Presence Of ctDNA Research Articles

Circulating tumor DNA as a predictor for response to treatment in BRAF V600E mutant malignant melanoma.

9564 Background: Available biomarkers LDH and S100B possess limited sensitivity and specificity to predict outcome in melanoma. In this pilot study we evaluated the use of circulating tumor (ct)DNA harboring BRAF and NRAS mutations as a predictive biomarker for treatment response and progression-free survival (PFS) in patients with locally advanced or metastatic melanoma. Methods: We analyzed 89 retrospective plasma samples from 32 unselected pts, and 158 samples from 12 pts included in a prospective trial (DRKS00009507). We included stage III disease with planned resection or stage IV disease before initiation or change of medical treatment. Blood samples were taken at baseline at d +8, d +28, and thereafter at 3 months intervals for up to two years. We developed a hydrolysis probe based, Locked Nucleic Acid assay to detect BRAF V600E and wild type ctDNA by droplet digital PCR. Results were correlated with LDH, S100B and PFS. Results: Sensitivity of BRAF V600E specific assay was 0.01% with a limit of Blank of 0.28 copies/well. Of 31 stage IV pts with retrospective samples, 23 were positive for BRAF V600E ctDNA at least once (74%). Positive pts had a mean of 9 (range: 1-17) and 483 (range: 0.1-16,388) BRAF V600E copies/mL for stage III and stage IV respectively. The presence of ctDNA at baseline predicted poor PFS (hazard ratio [HR] 1.487, 95% CI 0.34-6.45). A negative slope in BRAF V600E ctDNA was a favorable prognostic factor for PFS (hazard ratio [HR] 0.230, 95% CI 0.04-1.20) with a median PFS of 3.42 vs. 2.56 months (Range 1.87-8.9 vs. 0.89-5.02). Residual ctDNA at the first time point after initiation or change of treatment was related to a shorter PFS (hazard ratio [HR] 2.02, 95% CI 0.39-10.53). Based on 144 measurement pairs, BRAF ctDNA strongly correlated with S100 (r = 0.73) and LDH (r = 0.52). Conclusions: Residual ctDNA early after change or institution of treatment predicted tumor progression at first clinical response assessment. A positive to negative conversion or a decrease indicated a more favorable course. These data support the use of ctDNA as an early predictive marker for treatment response. We will examine whether two or more detected mutations indicate clonal heterogeneity and confer adverse prognosis. Clinical trial information: DRKS00009507.

Photodynamic effect of light-harvesting, long-lived triplet excited state Ruthenium(II)-polyimine-coumarin complexes: DNA binding, photocleavage and anticancer studies

Two coumarin based RuII-polyimine complexes (Ru-1 and Ru-2) showing intense absorption of visible light and long-lived triplet excited states (~12–15μs) were used for study of the interaction with DNA. The binding of the complexes with CT-DNA were studied by UV-vis, fluorescence and time-resolved nanosecond transient absorption (ns-TA) spectroscopy. The results suggesting that the complexes interact with CT-DNA by intercalation mode of binding, showing the binding constants (Kb) 6.47×104 for Ru-1 and 5.94×104 M−1 for Ru-2, in contrast no such results were found for Ru-0. The nanosecond transient absorption spectra of these systems in the presence of CT-DNA showing a clear perturbation in the bleaching region was observed compare to buffer alone. Visible light photoirradiation DNA cleavage was investigated for these complexes by treating with the supercoiled pUC19 DNA and irradiated at 450nm. The reactive species produced upon irradiation of current agents is singlet oxygen (1O2), which results in the generation of other reactive oxygen species (ROS). The complexes shown efficient cleavage activity, converted complete supercoiled DNA to nicked circular at as low as 20μM concentration in 30min of light irradiation time. Significant amount of linear form was generated by Ru-1 at the same conditions. Even though Ru-0 has significant 1O2 quantum yield but shown lower cleavage activity compared to other two analogs is due the miserable interaction (binding) with DNA. The cytotoxicity in vitro of the complexes toward HeLa, BEL-7402 and MG-63 cells was assessed by MTT assay. The cellular uptake was observed on BEL-7402 cells under fluorescence microscope. The complexes shown appreciable cytotoxicity towards the cancer cell lines.

Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer

Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencingbased assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8%) of 164 patients with negative ctDNA (hazard ratio, 18; 95% confidence interval, 7.9–40; P < 0.001). In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (hazard ratio, 11; 95% confidence interval, 1.8–68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.

