Utility Of Cell-free DNA Research Articles

Central conducting lymphatic anomaly: from bench to bedside.

Central conducting lymphatic anomaly (CCLA) is a complex lymphatic anomaly characterized by abnormalities of the central lymphatics and may present with nonimmune fetal hydrops, chylothorax, chylous ascites, or lymphedema. CCLA has historically been difficult to diagnose and treat; however, recent advances in imaging, such as dynamic contrast magnetic resonance lymphangiography, and in genomics, such as deep sequencing and utilization of cell-free DNA, have improved diagnosis and refined both genotype and phenotype. Furthermore, in vitro and in vivo models have confirmed genetic causes of CCLA, defined the underlying pathogenesis, and facilitated personalized medicine to improve outcomes. Basic, translational, and clinical science are essential for a bedside-to-bench and back approach for CCLA.

Navigation of Prenatal Care With Sex Discordance Between Cell-free DNA and Ultrasound Findings.

The utilization of cell-free DNA (cfDNA) screening has expanded rapidly across the age spectrum of pregnant persons. With cfDNA's widespread adoption, genetic fetal sex is now often known before a phenotypic assessment on anatomic survey. CfDNA detects sex discordance in 1/1500 to 2000 pregnancies. Upon detection of sex discordance, lab error or other factors should first be assessed. Once other causes have been ruled out, this may indicate an underlying disorder/difference in sex development. A multidisciplinary team should coordinate diagnosis, treatment, and support for the family. This review discusses the diagnostic workup, emphasizing the multidisciplinary counseling and management of disorder/differences in sex development.

Incidental Detection of Malignancies With Cell-Free DNA Screening.

Cell-free circulating DNA is an evolving technology with important clinical applications in both obstetric care and oncology. In the challenging patient with pregnancy and co-existing malignancy, the utility of cell-free DNA both for aneuploidy screening and cancer identification is an area of active research. Understanding the physiology associated with circulating cell-free DNA and subsequent laboratory evaluation is critical for clinicians caring for the obstetric patient with cell-free fetal DNA screening results suggestive of malignancy. Ongoing research is necessary to determine best practices for the evaluation and management of these patients with promising applications in the advancement of precision medicine.

Utility of cell-free DNA from bronchial washing fluid in diagnosis and genomic determination for radiology-suspected pulmonary nodules.

Bronchial washing fluid (BWF) is a less-invasive specimen. Due to the limited sensitivity of BWF cellular component diagnosis, the aim of this study was to explore the potential role of BWF supernatant as a source of liquid biopsy of lung cancer. This prospective study enrolled 76 suspected and 5 progressed lung cancer patients. Transbronchial biopsy tissues, BWF supernatant (BWF_Sup) and BWF precipitant (BWF_Pre) were tested by a targeted panel of 1021 genes. BWF_Sup cell-free DNA (cfDNA) was superior to tissue biopsy and BWF_Pre in determining mutational allele frequency, tumour mutational burden, and chromosomal instability. Moreover, BWF_Sup and BWF_Pre achieved comparable efficacy to tissue samples in differentiating malignant and benign patients, but only BWF_Sup persisted differentiated performance after excluding 55 malignancies pathologically diagnosed by bronchoscopic biopsy. Among 67 malignant patients, 82.1% and 71.6% of tumour-derived mutations (TDMs) were detected in BWF_Sup and BWF_Pre, respectively, and the detectability of TDMs in BWF_Sup was independent of the cytological examination of BWF. BWF_Sup outperformed BWF_Pre in providing more subclonal information and thus might yield advantage in tracking drug-resistant markers. BWF_Sup cfDNA is a reliable medium for lung cancer diagnosis and genomic profiles and may provide important information for subsequent therapeutic regimens.

Molecular profiling and utility of cell-free DNA in nonsmall carcinoma of the lung: Study in a tertiary care hospital.

