ctDNA Concentration Research Articles

Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing during Checkpoint Blockade Immunotherapy in Non-Small Cell Lung Cancer.

The evolutionary dynamics of tumor‐associated neoantigens carry information about drug sensitivity and resistance to the immune checkpoint blockade (ICB). However, the spectrum of somatic mutations is highly heterogeneous among patients, making it difficult to track neoantigens by circulating tumor DNA (ctDNA) sequencing using “one size fits all” commercial gene panels. Thus, individually customized panels (ICPs) are needed to track neoantigen evolution comprehensively during ICB treatment. Dominant neoantigens are predicted from whole exome sequencing data for treatment‐naïve tumor tissues. Panels targeting predicted neoantigens are used for personalized ctDNA sequencing. Analyzing ten patients with non‐small cell lung cancer, ICPs are effective for tracking most predicted dominant neoantigens (80–100%) in serial peripheral blood samples, and to detect substantially more genes (18–30) than the capacity of current commercial gene panels. A more than 50% decrease in ctDNA concentration after eight weeks of ICB administration is associated with favorable progression‐free survival. Furthermore, at the individual level, the magnitude of the early ctDNA response is correlated with the subsequent change in tumor burden. The application of ICP‐based ctDNA sequencing is expected to improve the understanding of ICB‐driven tumor evolution and to provide personalized management strategies that optimize the clinical benefits of immunotherapies.

Prospective trial of nivolumab (Nivo) plus radium-223 (RA) in metastatic castration-resistant prostate cancer (mCRPC) evaluating circulating tumor DNA (ctDNA) levels as a biomarker of response.

TPS267 Background: RA is a calcium-mimetic radiopharmaceutical emitter of alpha particles that has been approved for treatment of mCRPC. Radiation plus checkpoint inhibitors has demonstrated promising efficacy in previous clinical trials (PMID 27466265, 23535954). Alteration to PD-1 expression has been observed with radium-223, suggesting potential synergy with Nivo (PMID 29137877). ctDNA concentration may accurately reflect overall tumor burden and response to immune therapy. ctDNA testing after 6 weeks of therapy predicts efficacy of immunotherapy in patients with metastatic NSCLC and urothelial carcinoma (PMID 30093454) and metastatic gastric cancer (PMID 30013197). Reduction of ctDNA correlated with both radiographic progression free survival (rPFS) and overall survival. We hypothesize that RA + Nivo will be safe and decrease ctDNA, which may predict response to therapy earlier than conventional scans. Methods: This is a single-arm phase I/II investigator initiated trial (NCT04109729). Primary objectives: 1) Safety, 2) Change in ctDNA after 6 weeks treatment compared to baseline. Secondary objectives: 1) PSA-PFS; 2) PSA 50% response rate; 3) Time to skeletal related event; 4) Bone metabolism marker response. Inclusion criteria: symptomatic bone metastasis, mCRPC, adequate hematopoiesis. Exclusion criteria: visceral metastasis, history of autoimmune disease and current use of immune suppression therapy. A total of 36 patients will be enrolled. Cycles are 28 days. ctDNA concentration will be measured using GuardantOMNI research platform which evaluates 500 genes. Treatment: RA (55 kBq/kg IV) monotherapy lead in for two cycles followed by RA plus Nivo (480mg IV) for an additional 4 cycles. Nivo monotherapy then continues for up to 2 years. ctDNA collected prior to combination therapy and 6 weeks after. Restaging scans done every 2 cycles while on radium-223 and every 3 cycles while on nivolumab. Clinical trial information: NCT04109729.

Exploration of the Beta-Actin DNA Integrity Index as Early Genetic Marker of Presence of Breast Cancer

