bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasma ctDNA sequencing supplementary to tissue-based molecular profiling as a source of additional actionable information in advanced cancers.
Authors: Hancock Bradley Allen , Wong Swee Seong , Sinha Saloni , Barber Tom , Salisbury Benjamin A. , Bray Steven M. , Smallwood Lori A. , Schneider Bryan P. , Radovich Milan 
Published: May 2020

e15554 Background: The standard for tumor molecular profiling in precision oncology is the tissue-based biopsy. However, tissue biopsies are limited in the ability to capture the bulk of heterogeneity in advanced cancers. The integration of liquid biopsy diagnostics complementary to tissue-based sequencing may provide a broader perspective of heterogeneity and actionability. Methods: Patients with metastatic solid tumors were evaluated in the Indiana University Health Precision Genomics (PG) Program. We retrospectively analyzed existing genomic data from matched plasma and tissue that were collected within a 90-day window. Plasma sequencing was generated with a CLIA-certified test: FoundationACT/FoundationLiquid. Tumor tissue sequencing was performed either with CLIA-grade whole exome or a gene-panel. All sequencing data was reviewed by a molecular tumor board for the determination of a personalized treatment strategy. Molecular data for this analysis was concatenated and analyzed for mutations present in ctDNA but absent in tissue. Clinical actionability was defined based on clinical evidence of a biomarker to guide therapy or match a patient to a clinical trial. Results: 288 subjects with tissue- and plasma-derived mutation data were analyzed. Discordance, defined by the presence of a somatic mutation in the plasma that was not present in the tumor tissue, was detected in 156/288 (54%) of pairs. Interestingly, the discordance was clinically actionable in 26/288 (9%) subjects. In the 32 gene-level instances of actionable discordance, the 3 top mutated genes were PIK3CA (7), PTEN (4), and EGFR (4). Discordant mutations in ATM and CHEK2, which are actionable with PARP inhibitors, but also frequently mutated in clonal hematopoiesis of indeterminant potential (CHIP) represented 4/26 (15%) instances. A complete and formal CHIP analysis is ongoing. As a result of plasma discordance, targeted interventions were employed in 3/26 (12%) subjects targeting high TMB with immunotherapy, an ERBB2 mutation with ado-trastuzumab emtansine, and a BRCA1 mutation with olaparib. All 3 patients were non-responders. Conclusions: Discordant mutations that appear in ctDNA but not in matched tumor tissue is a common occurrence. The majority of these mutations are either not druggable or variants of unknown significance, but a significant subset would be deemed clinically actionable. Clinical efficacy of targeted intervention based on actionable liquid biopsy discordance warrants further investigation.
Correlation of levels of extracellular vesicles in peripheral and pulmonary blood plasma with pathological stages of lung cancer patients.
Authors: Kim Hyun Koo , Choi Byeong Hyeon , Quan Yu Hua , Rho Jiyun , Hong Sunghoi , Park Yong , Choi Yeonho , Park Ji-Ho , Yong Hwan Seok , Han Kook Nam , Choi Young Ho 
Published: May 2020

e15558 Background: Exosome concentration is known to be higher in cancer patients than in healthy individuals. In this study, we observed that the levels of exosomes differ in tumor-draining pulmonary blood and in peripheral blood in animal models and human subjects at different pathological stages of lung cancer. Methods: Ten rabbits and 40 humans formed the study cohorts. Blood was collected from a peripheral vein in all groups, and pulmonary blood was collected intraoperatively from all groups, except the healthy human controls. Quantitative analysis of exosomes was performed by nanoparticle tracking assay, CD63 enzyme-linked immunosorbent assay, and western blotting. Results: The peripheral blood of lung cancer-bearing animals and patients with lung cancer carried higher amounts of exosome than that from healthy controls ( p < 0.01 and p < 0.001, respectively). Moreover, pulmonary blood from lung cancer-bearing animals and patients had significantly higher exosome levels, compared to preoperative peripheral blood ( p < 0.01 and p < 0.0001, respectively). In patients, pulmonary exosome levels showed higher correlation with pathological stages of lung cancer than the peripheral exosome levels. Conclusions: Exosome levels increased with increasing grade of lung cancer, and this trend was more prominent in the pulmonary than in the peripheral blood.
A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors.
Authors: Murad Andre Marcio , Casali-da-Rocha José Claudio , Carneiro Juliana Garcia 
Published: May 2020

