bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2168: Testing the electrical properties of different cell lines using 3DEP reader and compare to TTFields response
Authors: Giladi Moshe , Zeevi Einav , Gotlib Karnit , Wenger Cornelia , Naveh Ariel , Bomzon Zeev , Kirson Eilon D. , Weinberg Uri , Kinzel Adrian , Palti Yoram
Published: August 2019

Background: Tumor Treating Fields (TTFields) are an approved anti-neoplastic treatment modality delivered via application of low intensity, intermediate frequency, alternating electric fields. The electrical properties of cells (such as permittivity and conductivity) determine the optimal frequency of TTFields that incurs the highest reduction in cell counts. Currently, there are no predictive markers for determining TTFields response or the optimal frequency to be applied for individual patient. The goal of this study is to determine the correlation between electrical properties of cells and TTFields’s optimal frequency and sensitivity. The 3DEP reader (LabTech) determines the electrical properties of cells, including permittivity and conductivity, by using Dielectrophoresis (DEP) force. DEP is a physical effect that generates a force on polarizable particles experiencing a non-homogeneous electric field and can therefore be used as a technique to analyse the way cells move within electric fields at different frequencies. Methods: The baseline electrical properties (permittivity and conductivity) of 17 cell lines from different tumor types were determined using the 3DEP reader (LabTech). The curves were analyzed using 2-way ANOVA. The optimal TTFields frequency for each cell line was determined by testing the cytotoxic effect of TTFields at various frequencies using the inovitro system. The electrical properties of cells were compared with the optimal TTFields frequency and sensitivity of each cell line. Results: The results demonstrate significant differences (p<0.001) between the lower frequency range of the 3DEP curves that corresponds to membrane capacitance of cells with TTFields optimal frequency of 150kHz (9 cell lines) and cells with TTFields optimal frequency of 200kHz (8 cell lines). Membrane capacitance was also a good predictor for TTFields sensitivity based on the differences in the curves in the low frequency range. Conclusions: The results presented in this study demonstrate that cell membrane capacitance correlates with TTFields optimal frequency and sensitivity. Based on the above, there is a strong rational to further explore the potential of measuring the electrical properties of cells as a predictive marker to help determine which patient will respond better to TTFields and the optimal TTFields frequency to be applied for each patient.

Citation Format: Moshe Giladi, Einav Zeevi, Karnit Gotlib, Cornelia Wenger, Ariel Naveh, Zeev Bomzon, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. Testing the electrical properties of different cell lines using 3DEP reader and compare to TTFields response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2168.
Abstract 2172: Quantification of ERK activity in cancer cell lysates and tumor extract using differential sensing methods
Authors: Zamora-Olivares Diana , Kaoud Tamer S. , Zeng Lingyu , Telles Mitchell , Anslyn Eric V. , Dalby Kevin N.
Published: August 2019

The activity of protein kinases in biological samples is typically estimated by determining the phosphorylation status of either the kinase itself, or a known cellular substrate. This is typically evaluated by multi-step western blotting, or proteomics procedures. Such procedures typically provide qualitative estimates of the modifications in question. While immunoblotting and proteomics procedures can be used in a quantitative manner, their complicated workflow diminishes reproducibility. Futhermore, modifications do not necessarily correlate closely with a protein kinase’s activity. The motivation for this work was to assess the potential of a peptide array to quantify ERK activity in cancer cell lines and tumor samples, without the need to suppress related kinase activities. This work shows that a library of cross-reactive peptide-based biosensors, along with chemometric analysis can be used to profile a kinase activity in cancer cell lines. Significantly, the array is suitable for quantifying unknown levels of ERK activity in unfractionated cancer cell lysates and tumor samples using a multivariate regression model. The predicted values provided by our model were found to be comparable to those obtained by immune complex kinase assays.

Note: This abstract was not presented at the meeting.

Citation Format: Diana Zamora-Olivares, Tamer S. Kaoud, Lingyu Zeng, Mitchell Telles, Eric V. Anslyn, Kevin N. Dalby. Quantification of ERK activity in cancer cell lysates and tumor extract using differential sensing methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2172.
Abstract 2174: A solution for simultaneous DNA & RNA extraction from single tissue without lysate splitting
Authors: Wei Han
Published: August 2019

Extraction of intact and high quality gDNA or RNA is one of the most common procedures in molecular biology labs. With the advance of NGS, scientists can analyze both genomic and transcriptomic information from the same sample. Techniques that offer the ability to isolate both DNA and RNA from a single biological sample are crucial for this application. There is still great need for improved protocols in this area especially for precious and low-yield samples. Currently, most researchers use TRIzol® to isolate RNA and DNA from the same tube by extracting DNA from the organic phase and RNA from the aqueous phase. Although this method can extract high-quality RNA, the DNA yield is often low. With many biological samples in short supply, it is crucial to maximize yields. We report here using Beckman Coulter reagents, a protocol to isolate both DNA and RNA from the same fresh/frozen sample. The method utilizes size selection to separate DNA and RNA. We tested this method on 4 different frozen mouse tissues and report consistent extraction performance across all 4 tissue types. There are two advantages for this method: (1) There is no need to split a lysate for separate preparation of DNA/RNA; thus, researchers will not lose any portion of DNA or RNA. (2) Due to the different size distributions between DNA and RNA, this method offers quick and efficient extraction.

