatasertib, an Akt inhibitor therapy. The phosphatase and tensin (PTEN) protein plays an important role in controlling cell survival and cell cycle progression as a negative regulator of the phosphoinositide 3-kinase/AKT pathway.

Methods: PTEN (SP218) rabbit monoclonal primary antibody was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform (PTEN (SP218) RxDx Assay). The PTEN (SP218) RxDx Assay was developed for use in formalin-fixed, paraffin-embedded tissue samples of mCRPC in a series of studies addressing sensitivity, specificity, robustness and precision. The scoring algorithm was defined using statistical analysis of clinical outcome data analysis, and the PTEN (SP218) RxDx Assay staining pattern and prevalence of PTEN loss of expression in a set of tissue samples. Inter-reader precision was established by 3 pathologists evaluating 90 mCRPC samples across the range of PTEN expression levels.

Results: The Ventana PTEN (SP218) RxDx Assay met all pre-defined acceptance criteria. mCRPC tissue samples are designated with PTEN-Loss status when ≥ 50% of viable malignant cells have no specific cytoplasmic staining with PTEN (SP218) in the presence of acceptable internal controls. Inter-reader precision in determining PTEN status resulted in agreement rates greater than 97%.

Conclusion: These results highlight the robustness and reproducibility of the Ventana PTEN (SP218) RxDx Assay. In the clinical outcome analysis, patients identified to have PTEN-loss status demonstrated clinically meaningful improvements when treated with ipatasertib. The clinical utility of the PTEN (SP218) RxDx assay will be further validated in additional patients in subsequent ipatasertib studies, including a Phase 3 study (IPATential150) beginning in 2017.

Citation Format: Crystal Stephens, Erica Harnish, Rebecca Bowermaster, Azita Djalilvand, Dustin Smith, Doris Kim, Steven Gendreau, Edmundo Del Valle. Development of a companion diagnostic assay for the detection of phosphatase and tensin (PTEN) protein loss and treatment with ipatasertib in metastatic castration-resistant prostatic cancer (mCRPC) [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 4531.
Abstract 4538: LiquidGxTMqPCR, an extremely sensitive qPCR-based liquid biopsy platform
Authors: Song Yang , Quintanilha Danielle , Chen Si , Huang Jun
Published: August 2018

In recent years, circulating cell-free tumor DNA has emerged as an excellent source of biomarkers for cancer detection, diagnosis and monitoring. One great challenge of detecting cell-free tumor DNA molecules is that they are usually found in a large background of wild-type DNA; therefore, assays with high sensitivity and low limit of detection are required. We have developed an extremely sensitive qPCR-based liquid biopsy platform, LiquidGxTM qPCR, to detect and track key driver mutations from patient blood. The platform takes advantage of our patent-pending technology BESTTM (Blocker-based Enrichment System of Tumor DNA) to specifically enrich mutant alleles from as low as 0.01% to more than 90%. With this platform, we currently have developed a CLIA-approved non-small cell lung cancer panel (EGFR T790M/L858R/C797S/Exon19Del, KRAS G12X/G13CD, BRAF V600E, ALK-EML4 fusion), and a colorectal cancer recurrence monitoring panel (KRAS G12X/G13CD, NRAS G12DV/Q61KR, PIK3CA E542K/E545K/H1047R, BRAF V600E). Our validation data show that the NSCLC panel has superior performance, with a LOD as low as 0.005%, analytical sensitivity more than 95% and analytical specificity of 100%. We have used this panel to test samples from nearly 200 NSCLC patients, and the results are highly concordant with those obtained by error-reducing NGS-based tests, despite some samples only detected via LiquidGxTM qPCR due to superior LOD. Other features of LiquidGxTM qPCR include (1) simultaneous enrichment of multiple mutants in the same reaction; (2) cost-effective and no special equipment required; and (3) fast turnaround time that takes as little as 0.5 days to get the results. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. With the technology, we have developed two cancer panels and this platform is also available as an assay development service.

Citation Format: Yang Song, Danielle Quintanilha, Si Chen, Jun Huang. LiquidGxTMqPCR, an extremely sensitive qPCR-based liquid biopsy platform [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 4538.
Abstract 4555: Potent Conditionally Active Biologic (CAB) PD-1 antibodies to reduce systemic toxicities associated with single agent and combination therapies
Authors: Chang Cathy , Frey Gerhard , Sharp Leslie L. , Liu Haizhen , Wang Jing , Xing Charles , Yalcin Safak , Ben Yong , Boyle William J. , Short Jay M.
Published: August 2018

PD-1 is an immune checkpoint inhibitor that limits T cell activity. The use of anti-PD-1 antagonist antibodies has demonstrated anti-tumor efficacy in mouse models and in a subset of human patients. However, blockade of PD-1 as a single agent and in combination with chemotherapy, targeted, and other immunomodulatory therapies has been limited clinically in part due to toxicities related to systemic immune-response related adverse events. In addition, there is increasing concern that systemic immune activation may lead to autoimmune-related adverse events in survivors.

