bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biomarkers for predicting cancer in women with a suspicious mammogram.
Authors: Espina Virginia A. , Vuong Ngoc , Luchini Alessandra , Mueller Claudius , Mack Denitra S , Smith Michael , May Jennifer , Liotta Lance A. , Hoefer Richard A. 
Published: May 2020

e15533 Background: Biomarker identification for early breast cancer diagnosis is confounded by comparing healthy control patients to patients undergoing surgical procedures and stress of a potential cancer diagnosis. We implemented a clinical research protocol that combines biomarker harvesting and identification with Breast Imaging-Reporting and Data System (BIRADS) results, within a cohort of women with a suspicious mammogram who donated samples prior to biopsy. The primary goals were to discover candidate novel plasma markers for stage I breast cancer versus benign lesions, and validate the markers by mass spectrometry and immunohistochemistry. Methods: 150 women found on screening mammography to have a BIRADS IV or V mammographic abnormality were enrolled in the IRB approved study, with one year follow-up. After informed consent, serum, plasma, and saliva specimens were obtained and frozen. The patient underwent image guided biopsy of the mammographic abnormality. Hydrogel nanoparticles were used to harvest and concentrate low abundance protein biomarkers from plasma. Proteins were identified by mass spectrometry. The BIRADS score and biopsy outcome were blinded to the laboratory researchers. Results: 37/150 women (median age 64, 73% ER+, 70% PR+) were diagnosed with biopsy-proven breast cancer. 15/37 had a family history of breast cancer. Within the context of stress of an abnormal mammogram and invasive biopsy, we identified 5478 plasma peptides. A model to predict endpoints that discriminate cancer vs no cancer was developed using cross-validation and lasso shrinkage method. The best fit multi-analyte ROC/AUC model of peptide spectral matches revealed 10 candidate peptides, including PLAA, TRAPPC9, PROS1, DDX41, ANKRD63, EGFLAM (AUC = 0.81), that discriminated cancer versus no cancer. The functional mechanisms of these proteins are calcium metabolism, GPI anchor biosynthesis, neural-immune crosstalk, DNA repair, and ubiquitin-mediated protein trafficking. Conclusions: Molecular profiling of blood can potentially complement imaging to improve diagnostic specificity in the setting of a suspicious mammogram. This unique trial design, enhanced by nanotechnology protein harvesting, identified potential novel cancer biomarkers in the presence of a suspicious mammogram. A confirmation trial is underway.
Potential utility of methylation levels detected from circulating tumor DNA (ctDNA) in predicting molecular residual disease (MRD) in patients with resected non-small cell lung cancer (NSCLC).
Authors: HU Hong , Li Hang , Ma Zelin , Xiang Jiaqing , Zhang Yawei , Lizaso Analyn , Hou Ting , Guan Xiaoqian 
Published: May 2020

e15535 Background: To improve the prognosis of resected lung cancer patients, growing efforts are being invested in finding the most optimal approach to detect MRD and predict relapse. In our study, we aimed to investigate the utility of ctDNA methylation profiling in detecting MRD from patients with resected early-stage NSCLC. Methods: Surgically-resected tumor tissues were obtained from 65 patients diagnosed with resectable stage IA-III NSCLC with various histological subtypes. Matched blood samples were also collected before surgery (baseline) and during regular follow-up after 2-8 weeks of surgery. Comprehensive somatic mutation and methylation level profile from circulating tumor DNA (ctDNA) were performed using unique molecular identifier-based targeted sequencing and targeted bisulfite sequencing, respectively. A tumor-informed MRD prediction model was constructed based on methylation levels obtained from the patient’s resected tumor tissue to calculate the corresponding methylation signal intensity from the matched plasma sample of the patient at baseline or other time points during follow-up, which is reflected as MRD score. Results: Of the 28 patients with baseline ctDNA methylation data, 28.6% (8/28) of the patients had elevated ctDNA methylation levels at first post-operative follow-up (F1) as compared to baseline, indicating the possibility of MRD. Meanwhile, of the 20 patients (71.4%, 20/28) who had reduced ctDNA methylation levels at F1, elevation of ctDNA methylation level was detected from 3 and 1 patients at second (F2) and third (F3) follow-up, respectively. Based on the MRD prediction model, 17.9% (5/28) of the patients had higher MRD scores at F1. Of the 23 patients with lower MRD scores at F1, 5, 1 and 1 patients had an elevation in MRD scores at F2, F3, and F4, respectively, which indicates a possible MRD. Disease relapse was radiologically confirmed after 10-16 months post-surgery in three patients with concomitant elevation of ctDNA methylation level and MRD score during follow-up between 2-9 months prior to radiologic relapse. Conclusions: Our results demonstrate that post-operative ctDNA methylation levels could be used to detect MRD in patients with resected NSCLC. Moreover, ctDNA methylation-based prediction model of MRD could serve as a potential model to predict relapse in early-stage as well as disease progression in advanced-stage NSCLC.
Detection of pancreatic cancer using a novel blood-based DNA methylation signature.
Authors: Munugala Nishant , Hammad Hytham , Vrba Lukas , Oshiro Marc , Wertheim Betsy , Roe Denise , Gavini Hemanth , Latura Leah , Pennington Daniel , Dalgai Shelby , Babiker Hani M. , Elquza Emad , Scott Aaron James , Nelson Mark , Futscher Bernard W. , Shroff Rachna T. 
Published: May 2020