Citation Format: Han Wei. A solution for simultaneous DNA & RNA extraction from single tissue without lysate splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2174.
Abstract 2176: Topical delivery of carvedilol-loaded transfersomes for photoprotection and skin cancer prevention
Authors: Chen Mengbing , Chen Mengbing
Published: August 2019

Ultraviolet (UV)-induced skin cancer is the most common cancer diagnosed in the United States and remains one of major public health concerns. Thus, there is an urgent need for the development of more effective strategies to prevent and treat skin cancer. We have previously demonstrated that carvedilol, an FDA approved β-blocker for cardiovascular diseases, could prevent UV-induced skin cancer both in vitro and in vivo. However, the β-adrenergic blockade effects of carvedilol might be a barrier for its repurposing as a cancer chemopreventive agent due to its cardiovascular side effects. In this work, we aimed to design a novel topical formulation, namely carvedilol-loaded transfersomes, which could maintain anti-cancer activities but avoid systemic cardiovascular effects. Carvedilol-loaded transfersomes were prepared using different phospholipids and surfactants at various ratios by a thin-film hydration method. Following optimization of preparation, the transfersomes were characterized in terms of particle size, zeta potential, and encapsulation efficiency. Skin permeation studies were performed using Franz vertical diffusion cells and skin dissected from SKH-1 hairless mice to examine the permeability of carvedilol. An optimal formulation was identified to compose carvedilol, Soy PC, and Tween-80 at a ratio of 1:3:0.5. The particle size, zeta potential and encapsulation efficiency for this formulation were determined to be 162.9±9.6 nm, 17.1±0.3 mV and 47.9±3.5%, respectively. Skin permeation studies showed that the specific carvedilol-loaded transfersomes could not penetrate through the skin layers in comparison with free carvedilol dissolved in acetone. Taken together, these results suggest that the transfersomal formulations of carvedilol may have desirable physical and biological characteristics for skin retention and local action. Ongoing studies are focused on further improvement of encapsulation efficiency and determination of its effectiveness against skin cancer.

Citation Format: Mengbing Chen, Mengbing Chen. Topical delivery of carvedilol-loaded transfersomes for photoprotection and skin cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2176.
Abstract 2188: Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells
Authors: Yamamura Masahiro , Yamauchi Akira , Katase Naoki , Sakaguchi Masakiyo , Katata Yosuke , Tanioka Hiroaki , Okawaki Makoto , Nagasaka Takeshi , Yamaguchi Yoshiyuki
Published: August 2019

Background & Aims: The new therapy for pancreatic cancer which has the poorest prognosis among malignant tumors is high demand nowadays. The overall 5-year survival rate in all stages of this type of cancer is less than 5%, even with the multimodality therapy, including surgery, chemotherapy and radiotherapy. Although gemcitabine (GEM) is a key drug in the treatment of advanced pancreatic cancer, the therapeutic effect is limited because of drug resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition has shown promise in the treatment of solid malignancies. Src kinases work as a signal switch for multiple molecular signal transduction pathways. The objective of this study is to evaluate an antitumor effect of three kinds of small molecular weight compounds (antimetabolite Gemcitabine, HSP90 inhibitor NVP-AUY922, and Src kinase inhibitor Dasatinib) on tumor growth and chemotaxis in pancreatic cancer cell lines. Methods: Pancreatic cancer cell lines were treated with each of the 3 compounds to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. Results: Of these compounds, HSP90 inhibitor inhibited cell proliferation in pancreatic cancer cells best in a dose- and time-dependent manner. Growth inhibitory effect of HSP90 inhibitor for pancreatic cancer cells were highest of the three. Further, HSP90 inhibitor inhibited the growth of gemcitabine sensitive and resistant pancreatic cancer cell lines. With flowcytometry and immunostaining, HSP90 inhibitor had higher apoptosis-inducing effect than gemcitabine. In the chemotaxis assay, HSP90 inhibitor inhibited the migration of pancreatic cancer cell best in the three compounds. In western blotting, HSP90 inhibitor suppressed the expression of multiple receptor tyrosine kinase proteins as well as the signal transduction in PI3K-Akt and RAS-MAPK pathways. Conclusion: In this study, HSP90 inhibitor was the best of 3 small molecular compounds and has proven effective in gemcitabine sensitive and resistant pancreatic cancer. The HSP90 inhibitor is useful as an antitumor drug by inhibiting proliferation and chemotaxis of pancreatic cancer cells through multiple kinase pathways.