The Conditionally Active Biologics (CAB) technology is a patented, proprietary platform that selects antibodies that reversibly bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of glycolytic tumor metabolism including the Warburg effect. Using our CAB technology, we have identified anti-PD-1 Abs that reversibly bind human PD-1, and enhance T cell response to bacterial super-antigen under in vitro conditions that are present in the tumor microenvironment but not in normal tissues.

CAB-PD-1 Abs demonstrated anti-tumor efficacy, including tumor regression, against MC38 colorectal tumor in human PD-1 knock-in mouse model in which the mouse PD-1 gene has been replaced with human PD-1. The anti-tumor activity of CAB-PD-1 Abs was similar to a reference PD-1 Ab.

In conclusion, our data is consistent with our work on CAB-EGFR-ADC, CAB-AXL and CAB-ROR2-ADC antibodies, and suggests that anti-PD-1 Abs generated using the CAB technology provide potent, effective biologics with potential for increased therapeutic index. Specifically, the CAB-PD-1 is an excellent candidate for evaluation as a treatment for human cancers as a single agent or in combination with other anti-cancer therapies including immuno-oncology agents.

Citation Format: Cathy Chang, Gerhard Frey, Leslie L. Sharp, Haizhen Liu, Jing Wang, Charles Xing, Safak Yalcin, Yong Ben, William J. Boyle, Jay M. Short. Potent Conditionally Active Biologic (CAB) PD-1 antibodies to reduce systemic toxicities associated with single agent and combination therapies [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 4555.
Abstract 4557: Dual targeting of innate and adaptive checkpoints on tumor cells limits immune evasion
Authors: Fu Yang-Xin , Liu Xiaojuan
Published: August 2018

CD47 on tumors inhibits phagocytosis, while PD-L1 limits T cell-mediated tumor killing. However, whether and how these two pathways coordinately evade immune responses are poorly understood. We reveal that both CD47 and PD-L1 on tumors coordinately evade innate and adaptive sensing. Targeted blockade of CD47 and PD-L1 on tumor cells with bispecific anti-PDL1-SIRPα showed better therapeutic efficacy than single blockades with reduced off-tumor targeting. Mechanistically, the dual-targeting approach significantly enhanced DNA sensing in dendritic cells (DC), DC cross-presentation and anti-tumor T cell response. Our data indicate that tumor cells evolve to utilize two-levels of checkpoints to evade anti-tumor immune responses, and tumor cell-specific dual-targeting of both innate and adaptive checkpoints represents a new strategy for increasing efficacy and reducing toxicity.

Citation Format: Yang-Xin Fu, Xiaojuan Liu. Dual targeting of innate and adaptive checkpoints on tumor cells limits immune evasion [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 4557.
Abstract 4562: Tumor B7-H3 (CD276) expression and survival of patients with pancreatic cancer
Authors: Inamura Kentaro , Yokouchi Yusuke , Kobayashi Maki , Inoue Yosuke , Saiura Akio , Takazawa Yutaka , Ishikawa Yuichi
Published: August 2018

Background: B7-H3 (CD276) belongs to a family of immune modulators that includes PD-L1 (B7-H1 or CD274), and has been associated with tumor immunosuppression. Although several studies have reported the association of B7-H3 expression with prognosis in pancreatic cancer, the results are conflicting and inconclusive. We therefore examined the association of tumor B7-H3 expression with prognosis of pancreatic cancer.

Methods: Using tissue microarrays of 150 consecutive cases of pancreatic cancer, we evaluated tumor B7-H3 expression by immunohistochemistry. We examined the prognostic association of B7-H3 expression, using the log-rank test. We also used Cox proportional hazards regression models to compute the mortality hazard ratio (HR), adjusted for covariates including pathological-stage, tumor differentiation, preoperative levels of CEA and CA19-9, and adjuvant chemotherapy.

Results: Tumor B7-H3 positivity (10% of tumor cells) was observed in 99 (66%) of 150 cases of pancreatic cancer. In a log-rank test, B7-H3 positivity was associated with higher pancreatic cancer-specific mortality (P = 0.0005). In a multivariable analysis, B7-H3 positivity was independently associated with higher pancreatic cancer-specific mortality (multivariable HR = 1.82; 95% confidence interval [CI] = 1.25-2.71; P = 0.0016). When we divided immunoreactivity of B7-H3 into four scores; i.e., score 0, negative; score 1, weak; score 2, moderate; score 3, strong, the immunoreactivity scores of B7-H3 were 51 (34%), 67 (45%), 20 (13%), 12 (8%) for score 0, 1, 2, 3, respectively. In a log-rank test, higher score of B7-H3 immunoreactivity was associated with higher pancreatic cancer-specific mortality (P = 0.0014). In a multivariable analysis, higher B7-H3 immunoreactivity was independently associated with higher pancreatic cancer-specific mortality (comparing score 3 versus score 0: multivariable HR = 2.78; 95% CI = 1.38-5.25; P for trend = 0.0011).

