bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-coverage whole genome sequencing of FFPE-derived-DNA and extracellular vesicle-associated DNA in patients with metastatic cancer.
Authors: Nguyen Bella Hai , Wong Nick , Semple Timothy , Ruhen Olivia , Wong Stephen Q , Leslie Connull , Mirzai Bob , Millward Michael , Meehan Katie , Lim Annette May Ling
Published: May 2019

e14523

Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log2 ratio ≥0.15 and losses < -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.
Circulating cell-free tumor DNA chromosomal instability and circulating tumor cells as tools to predict microvascular invasion of hepatocellular carcinoma.
Authors: Wang Jin , Liu Yanzhe , Chen Kuang , Liu Qu , Ziliang Qian , HU Minggen
Published: May 2019

e14524

Background: Extended hepatic surgical margins, especially anatomical resections, were suggested for hepatocellular carcinoma (HCC) patients with pathological microvascular invasion (MVI) to improve patient survival. Here we investigated plasma cell-free tumor DNA (ctDNA) and circulating tumor cells as a tool to evaluate MVI before surgery. Methods: 47 treatment-naïve patients with liver lesions were recruited since June 2018, Peripheral blood samples were collected before surgery for all the individuals. Plasma cfDNA was sent to low-coverage genome-wide sequencing, followed by chromosomal instability analyses by a customized workflow UCAD. Circulating tumor cells (CTC) were analyzed by CELLSEARCH@ System. MVI was reported by pathological examination after surgery. Results: Of the 47 patients recruited, there are 32 hepatocellular carcinoma (HCC), 8 intrahepatic cholangiocarcinoma (ICC), 4 benign lesions, 1 solitary fibrous tumor, 1 neuroendocrine tumor and 1 hepatic epithelioid cell tumor by pathological examinations. MVI was also reported for HCCs. Nineteen of 32 HCC patients (59.4%) was found with elevated chromosomal instability (CIN). The most frequent changed chromosomes include 1q, 6p and 8q. Nine of 19 (47.4%) elevated CIN patients were MVI positive by pathological examinations. Among them, 4 was confirmed as MVI = 2. The other 5 was MVI = 1. 11 of 13 (84.6%) patients with low CIN were MVI negative (MVI = 0) as reported by pathological examinations. The rest 2 were reported as MVI = 1. Two of 32 HCC patients (6.25%) was found with positive CTC (tumor cell count > 0). The patient with CTC = 2 was reported as MVI = 1. The other one with CTC = 1 was MVI = 0. Taking together, 20 patients (62.5%) were found either elevated ctDNA or positive CTC. 9 were confirmed as MVI negative. The overall positive predictive value is 45.0%. 12 patients (38.7%) were both ctDNA CIN low and CTC negative. 10 were confirmed as MVI negative. The negative predictive value (NPV) is 83.3%. Conclusions: Elevated circulating tumor DNA chromosomal instability has good PPV and NPV in the predicting of MVI. In such patients, anatomical hepatic resections might be recommended to improve survival.
Zero delay plasma vs. Streck cfDNA tubes: Does immediately centrifuging blood at the point of draw improve cell-free DNA signal-to-noise?
Authors: Sommer Greg , Fredriksen Laura , Iacovetti Gabriella , Hong Kyungjin , Schaff Ulrich
Published: May 2019

e14529

Background: Sample quality is a critical consideration for high fidelity cell-free DNA (cfDNA) testing. Oncological cfDNA tests used for liquid biopsy typically employ specialty blood collection tubes containing chemical preservatives to minimize degradation of samples prior to lab testing. Here we describe a newly developed device, Zero Delay Plasma– a portable centrifuge and disc system designed to immediately isolate cell-free plasma at the point of blood draw – and evaluate its performance against the Streck cfDNA collection tube. Methods: Whole blood was collected, processed, and stored at room temperature for up to 7 days with both the Zero Delay Plasma system and the Streck cfDNA blood collection tube. Sample hemolysis was measured via cell-free hemoglobin. Genomic contamination and cfDNA signal-to-noise were evaluated by qPCR and electrophoresis, comparing signal from target 150-200bp cfDNA to contaminating longer length genomic sequences in the sample. 2 sets of hemolysis experiments, 2 sets of electrophoresis experiments and 4 sets of qPCR experiments were conducted. Results: Plasma processed with the Zero Delay Plasma system yielded ~4X lower hemolysis levels, ~10X lower genomic contamination, and ~20X higher cfDNA signal-to-noise compared to the Streck cfDNA collection tube after 7 days of storage at room temperature. Conclusions: The Zero Delay Plasma system minimizes sample degradation and analytical background signal for cfDNA testing by immediately removing cells and other contaminants at the point of blood collection. Clinical evaluations are in process.
Assessing the impact of clonal hematopoiesis in disease monitoring using targeted cell-free DNA (cfDNA) sequencing technology.
Authors: Yaung Stephanie J. , Xi Liu , Woestmann Corinna , Ju Christine , Klass Daniel M. , Casey Fergal , Hinzmann Bernd , Heussel Claus Peter , Thomas Michael , Herth Felix , Muley Thomas , Wehnl Birgit , Palma John F. , Ma Xiaoju Max
Published: May 2019