Citation Format: Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Masakiyo Sakaguchi, Yosuke Katata, Hiroaki Tanioka, Makoto Okawaki, Takeshi Nagasaka, Yoshiyuki Yamaguchi. Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2188.
Abstract 219: The potential benefit of lower drug-antibody ratio (DAR) on antibody-maytansinoid conjugate in vivo efficacy
Authors: Sinkevicius Kerstin W. , Lanieri Leanne , Lee Jenny , Boule Steven , Yoder Nicholas C. , Hicks Stuart W. , Pinkas Jan , Ponte Jose F. , Gregory Richard J.
Published: August 2019

The majority of antibody-drug conjugates (ADCs) with maytansinoid payloads in development are linked via lysine residues and target an average drug-antibody ratio (DAR) of 3.5. These ADCs demonstrate substantial preclinical potency while maintaining good biophysical and pharmacological properties. However, some targets are expressed in normal tissues at levels that result in target-mediated drug disposition (TMDD), which can impact systemic and tumor ADC exposure and tumor penetration. For targets with TMDD, lowering the DAR of the ADCs may be advantageous. Since ADC tolerability is generally determined by the payload dose, lower DAR ADCs can be dosed at a higher antibody concentration, resulting in an increased conjugate exposure which may improve efficacy by saturating TMDD and/or increasing tumor delivery and penetration of the conjugate. In preclinical cell line xenograft studies, lysine-conjugated 2.0 and 3.5 DAR ADCs had comparable efficacy when dosed by payload concentration. However, the effect of TMDD cannot be captured with these models because the antibody in the ADCs does not cross-react with murine target antigen expressed on normal tissues. To supplement our understanding of the factors that influence ADC activity, we generated a cross-reactive model system that utilizes a chimeric anti-murine folate receptor α (FRα) antibody that binds with similar affinity to mouse and human FRα. Using this cross-reactive system, where the target is also expressed in normal tissues, 2.0 DAR conjugates were more efficacious than 3.5 DAR conjugates when dosed at matched payload concentrations in multiple xenograft models, suggesting that lower DAR can be an effective strategy to compensate for TMDD. Additional studies using xenograft models with varying antigen expression levels are ongoing, and studies designed to assess different conjugation methods of generating lower DAR ADCs are planned. This work highlights some of the many factors that impact ADC activity and demonstrates that the DAR of an ADC should be optimized for each target.

Citation Format: Kerstin W. Sinkevicius, Leanne Lanieri, Jenny Lee, Steven Boule, Nicholas C. Yoder, Stuart W. Hicks, Jan Pinkas, Jose F. Ponte, Richard J. Gregory. The potential benefit of lower drug-antibody ratio (DAR) on antibody-maytansinoid conjugate in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 219.
Abstract 2190: Repurposing the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma
Authors: Ding Andy S. , Casaos Joshua , Huq Sakibul , Brem Henry , Skuli Nicolas , Tyler Betty
Published: August 2019

Purpose: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is often diagnosed at an advanced stage, leading to poor disease-free and overall survival. Accumulating literature suggests that elevated protein expression of EZH2, eIF4E, and IMPDH correlates with poor prognosis in NPC. These three proteins are modulated by the FDA-approved antiviral drug ribavirin, which has recently been repurposed by our laboratory and others as a promising anti-cancer agent. Based on this intersection of molecular signature and drug targets, we investigated the potential use of ribavirin as a therapeutic agent for NPC.

Experimental Design: We assessed anti-neoplastic efficacy of ribavirin on human NPC cell lines in vitro using cellular growth assays, flow cytometry, and scratch wound assays. Mechanistic pathways involved were investigated using genomic expression datasets, western blots, and enzymatic activity assays. The effects of combining ribavirin with radiation were assessed via clonogenic assays and flow cytometry. Finally, we evaluated the effects of ribavirin on tumor growth in vivo using human cell line-derived xenograft models.

Results: Ribavirin significantly decreased NPC cellular proliferation and migratory capacity in addition to promoting cell cycle arrest and cell death. Modulation of the EZH2, Snail, eIF4E, and IMPDH pathways was observed in response to ribavirin treatment. Ribavirin significantly enhanced the cytotoxic effects of radiation therapy in NPC. Most importantly, ribavirin significantly reduced flank tumor growth in NPC xenograft models.

Conclusions: Our work suggests that ribavirin has potent anti-cancer effects in NPC and could represent a safe and promising addition to current NPC treatment regimens.

Citation Format: Andy S. Ding, Joshua Casaos, Sakibul Huq, Henry Brem, Nicolas Skuli, Betty Tyler. Repurposing the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2190.
Abstract 2203: WSD1227: A BBB penetrable VEGFR small molecule for the treatment of primary and metastatic brain tumors
Authors: Zhang Jinqiang , Zhong Wei , Mu Zhihua , Sun Claire
Published: August 2019

Anti-angiogenesis has been proved to be a good strategy for cancer treatment (such as in GBM, NSCLC, mBC, CRC, OC, etc.) with satisfactory performance to extracranial lesions, but not in intracranial lesions. Insufficient penetration across BBB could be one of factors limiting intracranial activity for those approved anti-VEGF agents. There is a need to develop a BBB penetrable VEGFR inhibitor to clinic to assess the efficacy and safety. Here we report that WSD1227, a novel, selective VEGFR inhibitor, displays potent activity, excellent CNS penetration and preclinical safety and efficacy. The IC50 of WSD1227 against VEGFR1/2/3 at 0.69/0.35/0.41nM versus against other targets such as PDGFRα IC50 22.9nM, PDGFRβ IC50 19.4nM, cKit IC50 383nM, FLT3 IC50 555 nM and CSF1R IC50 1062nM. WSD1227 treatment resulted in significant anti-tumor activity in VEGF related extracranial and intracranial mice models, with superior efficacy than bevacizumab. There is no increased risk of CNS hemorrhage in intracranial tumor bearing mice treated with WSD1227. In addition, WSD1227 is predicted to be CNS penetrable with decent PK in human. Taken together, our data provide a good rationale for WSD1227 to be developed toward clinic to investigate anti-angiogenetic therapies for management of patients with primary or metastatic brain tumors.