Conclusion: Tumor B7-H3 positivity and higher B7-H3 immunoreactivity are both independently associated with higher mortality of pancreatic cancer.

Citation Format: Kentaro Inamura, Yusuke Yokouchi, Maki Kobayashi, Yosuke Inoue, Akio Saiura, Yutaka Takazawa, Yuichi Ishikawa. Tumor B7-H3 (CD276) expression and survival of patients with pancreatic 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 4562.
Abstract 4567: Immunomodulatory action of SGI-110 and combination with γδ T cell immunotherapy in ovarian cancer
Authors: Natoli Marina , Karadimitris Anastasios , Ghaem-Maghami Sadaf
Published: August 2018

Ovarian cancer (OC) is regarded as being an immunogenic cancer and strategies to enhance immune function in patients are thought to confer benefit and improve prognosis. Immunotherapy has been shown to be most promising when combined with other therapeutic approaches. Recent evidence in the field and preliminary data from our group suggest that treatment with epigenetic drugs, such as DNA methyltransferase inhibitors (DNMTi), can lead to improved immune activation in several cancers including ovarian.

The purpose of this study is to test the hypothesis that treatment with next generation hypomethylating agent SGI-110 can induce the re-expression of immunomodulatory genes and endogenous retroviruses (ERVs), which shifts OC cells towards a more immunogenic expression profile.

OC cell lines SKOV3, Kuramochi and OVSAHO were treated with low dose SGI-110 and assessed for increased immunogenicity. Expression of immune biomarkers and ERVs was assessed at different time points at mRNA levels by qPCR and protein levels by Flow Cytometry. Treated cell lines were functionally tested for increased immunogenicity by co-culture with γδ T cells, which were isolated from healthy donors and expanded by zoledronic acid selection. MTT assays were performed to assess cell viability and T cell killing following co-culture. IFNγ and Granzyme B release from T cells and chemokine release from tumor cells were assessed by ELISA.

Our results show up-regulation of key immunogenicity biomarkers and ERVs in our panel of OC cell lines, following treatment with SGI-110. Co-culturing SGI-110-treated cell lines with γδ T cells resulted in increased tumor cell killing and T cell activity.

We are now analysing RNA-sequencing data of SGI-110-treated OC cell lines in an effort to dissect the immunomodulatory mechanism of action of this novel hypomethylating agent. Moreover, we plan to test the in vivo feasibility of a SGI-110/γδ T cell combination therapy, which if successful might be further optimised for a clinical trial.

Citation Format: Marina Natoli, Anastasios Karadimitris, Sadaf Ghaem-Maghami. Immunomodulatory action of SGI-110 and combination with γδ T cell immunotherapy in ovarian 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 4567.
Abstract 4575: Efficient and accurate NGS approach for liquid biopsy detection and monitoring
Authors: Tang Ke , Kasbek Christopher , Song Yang , Song Qingxuan , Chen Si , Almalki Nouran , Zhang Xu , Huang Jun T.
Published: August 2018

Liquid biopsy detection using ctDNA has great potential in cancer diagnosis, monitoring, and predicting survival. However, detection sensitivity and specificity remains a major challenge at the current stage. Here we utilized targeted next-generation sequencing (NGS) approach to develop a noninvasive test to identify SNVs, indels, and fusions present at low frequencies in lung cancer patients. Target regions of interest were sequenced using tagging of individual molecules, followed by amplicon library generation and massive parallel sequencing using Illumina platform. The variants were called and annotated using our in-house AGIS (Actionable Genomic Interpretation System) bioinformatics pipeline. This CLIA-approved NGS panel (LiquidGx™ Lung) detects 112 variants in 10 genes (AKT1, ALK, BRAF, EGFR, ERBB2, KRAS, MET, PIK3CA, RET, and ROS1) with detection limit of 0.1%. Mutations are detected with 94% sensitivity and 99.8% specificity in a short turnaround time of 4-6 days. To assess the performance in a clinical setting, accurate sequencing was performed on 210 lung cancer patients; the frequency of mutations identified of each gene closely mirrored the published lung cancer tissue biopsy data. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. Our technology is also available as an assay development service and can be easily adaptable to the diagnosis and monitoring of other cancer types.