e15546 Background: Pancreatic cancer (PC) is a high mortality malignancy typically found when curative surgery is not an option. Liquid biopsies are a minimally invasive option that may allow earlier detection of PC. Using a bioinformatic analysis of TCGA and GEO we identified a novel set of blood-based biomarkers to detect PC with high sensitivity and specificity (AUROC = 0.999) (Vrba et al, Epigenetics 2019). Here we tested the ability of this DNA methylation signature (MS) to distinguish metastatic PC from pancreatic cysts (Cy) and healthy controls (HC) through quantitative DNA methylation analysis of cell free DNA (cfDNA). Methods: A 10 gene DNA methylation marker set that identifies PC was used to evaluate the cfDNA component of the plasma of PC patients, Cy patients and HC. The ROC analysis and AUC calculations were performed using the R library pROC (Robin et al, BMC Bioinformatics 2011). Plasma samples were collected in cfDNA collection tubes from patients with benign Cy, PC, and HC. Clinical information was collected including age, gender, smoking history, CA 19-9 level, cyst size, presence of liver metastases, and survival status. After processing and cfDNA extraction, qRT-PCR methylation analysis was performed on these prospectively collected samples. The difference in median DNA MS per marker of the full 10-gene marker set between the above groups was analyzed by Wilcoxon rank sum test and ROC analysis. A Cox proportional hazard regression model was used to test the association between MS and survival. Results: A total of 66 samples were collected (PC = 10, Cy = 12, HC = 44). Clinical information was collected on all patients included in the analysis. Using liquid biopsies for cfDNA analysis and a novel set of DNA methylation biomarkers, MS was substantially stronger in PC (median, 13.2) patients than Cy (median, 3.5) patients (p = 0.0001). Importantly, while there was no clear association between MS and age, gender, smoking status, CA 19-9 levels, or presence of liver metastases, higher methylation levels were non-significantly associated with worse overall survival in PC patients (HR, 1.29; 95% CI, 0.97–1.72; p = 0.082). Conclusions: While this is a small sample size, early findings suggest that a DNA MS may reliably distinguish PC from benign Cy. The strength of signal may be a prognostic marker that could play a role in determining treatment approaches. Given its utility in predicting PC, future directions include testing this tool as a blood-based early detection mechanism as well as a predictor for recurrence or response to PC treatment.
Analytical validation of a tissue agnostic ctDNA MRD assay using tumor specific methylation and somatic variant profiles in early-stage CRC.
Authors: Artieri Carlo , Axelrod Haley , Baca Arthur , Burke Jordan , Chudova Darya , Dahdouli Mahmoud , Ghadiri Farsheed , Hartwig Anna , He Yupeng , Hite Dustin , Jaimovich Ariel , Juntilla Marisa , Kurata Jessica , Kong Yu , Nance Tracy , Ohshima Saiyou , Sharma Sweta , Westesson Oscar , Talasaz AmirAli 
Published: May 2020