e14530

Background: Somatic variants found in plasma cell-free DNA (cfDNA) may derive from either solid tumors or clonal hematopoiesis (CH). Little is known about how this may impact plasma-based longitudinal disease monitoring using targeted sequencing of circulating tumor DNA (ctDNA). Methods: To assess the potential impact of CH in disease monitoring, we evaluated monitoring algorithms by targeted sequencing with and without matched peripheral blood mononuclear cells (PBMC). Samples were collected from a prospective observational study, where 62 late stage lung adenocarcinoma subjects were treated with first-line chemo or chemoradiation therapy. Pre-treatment plasma cfDNA and matched PBMC were analyzed with the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), a sequencing panel of 198 kilobases targeting cancer genes. Median input amounts of 25 ng cfDNA and 50 ng PBMC DNA were sequenced to median deduplicated depths of 4582 and 6134, respectively. Results: A median of 120 single nucleotide variants were detected per cfDNA sample, with 93.1% of these identified in matched PBMC. Most PBMC-matched cfDNA variants were germline SNPs, with allele frequency (AF) ~ 50% or 100%. A median of 1 (range 0-5) PBMC-matched cfDNA variants per sample were detected with an AF < 10%, consistent with CH. The number of these variants was positively associated with age (p-value = 0.0039) and the most frequently mutated gene was TP53. The remaining somatic variants (i.e., in cfDNA and not PBMC) had an AF range 0.03-40.9%. These PBMC-informed variants (median of 7 per sample) were used in longitudinal monitoring in the first post-treatment plasma sample to assess early response to therapy. Association between ctDNA level and progression-free survival using the same monitoring algorithm yielded nearly identical results on somatic variants derived from filtering approaches independent of matched PBMC (HR 0.32; 95% CI 0.16 - 0.65; log-rank P = 0.0009) and the PBMC-informed method (HR 0.31; 95% CI 0.14 - 0.66; log-rank P = 0.0013). Conclusions: A targeted panel focused on solid tumors by design has limited impact from CH. For disease monitoring applications in a non-MRD setting, measuring multiple variants instead of a single variant further enables robust classifiers that can moderate the impact of variants, if any, from CH.
CTC enumeration and characterization as a pharmacodynamic marker in the phase I clinical study of APX3330, an APE1/Ref-1 inhibitor, in patients with advanced solid tumors.
Authors: Chu Lincy , Anderson Amanda K. L. , Landers Mark Andrew , Wang Yipeng , Kelley Mark R. , Messmann Richard Adam
Published: May 2019

e14531

Background: APE1/Ref-1 is a dual-function protein with a pleiotropic role in regulating transcription factors involved in cancer cell signaling via redox control, as well as responding to oxidative and base DNA damage. APX3330 is a highly selective inhibitor of the Ref-1 mediated redox function in tumors, while enhancing the neuronal protective function of APE1. APX3330 is undergoing clinical development as an anti-tumor agent that also protects and reverses oxidative damage to neurons. We now report on the CTC analysis conducted as part of the study (NCT0337508). Results of the clinical study are reported in a separate abstract. Methods: A total of 19 patients with 10 various solid tumors, including rectal, pancreatic, colon, endometrial, gall bladder, hepatocellular, prostate, melanoma, bladder, and ovarian cancers received APX3330 in escalating divided daily doses of 240, 360, 480, 600, and 720mg. Blood samples were collected from all patients prior to receiving APX3330 and after achieving steady-state serum concentrations of the drug. Samples were sent to Epic Sciences to analyze circulating tumor cells (CTCs) in peripheral blood via their CTC Platform. Results: Cumulatively, 37 samples from 19 patients were received. 35/37 (95%) samples passed technical quality control and were feasible for downstream analysis. 9/17 (53%) baseline samples (BL) had CTCs detected, while 9/18 (50%) on-treatment (OTx) draws had CTCs detected. 16 patients had BL and OTx samples that were further evaluated with the longitudinal analysis. Of these, 7/16 (44%), 6/16 (38%), and 3/16 (19%) patients showed a reduction trend, an increase trend, and no change in all CTC populations (delta greater than or equal to 0 CTC/ml), respectively. Patient follow-up is ongoing and the correlation of CTC biomarkers with clinical outcomes is pending. Conclusions: APX3330 is undergoing clinical evaluation as an anti-tumor agent that protects against and reverses CIPN. In this phase I study, 44% of evaluable blood samples showed a reduction in CTCs after initiation of treatment with APX3330. Additional studies are now being planned. Clinical trial information: NCT0337508.
Follow-up evaluation of outliers with elevated spermine-spermidine acetyltransferase-1 activity.
Authors: Maksymiuk Andrew W. , Tappia Paramjit S. , Ramjiawan Bram , Khatun Nazrina , Parveen Rahnuma , Ahmed Rashiduzzaman , Huang Gina , Bux Ahmed Rashid , Akhtar Parveen S. , Bach Horacio , Sitar Daniel
Published: May 2019