Citation Format: Ke Tang, Christopher Kasbek, Yang Song, Qingxuan Song, Si Chen, Nouran Almalki, Xu Zhang, Jun T. Huang. Efficient and accurate NGS approach for liquid biopsy detection and monitoring [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 4575.
Abstract 4580: Changes in the efficacy of anti-EGFR antibody drugs by exosomes derived from colorectal cancer patients
Authors: Morimura Rii , Tsukamoto Kei , Irie Shinji , Kitano Shiro , Shinozaki Eiji , Yamaguchi Kensei
Published: August 2018

Background: One of the biggest hurdles in cancer treatment is drug resistance. Exosomes are small membrane vesicles of endocytic origin, which contain mRNAs, DNA fragments, and proteins, and are released by many different cell types, including cancer cells. Several studies have shown exosome-mediated drug resistance mechanisms: drug export via the exosome pathway, neutralization of antibody-based drugs and exosome-mediated transfer of miRNAs. Here, we demonstrated the effect of exosomes derived from colorectal cancer (CRC) patients on the efficacy of anti-EGFR agent by in vitro cell-based assay.

Method: Exosomes were purified by ultracentrifugation from culture media of HCT116 cell line and the sera of CRC patients with stage IV pre- and post-treatment with cetuximab (Cmab). As a control, sera from healthy volunteers were used. The number of exosomes and amount of protein were determined by NanoSight and Qubit fluorometer, respectively. The isolated exosomes and 0.01 mg/mL and 0.1 mg/mL of Cmab were added to HT29 and NCI-H508, which stained with PKH dye previously. NCI-H508 is a Cmab super sensitive cell line. After culturing for 3 days, the cells were collected, stained with trypan blue, and the number of the remaining viable cancer cells was calculated with a cell counter. Cmab in the exosome fraction was quantified by ELISA.

Result: HT29 became drug ineffective depending on the number of exosomes produced by Cmab-insensitive HCT-116 harboring KRAS G13D mutation. In addition, exosomes in 9 of 18 cases of colorectal cancer patients changed NCI-H508 into a drug-ineffective state and also correlated with clinical information. In contrast, NCI-H508 was not converted into a drug-ineffective state by exosomes of healthy volunteers and Cmab responder patients. However, on the other hand, quantitative results by ELISA showed that Cmab in sera of some patients remained in the exosome fraction and inhibited the growth of NCI-H508. As a remarkable point, exosomes derived from healthy volunteers significantly promoted the efficacy of Cmab.

Conclusion: In this feasibility study, Cmab resistance in CRC patients may be reproducible by cell-based assay using exosomes. We believe that understanding this mechanism of action will contribute to drug discovery and personalized medicine in the future. Now, we are searching for biomarkers in these exosomes that change the efficacy of Cmab.

Citation Format: Rii Morimura, Kei Tsukamoto, Shinji Irie, Shiro Kitano, Eiji Shinozaki, Kensei Yamaguchi. Changes in the efficacy of anti-EGFR antibody drugs by exosomes derived from colorectal cancer patients [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 4580.
Abstract 4590: Larger family sizes improve stochastic error correction during untargeted searches of very-low frequency variant alleles in circulating cell-free DNA (ccfDNA)
Authors: Bhetariya Preetida J. , Nix David , Hellwig Sabine , Marth Gabor , Bronner Mary , Underhill Hunter
Published: August 2018

Detection of ccfDNA derived from non-metastatic solid tumors requires discrimination of very-low frequency true variants from NGS library preparation and sequencing noise that commonly occurs at a similar frequency (<1%). Correction of NGS-related errors is particularly important during searches of unknown tumor-associated variants with multi-gene panels. Unique molecular identifiers (UMIs) have been used during in silico error correction by molecular barcoding and performing a consensus base calling at each position from DNA labelled with the same UMI (i.e., PCR duplicates). In this study, we sought to determine the utility of UMIs for error correction of stochastic noise in ccfDNA. ccfDNA was isolated from the plasma of 11 healthy participants. An eight base-pair random barcode (UMI) was ligated prior to library amplification. Libraries were enriched using a custom capture probe set (128 genes; 128 kb) and then sequenced (HiSeq 2500). Reads were aligned, grouped based on UMI similarity, and a consensus read sequence was determined. The number of PCR duplicates that yielded a single consensus sequence was defined as family size (FS). For non-reference alleles detected at a variant allele frequency (VAF) ≥ 0.1% and ≤ 1.0%, there was a significant reduction in observed counts with increasingly larger FSs (Table 1). This effect was observed for both unique and common non-reference allele counts (Table 1). Of note, counts of unique non-reference alleles (stochastic noise) were consistently greater than the common non-reference alleles regardless of family size (Table 1). The use of larger family sizes reduced the stochastic noise that may confound detection of very-low frequency variants present in ccfDNA during searches for unknown tumor-associated variants. Strategies that generate larger family sizes or suppress early PCR errors may further improve NGS specificity to broaden liquid biopsy clinical applications.