Background: Circulating cell-free DNA (cfDNA) and its integrity index can be a fast and non-invasive (liquid biopsy) biomarker, this provides significant additional data for diagnosis, prognosis and therapy stratification in cancer patient. Methods: The circulating tumor DNA (ctDNA) concentration and integrity was investigated in the plasma from patients with breast cancer by a quantitative polymerase chain reaction (qPCR) and their diagnostic value for breast cancer etiology was evaluated Plasma samples were collected from 55 patients 40 patients with breast cancer, 5 patients each with other type of cancer (ovarian cancer, colon cancer, stomach cancer) and 20 healthy controls. Real-time PCR of β-actin gene were investigated using two primer sets (400 and 100bp) to amplify different DNA fragment lengths. The DNA integrity index was calculated as the ratio of q-PCR results of β-actin 400bp/100. Results: In all cancer patients the DNA concentrations were significantly higher p<0.0001, than those of the control group. The plasma DNA integrity was statistically significantly lower in breast cancer and colon cancer than the control groups (p<0.001). Conclusion: The plasma DNA concentration and integration test can serve as a new diagnostic marker for detection and monitoring of patients with breast cancer and colon cancer.

Induction of apoptosis increases sensitivity to detect cancer mutations in plasma

BackgroundThe study of cell-free DNA (cfDNA), namely the fraction derived from tumors (ctDNA), is a clinically relevant noninvasive biomarker for cancer management. However, the intrinsic low abundance of ctDNA in plasma limits its implementation in the clinic. Aim of the studyIn this study, the objective was to demonstrate that induction of apoptosis—the major source of ctDNA—increases ctDNA concentration, thereby increasing the sensitivity to detect clinically relevant mutations in plasma. MethodsIn vitro models were used to test the effect of docetaxel on the release levels of DNA from lung cancer cells. In vivo, Rag2−/−IL2rg−/− immunodeficient C57BL/6 xenografted mice were treated with docetaxel for 24 h or 48 h. Tumor tissue and blood were collected to evaluate the levels of apoptosis DNA release levels, respectively. ResultsWe observed increased levels of apoptosis in H1975 cells and a consequent increase in cfDNA released into the culture medium after docetaxel treatment. In vivo, the results show increased cfDNA concentration in plasma of xenografted mice after apoptosis stimulation. Importantly, treatment increased the sensitivity of detection of relevant cancer mutations, namely 24 h after treatment. ConclusionThis study provides new insights regarding the importance of timing for blood collection. In our experimental model, we demonstrate that blood collection should be performed 24 h after treatment (apoptosis induction), for optimal ctDNA analysis. Translating these results into the clinical setting is likely to increase sensitivity to detect tumor-derived mutations in plasma, might help guide the therapeutic decision, and optimize current liquid biopsy procedures for situations where tissue analysis is not possible.

Perioperative Dynamic Changes in Circulating Tumor DNA in Patients with Lung Cancer (DYNAMIC).

No study has investigated the precise perioperative dynamic changes in circulating tumor DNA (ctDNA) in any patients with early-stage cancer. This study (DYNAMIC) investigated perioperative dynamic changes in ctDNA and determined the appropriate detection time of ctDNA-based surveillance for surgical patients with lung cancer.Experimental Design: Consecutive patients who underwent curative-intent lung resections were enrolled prospectively (NCT02965391). Plasma samples were obtained at multiple prespecified time points including before surgery (time A), during surgery after tumor resection (time B-time D), and after surgery (time P1-time P3). Next-generation sequencing-based detection platform was performed to calculate the plasma mutation allele frequency. The primary endpoint was ctDNA half-life after radical tumor resection. Thirty-six patients showed detectable mutations in time A. The plasma ctDNA concentration showed a rapid decreasing trend after radical tumor resection, with the average mutant allele fraction at times A, B, C, and D being 2.72%, 2.11%, 1.14%, and 0.17%, respectively. The median ctDNA half-life was 35.0 minutes. Patients with minimal residual disease (MRD) detection had a significant slower ctDNA half-life than those with negative MRD (103.2 minutes vs. 29.7 minutes, P = 0.001). The recurrence-free survival of patients with detectable and undetectable ctDNA concentrations at time P1 was 528 days and 543 days, respectively (P = 0.657), whereas at time P2 was 278 days and 637 days, respectively (P = 0.002). ctDNA decays rapidly after radical tumor resection. The ctDNA detection on the third day after R0 resection can be used as the baseline value for postoperative lung cancer surveillance.

A novel view of the separate and simultaneous binding effects of docetaxel and anastrozole with calf thymus DNA: Experimental and in silico approaches.