e14536

Background: In the evolving role of biomarkers, proteinomic signatures related to up-regulation of polyamine synthesis including spermine/spermidine acetyltransferase-1 (SSAT-1) appear to be promising markers of malignancy. Acetylamantadine (AA) excretion, a measure of SSAT-1 up-regulation, has been shown to be a marker for malignant proliferation. In preliminary analyses of ostensibly normal individuals from Canada (Winnipeg) and Bangladesh (Dhaka), a proportion were identified to have evidence of SSAT-1 up-regulation above the expected non-malignant range (outliers). These outliers were assessed and followed for development of identifiable medical conditions. Methods: SSAT-1 up regulation was assessed in 60 ostensibly normal individuals by analysis of urinary excretion of AA after ingestion of amantadine 200 mg x 1 dose. AA was assayed using HPLC-mass spec techniques as previously described. Outliers who consented were followed-up by clinical examinations, routine biochemical and hematological tests and radiographs, where indicated. Results: Total AA average (median) excretion at 6 hrs in the Winnipeg cohort was 579+/-252 vs 1699+/-633 ng in the Dhaka cohort. Average urinary concentration at 6 hrs was 3.75+/-0.75 vs 21+/-20 ng/ml. In outliers consenting to follow-up, 1 case of invasive malignancy was identified, 6 cases of pre-malignant neoplastic change, chronic liver disease in 7 cases and chronic inflammation in 2 cases. In others, no malignant or inflammatory conditions have yet been identified. The range of SSAT-1 activity in individuals living in Bangladesh was significantly higher than in the Canadian cohort. Bangladesh volunteers live in an area of known to have high natural contamination with arsenic, a recognized carcinogen and they are exposed to high levels of air pollution. Conclusions: By this assay, high SSAT-1 activity has been confirmed in patients harbouring malignancy; however, pre-malignant and inflammatory conditions may result in a positive test. Follow-up of individuals with elevated SSAT-1 activity may reveal unrecognized malignancies, pre-malignant neoplasms, liver disease, inflammatory conditions and possibly false-positivity in individuals exposed to carcinogens such as arsenic. In further work, SSAT-1 up-regulation is being correlated with 5 other putative polyamine metabolite markers for accurate diagnosis of lung cancer.
Functional cell profiling (FCP) of ~100,000 CTCs from multiple cancer types identifies morphologically distinguishable CTC subtypes within and between cancer types.
Authors: Vansant Gordon , Jendrisak Adam , Sutton Ramsay , Orr Sarah , Lu David , Schonhoft Joseph , Wang Yipeng , Dittamore Ryan
Published: May 2019

e14553

Background: Different cancers subtypes can often be effectively treated with similar Rx classes (i.e. platinum or taxane Rx). Yet, within a disease patient therapy benefit can be variable. The origins of precision medicine derive from pathologic sub-stratification to guide therapy (e.g. SCLC vs. NSCLC). Using the Epic Sciences platform, we performed FPC analysis of ~100,000 single CTCs from multiple indications and sought to utilize high resolution digital pathology and machine learning to index metastatic cancers for the purpose of improving our understanding of therapy response and precision medicine. Methods: 92,300 CTCs underwent FCP analysis (single cell digital pathology features of cellular and sub-cellular morphometrics) were collected from prostate (1641 pts, 70,747 CTCs), breast (268 pts, 8,718 CTCs), NSCLC ( 110 pts, 1884 CTCs), SCLC ( 141 pts, 8,872 CTCs) and bladder (65 pts, 2079 CTCs) cancer pts. After pre-processing the raw data, a training set was balanced by sampling the same number of CTCs from each indication. K-means clustering was applied on the training set and optimized number of clusters were determined by using the elbow approach. After generating the clusters on the training set, the cluster centers were extracted from k-means, and used to train a k-Nearest Neighbor (k-NN) classifier to predict the cluster assignment for the remaining CTCs (test set). Results: The optimized # of clusters was 9. The % and characteristics of CTCs in each indication are listed below. BCa CTCs were more enriched in cluster c1, which had higher CK expression, while SCLC and some of mCRPC shared the small cell features (c5). Conclusions: Heterogeneous CTC phenotypic subtypes were observed across multiple indications. Each indication harbored subtype heterogeneity and shared clusters with other disease subtypes. Patient cluster subtype analysis to prognosis and therapy benefit are on-going. Analysis of linking of CTC subtypes genotypes (by single cell sequencing) and to patient survival on multiple indications is ongoing.[Table: see text]
RNA-based biomarker signatures in plasma as an independent predictor of outcome to chemotherapy in lung, colon, and breast cancers: Correlation of relative PD-L1 expression with immunotherapy outcomes.
Authors: Raez Luis E. , Usher Joshua L. , DANENBERG KATHLEEN , Sumarriva Daniel , Castrellon Aurelio Bartolome , Rabizadeh Shahrooz , Milillo Adriana , Hunis Brian , Reddy Sandip , Huang Liyang , Zikria Jennifer , Ferraro Pablo , Danenberg Peter
Published: May 2019