Table 1. Mean (SD) Non-Reference Allele Counts with a VAF between 0.1% and 1.0% in Cell-Free DNAFS >= 1FS >= 5FS >= 10FS >= 15FS >= 20ANOVA Results F(4,50)P-valueAll Variants5536 (1230)3988 (881)2621 (805)1028 (552)1028 (552)49.8<0.001Unique Variants3597 (1107)2635 (766)1112 (526)709 (434)709 (434)27.6<0.001Common Variants1939 (161)1353 (188)521 (141)318 (121)318 (121)186.1<0.001

Citation Format: Preetida J. Bhetariya, David Nix, Sabine Hellwig, Gabor Marth, Mary Bronner, Hunter Underhill. Larger family sizes improve stochastic error correction during untargeted searches of very-low frequency variant alleles in circulating cell-free DNA (ccfDNA) [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 4590.
Abstract 4591: Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study
Authors: Shishido Stephanie Nicole , Rodriguez_Lee Mariam , Kolatkar Anand , Xu Liya , Restrepo-Vassalli Sara , Welter Lisa , Carlsson Anders , Greenspan Emily , Hwang Shelley , Waitman Kathryn , Nieva Jorge , Bethel Kelley , Hicks James , Kuhn Peter
Published: August 2018

The liquid biopsy allows assessment of multiple biological analytes over time to provide dynamic temporal information with the potential for improving clinical management and guiding treatment decisions. While the promise of liquid biopsies for prediction and response monitoring are intensely investigated, the pre-analytic variables are of primary concern for its implementation in diagnostic clinical medicine, including such categories as collection method, shipping conditions, and sample storage. Here we utilize an integrated high-definition single cell analysis (HD-SCA) workflow for genomic analysis of rare cells and cfDNA from the liquid biopsy to characterize the effects of pre-analytical variation and reproducibility of data analysis from the same cohort of patients. The results presented here confirm consistent rare cell enumeration and morphometric characterization between 24h and 48h time to assay (TTA), with a high efficiency and capacity for both copy number variation (CNV) and single nucleotide variation (SNV) analysis at the single cell level. Additionally, the freezing process neither diminishes or increases the DNA quantity, nor does it affect the DNA quality for CNV or SNV analysis. The integration of genomic information is imperative to reveal essential disease-implicated mutational profiles, but effective molecular diagnostic tests require reproducible coverage over a broad dynamic range. The performance of the HD-SCA platform quantified here may be utilized as a guide for implementation into patient care and/or research biorepository processes.

Citation Format: Stephanie Nicole Shishido, Mariam Rodriguez_Lee, Anand Kolatkar, Liya Xu, Sara Restrepo-Vassalli, Lisa Welter, Anders Carlsson, Emily Greenspan, Shelley Hwang, Kathryn Waitman, Jorge Nieva, Kelley Bethel, James Hicks, Peter Kuhn. Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study [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 4591.
Abstract 4597: The Blood Profiling Atlas in Cancer: Accelerating progress through collaboration in liquid biopsy
Authors: Godsey Jim
Published: August 2018

Liquid biopsies have the potential to improve early cancer detection, reduce the need for

invasive tumor biopsies, and improve personalized oncology treatment. To help drive

development within this space, the Blood Profiling Atlas in Cancer (BloodPAC) was

created as an initiative born out of the 2016 White House Cancer Moonshot. The

BloodPAC seeks to create an open database commons for liquid biopsies with the goal of

accelerating the development and approval of safe and effective blood profiling

diagnostic technologies for patient benefit.

BloodPAC was established as an independent not-for-profit in February 2017 and has

made exciting progress in advancing the liquid biopsy landscape. Today, BloodPAC is

comprised of over 30 member groups with a diverse range of stakeholders, representing

government agencies, academia, pharmaceutical, and diagnostic companies as well as

disease specific foundations, payers groups and patient advocates. BloodPAC members

actively contribute datasets that include sequencing data from circulating tumor cells,

circulating tumor DNA (ctDNA), and exosome assays, alongside relevant clinical data

into the BloodPAC Data Commons. The BloodPAC Data Commons helps facilitate the

aggregation and harmonization of submitted datasets, allowing researchers to generate

study cohorts size to make statistically significant inference in a scalable, reproducible,

and secure way. Establishing a comprehensive knowledge base through the BloodPAC

Data Commons provides an invaluable reference for anyone entering into the liquid

biopsy field, helps develop the evidence required to support clinical utility, assay

reproducibility, and provides support for FDA approval and payer reimbursement.