DNA stands as the primary purpose of many anticancer drugs and according to the performed research on this field, some certain changes contain crucial functionalities in the regulated transcription of DNA. Therefore, the interaction between anticancer drugs and DNA play an important role in understanding their function and also provide a better groundwork for producing more efficient and newer drugs. Here, the interaction between Docetaxel (DO) and calf thymus DNA (ct DNA), in the presence and absence of Anastrozole (AN), has been examined through the usage of different methods that include isothermal titration calorimetry, multi-spectroscopic, viscometry, and molecular docking techniques. Interaction studies have been performed by preparing different molar ratios of DO with the constant ct DNA and AN concentration at pH = 6.8. The binding constants have been calculated to be 7.93 × 104 M-1 and 6.27 × 104 M-1, which indicate the strong binding of DO with ct DNA double helix in the absence and presence of AN, respectively. Thermodynamic parameters, which were obtained from fluorescence spectroscopy and isothermal titration calorimetry, have suggested that the binding of DO and AN to ct DNA as binary and ternary systems have been mainly driven by the electrostatic interactions. The relative viscosity of ct DNA has increased upon the addition of DO and AN, which confirms the interaction mode. A competitive binding study has reported that the enhanced emission intensity of ethidium bromide (EB) and acridine orange (AO), in the presence of ct DNA, have been quenched through the addition of DO and Anastrozole as binary and ternary systems. As it is indicated by these findings, DO is capable of displacing EB and AO from their binding site in ct DNA; hence, it can be concluded that DO and AN are able to intercalate into the base pairs of ct DNA in binary and ternary systems. Molecular docking studies have corroborated the mentioned experimental results.

OS1.4 Liquid biopsy of the CSF in a series of GBM patients: preliminary results

Abstract BACKGROUND Liquid biopsy (LB) by cerebrospinal fluid (CSF) can be useful to identify circulating tumour DNA (ctDNA), thus offering information about the heterogeneity of the neoplastic genome. The aim of our study is to assess the effectiveness of LB of the CSF in detecting ctDNA which mirrors the genetic profile of the tumoural tissue, and to investigate the clinical and radiological aspects influencing the availability of ctDNA. MATERIAL AND METHODS Tumoral tissue and CSF samples of 13 GBM patients undergoing surgery was collected. CSF was withdrawn from the very proximity of the tumoural surface before the excision. DNA extracted from tissue samples was analysed by qPCR to identify typical genetic alterations such as copy number variations (EGFR, PDGFRA, CDK4, MDM2, CDKN2A), and point mutations (TP53, PTEN, IDH, NRAS, PI3K1, pTERT). CtDNA extracted from CSF was analysed by droplet digital PCR to assess the presence of the alterations found in the matching tissue. Both contrast-enhanced (CE) and FLAIR volumes of the lesions were measured in the pre-surgical MRI. Linear and logarithmic regressions were employed for the statistical analysis. RESULTS From June 2016 to February 2017 we prospectively collected 13 GBM patients. Median age was 73 years. All lesions showed CE at the MRI; other radiological findings included necrosis (84.6%), oedema (76.9%), cortical, ventricular or meningeal involvement (76.9%, 30.8%, and 15.4%). Median volumes of CE and FLAIR lesions were 28.6 and 25.5 cm3, with a median FLAIR/CE ratio of 72.9. Surgery was subtotal (&lt;95%) in all patients. All GBM tissues were tested for the following alterations: EGFR, PDGFRA, CDK4, MDM2, CDKN2A; 76.9% were tested for TP53, PTEN, and IDH mutations; 38.5% for NRAS and pTERT mutations; 30.8% for PI3KR1 mutation. MGMT methylation was assessed in 12 cases (92.3%) and found in 7 (58.3%). Median CSF volume, ctDNA quantity and concentration were 0.45 mL, 59.64 ng, and 0.42 ng/μL. Processable DNA was found in 11 CSF specimens (84.6%), in 8 of which (61.5%) it carried the same alteration expressed by the tumoural cells of the matched tissue, while in 3 cases (23.1%) it seemed to have a different genetic profile; finally, in 2 cases it was not possible to detect any circulating DNA in the CSF. Preliminary data on 13 patients suggest that the ctDNA concentration in the CSF could be related to the FLAIR/CE ratio as measured in the MRI before surgery (p = 0.02). Other correlations between the molecular and the radiological features are still being exploring. CONCLUSION Our study confirms that LB of CSF can detect ctDNA carrying the same molecular profile harboured in the tumour. Therefore, it seems to be an accurate method to identify markers useful for the diagnosis and the monitoring of the disease. Additionally, our ongoing study is trying to demonstrate a potential correlation between radiological features of the tumour and availability of ctDNA in CSF.