e14567

Background: Levels of cell-free circulating RNA (cfRNA) in cancer patients (pts) indicate tumor gene expressions and thus may provide a means, not only of evaluating response, but also for monitoring and predicting outcome to therapy. Methods: Blood was drawn every 6-8 weeks from pts undergoing various treatments (tx). CfRNA was extracted from resulting plasma and generated random-primed cDNA. Total cfRNA was quantitated by qPCR of β-actin, and correlated with pts response (CR/PR/SD/PD) determined by CT scans. Changes in PD-L1 expression were used to monitor response to immunotherapy in lung cancer pts. 125 pts (50 lung, 51 breast, 24 colon) were enrolled. Among the three tumor types, a total of 84 pts completed 1-3 lines of tx with an average of 5 blood draws per pt. Results: Changes in total levels of cfRNA over time correlated with pts outcomes in all three tumor types and were independent of the specific therapies. Increasing (INC) levels of cfRNA were predictive of disease progression and decreasing (DEC) levels with benefit from therapy, with an overall concordance of 81% (68/84 pts). Changes in relative PD-L1 expression were associated with immunotherapy outcomes in lung cancer (INC associated with progression, DEC associated with benefit). Lung: There was a 79% (23/29) concordance between changes in cfRNA levels and pts response. Changes in relative PD-L1 expression were predictive of outcome to immunotherapy in 9/10 pts. Breast: There was an 83% (30/36) concordance between cfRNA levels and pts response. Colon: There was a 79% (15/19) concordance between cfRNA levels and pts response. Conclusions: A noteworthy concordance was observed between clinical response and changes in cfRNA levels in lung, breast and colon cancer pts, independent of chemotherapy regimen. Changes in relative PD-L1 gene expression correlated specifically with outcome to immunotherapy (90%). We conclude that changing cfRNA levels can indicate tx response, and PD- L1 could be used to monitor response to immunotherapy.
Tracking circulating cell-free tumor DNA in gastric cancer to detect early disease recurrence.
Authors: Yang Jian , Gong Yuhua , Lam Vincent K. , Shi Yan , Guan Yan-Fang , Zhang Yanyan , Ji Liyan , Chen Yongsheng , Zhao Yongliang , Qian Feng , Chen Jun , Li Pingang , Zhang Fan , Wang Jiayin , Yang Ling , Futreal Andrew , Zhang Jianjun , Yi Xin , Xia Xuefeng , Yu Peiwu
Published: May 2019

e14571

Background: Identifying locoregional gastric cancer patients who are at high risk for relapse after resection could facilitate early intervention. By detecting molecular residual disease (MRD), circulating tumor DNA (ctDNA) has been shown to predict post-operative relapse in several cancers. Here, we aim to evaluate MRD detection by ctDNA and its association with clinical outcome in resected gastric cancer. Methods: This prospective cohort study enrolled 46 patients with stage I-III gastric cancer that underwent resection with curative intent. Resected tumor samples and plasma samples were obtained for targeted deep sequencing and longitudinal ctDNA profiling. Results: ctDNA was detected in 45% of treatment-naïve plasma samples. T stage was independently associated with pre-operative ctDNA positivity (p = 0.006). All patients with detectable ctDNA in the immediate post-operative period eventually experienced recurrence. ctDNA positivity at any time during longitudinal post-operative follow-up was associated with worse DFS and OS (HR = 14.78, 955 CI, 7.991-61.29, p < 0.0001 and HR = 7.664, 95% CI, 2.916-21.06, p = 0.002, respectively), and preceded radiographic recurrence by a median of 6 months. Conclusions: In locoregional gastric cancer patients treated with curative intent, these results indicate that ctDNA-detected MRD identifies patients at high risk for recurrence and can facilitate novel treatment intensification studies in the adjuvant setting to improve survival.
The host response to immune checkpoint inhibitors: From mechanisms to therapeutic implications.
Authors: Fremder Ella , Jacob Eyal , Khononov Irena , Issler Eran , Raveh Shani , Dahan Nili , Bar Haim , Shaked Yuval
Published: May 2019

e14584

Background: One of the major challenges in clinical immuno-oncology today is predicting which patients will respond to treatment. Although the use of immune checkpoint inhibitors (ICIs) has significantly improved therapeutic outcomes in a subset of patients with advanced malignancies, the majority of patients do not respond to treatment and some even hyper-progress. This response pattern raises questions regarding the possible mechanisms of resistance to ICIs. In the last decade we and others have shown that the host, in response to almost any type of anti-cancer drug, generates pro-tumorigenic and pro-metastatic effects which in turn contribute to tumor relapse and resistance to therapy despite an initial response. However, such host effects have never been investigated using immune-oncology drugs. Methods: We used functional and computational methods on plasma samples from both mouse models and cancer patients to identify the host-mediated tumor promoting molecular pathways that are induced in response to ICI therapy. Results: We found that these pathways are composed of dominant factors that regulate pro-inflammatory, metastatic and proliferation activities and thus contribute to rapid tumor re-growth. In pre-clinical models, we have found that pharmacological blockade of specific dominant factors overcomes the tumor-promoting effects induced by the host in response to ICI therapy. Conclusions: Based on our collective findings, we propose that therapies inhibiting host-mediated pro-tumorigenic effects could be used in combination with ICIs as a strategy to minimize tumor hyper-progression and increase the susceptibility of resistant tumors to ICI therapy. Furthermore, our studies pave the way towards the discovery of new predictive biomarkers for identifying subjects who will benefit from ICI therapy.
Prognostic value of serum apolipoprotein B (ApoB) to apolipoprotein A-I (ApoA-I) ratio in hepatocellular carcinoma patients with transcatheter arterial chemoembolization treatment: propensity score-matched analysis.
Authors: Liu Meng meng , Chen Zhan-Hong , Chen Yu-tong , Wang Zi-xian , Wu Xiangyuan , Dong Min
Published: May 2019