Citation Format: Jinqiang Zhang, Wei Zhong, Zhihua Mu, Claire Sun. WSD1227: A BBB penetrable VEGFR small molecule for the treatment of primary and metastatic brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2203.
Abstract 2220: Benchmarking of somatic variant calling algorithms for detection of circulating tumor DNA
Authors: Bhetariya Preetida J. , Hellwig Sabine , Nix David A. , Marth Gabor T. , Bronner Mary P. , Underhill Hunter R.
Published: August 2019

Purpose: Establishing a reliable bioinformatics variant calling workflow is crucial for untargeted searches of low-frequency variants in plasma circulating cell-free DNA (ccfDNA). We sought to benchmark the performance of nine variant calling algorithms on simulated data and cancer patient ccfDNA sequencing samples.

Methods: A simulated tumor dataset was constructed by in-silico introduction of pathogenic variants (303 SNVs and 91 INDELs; VAF of 0.1-12%) into a healthy control sequencing sample (128 gene capture panel; 128kb; 4200X depth). An unspiked replicate served as a control. Tumor and control datasets were analyzed by nine somatic variant calling algorithms available in the public domain (Table 1). Sensitivity and specificity were calculated for each caller. Variant calling algorithms that proved most successful in synthetic data (Lofreq, Mutect) were applied to 24 EGFR-mutant lung cancer patient ccfDNA library samples captured and sequenced under conditions identical to the benchmarking data set. Droplet digital PCR (ddPCR) was used to independently determine the VAFs of two EGFR SNVs (T790M and L858R) and exon 19 deletions in all lung cancer libraries (detected range: 0.1-48.5%).

Results:

Table 1:Sensitivity (Sens.) and false discovery rate (FDR) as determined for 9 variant callers. For synthetic data, results are shown for 1.6% VAF (NC = no call)CallerSynthetic data (1.6% VAF)Lung cancer ccfDNASNVINDELSNVINDEL% Sens.% FDR% Sens.% FDR% Sens. <1% VAF% Sens. >1% VAF% Sens. <1% VAF% Sens. >1% VAFLofreq87.15.792.35.675.094.466.7100Mutect47.52.291.26.762.588.916.787.5Mutect235.63.296.71.1Strelka17.83.57.69.2FreebayesNCNCNCNCVarscanNCNCNCNCVardictNCNCNCNCLumpy13.986.17.792.1DellyNCNCNCNC

Conclusion: Performance of variant callers differed greatly, with Lofreq providing the best sensitivity at low VAF. In cancer ccfDNA libraries Lofreq detected most but not all low-frequency variants, indicating that ccfDNA diagnostics require optimized variant calling workflows to improve detection of variants with a VAF <1%.

Citation Format: Preetida J. Bhetariya, Sabine Hellwig, David A. Nix, Gabor T. Marth, Mary P. Bronner, Hunter R. Underhill. Benchmarking of somatic variant calling algorithms for detection of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2220.
Abstract 2233: Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples
Authors: Saunders Lauren , Hur Antonia , Niccum Brittany , Patel Asmita
Published: August 2019

Cell free DNA (cfDNA) consists of small (150 - 500 bp) DNA fragments that circulate in the blood. cfDNA levels tend to be low in healthy, non-pregnant patients, and increase in patients with cancer, pregnancy, or extensive damage to tissue. cfDNA is believe to derive mostly from apoptotic cells, and biomarkers for a variety of diseases have been found in cfDNA. As cfDNA is extracted from blood, it is a non-invasive way to detect disease; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. This study measures the efficacy of cfDNA as a biomarker detection medium by comparing mutations found in both FFPE tumor samples and paired cfDNA samples. This study determines whether the same biomarkers are found in each sample type, and which of those can be used as biomarkers in both and which are preferred biomarkers for only a single sample type. Trends in mutation detection with the two different sample types are discussed.

Citation Format: Lauren Saunders, Antonia Hur, Brittany Niccum, Asmita Patel. Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2233.
Abstract 2234: Monitoring of genetic change in circulating tumor DNA isolated from Her2 positive advanced gastric cancer patients
Authors: Kwon Woo Sun , Jung Seung-Hyun , Chung Yeun-Jun , Ahn Joong Bae , Chung Hyun Cheol , Rha Sun Young
Published: August 2019