Liquid Biopsy by Next-Generation Sequencing: a Multimodality Test for Management of Cancer

While liquid biopsy is still relatively a new concept, the advent of next-generation sequencing (NGS) technologies has recently generated a revolution in the field and will be the focus of this review. Circulating tumor DNA (ctDNA) derives from tumor cells and provides information about the genetic alterations of tumors. However, ctDNA concentration in plasma can be below the level of detection by conventional methods; therefore, screening for actionable genetic information is challenging. Clinical trials exploring targeted and untargeted sequencing to improve the outcomes of ctDNA detection are showing promising results, having reached a limit of detection as low as 0.001% of ctDNA in a background of normal circulating DNA. Most of the challenges related to the sensitivity of detection of ctDNA have been defeated by dint of NGS-based approaches. Despite all the efforts, these methods are still expensive, time-consuming, and require advanced skills for appropriate interpretation. Nevertheless, the technology is rapidly improving, and the expectations for the implementation of liquid biopsy into the clinical practice in the near future are high.

Abstract 913: Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma

Abstract Background Circulating tumor DNA (ctDNA) analysis is an emerging field for diagnosis, monitoring of treatment efficacy and detection of disease recurrence in various cancers. In bladder cancer, previous work has demonstrated prognostic value of ctDNA for early stage cancers and early detection of recurrence following radical cystectomy (CX), however more extensive investigation of the potential impact of ctDNA analysis on clinical decision making remains to be carried out. Methods A total of 68 patients with localized advanced bladder cancer to be treated with neoadjuvant chemotherapy (NAC) and CX were prospectively included. We performed RNA-Seq to identify RNA subtypes and whole exome sequencing to identify somatic mutations for ctDNA analysis. Based on allele frequency and sequence context, 16 somatic mutations were selected for each patient for multiplex PCR and subsequent ultra-deep sequencing (median coverage of 114,000x). The sample-level analytical sensitivity was determined to be &amp;gt;95% at 0.01% ctDNA concentration level when at least 2/16 mutations were detected. The protocol was applied to detect ctDNA in 618 blood samples procured at diagnosis, during NAC, before CX and during surveillance following CX. Results The presence of ctDNA was highly prognostic already at diagnosis before initiation of NAC (HR=31.7, p=0.001). Analysis of ctDNA dynamics during NAC revealed that clearance of ctDNA was associated with pathological downstaging (≤TaN0 at CX) and reduced disease recurrence. Conversely, continually detectable ctDNA during NAC was associated with lack of pathological downstaging and later disease recurrence (p=0.023). Our results suggest that ctDNA dynamics during NAC may offer superior predictive value compared to clinical parameters and tumor centric molecular markers such as DNA damage response mutations and RNA subtypes. Following CX, ctDNA analysis identified 92% of metastatic relapse (100% specificity) during disease monitoring (100% positive- and 98% negative predictive value). The positive lead-time over radiographic imaging was 100 days on average (range: -17-245 days). Conclusions ctDNA analysis of blood samples procured throughout the disease course of patients with bladder cancer demonstrates novel evidence of early risk stratification and monitoring of treatment efficacy, which adds to the already strong evidence of ctDNA-based recurrence detection. Collectively, this paves the way for novel ctDNA-based clinical trials. Citation Format: Emil Christensen, Karin Birkenkamp-Demtröder, Himanshu Sethi, Svetlana Shchegrova, Raheleh Salari, Iver Nordentoft, Hsin-Ta Wu, Michael Knudsen, Philippe Lamy, Sia V. Lindskrog, Ann Taber, Mustafa Balcioglu, Søren Vang, Zoe Assaf, Shruti Sharma, Antony S. Tin, Ramya Srinivasan, Dina Hafez, Thomas Reinert, Samantha Navarro, Alexander Olson, Rosie Ram, Scott Dashner, Matthew Rabinowitz, Paul Billings, Styrmir Sigurjonsson, Claus L. Andersen, Ryan Swenerton, Alexey Aleshin, Bernhard G. Zimmermann, Mads Agerbæk, Cheng-Ho J. Lin, Jørgen B. Jensen, Lars Dyrskjøt. Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma [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 913.