e14589

Background: The aim of our research was to assess the prognostic value of ApoB to ApoA-I ratio (ApoB/ApoA-I) in hepatocellular carcinoma (HCC) patients with transcatheter arterial chemoembolization (TACE) treatment. Methods: We collected clinicopathological data of 455 HCC patients with TACE treatments. The cutoff value of ApoB/ApoAI identified by receiver-operating curve (ROC) was 0.56. Correlation analysis was carried out to explore the relationship among ApoB/ApoA-I and other clinicopathological variables. Propensity score-matched (PSM) analysis was carried out to eliminate the unbalance of baseline characteristics of high and low ApoB/ApoA-I group. Finally, 278 patients were included in the PSM cohort, 139 patients in the high APOB/APOA-I group and 139 patients in the low APOB/APOA-I group. Univariate and multivariate analysis were conducted to explore the independent prognostic value of ApoB/ApoA-I in 455 patients and in the PSM cohort. Results: ApoB/ApoA-I was significantly correlated with AFP, NCCN T stage, distant metastasis status and TNM system(P < 0.05). Patients with AFP≥400ng, T3-4, distant metastasis and TNM III-IV had significantly higher serum ApoB/ApoA-I level than that of patients with AFP ≥ 400 ng/ml, T1-2, without metastasis and TNM I-II(P <0.05). Patients in high ApoB/ApoA-I group had significantly shorter overall survival compared to those in low ApoB/ApoA-I group in 455 HCC patients and in the PSM cohort (P < 0.01). Multivariate analysis indicated that ApoB/ApoA-I was an independent prognostic index for OS in 455 HCC patients and the PSM cohort (HR = 1.443, P = 0.006; HR = 1.564, P = 0.006, respectively.) Conclusions: Serum ApoB/ApoA-I is a novel independent prognostic factor for the HCC patients treated with TACE.
Concurrent targeted resequencing for translocations and mutations in tumor samples.
Authors: Chen Li
Published: May 2019

e14597

Background: Genetic variations are diverse, and proper nucleic acid templates should be analyzed for optimal outcomes. Previous NGS assays separately treat DNA and/or RNA, and are costly and produce limited information. Here we present a concurrent assay simultaneously converting both RNA and DNA templates in a single-tube format to streamline variant detection process. Methods: We developed a high-throughput targeted resequencing assay utilizing both DNA and RNA templates for mutation and fusion detection, and applied to a cohort of over 1000 lung tumor samples. Total nucleic acids from tissue samples were processed in a single-tube format throughout, and post-assay data analysis split RNA and DNA signals for corresponding variant calling. Sensitivity and specificity were sufficient for tissue samples. Results: In addition to common EGFR, KRAS, BRAF, PIK3CA mutations and ALK, ROS1, RET fusions and MET Exon 14 skipping, we also identified rare HLA-DRB1---MET and MSN---NTRK2 fusions. We found that MET exon 14 skipping was abundant, and that EGFR T790M is more associated with Exon 19 deletion than with L858R. We also found baseline mutation frequency for FFPE samples at 4-5%. Conclusions: Overall, this method is robust and convenient for clinical molecular diagnosis for tissue samples where variant types are diverse and time and budgets are constrained. Extreme caution is suggested for making positive calls below 5% MAF.
Real-world experience in advanced NSCLC using FDA approved NGS CDx.
Authors: Elkhouly Ehab , Zhou Li , Kurtis Boaz , Wang Lan , Israyelyan Anna
Published: May 2019

e14602

Background: The number one challenge in clinical biomarker testing for non-small cell lung cancer (NSCLC) has been the sample adequacy, given that the majority of patients are diagnosed based on minimally invasive sampling of tumor tissue. The increasingly evolving integration of high-throughput next-generation sequencing (NGS) has redefined molecular diagnostic lab practice allowing for time- and cost-efficient parallel testing of multiple biomarkers with limited specimen. Two NGS companion diagnostic (CDx) tests have been approved by FDA, FoundationOne CDx (F1CDx) and Oncomine Dx Target Test (Oncomine Dx), but with different sample acceptance criteria. The minimum tumor content requirement for F1CDx is 20% while it is 10% for Oncomine Dx. The surface area requirement for F1CDx is 25mm2 while there is no required minimum surface area for Oncomine Dx. The difference in sample acceptance criteria may have a major impact on the number of advanced NSCLC patient samples that can be accepted and tested successfully, affecting biomarker-directed therapy. Methods: The analysis of 153 NSCLC patient samples tested by Oncomine Dx at Cancer Genetics, Inc. CLIA-certified and CAP-accredited lab from October 2017 to September 2018 evaluated the sample acceptance rate, biomarker results, and testing turn-around time (TAT). Results: Based on the data, a significant portion of the samples submitted for routine clinical testing consists of extremely small specimen, including FNA cytology, with 42% less than 1mm2. 83% of samples are acceptable by Oncomine Dx criteria, compared to 14% by F1CDx. Of the 82 samples that would not be acceptable by F1CDx but generated results by Oncomine Dx, 5 were positive for EGFR exon 19 deletions and L858R, 1 for BRAF V600E, 1 for a ROS1 fusion, and the remaining 35 were positive for other actionable alterations. Average TAT was 9.2 days, consistent with recommendation by IASLC/AMP/CAP guideline of within 10 days. Conclusions: The real-world routine clinical testing of NSCLC patient samples demonstrated that a higher number of specimens with limited tumor tissue could be tested with the FDA-approved NGS CDx Oncomine Dx providing rapid TATs, actionable insights, and impacting the patient treatment outcomes.
Tumor volumetric correlation with plasma cell free DNA (cfDNA) mutation detection in metastatic lung cancers.
Authors: Haseltine Justin , Offin Michael , Myers Mackenzie L. , Makhnin Alex , Adamski Ariana , Li Henry , Li Mark , Shaffer Tristan , Henderson Samantha , Shen Ronglai , Ladanyi Marc , Arcila Maria E. , Jones David Randolph , Rusch Valerie W. , Rudin Charles M. , Isbell James M. , Li Bob T. , Rimner Andreas
Published: May 2019