Gastric cancer is the common solid cancer and a leading cause for cancer related mortality worldwide. Despite developments and improvement in diagnosis, surgical technique, chemotherapy and radiotherapy, median survival time of advanced/metastatic gastric cancer (AGC) patients who received systemic chemotherapy is between 15-18 months. Tumor heterogeneity is associated with heterogeneous genetic profiling, which lowers diagnostic precision and consequently becomes an obstacle to determining the appropriate therapeutic strategies for individual cancer patients. Therefore, predictive biomarkers for determining potential treatment strategy are needed to improve the prognosis of patients. Personalized medicine has emerged as the future of cancer care to ensure that patients receive individualized treatment specific to their needs. In order to provide such care, molecular techniques that enable oncologists to diagnose, treat, and monitor tumors are necessary. Liquid biopsy by genotyping circulating tumor DNA (ctDNA) has provided a non-invasive and real-time liquid biopsy approach in assessing tumor genomic alterations in clinical oncology. This ctDNA have been associated with poor prognosis and clinical outcome in various cancer including lung, breast and colorectal. In this study, we evaluated utility and genetic profiling of ctDNAs in AGC. Under IRB approval, blood samples were collected in Cell-Free DNA BCT® streck tubes from 14 Her2-positive. Depending on the treatment schedule, blood can be collected 2 to 5 times per patient sequentially. The isolated ctDNAs were analyzed to genetic profiling by OncoChase (95 genes) based on AmpliSeq. And tumor tissue genotyping by CANCER MASTER and compared different sample preparation conditions. Validation of blood-based tumor genomic profiling in additional multicenter outcome studies is necessary; however, ctDNA monitoring can provide clinically important actionable information for precision oncology approaches.

Citation Format: Woo Sun Kwon, Seung-Hyun Jung, Yeun-Jun Chung, Joong Bae Ahn, Hyun Cheol Chung, Sun Young Rha. Monitoring of genetic change in circulating tumor DNA isolated from Her2 positive advanced gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2234.
Abstract 2236: Development and validation of urine cfDNA preservative kit for detection of genomic alterations in cancer
Authors: Li Zheng , Tang Haoran , Zhang Tiantian , Liu Chunxiao , Zhu Shengnan , Zhang Ying , Xiang Binggang , Zhao Zhixin
Published: August 2019

Introduction: cfDNA (circulating cell-free DNA), short DNA fragments shredded from dying normal and tumor cells, has been proved capable of serving as molecular biomarkers for clinical decision making, including but not limit to tumor diagnosis, treatment and monitoring. Urine, by its non-invasive nature and unique biological characteristics, is far superior comparing to other liquid biopsy specimens, such as blood and cerebrospinal fluid, and has broad clinical implications for genitourinary cancers. The challenge for urine cfDNA test, is the instability of cfDNA, caused by nuclease, high content of urea, and contamination from genomic DNA released from nucleated cells and microbials in urine. Here, we have developed a robust and user friendly urine preservative collection Kit to maintain integrity of cfDNA in urine and to facilitate transportation of urinary samples to clinical laboratory.

Methods: Urine samples from 5 donors collected using the developed urine collection kit were tested after incubation under 4°C and 37°C, mimicking extreme storage or transporting environments, and were compared to untreated urine and treated urine by another commercial preserve buffer. cfDNA was extracted after incubation of 0, 4, 7 days and then quantified by ddPCR with 3 markers: EGFR as an endogenous DNA marker, PSA (synthetic oligos containing intron-deleted sequence, pre-incubation spike-in) as an exogenous DNA marker, ARv7 (synthetic oligos containing intron-deleted sequence, post-incubation spike-in) as an inner control for the extraction and detection assays.

Results While untreated urine showed significant decrease after incubation under 37°C, cfDNA derived from urine stored in developed urine collection tubes remained stable till day 7. There is no significant change on normalized copies of EGFR and PSA in the treated urine samples under 4°C for up to 7 days.

Conclusion: the developed Urine Collection Kit demonstrated great urine cfDNA preserving capacity under different temperatures for at least 7 days, on par with commercially available preserve buffer and even outperforming it under certain circumstances. The developed kit provides an easy-to-use solution of obtaining stable urine cfDNA for clinical use.

Citation Format: Zheng Li, Haoran Tang, Tiantian Zhang, Chunxiao Liu, Shengnan Zhu, Ying Zhang, Binggang Xiang, Zhixin Zhao. Development and validation of urine cfDNA preservative kit for detection of genomic alterations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2236.
Abstract 2237: Detection of CK-19 mRNA positive CTCs, isolated with a size-based microfluidics platform, in NSCLC patients under osimertinib therapy
Authors: Ntzifa Aliki , Kotsakis Athanasios , Georgoulias Vassilis , Lianidou Evi
Published: August 2019

Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) and resistant to first and second generation EGFR tyrosine kinase inhibitors (EGFR TKIs), receive therapy with osimertinib (third generation EGFR TKI). However, some patients experience resistance to osimertinib and disease progression. Molecular characterization of circulating tumor cells (CTCs) could offer great advantages as a non-invasive approach for disease monitoring. In this study, we isolated CTCs using a label-independent microfluidic-based platform to elucidate resistance mechanisms in these NSCLC patients.

Materials and methods: Peripheral blood (PB) (15mL) was obtained at different time points from 27 patients with advanced NSCLC under osimertinib therapy. CTCs were isolated using the ParsortixTM system (ANGLE, Plc) and further harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. RT-qPCR was performed for CK-19 and B2M (used as a reference gene) mRNA in the COBAS z480 system (Roche Diagnostics).

