bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 5690: Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA)
Authors: Artieri Carlo G. , Beauchamp Kyle A. , Vysotskaia Valentina S. , Welker Noah C. , Evans Eric A. , Chu Clement , Tezcan Haluk , Haque Imran S. 
Published: July 2017

Abstract
The evaluation of cfDNA allows novel approaches to noninvasive detection of actionable alterations, resistance mechanisms, and tumor monitoring in patients with cancer. Importantly, tumor-specific DNA fragments represent a small minority of the cfDNA and can be obscured by false positive (FP) variants introduced by chemical damage and sequencer error. To address this, we improved key processes in the design of NGS libraries, including a new molecular barcoding approach, that maximize molecular recovery while eliminating spurious variants. We engineered a set of Illumina sequencing chemistry compatible adaptors incorporating unique molecular identifiers (barcodes) enabling reconstruction of the sequence of both strands of the original DNA molecule. These barcodes incorporate a number of key design improvements as compared to published methodologies, which enhance sequencer cluster density, thereby increasing library diversity and molecular recovery. Our new design identified both chemical and sequencer errors, reducing incorrect base calls to rates below 5e-7. We validated our methodology for use in cfDNA using both dilution experiments and patient blood samples with known oncogenic alterations via a custom capture panel targeting actionable genomic alterations in a 55kb region. By identifying the molecular origin of each read, we found that the sensitivity of detection obtained from barcoded libraries followed ideal binomial sampling expectations. We obtained an average molecular depth of 1,000 molecules per site from the plasma extracted from a single blood collection tube, which corresponded to an 80% sensitivity of detection of known oncogenic single-nucleotide and indel mutations at 0.15% mutant allele frequency (MAF) in cfDNA with no FP calls. Furthermore, we successfully detected known gene-fusions at 0.5%, and amplifications (&gt;10 copies) down to 1% MAF. We designed and validated a custom-engineered error-correcting sequencing adapters, ideal for broad range of applications requiring high accuracy detection of ultra-low frequency alterations.
Note: This abstract was not presented at the meeting.
Citation Format: Carlo G. Artieri, Kyle A. Beauchamp, Valentina S. Vysotskaia, Noah C. Welker, Eric A. Evans, Clement Chu, Haluk Tezcan, Imran S. Haque. Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5690. doi:10.1158/1538-7445.AM2017-5690
Abstract 5697: Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads
Authors: Mizutani Kosuke , Kawakami Kyojiro , Fujita Yasunori , Horie Kengo , Kameyama Koji , Ito Masafumi , Deguchi Takashi 
Published: July 2017

Abstract
Purposes of the study: Exosome has been demonstrated to be a useful non-invasive biomarker for several cancers including prostate cancer. For better understanding cancer-derived exosome, their proteomic analysis is necessary. The aim of this study is to establish the method for isolation and analysis of prostate cancer-related exosomes. Experimental procedures: Total exosomes were isolated from the conditioned media of LNCaP cells by ultracentrifugation. Then, exosomes positive for CD9 or prostate specific membrane antigen (PSMA) were isolated by magnetic beads conjugated with anti-CD9 or -PSMA antibody, respectively. Isolated exosomes were subjected to proteomic analysis. The PSMA positive fraction was also collected by anti-PSMA beads from diluted serum of 3 prostate cancer patients and analyzed by proteomics. Results: A total of 126, 139 and 13 proteins were detected in the LNCaP exosome fractions that were isolated by ultracentrifugation, anti-CD9 beads and anti-PSMA beads, respectively. Out of 126 proteins identified in ultracentrifuge isolated exosome, 56 and 6 protein were found in exosomes that were isolated by anti-CD9 beads and anti-PSMA beads, respectively. Seven proteins were commonly detected in exosomes that were isolated by both beads. Fifty, 54 and 48 proteins were identified in the PSMA positive fraction from serum of 3 prostate cancer patients (P1: localized prostate cancer, P2: advanced prostate cancer and P3: castration resistant prostate cancer). Thirty-three proteins were detected in one patient, 22 in two patients and 25 in three patients. Most proteins detected in all patients were serum- or immunoglobulin-related proteins. The numbers of proteins that were detected only in P2 and P3 were 16 and 8, respectively. Six proteins were found in both P2 and P3 patients. Conclusion: In the present study, we analyzed prostate cancer-related exosomes that were isolated by immunoaffinity-based method. Although most proteins were serum- or immunoglobulin-related proteins, proteins that had been reported as a cancer marker were also detected, which could be candidates for exosomal marker of prostate cancer.
Citation Format: Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Kengo Horie, Koji Kameyama, Masafumi Ito, Takashi Deguchi. Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5697. doi:10.1158/1538-7445.AM2017-5697
Abstract 5698: Next generation sequencing of circulating-free DNA from advanced non small cell lung cancer patients using Gene Reader®
Authors: Casas Clara Mayo de las , Garzón Mónica , Ariza Nuria Jordana , Balada Ariadna , Bertran-Alamillo Jordi , García Beatriz , Villatoro Sergio , Aldeguer Erika , Rodriguez Sonia , Campos Raquel , Ramirez Santiago Viteri , Gonzalez-Cao Maria , Karachaliou Niki , Costa Rafael Rosell , Molina-Vila Miguel Angel 
Published: July 2017