Detecting and Tracking Circulating Tumour DNA Copy Number Profiles during First Line Chemotherapy in Oesophagogastric Adenocarcinoma.

DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing (lcWGS) of ctDNA from 30 patients with advanced OGA prior to first-line chemotherapy and on progression. SCNA profiles were detectable pretreatment in 23/30 (76.7%) patients. The presence of liver metastases, primary tumour in situ, or of oesophageal or junctional tumour location predicted for a high ctDNA fraction. A low ctDNA concentration associated with significantly longer overall survival. Neither chromosomal instability metrics nor ploidy correlated with chemotherapy outcome. Chromosome 2q and 8p gains before treatment were associated with chemotherapy responses. lcWGS identified all amplifications found by prior targeted tumour tissue sequencing in cases with detectable ctDNA as well as finding additional changes. SCNA profiles changed during chemotherapy, indicating that cancer cell populations evolved during treatment; however, no recurrent SCNA changes were acquired at progression. Tracking the evolution of OGA cancer cell populations in ctDNA is feasible during chemotherapy. The observation of genetic evolution warrants investigation in larger series and with higher resolution techniques to reveal potential genetic predictors of response and drivers of chemotherapy resistance. The presence of liver metastasis is a potential biomarker for the selection of patients with high ctDNA content for such studies.

Applying machine learning strategy for microsatellite status detection in plasma sample type.

e14219 Background: Microsatellite(MS) status is an important biomarker in cancer diagnosis . Most of available detection algorithms were based on figuring out number of validated allele types from control and cases, then determine the MS status by identify the difference. That would be suitable for tissue like samples due to their clear and sufficient tumor nucleic acid content. But for liquid biopsy such as plasma, lower ctDNA concentration and higher background ratio induced from laboratory and sequencing procedure make it necessary to develop novo method for plasma MS status detection. Methods: We developed an machine learning based approach to detect the MS status by using NGS sequencing data from plasma sample. 98 blood cell , 12 tumor tissue samples with PCR confirmed MSS status and 87 plasma samples from 134 patients were used to build normal baseline, and check the sequencing stability and population polymorphism of 207 candidate SSR locus in our panel. 97 locus then passed and were chosen as original biomarkers for model construction. For each coming sample, the allele probability distribution were obtained for each locus. Followed by calculating the Kullback–Leibler Divergence (KLD) value between target and baseline samples for each locus, the KLD value was used as input for feature selection and machine learning model fitting. Finally a leave one out strategy was used for model performance evaluation. Results: We obtained 17 most relevant SSR locus with suitable for MS status analysis of plasma sample. By using a SVM model fitted with 49 real patients sample data (21 MSI and 28 MSS) , The MS status prediction of plasma sample was of high sensitivity and specificity which were 90.47% and 96.42% respectively. Conclusions: Our results suggest that our MSI calculation model by using KLD value and SVM method is appropriate for the plasma sample. Further large-scale prospective studies are needed to validate our model.

New DNA-sensor based on thiacalix[4]arene-modified polydiacetylene particles

New p-tert-butyl thiacalix[4]arene derivative in the 1,3-alternate stereoisomeric form containing two diethylenetriamine groups and pentacosa-10,12-diynoic moieties on the opposide sides of macrocyclic cavity was synthesized using the copper(i)-catalyzed azide-alkyne cycloaddition. According to the dynamic and electrophoretic light scattering data, the synthesized macrocycle forms submicron particles with the sizes 200 nm and ζ-potential equal to 43 mV. The critical aggregation concentration of the macrocycle was 0.019 mmol L−1. The obtained macrocycle intercalates into calf thymus DNA (CT DNA) to form a lipoplex. Using ethidium bromide as a fluorescent probe intercalation of obtained macrocycle into CT DNA with following formation of a lipoplex with the ζ-potential equal to −30 mV was found. The macrocycle was used for the synthesis of mixed polydiacetylene particles with N-(2-aminoethyl)pentacosa-10,12-diynamide (PCDA) as a base lipid. The highest degree of polymerization is achieved in the system with the macrocycle to PCDA ratio equal to 1 : 4. Macrocycle embedding into the polydiacetylene particles significantly increases their colorimetric response to CT DNA. The response to CT DNA as a change in the color of a solution of particles from blue to red is seen by naked eye at the CT DNA concentration starting from 20 µmol L−1, which makes the obtained particles promising for bioanalytical application.