Results: PB samples were obtained at baseline (n=25), after one cycle of treatment with osimertinib (n=23), every 3 months (n=83) and at disease progression (PD) (n=16). CK-19 positive CTCs were detected in 3/25(12%) baseline samples, 24/83(28.9%) during treatment at different time points; 5/23(21.7%) after one cycle of treatment, 8/23(34.8%) after 3 months, 3/17(17.6%) after 6 months, 4/12(33.3%) after 9 months, 1/2(50%) after 12 months, 3/5(60%) after 15 months; and 5/16(31.2%) at PD.

Conclusions: CK-19 mRNA-positive CTCs were isolated using an epitope-independent enrichment microfluidic device. Further molecular characterization of these cells at the gene expression, DNA mutation, DNA methylation and miRNA level will provide biomarkers for the elucidation of resistance mechanisms in NSCLC patients treated with osimertinib.

Citation Format: Aliki Ntzifa, Athanasios Kotsakis, Vassilis Georgoulias, Evi Lianidou. Detection of CK-19 mRNA positive CTCs, isolated with a size-based microfluidics platform, in NSCLC patients under osimertinib therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2237.
Abstract 2250: Combinational markers of neoantigen intratumor heterogeneity and neoantigen burden predict progression-free survival for lenvatinib plus PD-1 blockade in patients with intrahepatic cholangiocarcinoma
Authors: Lin Jianzhen , Liu Yiying , Yang Xu , Bai Yi , Long Junyu , Long Junyu , Li Yongchao , Wang Jiaqian , Wang Dongxu , Yang Xiaobo , Guan Mei , Huo Li , Pan Jie , Hu Ke , Gao Zhibo , Zhao Haitao
Published: August 2019

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant neoplasm derived from biliary epithelial cells. Although many targetable gene alterations have been identified, there has no molecular targeted drug available for these patients at present, which may be due to the high intratumor heterogeneity (ITH) of ICC. Previous studies have demonstrated that some ICC patients could be benefited from immune checkpoint inhibitors (ICIs), but the predictive biomarkers are still unknown. We used whole exome sequencing to evaluate the impact of neoantigen ITH and tumor neoantigen burden (TNB) in 45 ICC patients, in which 15 patients were treated by anti-PD1 inhibitor combined with lenvatinib. The median neoantigen and clonal neoantigen load of these patients were 0.65 and 0.29 per Mb, respectively. The median neoantigen ITH index was 0.45, with a range from 0.02 to 1. Survival analysis of those patients who received the combined treatment revealed significantly longer progression-free survival (PFS) in patients with higher TNB and low neoantigen ITH (9.5 vs 3.5 months, p=0.00583). Among the 30 patients without anti-PD1 combined therapy, there were 36.7% (11/30) ICC patients with high TNB and low neoantigen ITH, indicating these patients might be benefited from this combined therapy. One of these 45 patients had the highest TMB and TNB with high MSI and dMMR has the longest PFS (12 months) after the combined treatment. Besides, we found that the clonal KRAS mutation (G12D) occurred exclusively in patients with shorter PFS and OS and lower TMB and TNB, while the FLG mutation occurred only in patients with longer PFS and OS and higher TMB and TNB. These results indicated the potential relationship between mutations in KRAS and FLG and the levels of TMB and TNB, which needs to further investigation. Therefore, these data revealed that the high TNB and lower neoantigen ITH might be a promising marker for predicting the efficacy of the combination therapy of anti-PD1 inhibitor and lenvatinib in ICC patients.

Citation Format: Jianzhen Lin, Yiying Liu, Xu Yang, Yi Bai, Junyu Long, Junyu Long, Yongchao Li, Jiaqian Wang, Dongxu Wang, Xiaobo Yang, Mei Guan, Li Huo, Jie Pan, Ke Hu, Zhibo Gao, Haitao Zhao. Combinational markers of neoantigen intratumor heterogeneity and neoantigen burden predict progression-free survival for lenvatinib plus PD-1 blockade in patients with intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2250.
Abstract 2253: Rapid personalized selection of immune checkpoint blockage agent combinational treatment for an adenocarcinoma patient
Authors: Lee Kang-Yun , Chen Chien-Chung , Huang Won-Ting
Published: August 2019

The combination of immune checkpoint blockage (ICB) agent and chemotherapy for cancer treatment is gaining lots of attention lately. However, there is no clear biomarker or index for such treatment, particularly after tumor gaining resistance to ICI. In this study, an improved hollow fiber assay (HFA) was utilized based upon a novel substrate of microtube array membrane (MTAM) to test the possible patient response towards the combined treatment of immunotherapy and chemotherapy. In this case study, biopsy of a stage 4 adenocarcinoma (cT2N3M1b) patient, with the disease progression after a durable response to prior pemetrexed and Nivolumab, was retrieved and treated with enzyme for a homogenous cell suspension. Pieces of MTAM preloaded with 106 of cancer cell along with patient’s peripheral blood mononuclear cell (PBMC) were implanted subcutaneously in bulb C mice. These mice were grouped and subjected to two different ICB combinational treatments (1) Nivolumab with Paclitaxel and (2) Nivolumab with Pemetrexed. 10 days later MTAMs were explanted and cancer cells were subject to MTT assay for their viability. The results of two treatments clearly demonstrated the better efficacy of Nivolumab and Paclitaxel towards this patient cancer cells. Clinically, CT images taken before and after a two-month treatment show positive response towards the combinational treatment of Nivolumab and Paclitaxel. This case study suggests a rapid clinical regiment prediction/selection of ICB combinational treatment is possible in two weeks using MTAM/HFA and provides reasonable evident for the corresponding treatment.