Abstract
Background: Stand alone tests such as PCR-derived techniques, FISH or IHC are usually employed to determine clinically relevant alterations in non-small cell lung cancer (NSCLC). However, they target single genes and proteins. Mutiplex techniques can reduce the turnaround time and quantity of sample in this setting, but require a careful validation.
Methods: A total of 41 cfDNA samples from serum and plasma from advanced NSCLC p were analyzed with the Actionable Insights Tumor Panel, which covers mutations in 15 clinically relevant genes, using the Gene Reader platform (Qiagen). The samples had been previously genotyped for EGFR, KRAS and BRAF mutations by stand alone, PNA-Taqman assays. Paired biopsies were available in 37 cases. The remaining 4 corresponded to p.T790M-positive blood samples of p progressing to EGFR TKIs.
Results: Of the 41 samples taken into the GeneReader workflow, some had a DNA input concentration below specifications, in spite of this limitation, good results were obtained. 14 mutations were fully concordant between tissue, Taqman and GeneReader and the four p.T790M mutations were concordant between Taqman and GeneReader. Five mutations present in tissue were detected by GeneReader and not by Taqman and 11 mutations detected by Taqman were below the 1% detection threshold of GeneReader. Finally, 12 mutations present in tissue were not detected in cfDNA by any of the assays. Concordance between the stand alone tests and the Gene Reader in cfDNA was 64%, raising to 84% if mutations &lt;1% allelic fraction were excluded.
Conclusions: Application of NGS to liquid biopsies is challenging and requires a careful validation. However, once fully validated, NGS will probably become the methodology of choice for cfDNA analysis in NSCLC patients at presentation and at progression.
Citation Format: Clara Mayo de las Casas, Mónica Garzón, Nuria Jordana Ariza, Ariadna Balada, Jordi Bertran-Alamillo, Beatriz García, Sergio Villatoro, Erika Aldeguer, Sonia Rodriguez, Raquel Campos, Santiago Viteri Ramirez, Maria Gonzalez-Cao, Niki Karachaliou, Rafael Rosell Costa, Miguel Angel Molina-Vila. Next generation sequencing of circulating-free DNA from advanced non small cell lung cancer patients using Gene Reader® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5698. doi:10.1158/1538-7445.AM2017-5698
Abstract 5702: A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers
Authors: Dong Liqin , Khetrapal Pramit , Rodney Simon , Tan Wei Shen , Rezaee Sheida , Winter Patricia de , Kelly John , Feber Andrew 
Published: July 2017

Abstract
Bladder cancer (BC) is the 5th most common cancer in Western societies with a rising global incidence. The major challenge to improving patient outcomes is to better identify patients at risk for recurrence, progression, and metastasis, and to monitor treatment response. A non-invasive monitoring assay is needed in order to interrogate the molecular features and clinical tumor heterogeneity of bladder cancers, which will potentially lead to better stratified patient management.
We developed a targeted amplicon-based re-sequencing workflow for detecting and tracking genomic mutations in cfDNA using dual molecular indexed primers for specific amplification of a panel of 20 of known (TERT, FGFR3, PIK3CA, HRAS) and novel BC mutations. Only 3hr time is needed for the total workflow for the library generation and followed by next generation sequencing (NGS) performed on Illumina MiSeq sequencing platform. Data analysis and variants calling was performed.
After a thorough test, optimization, and validation of the targeted panel and the workflow on selected samples of matched bladder tumor-blood pairs and controls, 45 samples were included for testing the workflow, which includes samples of 16 matched bladder tumor-blood pairs, technical duplicates of cfDNA DNA Reference Standards (Horizon Discovery) with mutations at known allelic frequencies. Technical duplicates of cfDNA extracted from blood from patients with metastatic BC (pre and post chemotherapy) were also included in this study. Somatic variants from matched tumor-blood pairs were detected. We showed that detection sensitivity at 1.0% allelic frequency was achieved. Our preliminary results demonstrated that we were able to detect mutation burden from cfDNA samples and track mutation burden change in response to chemotherapy for patients with metastatic BC.
Here we developed a sensitive, non-invasive targeted NGS workflow to detect somatic mutations for bladder cancers from both tumor DNA and cfDNA samples. Our early results demonstrated that we can detect and track mutational changes from patients with metastatic BC.
Citation Format: Liqin Dong, Pramit Khetrapal, Simon Rodney, Wei Shen Tan, Sheida Rezaee, Patricia de Winter, John Kelly, Andrew Feber. A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5702. doi:10.1158/1538-7445.AM2017-5702
Abstract 5703: An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic breast cancer
Authors: Liu Jiasu , Wang Yaohuai , Sun Shengrong , Yu Dehua Derek 
Published: July 2017