Lung carcinoma accounts to the most common cause of cancer globally. Optimal management of nonsmall cell lung carcinoma (NSCLC) requires prognostic biomarkers that help in targeted therapy and identification of tumor subsets with a distinctive molecular profile that can foretell response to therapy. Quantitative analysis of circulating cell-free DNA is considered as a possible aid for lung cancer screening. The main aim of our study was detection of the clinicopathological spectrum of NSCLC, immunohistochemical (IHC) study of lung adenocarcinoma with epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and molecular expression of EGFR mutation using Formalin fixed paraffin embedded tissue (FFPE) and cell-free DNA (cfDNA) from blood samples. It was a prospective and observational study conducted in the Department of Pathology in association with the Department of Chest Medicine in a tertiary care hospital for 18 months, done on 50 patients. Histological subtyping of lung carcinomas was done, followed by IHC analysis using P40, thyroid transcription factor (TTF1), EGFR, and ALK. Molecular analysis for EGFR mutation was done using FFPE and cfDNA from the patient's blood samples. On histological subtyping, majority (66%) of the cases were found to be adenocarcinoma. All adenocarcinoma (66%) cases show TTF1 positivity and all squamous cell carcinoma (32%) cases show P40 positivity. All the ALK-positive (6%) cases were never smokers and histologically diagnosed as adenocarcinoma. About 58% of the NSCLC cases were found to be EGFR IHC positive. Formalin-fixed paraffin tissue (FFPE) showed EGFR mutation in 32% cases, of which majority were deletion (19, 28%) and rest (4%) of the cases involving mutation in exon 21. From cfDNA, mutations were noticed in 16% of the cases where majority involved deletion 19 (12%), whereas the rest of the cases were positive for missense mutation in exon 21 of the EGFR gene (2%) and compound heterozygous mutation involving deletion 19 and missense mutation for exon 21 (2%). On correlation of EGFR mutation studies from FFPE with that of cfDNA analysis, the study was statistically significant (P = 0.000). This study reports clinicopathological, immunochemical, and molecular analysis of EGFR among NSCLC cases. EGFR mutation detection from cfDNA has its advantage of being a noninvasive technique to avoid rebiopsy in cases of the progressive disease to detect resistance to a drug and emergence of a newer mutation. Mutation detection from FFPE samples still remains the gold standard for targeted therapy using EGFR tyrosine kinase inhibitors. ALK rearrangement detection using IHC serves as an adjunct to EGFR diagnosis.

High Cell-Free DNA Integrity Is Associated with Poor Breast Cancer Survival.

Simple SummaryA recent point of focus in breast cancer (BC) research has been the utilization of cell-free DNA and its concentration (cfDConc) and integrity (cfDI) as potential biomarkers. Though the association of cfDConc and BC survival is already recognized, studies on the prognostic value of cfDI have had contradictory results. The aim of this study was to investigate the prognostic potential of cfDConc and cfDI in Eastern Finnish BC cases with a non-metastatic disease. While the prognostic value of cfDConc remained non-significant in our analyses, high cfDI was an independent prognostic factor for poor overall survival (OS) and breast cancer-specific survival (BCSS). Inclusion of cfDI in the multivariate logistic regression model improved the predictive performance of the model, thus suggesting that the combined use of traditional tumor features and liquid biopsy could help to discriminate BC patients with poor OS and BCSS more accurately at the time of diagnosis.Background: A recent point of focus in breast cancer (BC) research has been the utilization of cell-free DNA (cfDNA) and its concentration (cfDConc) and integrity (cfDI) as potential biomarkers. Though the association of cfDConc and poor survival is already recognized, studies on the prognostic value of cfDI have had contradictory results. Here, we provide further evidence to support the use of cfDI as a potential biomarker. Methods: We selected 204 Eastern Finnish BC cases with non-metastatic disease and isolated cfDNA from the serum collected at the time of diagnosis before any treatment was given. The cfDConc and cfDI were measured with a fluorometer and electrophoresis and analyzed with 25 years of survival data. Results: High cfDConc was not an independent prognostic factor in our analyses while high cfDI was found to be an independent prognostic factor for poor OS (p = 0.020, hazard ratio (HR) = 1.57, 95% confidence interval (CI) 1.07–2.29, Cox) and BCSS (p = 0.006, HR = 1.93, 95% CI 1.21–3.08)). Inclusion of cfDI in the multivariate logistic regression model improved the predictive performance. Conclusions: Our results show high cfDI is an independent prognostic factor for poor OS and BCSS and improves the predictive performance of logistic regression models, thus supporting its prognostic potential.