e15549 Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.
Lung cancer exosome specific protein 1(LESP-1) as a potential factor for diagnosis and treatment of non-small cell lung cancer.
Authors: Kim Hyun Koo , Jeong Hyesun , Choi Byeong Hyeon , Quan Yu Hua , Rho Jiyun , Park Ji-Ho , Park Yong , Choi Yeonho , Han Kook Nam , Choi Young Ho , Hong Sunghoi 
Published: May 2020

e15550 Background: Exosomes are endosome-derived nano size (30-150 nm) extracellular microvesicles released from many cell types including cancer cells and encapsulated by cell membrane play a key role for cell to cell communication. Use of exosomes as a biomarkers in lung cancer is a rising nanotechnology in a liquid biopsy. We explored a role of an exosome specific marker and the relationship between pathological stage of lung cancer patients who underwent surgery and lung cancer specific exosome markers. Methods: Conditioned medium from non-small cell lung cancer (NSCLC) cells and human pulmonary alveolar endothelial cell (HPAEpiC) was collected and exosomes were isolated by size exclusion chromatography. Proteomics analysis was performed to investigate lung cancer specific proteins. Written informed consent was obtained from all human subjects (17 controls and 54 patients), approved by the Korea University Guro Hospital Institutional Review Bored. Plasma exosomes were isolated using dual size exclusion chromatography. We validated proteomics results with lung cancer exosome-specific protein 1 (LESP-1) ELISA assay and western blot assay in lung cancer patients with healthy control. Cancer cell lines with pCMV-CD63-GFP were transduced by lentiviral vectors containing LESP-1 shRNA. The distribution of GFP+ exosomes and cell migration were examined by immunocytochemistry (ICC) and cell migration assay. Results: We identified LESP1 by the proteomics analysis of exosomes from NSCLC cell lines, but not in HPAEpiC cell. Level of LESP1 was dramatically increased in exosomes from NSCLC cell lines and from NSCLC patients. LESP-1 concentration increased in lung cancer patients than healthy controls ( p < 0.0001) and increased according to the grade of lung cancer stage in peripheral blood ( p < 0.0001). Western blot results confirmed the presence of the LESP1 with higher intensity band at each grade of lung cancer stage than healthy controls. Interestingly, we found that the number of GFP+ exosomes was decreased and cell migration was inhibited when the LESP1 was suppressed in NSCLC cells. Conclusions: The LESP-1 in exosomes was highly expressed in blood plasma of lung cancer patients, and the exosome release and cancer cell migration was inhibited by the LESP-1, which suggest that LESP-1 could be a feasible factor for diagnosis and treatment of non-small cell lung cancer.
Natural killer cells and their activity as a potential biomarker for predicting response to checkpoint inhibitors in non-small cell lung cancer.
Authors: Song Paul Y. , Cho Yong-Hee , Choi Myeong Geun , Kim Dong Ha , Choi Yun Jung , Kim Seon Ye , Sung Ki Jung , Lee Jae Cheol , Kim Sang-Yeob , Rho Jin Kyung , Choi Chang-Min 
Published: May 2020