Abstract
Activating mutations of estrogen receptor α (ESR1) are attributed to acquired resistance to estrogen-deprivation therapy in patients with metastatic breast cancer (MBC). Thus, detection of these mutations is clinically critical for monitoring early progression and effective therapeutic options in treatment of BC patients under hormonal therapy. However, current investigations for detection ESR1 mutations mainly focus on digital PCR technology due to low abundance of the cfDNA in plasma samples, which is not a currently feasible method for clinical diagnosis.
Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection of ESR1 four mutations (Y537S, Y537C, Y537N, and D538G) in a single tube reaction. In this real-time PCR system, the wild-type ESR1 DNA is completely blocked by a modified probe with a higher annealing temperature, and the mutant gene is selectively amplified by the modified ARMS primers. The assay was found to be highly specific and sensitive. With this assay, as low as 0.1% mutant DNA template in the background of a total of 10ng wild-type genomic DNA could be detected.
By using this assay, we analyzed the ESR1 mutations in 50 Chinese MBC FFPE and plasma samples. The ESR1 mutations were detected in 3 FFPE and 6 plasma samples, which were confirmed by pyrophosphate sequencing.
The multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations with potential for clinical application.
Citation Format: Jiasu Liu, Yaohuai Wang, Shengrong Sun, Dehua Derek Yu. An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5703. doi:10.1158/1538-7445.AM2017-5703
Abstract 5704: Ultrasensitive mutational analysis in cell-freeDNA by targeted next-generation sequencing with molecular tags
Authors: Tam Nga Wan Rachel , Diala Irmina , Chen Xiaoji , Darbonne Walter 
Published: July 2017

Abstract
Circulating cell-free DNA (cfDNA) in plasma offers a non-invasive approach to monitor tumor molecular profiling in real-time at multiple time-points, detection of emerging genomic alterations associated with drug resistance and clarifying cancer prognosis and diagnosis of cancer recurrence or progression. Therefore, it is crucial to improve our current NGS technology to detect low frequency alleles in cfDNA.
There are many technical challenges in NGS technology, including base errors generated from library preparation and sequencing, making it hard to distinguish real mutations from false positives produced from amplification sequencing and base-calling processes.
We have adopted a more sensitive approach to overcome this challenge. We used short, random DNA sequences called unique molecular tags (UMTs) during library preparation to label input DNA. This technique can remove background errors, false positives during data analysis, providing high confident mutation calls with better sensitivity down to 0.1% (depends on DNA input amount).
We will present data showing validation of this NGS platform using commercially available DNA control, archival FFPE tissue and cfDNA matched samples and demonstrate robust sensitivity and specificity by using the off-the-shelf Archer Reveal ctDNA panel for Illumina covers 28 oncogenes. This molecular-tagged NGS panel can detect mutations down to 0.1% allelic frequency. This robust NGS platform is being used in our lab to analyze cfDNA samples, enabling the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient’s tumor evolution in real time, and aid in the development of personalized therapies for cancer patients.
Citation Format: Nga Wan Rachel Tam, Irmina Diala, Xiaoji Chen, Walter Darbonne. Ultrasensitive mutational analysis in cell-freeDNA by targeted next-generation sequencing with molecular tags [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5704. doi:10.1158/1538-7445.AM2017-5704
Abstract 5707: STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate cancer
Authors: Mohanty Sambit K. , Yagiz Kader , Luthringer Luthringer , Amin Mahul B. , Alkan Serhan , Cinar Bekir 
Published: July 2017