This presentation will discuss current progress made within the BloodPAC as an

innovative consortium along with the development of the BloodPAC Data Commons,

lessons learned, and upcoming next steps. The discussion will focus on examining the

BloodPAC consortium's strategy over the past year, the group's work in developing pre-

analytical standards, an overview of the contributed data, as well as the collaborative

process that has been established with the FDA and CAP to maximize the organization's

impact and effectiveness.

Citation Format: Jim Godsey. The Blood Profiling Atlas in Cancer: Accelerating progress through collaboration in liquid biopsy [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 4597.
Abstract 4624: The sTRA glycan is complementary to CA19-9 as a serologic biomarker of pancreatic cancer
Authors: Liu Ying , Barnett Daniel , Staal Ben , Huang Ying , Partyka Katie , Zeh Herbert , Singhi Aatur , Drake Richard R. , Brand Randall E. , Haab Brian B.
Published: August 2018

The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic cancer, but not enough to be reliable for detection, diagnosis, or monitoring of the disease. We hypothesized that a glycan called sTRA complements CA19-9, and that the two biomarkers in combination provide added information over either biomarker individually. As an individual marker, the sTRA assay performed equivalently with CA19-9 for distinguishing cancers from benign pancreatic diseases, but in combination, CA19-9 and sTRA showed exclusive elevations in significantly different groups of patients. Using high-specificity cutoffs giving one false-positive detection in each marker, sTRA was elevated in 34/46 (74%) patients with low CA19-9 in stage III/IV cancers and 28/70 (40%) with low CA19-9 in stage I/II cancers. A three-marker panel consisting of CA19-9 and two versions of the sTRA assay gave 81% sensitivity and 88% specificity (84% accuracy) in a training set of 72 stage I-IV cancers and 75 benign disease controls, compared to 57% sensitivity and 92% specificity (75% accuracy) for CA19-9 alone. The application of the fixed three-marker classifier to a test set of 25 cancers and 25 benign controls gave 80% sensitivity and 84% specificity, compared to 56% sensitivity and 96% specificity for CA19-9 alone. In an analysis of patient-matched tumor tissue and plasma samples, we found that the tumors secreting both sTRA and CA19-9 into the blood tended to be conventional PDAC with well-to-moderate differentiation and heavy marker staining in the ductal lumens, but the tumors secreting only sTRA showed sTRA staining in glands with extremely large and unpolarized nuclei and vacuolated or foamy cytoplasm. Those without plasma elevations in either marker did not have glandular features of typical adenocarcinoma. Surprisingly, about half of the patients without plasma elevations of CA19-9 nevertheless showed strong tumor staining. Such tumors expressed CA19-9 mostly in poorly differentiated patches of cells with compact nuclei and cytoplasm. Thus, sTRA and CA19-9 are complementary serologic biomarkers that together could increase the diagnostic accuracy for PDAC and potentially identify distinct subgroups of tumors.

Citation Format: Ying Liu, Daniel Barnett, Ben Staal, Ying Huang, Katie Partyka, Herbert Zeh, Aatur Singhi, Richard R. Drake, Randall E. Brand, Brian B. Haab. The sTRA glycan is complementary to CA19-9 as a serologic biomarker of pancreatic 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 4624.
Abstract 465: Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis
Authors: Im Keon-Il , Nam Young-Sun , Kim Nayoun , Song YuneJin , Jeon Young-Woo , Lim Jung-Yeon , Cho Seok-Goo
Published: August 2018

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here, we show that high-mobility group box 1 (HMGB1), a “danger signal” that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal-layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF-α, IL-1β and MIP-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM.

Citation Format: Keon-Il Im, Young-Sun Nam, Nayoun Kim, YuneJin Song, Young-Woo Jeon, Jung-Yeon Lim, Seok-Goo Cho. Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis [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 465.
Abstract 4652: Targeted nanomedicines improve therapeutic indices of RAF and MEK inhibitors
Authors: Heller Daniel A. , Shamay Yosi
Published: August 2018

Potent kinase inhibitors have been developed to treat tumors, but they suppress signaling in normal cells so that their dosing and ability to inhibit the pathway are limited by toxicity, resulting in a narrow therapeutic index. We developed a novel drug delivery strategy which efficiently encapsulates and targets kinase inhibitors to the tumor microenvironment while obviating the sources of dose-limiting toxicities. The platform employs nanoparticle scaffolds that we modified to target caveolae and P-selectin, that can both be overexpressed on tumor vasculature. We assessed the targeted drug delivery properties of the nanoparticles, encapsulating the RAF inhibitor sorafenib and MEK inhibitor trametinib. Strikingly, we found selective tumor uptake and exceptional net anti-tumor efficacies in patient-derived xenografts and genetically modified mouse models. The nanomedicine strategy also prevented the inhibition of ERK phosphorylation in the skin, demonstrating that this targeting strategy may abrogate dose-limiting toxicities of kinase inhibitors to improve their therapeutic indices.