Determination of DNA based on fluorescence quenching of terbium doped carbon dots.

This work describes the preparation of carbon dots doped with terbium(III) (Tb-CDs) via a hydrothermal method, starting from terbium ion and ethylenediamine. The size, composition and spectral properties of the Tb-CDs were characterized by transmission electron microscopy, infrared spectra, and fluorescence spectra. The results show that doping of the CDs with Tb(III) reduces the particle size and results in more uniform particles, while fluorescence (at excitation/emission peaks of 380/475nm) is strongly enhanced. The interaction between Tb-CDs and ct-DNA results in fluorescence quenching of Tb-CDs. The findings were exploited to design a quenchometric method for the determination of ct-DNA. The signal drops linearly in the 80ng·mL-1 to 50μg·mL-1 ct-DNA concentration range, and the detection limit is 53ng·mL-1. The method was applied to the determination of ct-DNA in spiked samples and gave satisfactory results. The possible fluorescence quenching mechanism (which is mainly static)was investigated using theStern-Volmer equation and thermodynamic equations. Graphical abstract A kind of carbon dots doped with terbium(III) (Tb-CDs) were prepared via a hydrothermal method, using terbium ion and ethylenediamine as precursor. Doping with Tb(III) reduced the particle size of CDs and results in uniform particle size and stronger fluorescence. The interaction between the Tb-CDs and dsDNA results in quenching of the fluorescence of Tb-CDs and can be applied to determination of dsDNA.

Circulating tumour DNA is a potential biomarker for disease progression and response to targeted therapy in advanced thyroid cancer

BackgroundConventional biomarkers in thyroid cancer are not disease specific and fluctuate in advanced disease, making interpretation difficult. Circulating tumour DNA (ctDNA) has been shown to be a useful biomarker in other solid tumours. This is a multimutational study of ctDNA over multiple timepoints, designed to test the hypothesis that ctDNA is a potential biomarker in patients with advanced thyroid cancer. MethodsMutational analysis of archival tumour tissue was performed using NGS with a targeted gene panel. Custom TaqMan assays were designed for plasma ctDNA testing using digital droplet polymerase chain reaction. Concentrations of detected ctDNA were correlated with the conventional biomarker concentration and axial imaging status defined by the Response Evaluation Criteria in Solid Tumours criteria. ResultsTumour tissue from 51 patients was obtained, with the following histologies: 32 differentiated (differentiated thyroid cancer [DTC]), 15 medullary (medullary thyroid cancer [MTC]), three poorly differentiated and one anaplastic. NGS analysis detected variants in 42 (82%) of cases. Plasma was assayed for these patients in 190 samples, and ctDNA was detected in 67% of patients. Earlier detection of disease progression was noted in three patients with MTC. In two cases (PTC and ATC), where conventional biomarkers were not detectable, ctDNA was detected before disease progression. Changes in ctDNA concentration occurred earlier than conventional markers in response to disease progression in multiple patients with DTC receiving targeted therapies. ConclusionThe majority of patients with advanced thyroid cancer had detectable ctDNA. ctDNA measurement may offer superiority over conventional markers in several scenarios: earlier detection of progression in MTC; as an alternative biomarker when conventional markers are not available; more rapid assessment of the disease status in response to targeted therapies, thereby potentially allowing prompter discontinuation of futile therapies. These early results support the hypothesis that ctDNA may be a clinically useful biomarker in thyroid cancer.