e15562 Background: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGF(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), PIK3CA (mutations), ERBB2(CNV- copy number variation), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Therefore, our genes can be used in panels, both in therapeutically applied genotyping and in detecting molecular responses as well as in secondary resistance to tumors such as NSCLC, breast, cervix. rectal, GIST, melanoma and pancreas. Methods: dd-PCR was performed using the QX200 system (BIO-RAD, Hercules). The extraction of DNA and RNA was done using the magnetic beads technique of Thermofisher's MagMAX Kit. All samples were tested in duplicate. Up to now, 54 metastatic cancer patients were tested: NSCLC - 14 (26%), breast - 11 (20%), colorectal - 11 (20%), melanoma - 9(17%), pancreas - 4(7%), ovarian - 3(6%) and salivary glands - 2 (4%). Results: Significant genomic alterations were detected in 19 (35%) patients: 5 (9%) mutations in the KRAS G12V gene (all in colorectal cancer), 5 (9%) ERBB2 amplification (breast cancer), 2 (3.65%) EGFR L858R mutations (NSCLC), 2 (3.5%) EGFR del19 mutations (NSLC), 2 (3.5%) EGFR T790M mutations (NSCLC), 2 (3.5%) BRAF-V600E mutations (colon and melanoma), 1 (1.8%) ALK-EML4 fusion (NSCLC). The MAF (Mutant Allele Fraction) ranged from 0.9% to 24%. In all cases, the results were decisive for the indication or change of a targeted therapy. Median turnaround time was 36 hours and the average cost of the panels was around 500 USD (median of 4 genes per panel). Conclusions: Our results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.
Mutational landscape of AKT1/2/3 in Chinese patients with solid tumors.
Authors: Kong Hui , Fang Weijia , Mou Haibo , Chen Feng Xiao , Liu Lingxiang , Wei Jia , Cui Shiyun , Wei Yongzhong , Li Juming , Du Juan , Ling Qi , Tang Xiaofeng , Qian Yigang , Yu Songfeng , Xue Liang , Wang Ting , Xiao Min , Pan Qiuyue , Zhang Shiyue , Zhang Lin 
Published: May 2020

e15566 Background: As a central component of PI3K/AKT pathway, AKT serves as an attractive target of anti-cancer strategy with various AKT inhibitors, which show great promise in phase I/II clinical trials. This study aimed to investigate AKT1/2/3 status in different types of cancers by using next generation sequencing (NGS). Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from 10,010 Chinese patients with solid tumors and subjected to next-generation sequencing (NGS)-based 450 gene panel testing carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Genomic alterations, tumor mutational burden (TMB) values, and microsatellite instability (MSI) status were assessed with a mean coverage of 1000X, including single base substitutions, short and long insertion/deletions, copy number variations, gene fusions, and rearrangements. Genomic data and immune checkpoint inhibitors (ICIs) treatment outcome of a cohort of 1610 patients with solid tumors were derived from cBioPortal (MSKCC, Nat Genet. 2019). Results: AKT1/2/3 were found to be mutually exclusive with each other and accounted for 3.4% in this cohort. The frequencies of AKT1/2/3 variations were 1.1%, 1.6%, and 0.8%, respectively. The most common co-altered genes associated with AKT1/2/3 variations were TP53 (69.4%), PIK3CA (19.3%), KRAS (19%), CCNE1 (18.4%), CDKN2A (16.6%), and 11q13 (6.5%). AKT1/2/3 variations were significantly associated with higher TMB, and independent of MSI status. Outcome data from the MSKCC cohort showed that patients with AKT1/2/3 variations had a remarkable clinical benefit to ICIs treatment compared to patients with wild-type AKT1/2/3 in overall survival (OS) (NA vs 18 months, p = 0.009). Furthermore, AKT1/2/3 variations were independent risk factors of OS (HR: 0.55, 95%CI: 0.34-0.87, p = 0.012). Conclusions: The prevalence of AKT1/2/3 somatic alterations across different types of solid tumors in China was 3.4%. AKT1/2/3 variations were associated with an increased TMB and favorable response to ICIs, suggesting that A KT1/2/3 variations may be biomarkers for guiding anti-AKT agents and ICI treatment.
Utilizing single-cell-subtype analysis via advanced microdissection methods and the impact on precision medicine.
Authors: Johann Donald Joseph , Laun Sarah , Stephens Owen , Weigman Robert , Shin Ikjae , Roberge Adam , Greisman Valerie , Steliga Matthew V , Muesse Jason , Peterson Erich , Emmert-Buck Michael , Tangrea Michael 
Published: May 2020