Abstract 551: Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer

Abstract Loss of expression of the PTEN gene is associated with poorer prognosis in many solid tumors. While cfDNA-based liquid biopsy testing are gradually adopted as the routine testing method in the clinical setting, most of them are unable to reliably detect copy number loss and/or are limited reporting copy number amplification events only. Method: We have developed a capture-based CLIA-certified NGS assay, PredicineCARE, to robustly identify tumor-derived aberrations including SNVs, Indels, fusions, copy number amplifications and deletions from blood samples. Analytical validation of the assay was performed with commercial standard reference materials and contrived cell lines. Copy number loss of PTEN was also confirmed by Immunohistochemistry (IHC) assay in 15 clinical samples. Clinical utility of PTEN copy number loss was further established by its significant association with poor outcome in clinical patients with metastatic castration-resistant prostate cancer (mCRPC). Results: Analytical validation of the PredicineCARE assay demonstrated its super sensitivity and robustness of detecting homozygous copy number loss at approximately 10% tumor fraction. Both the sensitivity and Positive Prediction Value reached 100% when overall gene copy number is below 1.75 copies. When comparing to tissue IHC results reviewed by two independent pathologists, PredicineCARE liquid biopsy assay revealed 86.7% concordance with IHC in detecting PTEN loss (80% sensitivity, 100% specificity, and 100% PPV). The copy number loss was further confirmed by low-pass whole-genome sequencing of cfDNA samples. When the assay was applied to profile mCRPC patients, copy number loss of PTEN was detected at the prevalence rate of 19.2% (10/52) and 35.9% (23/64) respectively. Notably, patients harboring PTEN copy number loss showed significantly worse overall survival in the two cohorts independently (log-rank P = 0.0001 and 0.0004 respectively). Conclusion: We have developed PredicineCARE for clinical use, a highly sensitive cfDNA-based assay to detect gene copy number variants in addition to gene mutations and fusions. Its ability to detect gene copy number loss in low fraction of tumor contents as a valuable molecular biomarker holds great potential for clinical applications and patient selections in personalized precision oncology, particularly for investigational drugs targeting the AKT-PI3K-PTEN pathway. Citation Format: Xiaoxi Dong, Minghua Zhang, Tiantian Zheng, Lu Tan, Feng Xie, Amy Xiaohong Wang, Pan Du, Shidong Jia, Chris Lu, Jianjun Yu. Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 551.

23P Clinical utility of cell free DNA in pleural lavage and plasma in resectable NSCLC: A pilot study

23P Clinical utility of cell free DNA in pleural lavage and plasma in resectable NSCLC: A pilot study

Bile-Based Cell-Free DNA Analysis Is a Reliable Diagnostic Tool in Pancreatobiliary Cancer.