e15559 Background: Despite the increased promise of checkpoint inhibitors in the treatment of non-small cell lung cancer, the majority of patients do not respond and the potential for severe toxicity is significant. Unfortunately, the predictive value of PD-L1 expression has not always proven to be definitive and the overall need to find better biomarkers that will optimize the selection of patients who will best respond to therapy remains. Recent evidence has pointed to Natural Killer (NK) cells as an essential mediator in overall response to checkpoint inhibitors. We explore whether NK cell populations and/or their activity have any correlation with response. Methods: Peripheral blood mononuclear cells (PBMCs) from nine patients with Stage IV non-small cell lung cancer (four adenocarcinoma, five squamous cell carcinoma) were collected pre and post six-weeks of checkpoint immunotherapy (six received pembrolizumab, and three received nivolumab). Overall, four of nine (44.5%) were diagnosed as SD or PR via RECIST 1.1. The immune cell composition of the PBMCs of all nine patients was analyzed using multi-variated single-cell analysis using mass cytometry (CyTOF) with an optimized 32-marker panel. Natural Killer (NK) cell activity was measured with the NKVue kit which detects NK-secreted IFN-γ levels using a quantitative sandwich ELISA from NK cells exposed to a specific recombinant cytokine. Results: The overall percentages of NK cell populations found in the immune cells of PBMCs were prominently elevated in patients who responded (SD or PR) to checkpoint therapy compared to those who had progressive disease (non-responders). While there were no significant differences in the population of other immune cells between these two groups, the overall NK cell activity in patients who responded was highly elevated compared to the NK cell activity in non-responders. From the analysis of NK subsets, there were no differences in the population of early NK cells between the two groups, but the functionally differentiated late NK cells were prominently high in the responder group. Conclusions: Natural Killer (NK) cells have been recently implicated to play a key role in anti-tumor response to checkpoint inhibitors and our results suggest that the overall number of NK cells and their activity could prove to be an independent and reliable predictive tool/biomarker in patients with NSCLC.
Greater than two coexisting mutations in KRAS and NRAS identified in the circulating tumor DNA fraction of liquid biopsies by NGS and confirmed with ddPCR.
Authors: Boulos Hala , Tell Robert , Beaubier Nike , Blidner Richard 
Published: May 2020

e15563 Background: Liquid biopsies are increasingly utilized as a non-invasive tool in precision oncology to assess tumor mutational profiles in order to select targeted therapies, detect treatment resistance, and monitor disease progression in cancer patients. Additionally, liquid biopsies may provide a more comprehensive representation of tumor heterogeneity than standard tissue biopsies. However, limitations such as scarcity of circulating tumor DNA (ctDNA) and/or variants at low frequencies can be technically challenging to detect by next-generation sequencing (NGS) assays. Here, we use NGS to detect greater than two KRAS/NRAS mutations coexisting in single samples at low variant allele frequencies (VAFs). Methods: The Tempus xF liquid biopsy NGS assay is designed to detect actionable oncologic targets spanning 105 genes in plasma. The assay was validated to reliably detect single-nucleotide variants at 0.25% VAF, indels and copy number variants at 0.5% VAF, and fusions at 1% VAF with 96.2%-100% specificity and 97.4%-100% sensitivity. Pre-designed digital PCR assays were modified to measure 10ng of cell-free DNA (cfDNA) on a droplet-digital PCR (ddPCR) platform. Results: Overall, we report 100% positive predictive value and high correlation between ddPCR results and xF VAF, as well as in individual variants, such as KRAS G12D. Unexpectedly, we detected more than two coexisting KRAS/NRAS mutations at a low VAF in the plasma samples. To orthogonally confirm these results, ddPCR was deployed to independently measure the presence of each cfDNA variant with a sensitivity of 0.09% VAF. Subsequent ddPCR analysis of all targeted variants were concordant with NGS results. Conclusions: The occurrence of multiple KRAS and NRAS mutations in a single sample is quite uncommon and may be falsely interpreted as an NGS artifact. However, verification of this phenomenon by ddPCR confirmed the validity of the NGS liquid biopsy approach. These results highlight the capability of the Tempus xF assay to detect low-frequency variants, including those that fall below the validated detection threshold, which is essential for the diagnosis of early disease.
Assessment of 5-methylcytosine and 5-hydroxymethylcytosine technologies in contrived ctDNA-like samples.
Authors: Raiber-Moreau Eun-Ang , Hadfield James , Hodgson Darren , Wijdicks Marilou 
Published: May 2020