Citation Format: Daniel A. Heller, Yosi Shamay. Targeted nanomedicines improve therapeutic indices of RAF and MEK inhibitors [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 4652.
Abstract 4654: A cell-based translocation assay system identifies active fractions from plant that inhibit growth of FGFR3 overactivation cancers
Authors: Lee Yi-Ching , Lin Yun-Wen
Published: August 2018

Fibroblast growth factor receptor 3 (FGFR3) is one of four tyrosine kinase receptors that bind and respond to fibroblast growth factors (FGFs). Gain-of-function mutations in FGFR3 cause a group of skeletal dysplasias. The overactivation of FGFR3 in somatic cells is reported to be associated with human cancers, including multiple myeloma (MM), urothelial bladder carcinoma, cervical carcinoma, as well as benign seborrheic keratosis. Patients with FGFR3 overactivation have demonstrated an overall poor prognosis, and the treatment remains a substantial challenge. Several tyrosine kinase inhibitors (TKIs) have been proposed for treatment of FGFR3-activating cancers and some of them are currently in clinical trials. However, the side effects and a modest efficacy of the compounds towards FGFR3 promote the need to identify high-selectivity and high-potency specific FGFR3 inhibitors. We established a cell-based protein translocation assay system that can monitor FGFR3 activity and be used for high-throughput screening of complex mixtures. With this system we identified plant ethanol extract as a FGFR3 inhibitor and performed bioassay-guided fractionation to identify potent active fractions. The functionality of extract and active fractions was validated in vitro in FGFR3-activated primary multiple myeloma cells. The active fractions can preferentially inhibit the proliferation of MM with FGFR3 activation compared with MM without FGFR3 activation and inhibit the FGFR3 downstream signaling pathways. Furthermore, we demonstrated that the active fractions enhance FGFR3 degradation but not FGFR1, 2, 4 in a Western blot analysis. Our results demonstrate the applicability of this discovery approach. The identified plant extracts and active factions hold therapeutic potential for the treatment of FGFR3-activated skeletal dysplasias and cancers.

Citation Format: Yi-Ching Lee, Yun-Wen Lin. A cell-based translocation assay system identifies active fractions from plant that inhibit growth of FGFR3 overactivation cancers [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 4654.
Abstract 4656: Development of an RNA-based cancer therapeutic targeting the let-7-LIN28 interaction
Authors: Myoung Sunghyun , Savinov Sergey , Chen Lan , Chopra Gaurav , Avramova Larisa , Welch James , Loren Bradley , Thompson David , Kasinski Andrea L.
Published: August 2018

Based on the knowledge that microRNAs (miRNAs) are dysregulated in diseases such as cancer, various attempts have been explored to develop miRNA-based cancer therapeutics. Although many strategies have been used to restore the levels of therapeutically relevant microRNAs, clinical delivery of the processed or mature miRNA to cancer cells still remains a challenge. To overcome this challenge, innovative methods are being explored to increase the pools of tumor-suppressive miRNAs, such as enhancing miRNA biogenesis. This is especially true for the let-7 family of tumor-suppressive miRNAs, which has an additional layer of regulation over the canonical miRNA biogenesis pathway. In particular, the DROSHA and DICER cleavage steps are blocked when unprocessed let-7 is bound by the RNA-binding protein LIN28. LIN28 interacts specifically with unprocessed let-7 via a sequence-specific motif, GGAG, contained in most let-7 family members. Because previous attempts to restore mature let-7 levels through increasing let-7 pools have been quite successful at reducing tumor burden, identifying novel and clinically relevant ways to increase the level of this tumor-suppressive miRNA represents a critical need. In this study, we hypothesize that small-molecule inhibitors that disrupt the LIN28-let-7 interaction will lead to enhanced processing and increased levels of mature, tumor-suppressive let-7. Thus, an in vitro high-throughput fluorescence polarization screen has been conducted using His-tagged LIN28 and a Cy5 fluorophore-tagged let-7 RNA probe. The nine-nucleotide RNA probe was designed through in silico modeling to include the GGAG motif, which associates with LIN28 through the zinc knuckle domain of LIN28. Due to the size difference between unbound let-7 probe and LIN28-bound let-7 probe, a wide window of polarization values was achieved, resulting in a Z′ score of 0.61, indicating that the assay was robust to proceed with the screen. The screen was performed against 23,680 compounds, which consist of FDA-approved LOPAC and blood-brain barrier-permeable CNS library compounds. Eight compounds were identified as positive hits from the screen. These compounds are currently being characterized in secondary assays both in vitro and in cell culture. The significance of this research is that small-molecule inhibitors may provide novel therapeutic strategies to increase the pool of mature, tumor-suppressive let-7 in tumors with elevated LIN28.