Simple SummaryTo elucidate and compare the value of plasma and bile as liquid biopsy source, cfDNA from 80 patients with pancreatobiliary cancers or non-malignant biliary obstructions was subjected to panel-based next generation sequencing (NGS). Results showed high correspondence in mutational profiles of bile-derived cfDNA and matched tissue samples, and the method proved superior to traditional plasma-based liquid biopsy techniques and with higher sensitivity than routine biomarkers such as CA19-9.Currently available serum biomarkers for pancreatobiliary cancers lack sensitivity and specificity and ultimate diagnosis still requires invasive procedures for histological confirmation. The detection of tumor-specific genetic aberrations with utilization of cell free DNA (cfDNA) is a less invasive approach than traditional tissue biopsies; however, it has not been implemented into clinical routine. In this study, we investigated bile as a liquid biopsy source in pancreatobiliary cancers and compared its potential as cell-free DNA source to plasma. Blood (n = 37) and bile (n = 21) samples were collected from patients affected by pancreatic ductal adenocarcinoma (PDAC) and extrahepatic cholangiocarcinoma (CCA) or with non-malignant biliary obstructions (blood n = 16; bile n = 21). Panel-based next generation sequencing (NGS) and digital droplet PCR (ddPCR) were applied for tumor mutation profiling. NGS results from matched tumor tissues (n = 29) served as comparison. Sequencing of cfDNA from bile resulted in detection of 96.2% of the pathogenic tumor mutations found in matched tissue samples. On the other hand, only 31.6% of pathogenic tumor mutations found in tissue could be detected in plasma. In a direct comparison, only half of the mutations detected in bile cfDNA were concordantly detected in plasma from the same patients. Panel NGS and ddPCR displayed comparable sensitivity. In conclusion, bile is a suitable source of cfDNA for the diagnosis of pancreatobiliary cancer and performs more reliably than plasma. Although primary diagnosis still requires histologic confirmation, bile-derived cfDNA could offer an alternative if tissue sampling is not feasible and might allow less invasive disease monitoring.

Abstract 719: Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via targeted, whole exome, and whole genome multi-region sequencing

Abstract Early cancer diagnosis, especially while the disease is localized and before symptoms appear, results in significantly higher survival rates compared to late-stage diagnosis. At the time of diagnosis, it is also common to find multiple foci within the prostate in men with localized disease. Cell-free DNA (cfDNA) are short DNA fragments released into circulation by dying cells, which may reflect underlying disease biology and simultaneously allow for the identification of genetically distinct tumor subclones. The objectives of this study are to determine if cfDNA concentration can be used to distinguish between healthy individuals from patients with localized or metastatic castration-resistant prostate cancer (mCRPC), and if somatic mutations identified in tumor tissue are detectable in cfDNA. This study included samples from 277 individuals: 41 healthy donors, 112 patients with localized prostate cancer who underwent radical prostatectomy (RP) at UCSF, and 124 mCRPC patients. Whole peripheral blood was collected in EDTA tubes or PAXgene Blood ccfDNA tubes. Blood and matched tissue from adjacent normal seminal vesicles and multiple tumor regions (1-9 samples per patient) were collected from patients undergoing RP. Extraction of cfDNA was performed on double spun plasma using the Qiagen QIAamp Circulating Nucleic Acid Kits. After extraction, the concentration and fragment length distribution were measured with a Bioanalyzer 2100 Instrument. Fifty-seven samples (multiple tumor tissue foci and matched blood) from nine patients with localized disease were subjected to whole exome sequencing, and 22 samples from five patients were subjected to whole genome sequencing. All samples from 14 patients underwent targeted sequencing with a 2.5Mb panel generated via machine learning on TCGA prostate cancer sequence data. Somatic variant calling was performed with Broad Institute's Terra platform (GATK4/MuTect2) for tumor tissue and with the Curio platform for cfDNA to build consensus sequences leveraging duplex unique molecular tags. To estimate tumor fraction in cfDNA, ichorCNA was used to profile low pass whole genome sequence data. Total cfDNA concentration was able to distinguish between healthy and metastatic (p = 0.001) participants, adjusted for age; and also between localized and metastatic groups (p < 0.001), adjusted for age and PSA. Among the patients with localized disease, cfDNA concentration was not associated with age, PSA, Gleason score, or Decipher Score (a 22 gene metastasis risk-predicting RNA gene expression signature), suggesting the potential for this marker to be independent of factors that are commonly used to assess disease burden and risk of progression. Targeted sequencing of prostate tumors resulted in an average of 16 mutations with a range of 1 to 128 mutations per tissue region. The cfDNA allele frequencies for mutations identified in tumor tissue ranged from 0.5% to 20%. Four patients with variant detection in cfDNA also experienced biochemical recurrence. While copy number analysis identified clonal and potentially subclonal alterations in WGS tumor tissue, no alterations were found in WGS cfDNA. Further analysis of potential factors influencing variant detection in cfDNA (adverse pathology, starting amount of DNA, coverage) will be performed. Citation Format: Emmalyn Chen, Clinton L. Cario, Lancelote Leong, Karen Lopez, César Márquez, Carissa Chu, Patricia S. Li, Erica Oropeza, Imelda Tenggara, Janet Cowan, Jeffry P. Simko, Daniel K. Wells, Robin Kageyama, June M. Chan, Terence Friedlander, Rahul Aggarwal, Felix Feng, Pamela L. Paris, Peter R. Carroll, John S. Witte. Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via targeted, whole exome, and whole genome multi-region sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 719.