e14610

Background: In patients with metastatic lung cancers, analysis of cfDNA is increasingly used to detect oncogenes, frequently allowing matched targeted therapy. Mutation detection in cfDNA may be influenced by several factors, such as disease burden. We hypothesized that volumetrics, as routinely analyzed during radiation simulation, correlates with mutation detection in cfDNA. Methods: Patients underwent cfDNA analysis between 10/2016 and 1/2018. Those with metastatic NSCLC, adequate imaging for volumetric analysis (PET/CT and MRI brain), and who were treatment naïve at plasma draw were included. Plasma underwent a 21-gene next generation sequencing assay (Resolution Bioscience). A single observer segmented tumor volumes. Volume of disease was correlated with mutation detection in cfDNA for all 21 genes including the 8 NCCN oncogenes (EGFR, ALK, ROS1, BRAF, KRAS, HER2, RET, MET) with univariate Mann-Whitney. Results: Of the 210 pts with plasma, 52 met inclusion criteria. There were 22 males (42%), median age 67yo (IQR 59-74), 24 former smokers (median pack-years: 25), 44 adenocarcinomas (85%), 21 matched to targeted therapy based on cfDNA results (40%), and median cfDNA extracted concentration was 1.56 ng/mL (IQR 1.1 – 2.6 ng/mL). Involved sites included lymph nodes (n = 43), bone (n = 25), brain (n = 15), liver (n = 13), and adrenals (n = 12). Median total tumor volume was 67.0 mL (IQR 36.6-184.6 mL). When evaluating all 21 genes, cfDNA was mutation positive in 40 pts and negative in 12 pts. Median tumor volume was 77.6 mL and 40.4 mL for positive and negative, respectively (p = 0.12). For the 8 NCCN oncogenes, cfDNA was positive in 18 pts and negative in 34 pts. Median tumor volume was 105.3 mL and 67.0 mL for positive and negative, respectively (p = 0.40). Conclusions: Volumetric analysis showed there was a non-significant trend toward larger tumor volume among pts with positive cfDNA mutation detection. As the use of cfDNA continues to expand into earlier disease stage settings, volumetrics at diagnosis may become an increasingly important tool to help predict cfDNA mutation detection to guide targeted therapy. Further studies of volumetrics and cfDNA analysis are warranted.
Analytical validation of the Oncomine Pan-Cancer Cell-Free Assay in a CLIA- and CAP-regulated laboratory for detection of solid tumor-derived variants in blood plasma.
Authors: Williamson John B. , Solano Luis , Yuki Akemi , Burkhart Vanessa D. , Chitwood James , Cao Ru , Schageman Jeoffrey , Saunders Hannah , Cheng Angie , Ha Thomas , Ojcius Cecilia , White Roxana , McCall Kimberly , Salazar Suzanne , Bramlett Kelli , Tian Huan , Ramsamooj Rajendra
Published: May 2019

e14614

Background: Assessing tumor-derived somatic variants from blood plasma provides minimally invasive tumor profiling, decreased cost relative to traditional tissue biopsy and rapid turn-around-time. We describe here the analytical validation of the Oncomine Pan-Cancer Cell-Free assay in a CAP-accredited, CLIA-certified laboratory. The assay is designed to detect somatic DNA single-nucleotide variants (SNV), insertions/deletions (INDEL), copy number variants (CNV), and gene fusions in cell-free total nucleic acid (cfTNA) across 52 genes. For research use only. Not for use in diagnostic procedures. Methods: We assessed the sensitivity, specificity, accuracy, and precision of the assay. Pre-characterized reference materials with alterations at known allelic frequencies (AF) were used to establish performance characteristics for each variant class followed by verification on clinical specimens. Whole blood (n = 73) from healthy (10), breast cancer (9), colorectal cancer (32), and lung cancer (22) donors were collected in K2EDTA tubes and plasma was separated within 8 hours of collection. cfTNA was isolated using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit on the KingFisher Flex. Libraries were prepared following kit instructions. Templating and sequencing were performed using the Ion 550 Kit on the Ion Chef and S5 XL systems. Alignment to hg19 and variant calling were performed using Torrent Suite and Ion Reporter software. Results: We observed a sensitivity of 80% at 0.1% AF and > 99.9% at 0.5% AF for SNV/INDEL. Sensitivity was > 99.9% at 1.34 fold-change for CNV, and > 99.9% at 0.4% fusion fraction. Specificity and accuracy were > 99% for all variant classes. Precision was 98% for SNV/INDEL and > 99.9% for CNV/fusions. Analysis of donor plasma demonstrated clinical feasibility; on average, we detected 1 hotspot SNV/INDEL (range 0-4) across the 3 cancer types. For SNV/INDEL in plasma where matched tissue genotyping was available (n = 17), we observed > 99% concordance (AF range 0.09%-52%). Conclusions: We have demonstrated that this assay is a sensitive method for evaluating tumor-derived somatic variants in blood plasma. It may provide an alternative and minimally invasive method for mutation profiling.
Mapping the activation landscape of the MAPK\ERK pathway for variants of unknown significance (VUS) using convolutional neural networks (CNN).
Authors: Tarcic Gabi , Zimmerman Lior , Aizenman Arie , Barbash Zohar , Zelichov Ori , Vidne Michael
Published: May 2019