Abstract
Background: Currently, there is no effective pharmacotherapy for metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying mCRPC are not clearly understood, thus hindering rational-based drug design. Evidence suggests that signal transducer and activator of transcription 3 (STAT3) and 5a/b (STAT5a/b), key components of the JAK/STAT pathway, play a significant role in aggressive PC. However, expression of STAT3/5 in prostate tumor samples have not been studied. Here, we evaluated the possible role of STAT3/5a in aggressive PC.
Materials and Methods: Expression of STAT3 and STAT5a in high grade PC (HGPC, n=15) and in benign prostatic hyperplasia (BPH, n=15) tissue sections were evaluated by immunohistochemistry (IHC) according to the protocol approved by Institutional Review Board. Age ranged from 59 to 95 years (median = 81) in the former and 57 years to 86 years (median = 68 years) in the latter category. In addition, the effects of STAT inhibitor, Pimozide, on androgen-sensitive LNCaP and its castration-resistant subline C4-2 PC cell growth were assessed in vitro.
Results: A strong nuclear immunoreactivity for STAT3 and STAT5a in 93% (n=14) and 80% (n=12) of HGPC cases, respectively, were observed. Only one BPH case showed strong STAT3 expression. Focal and weak positivity for STAT3 and STAT5a were noted in 47% (n=7) and 67% (n=10) of BPH cases. None of the BPH cases showed strong STAT5a expression. HGPC cases showed significantly higher STAT3 positivity compared with BPH (p=0.01). No significant differences were observed for STAT5a expression between HGPC and BPH group (p=0.4). However, multifocal or diffuse and strong STAT3 (p&lt;0.0001) and STAT5a (p&lt;0.0001) expression was significantly higher in HGRPC than BPH. Pimozide exposure significantly inhibited LNCaP cell proliferation at 10 μM and C4-2 cell proliferation at 20 μM concentration.
Conclusion: Our results demonstrate that increased expression of STAT3 and STAT5a may serve as a predictive biomarker for efficacy of the JAK/STAT targeted therapy. Since JAK/STAT and androgen receptor signaling are functionally synergistic in contributing PC progression, the inhibition of JAK/STAT-alone or in combination with AR may lead to a novel treatment modality for mCRPC.
Citation Format: Sambit K. Mohanty, Kader Yagiz, Luthringer Luthringer, Mahul B. Amin, Serhan Alkan, Bekir Cinar. STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5707. doi:10.1158/1538-7445.AM2017-5707
Abstract 5714: AXL and GAS6 Co-expression in lung adenocarcinoma as a prognostic classifier
Authors: Seike Masahiro , Noro Ritaro , Kim Cheol-Hong , Zou Fenfei , Kubota Kaoru , Gemma Akihiko 
Published: July 2017

Abstract
AXL, a receptor tyrosine kinase implicated in cell survival, proliferation, and migration, is also associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor therapy. However, its prognostic significance in lung adenocarcinoma (AD) remains unclear. We therefore evaluated the prognostic significance of the expression of AXL and/or its ligand, growth arrest-specific 6 (GAS6), in completely resected lung AD. We evaluated the relationship between AXL, GAS6, and vimentin expression, as determined by immunohistochemistry (IHC) analysis, with overall survival and disease-free survival in 113 patients with stages I-III lung AD. Protein expression was also assayed using western blot analysis in 10 lung AD cell lines. AXL-positive (AXL+), GAS6-positive (GAS6+), or AXL+/GAS6+ staining was significantly associated with vimentin-positive (vimentin+) expression. AXL+/GAS6+ and vimentin+ showed a negative tendency toward an association with EGFR mutation. AXL+, GAS6+, or AXL+/GAS6+ statuses significantly correlated with poor overall survival. In stage I cases, an AXL+/GAS6+ status significantly correlated with poor overall survival and disease-free survival, especially in cases with wild-type EGFR. In multivariate analysis, AXL/GAS6 classifications in stage I as well as in stages I-III lung AD were found to be independent factors for poor patient outcomes. Unlike lung AD cell lines with mutant EGFR, those with wild-type EGFR closely correlated with AXL and vimentin expression by western blotting. AXL+ and GAS6+ expression is relevant to a poor prognosis in resected lung AD patients at stage I. AXL/GAS6 might serve as crucial predictive and prognostic biomarkers and targets to identify individuals at high risk of post-operative death.
Citation Format: Masahiro Seike, Ritaro Noro, Cheol-Hong Kim, Fenfei Zou, Kaoru Kubota, Akihiko Gemma. AXL and GAS6 Co-expression in lung adenocarcinoma as a prognostic classifier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5714. doi:10.1158/1538-7445.AM2017-5714