Abstract 1371: Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via whole exome and whole genome, multi-region sequencing

Abstract Early cancer diagnosis, especially while the disease is still localized and before symptoms appear, results in significantly higher survival rates compared to late-stage diagnosis. At the time of diagnosis, it is also common to find multiple foci within a single prostate gland in men with localized disease. Cell-free DNA (cfDNA) may not only reflect underlying disease biology, but also simultaneously allow for the identification of genetically distinct tumor subclones. The objectives of this study are to determine if 1) cfDNA levels are able to distinguish between healthy individuals from patients with localized or metastatic castration-resistant prostate cancer (mCRPC), and 2) if somatic mutations identified in tumor tissue are detectable in cfDNA and representative of the distribution observed in tumor tissue. This study includes samples from 130 individuals at UCSF: 21 healthy donors, 100 patients with localized prostate cancer who underwent radical prostatectomy (RP), and 9 mCRPC patients. Blood samples and matched tissue from adjacent normal seminal vesicles and multiple tumor regions (1-9 samples per patient) were collected from patients undergoing radical prostatectomy. CfDNA was extracted from plasma, and the concentration and fragment length distribution were measured with a Bioanalyzer 2100. Comparisons of cfDNA levels between groups were assessed with a Welch’s t-test due to the potential for unequal variances. Fifty-seven samples from nine patients have been subjected to whole exome sequencing, and 22 samples from five patients have been subjected to whole genome sequencing at ~40x coverage. Somatic variant calling was performed with Broad Institute’s Firecloud platform (GATK4/MuTect2) for tumor tissue and with the Curio platform for cfDNA to build consensus sequences leveraging unique molecular tags. CfDNA levels are able to distinguish between healthy and localized (p = 0.005), as well as healthy and metastatic groups (p = 0.043). Tumor foci within a patient’s prostate gland are genetically heterogeneous, with the majority of somatic mutations private to tumor regions and a subset of mutations at the intersection of these regions. Preliminary analyses result in an average of 72 somatic SNVs and indels per tumor tissue region, with ~10% overlap between regions in the same patient. Additional mCRPC and follow-up blood samples are being collected from all patients. The association between cfDNA levels prior to RP surgery and biochemical recurrence will be investigated when follow-up collection concludes. Further analysis of mutational concordance, clonality, and copy number variation between tumor tissue DNA and cfDNA, along with clinical data, will be performed. Citation Format: Emmalyn Chen, Clinton Cario, Lancelote Leong, Karen Lopez, Patricia Li, Erica Oropeza, Imelda Tenggara, Janet Cowan, Jeffry Simko, Daniel Wells, Robin Kageyama, June Chan, Terence Friedlander, Pamela Paris, Peter Carroll, John Witte. Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via whole exome and whole genome, multi-region sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1371.