e14615

Background: The MAPK\ERK signaling pathway is a major determinant in the control of diverse cellular processes. This pathway is often up-regulated in human tumors and as such has been an attractive target for the development of anticancer drugs. Mapping the activation landscape of MAPK\ERK is a necessary step in understanding tumor progression and determining the significance of a mutation as well as the relevance of available treatments. Here we present a CNN model, combined with an in-vitro system to investigate the activation landscape of various members of the MAPK\ERK pathway Methods: A novel multi-resolution multi-channel deep neural network was trained to classify images of cells that were transfected with known activating mutated forms of MAPK activating proteins versus WT forms of the proteins. The cells were also transfected with a fluorescently tagged ERK2 expression construct that was one of the three channels imaged by a fluorescent microscopy system. The trained network was subsequently tested on its ability to score images of cells that were transfected with VUS forms of the proteins, or images of cells that were transfected with VUS forms of MAPK proteins and were incubated with the relevant targeted therapies Results: We analyzed 6 proteins activating MAPK\ERK carrying over 100 unique VUS as well as many known oncogenic mutations and determined the pathogenicity score outputted by the network. Analysis of the performance of our model showed that our predictions exceed 95% accuracy and AUC > 0.95. Interestingly, analysis of deeper layers of the network revealed that each gene and mutation exhibit a unique ERK2 activation signal which is considerably different between active and non-active mutations as well as the ability to recognize what mutated gene was transfected, which means that each mutated gene induces different phenological attributes to the cells Conclusions: The use of deep-learning algorithms is providing increasing clinical relevance in several medical fields, most notably pathology. We show that using a cell-based assay combined with high throughput microscopy and CNN based analysis can be used to determine the activity of a wide set of mutations and potentially assist the prediction of targeted therapy outcome. Our results highlight the role of functional interpretation of molecular profiles, enabling more accurate prediction of oncogenic mutations.
Notch family gene mutations associate with high tumor mutational burden in diverse cancers.
Authors: Jiang Da , Niu Zhaojian , Zhang Jianli , Wang Yanlei , Shang Liang , Li Bixun , Guo Jun , Wang Baogui , Zhao LingQin , Wang Wenjing , Cui Qiang , Pan Xiaodong , Hu Jinwei , Zhang Shiyue , Wang Kai
Published: May 2019

e14616

Background: The Notch pathway has an important role in tumorigenesis in many types of cancer. Clinical trials with Notch inhibitors are ongoing. Recently, Notch pathway has been reported to be part of tumor immunity and patients with NOTCH1 mutations (mut) showed better outcome to PD-1 inhibitors, however, the underlying mechanism is unknown. Methods: Next generation sequencing (NGS) panel of 450 cancer genes was performed on FFPE tissue and matched blood samples from 1341 solid tumors, including hepatocellular carcinoma (HCC, n = 644), esophageal carcinoma (EC, n = 255), breast cancer (BC, n = 175), small cell lung cancer (SCLC, n = 141), head and neck cancer (HNC, n = 77). Notch family gene (NOTCH1/2/3/4) muts were analyzed. Genomic alterations including single base substitution, short and long insertions/deletion, copy number variation, gene fusion and rearrangement were assessed. Tumor mutational burden (TMB) was calculated in all patients by NGS algorithms. TMB high (TMB-H) was defined as TMB values ≥75% tumors in each tumor type. Results: For all patients, Notch family gene mut (substitution/indel/truncation) were found in 27% EC, 19% SCLC, 12% HNC, 6% BC and 5% HCC. The median TMB was 6.1 muts/Mb. In general, tumors with Notch family gene mut had significantly higher median TMB in pan-cancer cohort when compared with tumors without Notch family gene mut (9.2 vs. 6.1 muts/Mb, p < 0.001). Specifically, tumors with NOTCH1 mut had higher TMB in HCC (8.5 vs 5.5, p = 0.034), in BC (23.2 vs. 4.6, p = 0.006) and in SCLC (14.0 vs. 9.4, p = 0.002), while NOTCH2 mutant tumors presented higher TMB in HCC (18.5 vs. 5.5, p = 0.026), in EC (14.7 vs. 6.9, p = 0.001) and in SCLC (13.2 vs. 4.6, p = 0.026). We found 65% of NOTCH1 mut were located in epidermal growth factor (EGF)-like repeats domain. Classes of the NOTCH1 mutation type were substitution/indel (non-frameshift) (58%) and truncation (42%), while NOTCH2 mut were 88% and 12%. In TMB-H tumors, substitution/indel is the predominant mutation type of Notch family gene (NOTCH1 61%, NOTCH2 100%). Conclusions: Tumors with Notch family gene mut presented higher TMB in multiple cancer types, indicating a potential strategy for targeted and immunotherapy in NOTCH mutant cancers.
Characterization of OASEP1 as a biomarker and therapeutic target for oral cancer.
Authors: Takano Atsushi , Yoshitake Yoshihiro , Shinohara Masanori , Daigo Yataro
Published: May 2019