The association of KRAS mutation in circulating-tumor DNA and survival in patients with pancreatic cancer

Circulating-tumor DNA (ctDNA) has been known to be released from tumor cells and investigated potential biomarkers for therapeutic responses. However, the role of ctDNA in pancreatic cancer has not been well studied. Here we selected KRAS mutation which has been known common over 95% of pancreatic ductal adenocarcinoma (PDA) and evaluated applicability as a prognostic marker through the quantitative analysis of ctDNA and KRAS mutation in the patients with PDA. Total of 147 PDA patients were enrolled in the study. The concentration and fraction of KRAS mutation were measured by KRAS screening multiplex droplet digital PCR kit (Biorad, USA) in plasma. Median of ctDNA concentration, KRAS mutant concentration and fractional abundance were 425 ng/mL, 0.11 copies/uL and 0.35 %, respectively. KRAS mutant concentration and fractional abundance showed the association with poor survival in OS (P =0.007 and P < .001). When we analyzed the receiver operating characteristic (ROC) curve to determine whether KRAS mutation in ctDNA have additive benefits with well-known tumor markers CA19-9, combined with KRAS mutation concentration or KRAS fractional abundance, the value of area under the curve (AUC) was significantly higher than the value calculated as CA19-9 alone. This study represents that KRAS mutant concentration and fractional abundance in ctDNA could be prognostic marker in pancreatic cancer.

Abstract IA17: Physical and in silico methods for improving sensitivity of detection of ctDNA for personalized medicine in high-grade serous ovarian cancer

Abstract Early detection of high-grade serous ovarian cancer (HGSOC) remains a major challenge owing to early metastasis from the fallopian tube and lack of specific symptoms or clinical signs. Noninvasive analysis of circulating tumor DNA (ctDNA) for common mutations has the potential to provide highly specific tests for detecting ovarian cancer. We have previously shown that TP53 ctDNA is highly correlated with total volume of disease using 3D volumetric reconstruction of CT images. However, detection of ctDNA requires ctDNA concentration to be greater than 0.5% for adequate sensitivity. Improved methods for detection of ctDNA are also required for reliable response monitoring during treatment and detection of minimal residual disease after surgery and systemic therapy. I will discuss recent approaches in our lab that have investigated whether size selection of DNA fragments from plasma can increase the detection of ctDNA by enrichment away from cell free DNA of non-tumor origin. The majority of HGSOC ctDNA is in a size range of 90-150 bp based on whole-genome sequencing of cell free DNA from women with relapsed HGSOC and from HGSOC mouse xenografts. Using physical size selection in 35 patients with recurrent HGSOC and 16 additional patients with other cancer types yielded enrichment of mutated DNA fractions by a median of 4.3-fold allowing improved analysis by targeted, whole-exome, and whole-genome sequencing. This allowed identification of new mutations that otherwise were not observed. Size selection allows detection of tumor alterations masked by non-tumor DNA in plasma, and could improve the sensitivity of liquid biopsy for applications in early diagnosis, detection of minimal residual disease and for establishing mutational signatures from ctDNA. Citation Format: James D. Brenton. Physical and in silico methods for improving sensitivity of detection of ctDNA for personalized medicine in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA17.

Circulating tumor DNA detectable in early- and late-stage colorectal cancer patients.

Characterization, diagnosis, and treatment of colorectal cancers (CRC) is difficult due to limited biopsy information, impracticality of repeated biopsies, and cancer biomarker fallibility. Circulating tumor DNA (ctDNA) has recently been investigated as a non-invasive way to gain representative gene mutations in tumors, in addition to monitoring disease progression and response to treatment. We analyzed ctDNA mutations and concentrations in 47 early- and late-stage CRC patients using a targetted sequencing approach using a panel that covers 50 cancer-related genes. ctDNA mutations in 37 genes were identified in 93.6% of the patients (n=47). The results showed that TP53, PIK3CA, APC, and EGFR were the most frequently mutated genes. Stage IV patients had significantly higher ctDNA concentration than Stage I patients, and increased ctDNA concentration correlated with increased tumor size. Additionally, ctDNA detection was found to be a greater predictor of disease when compared with five known commonly used tumor biomarkers. The present study supports the use of ctDNA as a liquid biopsy to gain clinical tumor information that may facilitate early diagnosis and treatment and improve CRC patient prognosis.