Citation Format: Sunghyun Myoung, Sergey Savinov, Lan Chen, Gaurav Chopra, Larisa Avramova, James Welch, Bradley Loren, David Thompson, Andrea L. Kasinski. Development of an RNA-based cancer therapeutic targeting the let-7-LIN28 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 4656.
Abstract 4666: An evaluation of breast cancer cellular membrane lipid-extracted nanoliposomes (CLENs) in relation to formulation design, stability, mechanism of cellular entry and cardioprotective function in vitro
Authors: Alharbi Hanan M. , Campbell Robert B.
Published: August 2018

One of the longstanding issues limiting the use of chemotherapeutic agents is the lack of tumor specificity, which leads to systemic toxicity. Therefore, various targeted nanocarriers have been developed to concentrate the cytotoxic drug agents at tumor regions to avoid uptake by normal healthy tissues. Previous studies from our laboratory have demonstrated that cell membrane lipid-extracted nanoliposomes (CLENs) are capable of selective targeting compared to nanoliposomes consisting of mainly conventional lipid materials. Also, CLENs were relatively nontoxic when employed at concentrations traditionally used to evaluate nanoparticles in vitro. However, parameters such as lipid composition, size, and surface charge all have a direct impact on liposomal cellular uptake. In this study, we prepared different CLENs different molar ratios of natural and cellular-derived lipids extracted directly from breast cancer cells (4T1), cholesterol, and DPPE-PEG-5000. The formulations were used to investigate the mechanisms of CLENs uptake and determine the underlying mechanisms regulating cellular entry. In the study, CLENs were used to evaluate cytotoxicity and cellular uptake for both target and off-target cell populations. CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) were able to retain doxorubicin in relevant therapeutic concentrations. CLEN formulations were able to exert selective cytotoxic drug effects against different target breast cancer cells in vitro. 4T1 CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) demonstrated greater binding to 4T1 (target) cells compared to CLENs prepared using 4T1 lipid extracts alone. The binding was not only temperature- and time-dependent, but also composition- and cell type-dependent. Off-target effect studies showed that 4T1 CLENs were taken up minimally by off-target cells (including normal breast fibroblasts and normal myocytes), compared to controls. Our studies collectively support the use of lipid extracts derived from target cells for selective drug targeting and enhanced cytotoxicity. Future studies will explore underlying mechanisms of cellular entry and cardioprotective function.

Citation Format: Hanan M. Alharbi, Robert B. Campbell. An evaluation of breast cancer cellular membrane lipid-extracted nanoliposomes (CLENs) in relation to formulation design, stability, mechanism of cellular entry and cardioprotective function in vitro [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 4666.
Abstract 4669: Role of CD8+ tissue-resident memory T (TRM) cells in anti-tumor T-cell response and cancer immunotherapy
Authors: Mami-Chouaib Fathia
Published: August 2018

Tissue-resident memory T (TRM) cells, very often defined by the expression of CD103 integrin, correspond to a new subset of long-lived memory T lymphocytes that reside in tissues and are highly protective during localized reinfections. CD8+ TRM cells also frequently reside in human epithelial tumors and play an essential role in anti-tumor T-cell responses. In this context, our previous results indicated that the interaction of CD103 integrin with its ligand, the epithelial cell marker E-cadherin, favors adhesion of TRM cells to their specific epithelial cancer cells and participate to the potentiation of T-cell receptor (TCR)-mediated cytotoxic activity and cytokine production. We also demonstrated that an enhanced CD103+ tumor-infiltrating lymphocytes (TIL) subset correlates with improved early-stage non-small cell lung carcinoma (NSCLC) patient survival and increased intraepithelial T-lymphocyte infiltration. Notably, TRM cells are enriched with tumor-specific T cells and express high levels of inhibitory receptors, including PD-1 and Tim-3, making them an attractive target for cancer immunotherapy, such as checkpoint blockade. Moreover, we provided evidence that CD103 is involved in T-cell recruitment within epithelial tumor islets and in local early T-cell signaling. Our more recent results indicated that integrin-linked kinase (ILK) and paxillin adaptor protein are directly involved in CD103 bidirectional signaling events and defined the CD103 cytoplasmic domain that controls TRM adhesion, migratory behavior and TCR-mediated T-cell functions. Data emphasizing the contribution of CD8+/CD103+ TRM cells in promoting intra-tumoral cytotoxic T lymphocyte (CTL) responses and in success of cancer immunotherapy will be discussed.

Citation Format: Fathia Mami-Chouaib. Role of CD8+ tissue-resident memory T (TRM) cells in anti-tumor T-cell response and cancer immunotherapy [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 4669.