e15571 Background: Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Patients diagnosed with advanced non-small cell lung cancer (NSCLC) are commonly of an older age and have significant co-morbidities. This frequently causes clinical dilemmas regarding the ability to obtain adequate amounts of tissue for tumor genotyping. In these cases, the tumor tissue may have been obtained by an image-guided biopsy, and the diagnosis of NSCLC proper determined via cytology. In certain instances, adequate tissue for tumor genotyping and/or a more advanced mutational analysis to identify oncogenic drivers may not be available. Methods: Following pathology review, formalin fixed paraffin embedded (FFPE) specimens were subjected to advanced immuno-based laser capture microdissection (LCM), DNA was extracted and prepped for droplet digital PCR (ddPCR) and run with a series of positive and negative controls. Results: Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from FFPE specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Conclusions: Synergy between microdissection and ddPCR enhances the molecular analysis. We demonstrate a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time and cost-efficient manner, using immunostain targeting as an example.
Application of automated segmentation and classification of PD-L1 immunohistochemistry images in cancer treatment prediction.
Authors: Zhou Xinrui , Zhang Xiaochen , Wei Jinwang , Liu Jun , Dai Chun , Wang Guan , Xu Qiang 
Published: May 2020

e15572 Background: Therapies targeting immune checkpoints, such as the programmed cell death-1 (PD-1) / programmed cell death ligand 1 (PD-L1) pathway, have become successful in treating several cancer types, particularly those with high PD-L1 expression. Patient response rates, however, are variable, demanding a more sensitive companion diagnostic test than the current method of manual scoring of PD-L1 immunohistochemistry images. The low predictive value of manual PD-L1 scoring is at least partially compounded by the pathologist-to-pathologist or hospital-to-hospital variation. To eliminate the variation caused by human factors, we developed an alternative PD-L1 scoring method based on automated image analysis and assessed its predictive and prognostic values. Methods: Archival or fresh tumor biopsies were analyzed for PD-L1 expression by immunohistochemistry. Digital images were automatically scored as PD-L1 positive or negative using a custom algorithm implemented in Python and Tensorflow, which uses a fine-tuned, ImageNet pre-trained, Inception-ResNet-V2 model as the binary classifier along with a modified U-net image segmentation model to determine the final prediction. Supervised machine learning was used to control for heterogeneity introduced by PD-L1 positive inflammatory cells in the tumor microenvironment. Samples from 256 patients were collected and used in algorithm training and verification by random assignment with an 8:2 split ratio. Results: In total, 10,000 images with balanced PD-L1 positive and negative distributions were obtained. A highly significant correlation of PD-L1 scores was found between pathologist-based consensus reading and automated analysis using our custom algorithm (R = 0.97, p < 0.0001). The automated analysis reached 0.94 sensitivity and 0.96 specificity. Additionally, it showed excellent reproducibility (R = 1.0, p < 0.0001) in independent trials executed on different workstations, which is far superior to the manual scoring performed by human examiners. Conclusions: Our automated PD-L1 evaluation algorithm significantly reduces scoring variability. It may help to identify cancer patients who can have optimal response to PD-1/PD-L1 related immune therapy and therefore facilitate patient stratification in clinical practice.
Enabling variant calling in challenging FFPE samples by coupling a novel library preparation chemistry with exome sequencing.
Authors: Min Bosun , Das Chakravarty Ushati , Hong Manqing , Star Ekaterina , Huang Hsiao-Yun , Barnes Tim , Chen Tzu-Chun , Lopez Ramses , Scott Karissa , Lewis Lynette A , Groenewold Steve , Ren Longhui , Zhou Jinglie , Lai Kevin , Sheu Jessica , Wang Xiaohui , Lau Patrick J , Henck Steven 
Published: May 2020

e15584 Background: Comprehensive tumor profiling using NGS is fundamentally transforming oncology research. However, converting archival tissue samples into libraries is often challenging due to the low quantity and quality of DNA. Here we present accurate detection of variants in the human exome using the novel chemistry of the xGen Prism DNA library preparation kit, optimized for low-input and degraded samples, with xGen Research Exome v2.0 hybrid-capture enrichment. Methods: The IDT Exome v2 panel was used to carry out targeted sequencing of Prism DNA libraries generated from archival FFPE samples. The unique library preparation is enabled by an engineered mutant ligase and proprietary adapters that prevent chimeras and suppress dimer-formation, thereby maximizing the conversion of input DNA to sequencing libraries. Results: We achieved high yields of library (300-400 ng) from input amounts as low as 25 ng for severely damaged FFPE samples (DIN 1-3), > 90% on-target rates and uniform depth of coverage ( > 96% bases covered at > 20X and > 98% bases covered at > 10X) for FFPE samples across a wide range in quality. We also observed minimal exon drop-outs in difficult-to-target genes for severely damaged FFPE material. To validate the variant calling performance of the Prism-Exome workflow, we used the Horizon OncoSpan FFPE reference control which contains 1-92% AF SNVs and Indels and achieved > 98% sensitivity across ~250 SNVs and Indels. Conclusions: This study demonstrates that the xGen Exome Research v2, when combined with xGen Prism DNA library preparation, provides researchers with a complete human exome FFPE-sequencing solution with robust performance across FFPE samples of varying quality.
Treatment of breast, ovarian and lung cancer cells by inducing apoptosis with a deep eutectic solvent prepared from 2-deoxy-D-glucose and metformin: Cancer cell metabolism as a drug target.
Authors: Vuoti Sauli , Eemil Jaakko , Narasimha Kumar , Reinikainen Kai 
Published: May 2020