Abstract SY31-01: Development and validation of a clinical-grade cell-free DNA profiling assay: MSK-ACCESS

Abstract Cell-free DNA profiling noninvasively reveals the underlying tumor genomic profiles to guide treatment decisions, especially important when the tumor is in a site that is difficult or risky to access. It has the potential to guide treatment stratification, monitor disease responses, identify resistance mechanisms and detection of minimal residual disease or relapse.Clinical implementation of molecular profiling assay requires the establishment of robust quality controls throughout the process including pre-analytical procedures, such as sample collection and processing, analytical steps such as molecular assay specification and bioinformatics algorithm, as well as data reporting. This talk will describe an academic institutional effort to develop and validate a clinical-grade cell-free DNA profiling assay for patient care. We will discuss considerations behind the development, including factors such as analytical sensitivity required for individual clinical applications, operational and logistical efficiency, regulatory requirement and clinical annotation to guide decisions. We will also present the validation studies required by the New York State Department of Health to demonstrate robustness of the assay before clinical deployment. The assay we have developed, called MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), is a custom next-generation sequencing assay covering selected exons from 129 cancer-related genes for ultra-sensitivity detection of somatic mutations from plasma. Using ultra-high depth (>15,000x) sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. We validated MSK-ACCESS using plasma samples collected from healthy non-cancer individuals and cancer patients with known mutations in selected genes of interest. We evaluated the analytical performance of the assays such as accuracy, intra- and inter-assay reproducibility using control samples and samples with known mutation data established by orthogonal assays. With the growing interest on cell-free DNA profiling for molecular diagnostics, it is crucial to ensure robustness of each assay to deliver high-quality data to guide decisions. Establishing analytical performance is the first step precedes large-scale prospective trials to further demonstrate clinical utility of cell-free DNA as a biomarker. Citation Format: Dana Wai Yi Tsui. Development and validation of a clinical-grade cell-free DNA profiling assay: MSK-ACCESS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY31-01.

Cell free DNA: A Novel Predictor of Neurological Outcome after Intravenous Thrombolysis and/or Mechanical Thrombectomy in Acute Ischemic Stroke Patients.

PurposeSeveral blood markers have been evaluated in stroke patients, but their role remains limited in clinical practice. This study was designed to evaluate the utility of cell free DNA (cf DNA) in stroke patients undergoing therapeutic intervention in the form of mechanical thrombectomy in acute ischemic stroke patients.Materials and MethodsTwenty-six patients with ischemic stroke who were managed with interventions like intravenous thrombolysis (IVT) and mechanical thrombectomy were recruited consecutively in this study. The cf DNA was extracted by using circulating nucleic acid kit and measured by real-time quantitative PCR assay for β-globin gene. The neurological outcome was measured by modified Rankin scale (mRS) score at three months after the onset of symptoms.ResultsCf DNA levels correlated with severity of stroke at the time of admission (r=0.421, P=0.032) and poor outcome at three months (r=0.606, P=0.001). Therapeutic intervention in the form of mechanical thrombectomy or IVT was associated with improved outcome in patients with cf DNA <10,000 kilogenome-equivalents/L (P=<0.05).ConclusionCf DNA level correlated well with the 3 month outcome in acute ischemic stroke patients. It can be a potential supplementary marker to predict neurological outcome after therapeutic intervention.

PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA.

PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample (n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers.

Cell-free DNA as a novel marker in cancer therapy.

Advances in next-generation sequencing have provided new insights and new therapeutic options for patients with cancer. However, assessment of genomic aberrations, which is required for precision medicine, has been challenging because of the difficulties in capturing intratumoral heterogeneity and in real-time assessment of tumors. Recent advances in technology have enabled detection and analysis of cell-free DNA in cancer patients, which provides real-time assessment of tumor evolution. Here, we review the recent advances in our understanding of the clinical utility of cell-free DNA and the future directions for its use in cancer management.