e14627

Background: The improved understanding of cancer-specific oncoproteins could lead to the development of new biomarkers or therapeutic strategies for oral cancers. Methods: We conducted this study as follows: i) Identification of up-regulated genes in oral cancers by means of cDNA microarray, ii) Validation of clinicopathological significance of their protein expression by tissue microarray, iii) Examination of the growth on cancer cells by siRNA, iv) Elucidation of their biological effects on tumor growth. Results: We selected a secreted protein, OASEP1 (oral cancer-associated serum protein 1) as a candidate. Immunohistochemical staining showed that OASEP1 protein expression in cytoplasm was observed in 73 (74.4%) of 98 oral cancers that had undergone curative surgery. In addition, high level of OASEP1 expression was associated with poor prognosis for oral cancer patients ( P = 0.0158, by log-rank test). Suppression of OASEP1 expression by siRNA for OASEP1 significantly suppressed the growth of oral cancer cell lines partly through apoptosis as detected by flow cytometric analysis, apoptosis assays, and time lapse imaging. In addition, siRNAs for a candidate receptor of OASEP1 also significantly inhibited its expression and the growth of oral cancer cells, indicating that the functional interaction between OASEP1 and its receptor could regulate the growth of oral cancer cells. Conclusions: Our data suggest that OASEP1 is a possible prognostic biomarker and therapeutic target for oral cancer.
A novel function for cyclin D1 as a transcriptional role in oncogenesis and tumor development by ChIP-Seq and RNA-Seq.
Authors: Xiong Yudi , Zhou Yunfeng
Published: May 2019

e14628

Background: Cancer is a major public health problem and remains one of the most leading causes of death worldwide. Aberrations in cell cycle control is defined as one of the hallmarks of cancer.While cyclin D1 is an essential protein to cell cycle which promote G1 phase into S phase, and frequently overexpressed in many human cancers.Although the participation in cell cycle and tumorigenesis has not been well characterized,new functions have been identified in transgenic mice models, including the transcription of genome,the development of chromosome instability and DNA repair.However,it remains unclear that the function of cyclin D1 indenpend of involving cell cycle.In this research,our aim is to find the function of cyclin D1 in transcription in human cancers. Methods: We chose the human cervical cancer cell line,C33A as our main research object.Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq),to find out the genes differentially expressed in C33A,cyclin D1 knock-in C33A and cyclin D1 knock-down C33A. Results: We detected 5590 genes involved in binding with cyclin D1 by ChIP-seq,screened 2811 genes up-regulation,2727 genes down-regulation in cyclin D1 knock-in cells,2244 genes up-regulation,1738 genes down-regulation in cyclin D1 knock-down cells by RNA-seq.Through analysis of the results of ChIP-seq and RNA-seq,we finally identified 422 genes differentially expressed at the RNA level.These genes are highly enriched in Gene Ontology categories and involve in diverse cellular functions via KEGG classification , including translation,replication and repair,signal transduction,cell growth and death. Conclusions: These findings suggested that cyclin D1 can serve both to activate and downregulate gene expression as a transcriptional role directly binding with genome DNA,which means that cyclin D1 may be a key protein during oncogenesis and tumor development.
DDP4 inhibitors as novel agents in improving survival in diabetic patients with CRC and lung cancer: A SEER-Medicare study.
Authors: Bishnoi Rohit , Hong Young-Rock , Shah Chintan , ali azka , Skelton William Paul , Huo Jinhai , Dang Nam H. , Dang Long H.
Published: May 2019

e14633

Background: DPP4 is a cell surface protein that is widely expressed on different tissues and can play important role in tumor biology by acting as a tumor suppressor or activator, depending upon the level of expression and interaction with the microenvironment and chemokines. DPP4 inhibitors are a class of drug used in the clinical setting to treat diabetes mellitus type II (DM-II) while recent research has suggested that they play a role in regulating tumor growth. Methods: We conducted this large population database Surveillance Epidemiology and Endpoint Research (SEER-Medicare) study to evaluate the role of DPP4 inhibitors on the overall survival (OS) of patients with colorectal and lung cancers. Results: Our results showed that diabetic patients with colorectal cancer (CRC) or lung cancer who were treated with DPP4 inhibitors exhibited a statistically significant survival advantage (HR of 0.89; 95% CI: 0.82-0.97, p = 0.007) that remained significant after controlling for all other confounders. When DPP4 inhibitors were used in combination of metformin, which is already known to improve OS in many cancers, the survival advantage was even more pronounced (HR of 0.83; 95% CI: 0.77-0.90, p < 0.0001). When results were analyzed for CRC-only cohort, the use of DPP4 inhibitors alone had a very positive survival advantage trend (HR of 0.87; 95% CI: 0.75-1.00, p = 0.055) whereas the combined use of DPP4 inhibitors and metformin was associated with statistically significant survival advantage (HR of 0.77; 95% CI: 0.67-0.89, p = 0.003). Similarly, in the lung cancer cohort, the DPP4 inhibitor group exhibited a potential survival advantage (HR of 0.93; 95% CI: 0.83-1.03, p = 0.153), while lung cancer patients treated with the combination of DPP4 inhibitors and metformin showed statistically significant survival advantage (HR of 0.88; 95% CI: 0.80-0.97, p = 0.010). Conclusions: These results again establish that DPP4 inhibition in CRC and lung cancer is associated with the suppression of tumor growth and improved OS. The beneficial effects of DPP4 inhibitor treatment are additionally improved when DPP4 inhibitors are used in combination with metformin. While the exact mechanism involved in the observed benefit of DPP4 inhibitor treatment in cancer remains to be elucidated, one possibility may be due to the effect of DPP4 inhibition on immunoregulation of cancer.