e15599 Background: Targeting cancer cell metabolism has gained attention as a future strategy to fight cancer. A characteristic of tumor cells is the elevated aerobic glycolysis for energy production. It has been shown that 2-deoxy-D-glucose (2DG) inhibits glycolysis and induces apoptotic cell death in different tumor types. The anti-diabetic drug metformin has been used in combination to enhance the inhibitory effect. So far, the attempts to combine both compounds in a clinical setting have been limited by the requirement of concentrations higher than those accessible in blood plasma of human beings. Deep eutectic solvents (DES) are solvent mixtures prepared from hydrogen bond donors and acceptors, wherein pharmaceutically active compounds can also be one of the components. Besides having unique physicochemical properties, DESs have been reported to demonstrate or enhance anticancer properties. Methods: We developed a DES mixture from 2DG and MET using a method based in mechanical grinding. We investigated the anticancer activity of conventional and DES mixtures of MET and 2DG, and used the mixtures to inhibit the growth, migration and invasion of cancer cells, and induce cell cycle arrest in vitro. Results: MET and 2DG, alone and in combination, induced apoptosis in the H460, SKOV-3, MDA-MB-231 and HCC1806 (TNBC) cell lines. Induction of apoptosis was further quantified by measurement of the loss of mitochondrial membrane potential and cleavage of PARP. DES mixtures had the highest impact on cell viability, exceeding the effect of 2DG/MET as single agents or combinations at all clinically relevant concentrations. The DES mixture with the lowest concentration to induce apoptosis consisted of 100 µM 2DG and 200 µM MET. A conventional solution with similar concentrations showed no activity. Conclusions: We developed a DES from 2DG/MET, which significantly reduced the viability of several types of cancer cells, surpassing the effect of single components or mixtures. The DES does not necessitate the use of additional solvents and could be used to develop clinical applications for targeting cancer cell metabolism.
Preclinical evaluation of CLX-155, a novel prodrug of 5-FU for the treatment of cancer.
Authors: KB Sunil Kumar , Kandula Mahesh , P Sivanesan 
Published: May 2020

e15614 Background: CLX-155 is a novel prodrug of 5-FU, developed by adding two acetyl groups (ester bond) and a caprylic acid moiety (amide bond) to 5'-DFCR ring structure. CLX-155 is designed to have a differentiated ADME profile, with improved safety and efficacy over capecitabine. Here, we present the preclinical data of CLX-155 in comparison with capecitabine. Methods: Solubility/stability of CLX-155 was studied in buffers, simulated gastrointestinal fluids and liver microsomes. In vitro metabolite profiling in hepatocytes was done following standard protocol. An oral (gavage) dose range finding (DRF) toxicity, comparative oral pharmacokinetic study and antitumor activity in HCT-116 human colon cancer cell xenograft model was conducted in mice. DRF study in mice evaluated the toxicity of CLX-155 when administered at once daily doses of 100 to 1000 mg/kg/day for 7-days. The oral pharmacokinetics CLX-155/metabolites (5'-DFCR, 5'-DFUR and 5-FU) was determined in mice at MTD dose of 500 mg/kg and for capecitabine at 1000 mg/kg. Antitumor activity of CLX-155 was evaluated in Foxn1 athymic mice at 125, 250 and 500 mg/kg/day and for capecitabine at 1000 mg/kg/day. Treatment was performed for three consecutive weeks (5 days on; 2 days off per week). Results: The results of in-vitro studies confirmed an acceptable profile for further development of CLX-155. Maximum tolerated dose of CLX-155 in mice was 500 mg/kg/day. The key toxicity findings were consistent with the mechanism of action and comparable with capecitabine. Oral PK study in mice showed a lower plasma Cmax for 5'-DFCR, 5'-DFUR and 5-FU. Plasma AUCs of the metabolites were close to or higher than that of capecitabine, indicating an extended absorption or altered metabolism in comparison with capecitabine. CLX-155 caused significant, tumor growth inhibition at all the dose levels tested. Complete tumor regression was seen in 2/10 animals at 500 mg/kg/day of CLX-155. CLX-155 was better tolerated in the mouse xenograft study, no mortality in CLX-155 versus 2/10 in capecitabine group. Conclusions: CLX-155 has an excellent safety and a differentiated ADME profile in relation to capecitabine. This translated into an improved in-vivo antitumor activity for CLX-155 in the HCT 116 xenograft model in relation to capecitabine. Overall data indicate that CLX-155 could offer significant improvements over the currently approved capecitabine in terms of dose size, frequency of administration, safety and interpatient variability in pharmacokinetics.