e15585 Background: Epigenomic changes to DNA are early driving events in tumorigenesis and are specific markers of the tumour and its microenvironment. Cell-free DNA that is released from the tumour microenvironment can potentially be used for the detection of tumour-specific epigenomic changes for cancer liquid biopsy testing. In this study, we assessed the robustness of 5-methylcytosine and 5-hydroxymethylcytosine technologies in contrived ctDNA-like samples from NSCLC. Additionally, we assessed the limit of detection of the tumour fraction within the tested dilution range looking at a subset of differential epigenomic marks. Methods: 4 NSCLC tumour and buffy coat DNA samples were commercially obtained and processed to generate contrived tumour DNA dilution mimicking circulating tumour DNA at varying tumour fractions. Samples were sent in triplicate to multiple epigenomic assay providers for analysis. The robustness i.e. reproducibility and limit-of-detection within the tested range was determined. Results: Technical replicates all passed method-specific quality controls and were highly reproducible. Cancer specific differentially-methylated or hydroxymethylated regions were determined for each individual tumour sample as a “sample-specific cancer signature”; when applied to the tumour dilution samples, these signatures were capable of distinguishing all tumour dilutions down to 0.05% from normal background DNA. Detection of differential methylation and hydroxymethylation regions was reproducible across technical replicates for all four NCSLC samples at > 0.05%. Generation of pooled methylation “signatures” derived from commercially available healthy plasma and cancer tissue datasets could still detect cancer DNA in these four NCSLC samples at > 0.1% tumour fraction. Conclusions: Our pilot study showed that all assessed technologies generated reproducible, robust data and could detect ctDNA at even clinically relevant levels for early stage cancer. One limitation of this study was the small number of “n” and the use of sonicated contrived sample that may induce artificial bias and reduce sensitivity highlighting a requirement for the development of reference material in the community. The ability to detect cancer-specific epigenomic changes from liquid biopsy with high sensitivity offers new promising avenues for early detection of cancer, which can increase the chances of successful treatment.
Distinct molecular profiles of interval and screen-detected tumors in a real-world breast cancer registry.
Authors: Wilks Sharon , McIntyre Kristi , Han Linda K. , Cairo Michelina Marie , Barone Julie , Haan Josien , Mittempergher Lorenza , Yoder Erin , Menicucci Andrea , Wang Shiyu , Audeh William ,   FLEX Investigators' Group
Published: May 2020

e15587 Background: Interval breast cancers (BC) are detected between routine screening mammograms and are associated with worse prognosis, requiring more aggressive treatment compared to screen-detected BC identified during scheduled mammograms. Identifying molecular differences between interval BC and screen detected BC may lay the foundation for developing novel therapies. In this study, we compared gene expression profiles of interval BC to screen-detected BC. Methods: This analysis included a subset of 2260 patients enrolled in the FLEX Registry (NCT03053193), an ongoing, prospective study evaluating primary tumor samples from stage I-III BC patients who receive 70-gene risk of recurrence testing (70GS), 80-gene molecular subtyping (80GS), and consent to collection of clinically annotated full genome data. Interval BC were diagnosed < 12 months following a normal screening mammogram. Breast tumors were classified by 70GS as having a Low Risk (LR) or High Risk (HR) of distant metastases. Tumors were classified as Luminal, HER2, or Basal type by 80GS. Differential gene expression analysis was performed with limma and subsequent pathway analysis with DAVID and GSEA. Differences in the proportion of 70GS or 80GS results, 70GS index, and Ki67 were assessed by Chi-squared test or t-test. Results: In this study, 81% (1834/2260) of patients had screen-detected BC and 19% (426) had interval BC. A higher proportion of interval BC (51%) were HR compared to screen-detected BC (44%; p = 0.01). Most LR tumors were invasive ductal carcinoma (78% interval and 73% screen-detected) and over 99% were Luminal type. Between the two LR groups, 70GS indices were similar and there was no significant difference in transcriptional profiles. Basal and HER2 subtypes were more frequent among HR interval BC compared to screen-detected BC (p = 0.03). HR interval BC had 70GS indices of higher risk compared to HR screen-detected BC (p = 0.02). Differentially expressed genes in HR interval BC compared to HR screen-detected BC were associated with MYC signaling and mitosis, which was concordant with higher Ki67 by IHC (p = 0.007). Conclusions: This real-world data analysis shows interval BC are not all biologically High Risk and can be further stratified by the 70GS, aiding in treatment decisions. Preliminary results suggest that following 70GS LR classification, there is no biological difference between interval BC and screen-detected BC. In contrast, there are distinct biological processes associated with HR interval BC, which may have implications in the management of these cancers. Clinical trial information: NCT03053193.