Controls to validate plasma samples for cell free DNA quantification

Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use.The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology.

Abstract 3000: New epigenetic biomarker for colorectal cancer.

Abstract DNA is released and circulate in the blood of cancer patients. Epigenetic changes in the levels of circulating DNA has been associated with tumor burden and malignant progression. Public physical examination recommends occult fecal blood test for colorectal cancer (CRC) screening but its false positive rate is too high. As for serum marker for CRC including CEA and CA19-9, their sensitivity are nor satisfactory especially in early stage CRC patients and therefore, the potential use of circulating DNA for CRC screening has emerged. The aim of this study is the clinical utility of cell-free DNA as blood biomarkers. Based on our previous report about the new methylation markers for CRC on a genome-wide scale, we selected 4 methylation markers positive for CRC and established CRC screening panel. First, we collected plasma DNA from blood samples of 100 CRC patients preoperatively and 50 age-matched healthy volunteers. Then, we performed pyrosequence about plasma DNA after bisulfite treatment. Using this cancer screening panel, 82 CRC patients were positive for at least 1 marker. On the other hand, no control samples were positive for any markers and therefore, sensitivity and specificity using this cancer screening panel for CRC were 82.0% and 100%, respectively. According to TNM classification, positive rate for earlier stage including 1 and 2 patients were lower than advanced CRC patients. Similarly, the patients with abnormal serum CEA level harbored higher positive rate for the cancer panel, but serum CA19-9 level which is another CRC marker was not associated with positive rate. Two markers of the panel were relatively high in old age although they were negative for control samples. Considering that no hypermethylation was detected in control samples, this cancer panel is superior to preexisting blood tumor markers for CRC and available for cancer screening. Citation Format: Kiyoko Takane, Yutaka Midorikawa, Koichi Yagi, Ayako Sakai, Hiroyuki Aburatani, Tadatoshi Takayama, Atsuhi Kaneda. New epigenetic biomarker for colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2013-3000

Circulating tumor DNA in plasma of breast cancer patients from India

e22213 Background: Recently, breast cancer has become the most common cancer among women in all urban population in India. Annually about 80000 new cases and 40000 deaths occur and majority of breast cancers are pre-menopausal. Conventional diagnostic methods are not very sensitive especially in early stages of cancer. This necessitated a more sensitive and reliable method for early diagnosis leading to effective treatment, better prognosis and survival. Recently, the level of cell free circulating tumor DNA in blood plasma or serum of patients with variety of tumors are being considered as reliable non-invasive diagnostic tool but no study has been done in India. The present study has therefore been undertaken to evaluate clinical utility of cell free DNA as potential biomarkers for early diagnosis and management of breast cancer. Methods: 25 newly diagnosed untreated breast cancer patients and 25 healthy subjects having no sign of significant medical illness with informed consent were enrolled for the study. 9 patients after chemotherapy were also included in the study. Blood plasma collected from both patients and controls were employed for DNA isolation, using Qiagen kit. Concentration of cell free plasma DNA was analyzed by 3 methods viz. nanodrop spectro-photometry, integrated density value (IDV) of PCR products of Exon 7 of p53 gene and quantitative real time PCR (cycles threshold converted to genome equivalent). All values of DNA concentration obtained by three methods used as continuous variables and receiver operating characteristic (ROC) were plotted and the cut-of value was determined at 90% sensitivity and 100% specificity level of ROC. Results: Mean free plasma DNA concentration as determined by both Q-RT PCR and IDV in cancer patients was found to be significantly higher in advanced stage breast cancer patients than in controls (genome equivalent 18850 vs 431; IDV 17912 vs 4197; p=0.001). However, no significant difference could be observed in early stage disease as compared to controls possibly due small sample size. Conclusions: Free Plasma DNA concentration is a reliable molecular marker for detection of breast cancer and can serve as a prognostic indicator leading to its potential clinical application either alone or in combination with other conventional methods. No significant financial relationships to disclose.