A method for treatment monitoring using circulating tumour DNA in cancer patients without targetable mutations.

BackgroundThe potentials of circulating tumour DNA (ctDNA) have been studied for non-invasive disease monitoring in patients with targetable mutations. However, the majority of cancer patients harbour no targetable mutations. A workflow including targeted next-generation sequencing (NGS) and droplet digital PCR (ddPCR) could be used for monitoring treatment in these patients. Thus, our aim was to evaluate the workflow for ctDNA monitoring in a cohort of non-small cell lung cancer patients.MethodsForty patients were prospectively included. Plasma samples were collected prior to and during treatment. NGS (Ion AmpliSeq Colon and Lung Cancer panel v2) was performed on ctDNA from pre-treatment samples. The identified mutations were monitored by ddPCR in consecutively collected samples.ResultsMutations were detected in 21 patients. The most commonly mutated genes were TP53 (N=20) and KRAS (N=13). Treatment was discontinued due to non-response in 18 patients. In 16 of these, a simultaneous increase in ctDNA concentration was observed. A twofold ctDNA concentration increase confirmed in a second successive sample predicted non-response on the following imaging in 83% of patients (10/12).ConclusionctDNA monitoring can be used for early detection of non-response in patients without targetable mutations, and therefore could supplement imaging data for treatment monitoring in this subset of patients.

DNA-Induced Novel Optical Features of Ethyl Viologen-Tethered Perylenediimide Triad

Perylenediimide (PDI) chromophores with redox-active groups have facilitated the construction of several novel functional materials. For the first time, unusual spectroscopic changes and differential binding behavior of a tetracationic ethyl viologen tethered PDI derivative, PDEV, with calf-thymus DNA (ct-DNA) is reported here and is ascertained as due to the DNA compaction. From an initial aggregation on the DNA backbone, resulting in quenching in the emission intensity (turn off), with increase in ct-DNA concentration, a novel and strong fluorescence band (turn on) emerged in the blue region. Distinct absorption spectral changes having sharp features in vibronic patterns, increased fluorescence lifetime (from 0.33 to 4.5 ns), anisotropy values are also displayed on titration with ct-DNA corroborating the changes in microenvironment of PDEV in commensurate with structural transition from coil state to compact state. The structural changes in the DNA are also established from the circular dichroism spectra, viscosity measurements, and DNA melting data. The compaction is also directly visualized in the atomic force microscopy, scanning electron microscopy, and fluorescence microscopy images. Such compact DNA morphologies with redox-responsive donor–acceptor moieties are potential to create desired nanostructures with stimuli-responsive functionalities such as on–off switch, biosensor, drug delivery, and other optoelectronic devices.

Spectrophotometric study on binding of 2-thioxanthone acetic acid with ct-DNA

Thioxanthone and its derivatives are the most remarkable molecules due to their vast variety of application such as radiation curing that is, until using them as a therapeutic drug. Therefore, in this study it was intended to use 2-Thioxanthone acetic acid with and without NaCl in Tris HCl buffer solution (pH:7.0) to represent the interaction with ct-DNA. The UV–vis absorption spectra of TXCH2COOH in the presence of ct-DNA showed hypochromism and the intrinstic binding constant (Kb) was determined as 6 × 103 L mol−1. The fluoresence intensity of TXCH2COOH with ct-DNA clearly increased up to 101% which indicated that the fluorescence intensity was very sensitive to ct-DNA concentration. The binding constant (K) and the values of number of binding sites (n) and were calculated as 1.8 × 103 L mol−1 and 0.69, respectively. When the quenching constants (Ksv) of free TXCH2COOH and TXCH2COOH, which were bonded with ct-DNA were compared, slightly changed values of Ksv were seen. Moreover, displacement assay with Hoechst 33,258 and viscosity measurements in the presence and absence of NaCl salt also confirmed the binding mode which noted the electrostatic interaction following groove binding between TXCH2COOH and ct-DNA. Last but not least, the salt effect was examined on ct-DNA binding with TXCH2COOH. The results of the experiments indicated that the groove binding was strengthened by NaCl whereas in the high NaCl concentration, the binding ability of TXCH2COOH to ct-DNA was inversely affected.