Citation Format: Kang-Yun Lee, Chien-Chung Chen, Won-Ting Huang. Rapid personalized selection of immune checkpoint blockage agent combinational treatment for an adenocarcinoma patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2253.
Abstract 227: BPRDP056, a novel small molecule drug conjugate specifically targeting phosphatidylserine for cancer therapy
Authors: Hsu Chia-Yu , Chen Yun-Yu , Lo Chen-Fu , Chiu Tai-Yu , Chen Ching-Ping , Huang Chen-Lung , Huang Chung-Yu , Wang Min-Hsien , Chao Yu-Sheng , Shih Joe C. , Yeh Teng-Kuang , Tsou Lun K. , Chen Chiung-Tong
Published: August 2019

Zinc(II)-dipicolylamine (Zn-DPA) have been described to specifically complex with phosphatidylserine (PS), which has a higher level at the external surface of cancer cells in tumorigenic condition. BPRDP056 is a Zn-DPA-SN38 conjugate designed to provide a PS-targeting drug delivery of cytotoxic payload SN-38 at the tumor microenvironment, thereby, decrease the dosage of SN38, while induce apoptosis in cancer cells. The in vivo therapeutic efficacy of BPRDP056 against the growths of human tumors has been shown significant in mice subcutaneously bearing a tumor type of pancreas, prostate, colon, liver, breast and glioblastoma, as well as in mice with an orthotopic pancreatic tumor. BPRDP056 shrunk tumors at a lower dosing intensity (~20%) of SN38 compared to CPT-11 in all models tested. Micro-Western assays showed that BPRDP056 exhibited apoptotic cell death signal levels similar to those of CPT-11 in the treated tumors in mice. Furthermore, pharmacokinetic and preliminary toxicology studies showed that BPRDP056 has a good stability in circulation with an acceptable therapeutic safety window in mice. BPRDP056 has been demonstrated with a tumor targeting ability and thus increases the cytotoxic payload SN38 concentration in situ for improved efficacy. Its therapeutic spectrum against malignant neoplasm will be expected to cover the PS-rich tumor microenvironment of all cancer types. BPRDP056 is a first-in-class Small Molecule Drug Conjugate for anti-cancer therapy.

Citation Format: Chia-Yu Hsu, Yun-Yu Chen, Chen-Fu Lo, Tai-Yu Chiu, Ching-Ping Chen, Chen-Lung Huang, Chung-Yu Huang, Min-Hsien Wang, Yu-Sheng Chao, Joe C. Shih, Teng-Kuang Yeh, Lun K. Tsou, Chiung-Tong Chen. BPRDP056, a novel small molecule drug conjugate specifically targeting phosphatidylserine for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 227.
Abstract 228: KSPi-ADCs: ADCs with novel kinesin spindle protein inhibitor payloads and a tailor-made linker chemistry
Authors: Lerchen Hans-Georg , Stelte-Ludwig Beatrix , Sommer Anette , Berndt Sandra , Rebstock Anne-Sophie , Johannes Sarah , Marx Leo , Mahlert Christoph , Greven Simone , Dietz Lisa , Joerissen Hannah , Weinmann Hilmar
Published: August 2019

Inhibitors of kinesin spindle protein (KSP/Eg5) have raised great interest because of their high antitumor potency which, however, could not be transferred into highly efficient clinical regimens due to dose limiting side effects such as neutropenia. We have developed a new, highly potent pyrrole subclass of KSPis as a novel payload class in ADCs. To increase the therapeutic window, a tailor-made effector chemistry has been designed. The tumor associated protease legumain (LGMN) is utilized for ADC cleavage to provide active metabolites with high and long-lasting exposure in tumors, thus matching the KSPi mode of action. LGMN is a lysosomal asparaginyl endopeptidase overexpressed in solid tumors which is associated with invasion, metastasis and poor survival. In our studies, we show an efficient LGMN mediated linker cleavage and payload release from different ADCs. In addition, the unique LGMN substrate sequence allows for high cleavage specificity and discrimination versus other proteases such as elastase, which has been associated with an undesired extracellular cleavage of e.g. vc-MMAE ADCs resulting in neutropenia. Variations of the linker chemistry were performed to retain high potency of the KSPi-ADCs against tumor cells while decreasing the activation of KSPi-ADCs in cells of healthy organs such as the liver. From this approach, highly potent KSPi-ADCs with improved tumor/organ ratios of active metabolites were obtained. In conclusion, ADC metabolism under legumain control is a versatile strategy to provide KSPi-ADCs with high potency and an improved safety profile.

Citation Format: Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Anette Sommer, Sandra Berndt, Anne-Sophie Rebstock, Sarah Johannes, Leo Marx, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Hilmar Weinmann. KSPi-ADCs: ADCs with novel kinesin spindle protein inhibitor payloads and a tailor-made linker chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 228.
Abstract 2283: Accurate and efficient Next-Generation Sequencing approach for liquid biopsy detection and monitoring
Authors: Chen Cai , Kasbek Christopher , Song Yang , Chen Si , Almalki Nouran , Li Jiangyu , Wan Yifei , Huang Jun , Ding Wei
Published: August 2019

Liquid biopsy detection using ctDNA has great potential in cancer diagnosis, monitoring, and predicting survival. However, detection sensitivity and specificity remain major challenge at the current stage. Here we utilized a targeted next-generation sequencing (NGS) approach to develop a noninvasive test to identify single nucleotide variations (SNVs), indels, fusions, copy number variations (CNVs) and microsatellite instability (MSI) present at low frequencies in different solid cancer patients. Target regions of interest were sequenced using tagging of individual molecules, followed by amplicon library generation and massive parallel sequencing using an Illumina platform. The variants were called and annotated using our in-house Actionable Genomic Interpretation System (AGIS) bioinformatics pipeline. This CLIA-approved NGS panel (LiquidGx™) detects more than 170 variants in 17 genes which were chosen mostly based on NCCN clinical actionable guidelines (AKT1, ALK, BRAF, EGFR, ERBB2, HRAS, KIT, KRAS, MAP2K1, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, ROS1 and TP53) with detection limit of 0.1%. Mutations are detected with 94% sensitivity and 99.8% specificity in a short turnaround time of 3-5 business 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. Fusions are detected from exosomal RNA, which could increase the sensitivity and/or concordance to tumor. For spike-in data validation, the limit of detection (LOD) of CNV can reach 2.2 -2.5 total gene copies. In addition to actionable mutations for targeted therapy, LiquidGx™ provides MSI status, which can predict a predisposition to mutations as a result from impaired DNA mismatch repair (MMR) and help predict if a tumor will respond to anti-PD-1 therapy. The LOD of our MSI detection is 2-4%. 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 different cancer types.

Note: This abstract was not presented at the meeting.

Citation Format: Cai Chen, Christopher Kasbek, Yang Song, Si Chen, Nouran Almalki, Jiangyu Li, Yifei Wan, Jun Huang, Wei Ding. Accurate and efficient Next-Generation Sequencing approach for liquid biopsy detection and monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2283.
Abstract 2289: Prognostic impact of progastrin levels in blood compared to MSKCC based clinical prognosis in metastatic renal cell cancer patients
Authors: Kohli Manish , Tan Winston , Payen Lea , Langlois-Jacques Carole , Liaud Pierre , Maucort-Boulch Delphine , Joubert Dominique , Prieur Alexandre
Published: August 2019

Background and Aims: Progastrin is a tumor promoting peptide which is detectable in the blood of patients with different cancers (Prieur et al. AACR 2017, Prieur et al. ASCO 2017). Progastrin gene is a direct target of the WNT/ß-catenin oncogenic pathway involved in tumorigenesis of many organs, but it is unknown if it has any prognostic significance in metastatic renal cell cancer (mRCC) patients. We evaluated progastrin as a prognosis marker along with known markers of prognosis in mRCCC.

Methods: 145 patients with mRCCC were enrolled in this study and blood samples were drawn after consent. Progastrin was measured using the ELISA cancerREAD®. Progastrin concentrations in the 145 mRCCC patients (test set) was assessed against 213 samples from asymptomatic volunteers from the French blood establishment (control set). The prognostic impact of progastrin levels was determined with overall survival (OS) using Cox proportional hazards and also compared to MSKCC based clinical prognosis (good;intermediate; poor). Statistical significance was considered at P≤0.05.

Results: The median follow-up of the cohort was 5.45 years (IQR: 1.87-9.95) and at the time of analysis 98/145 patients had died from disease progression. Plasma progastrin was detected in 95% of the patients (cut-off value 1 pM, range 0 to 272 pM, median value of 7.2 pM; IQR 3.20-19.71) compared to the control set (median value=0.61 pM; IQR 0.00-1.58). The Receiver Operating Characteristic analysis indicated an area under the curve of 0.92 (p<0.0001; 95% CI 0.89 to 0.94). At the univariate level, MSKCC scores (good; intermediate; poor categories) in this cohort was associated with OS and was prognostic (p<0.0001). We detected progastrin levels were higher with MSKCC score poor prognostic (p<0.0002) (median 30.39 pM; IQR 9.31-57.20). Elevated progastrin was also independently associated with poor survival (p<0.0001) and a multivariate model of MSKCC taken with progastrin levels remained significantly associated with poor survival (p<0.0001).

Conclusion: Elevated progastrin levels in mRCCC is correlated with poor survival and further refines clinically used MSKCC prognostic scores. Progastrin assay is a simple and inexpensive blood test that might define subsets of mRCCC patients with poor survival who need to be identified for aggressive treatments.

Citation Format: Manish Kohli, Winston Tan, Lea Payen, Carole Langlois-Jacques, Pierre Liaud, Delphine Maucort-Boulch, Dominique Joubert, Alexandre Prieur. Prognostic impact of progastrin levels in blood compared to MSKCC based clinical prognosis in metastatic renal cell cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2289.