Ultrasensitive colorimetric detection of circulating tumor DNA using hybridization chain reaction and the pivot of triplex DNA

This work presents an amplified colorimetric biosensor for circulating tumor DNA (ctDNA), which associates the hybridization chain reaction (HCR) amplification with G-Quadruplex DNAzymes activity through triplex DNA formation. In the presence of ctDNA, HCR occurs. The resulting HCR products are specially recognized by one sequence to include one GGG repeat and the other containing three GGG repeats, through the synergetic effect of triplex DNA and asymmetrically split G-Quadruplex forming. Such design takes advantage of the amplification property of HCR and the high peroxidase-like catalytic activity of asymmetrically split G-Quadruplex DNAzymes by means of triplex DNA formation, which produces color signals in the presence of ctDNA. Nevertheless, in the absence of ctDNA, no HCR happens. Thus, no triplex DNA and G-Quadruplex structure is formed, producing a negligible background. The colorimetric sensing platform is successfully applied in complex biological environments such as human blood plasma for ctDNA detection, with a detection limit corresponding to 0.1 pM. This study unambiguously uses triplex DNA forming as the pivot to integrate nucleic acid amplification and DNAzymes for producing a highly sensitive signal with low background.

Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients

Cell-free circulating tumor DNA (ctDNA) in plasma has been used as a potential noninvasive biomarker for various tumors. Our study was performed to evaluate the clinical implications of ctDNA detection in patients with metastatic pancreatic cancer. First, we attempted to prospectively screen a panel of 60 genes in cell-free DNA (cfDNA) from ten metastatic pancreatic cancer patients via exome sequencing. Second, droplet digital PCR (ddPCR) was used to identify potential mutations in a cohort of 188 patients with metastatic pancreatic cancer. Finally, to preliminary evaluate the potential role of ctDNA in monitoring tumor responses following chemotherapy, we detected the presence of ctDNA in serial plasma samples from 13 metastatic pancreatic cancer patients (Clinical trial: NCT02017015). The analysis revealed five somatic mutations at BRCA2, EGFR, KDR and ERBB2 gene loci. The frequencies of ctDNA mutation at BRCA2, KDR, EGFR, ERBB2 exon17 and ERBB2 exon27 were 11.7%, 13.8%, 13.3%, 13.3% and 6.4% respectively. Univariate and multivariate analyses identified the ERBB2 exon17 mutation (p = 0.035, HR = 1.61) as an independent factor associated with overall survival among metastatic pancreatic cancer patients. Furthermore, the rate of coincident detection of ctDNA and response to treatment as assessed by CT imaging was 76.9% (10 of 13 cases), and the presence of ctDNA provided the earliest measure of treatment in 6 of 10 patients (60%). ctDNA sequencing may have clinical value for determining metastatic pancreatic cancer treatment and monitoring the tumor response.

Abstract B24: The significance of postoperative follow-up of patients with advanced colorectal cancer using circulating tumor DNA: Selected case studies

Abstract Introduction: One of the most notable applications for circulating tumor DNA (ctDNA) detection in peripheral blood of patients with metastatic colorectal cancer (mCRC) is a long-term postoperative follow-up. Sometimes referred to as a “liquid (re)biopsy” it can be performed frequently at relatively short intervals (months or even weeks) with minimal invasivity. The presence of the disease and the actual extent of the tumor burden (tumor mass) within the patient body can be monitored. This is of particular importance especially when evaluating radicality of surgical tratment, monitoring the response to systemic anticancer therapy as well as for early detection of disease progression or recurrence. In this presentation we demonstrate clinical utility of ctDNA detection in a postoperative follow-up setting on several cases of mCRC patients. Patient cohort: A total of 91 patients undergoing resection of liver metastases were enrolled into the multicentre study involving three surgical centers. In addition to the baseline ctDNA testing a longitudinal observation was conducted for 16 patients by evaluating ctDNA presence (i) prior to surgery, (ii) a week after surgery and (iii) at subsequent intervals over the next several months after the surgery. The presence and the levels of ctDNA were correlated with radicality of surgical treatment and the actual clinical status of the patient. Five separate cases of patients who were followed for over a year by means of ctDNA monitoring are presented. Results: The R0 (curative) resection correlated with postoperative ctDNA negativity in 4 out of 5 cases of surgical procedures (80%). One of the patients with R0 surgeries, a radio-frequency ablation (RFA) of small liver metastases, continued displaying ctDNA and, subsequently, was diagnosed with the disease recurrence within the scar 3 months after the surgery. A post-surgical ctDNA positivity correlated with one case of R1 (residual tumor) resection. Four patients did show a postoperative relapse of the disease, which was documented in 3 of the cases by increasing levels of ctDNA even as the imaging examination result was negative. In one patient showing remission with no recurrence of tumor the ctDNA remained steadily negative during the entire follow-up period. Conclusion: CtDNA detection in patients with metastatic CRC is a viable tool for long-term follow-up and early detection of disease recurrence as well as in confirmation of the radicality of surgical treatment. The approach exhibits high specificity and at the minimal invasivity low burden to the patient. Supported by Ministry of Health of the Czech Republic, grant nr. 15-27939A. Citation Format: Lucie Benesova, Barbora Belsanova, Tereza Halkova, Pudil Jiri, Miroslav Ryska, Miroslav Levy, Jaromir Simsa, Filip Pazdirek, Jiri Hoch, Milan Blaha, Marek Minarik. The significance of postoperative follow-up of patients with advanced colorectal cancer using circulating tumor DNA: Selected case studies. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B24.

Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker.

Despite recent therapeutic advances, prognosis of patients with pancreatic adenocarcinoma remains poor. Analyses from tumor tissues present limitations; identification of informative marker from blood might be a promising alternative. The aim of this study was to assess the feasibility and the prognostic value of circulating tumor DNA (ctDNA) in pancreatic adenocarcinoma. From 2011 to 2015, blood samples were prospectively collected from all consecutive patients with pancreatic adenocarcinoma treated in our center. Identification of ctDNA was done with next-generation sequencing targeted on referenced mutations in pancreatic adenocarcinoma and with picoliter droplet digital PCR. A total of 135 patients with resectable (n = 31; 23%), locally advanced (n = 36; 27%), or metastatic (n = 68; 50%) pancreatic adenocarcinoma were included. In patients with advanced pancreatic adenocarcinoma (n = 104), 48% (n = 50) had ctDNA detectable with a median mutation allelic frequency (MAF) of 6.1%. The presence of ctDNA was strongly correlated with poor overall survival (OS; 6.5 vs. 19.0 months; P < 0.001) in univariate and multivariate analyses (HR = 1.96; P = 0.007). To evaluate the impact of ctDNA level, patients were grouped according to MAF tertiles: OS were 18.9, 7.8, and 4.9 months (P < 0.001). Among patients who had curative intent resection (n = 31), 6 had ctDNA detectable after surgery, with an MAF of 4.4%. The presence of ctDNA was associated with a shorter disease-free survival (4.6 vs.17.6 months; P = 0.03) and shorter OS (19.3 vs. 32.2 months; P = 0.027). ctDNA is an independent prognostic marker in advanced pancreatic adenocarcinoma. Furthermore, it arises as an indicator of shorter disease-free survival in resected patients when detected after surgery. Clin Cancer Res; 23(1); 116-23. ©2016 AACR.

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study.

BackgroundCirculating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive “real-time” biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC).Methods and FindingsWe recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01–3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30–3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66–6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations.ConclusionsIn this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.

Diagnostic and prognostic value of circulating tumor DNA in gastric cancer: a meta-analysis.

BackgroundCirculating tumor DNA (ctDNA) has offered a minimally invasive approach for detection and measurement of gastric cancer (GC). However, its diagnostic and prognostic value in gastric cancer still remains unclear.ResultsA total of 16 studies comprising 1193 GC patients met our inclusion criteria. The pooled sensitivity and specificity were 0.62 (95% confidence intervals (CI) 0.59−0.65) and 0.95 (95% CI 0.93–0.96), respectively. The AUSROC (area under SROC) curve was 0.94 (95% CI 0.89–0.98). The results showed that the presence of certain ctDNA markers was associated with larger tumor size (OR: 0.26, 95% CI 0.11–0.61, p = 0.002), TNM stage (I + II/III + IV, OR: 0.11, 95% CI 0.07−0.17, p = 0.000), as well as H. pylori infection. (H.p negative/H.p positive, OR: 0.57, 95% CI 0.36–0.91, p = 0.018). Moreover, there was also a significant association between the presence of ctDNA and worse overall survival (HR 1.77, 95% CI 1.38−2.28, p < 0.001), as well as disease-free survival (HR 4.36, 95% CI 3.08−6.16, p < 0.001).Materials and MethodsPubmed, Embase, Cochrane Library and Web of Science databases were searched for relating literature published up until November 30, 2016. Diagnostic accuracy variables were pooled by the Meta-Disc software. Engauge Digitizer and Stata software were applied for prognostic data extraction and analysis.ConclusionsOur meta-analysis indicates the detection of certain ctDNA targets is significantly associated with poor prognosis of GC patients, with high specificity and relatively moderate sensitivity.

Circulating tumor DNA detection in lung cancer patients before and after surgery.

Circulating tumor DNA (ctDNA) in peripheral blood is a “liquid biopsy” that contains representative tumor information including gene mutations. Additionally, repeated ctDNA samples can be easily obtained to monitor response to treatment and disease progression, which may be especially valuable to lung cancer patients with tumors that cannot be easily biopsied or removed. To investigate the changes in ctDNA after surgical tumor resection, tumor and blood samples obtained before and after surgery were collected prospectively from 41 non-small lung cancer (NSCLC) patients. Somatic driver mutations in tumor DNA (tDNA) and pre- and post-op plasma ctDNA sample pairs were identified by targeted sequencing in several genes including EGFR, KRAS, and TP53 with an overall study concordance of 78.1% and sensitivity and specificity of 69.2% and 93.3%, respectively. Importantly, the frequency of 91.7% of ctDNA mutations decreased after surgery and these changes were observed as little as 2 days post-op. Moreover, the presence of ctDNA had a higher positive predictive value than that of six tumor biomarkers in current clinical use. This study demonstrates the use of targeted sequencing to reliably identify ctDNA changes in response to treatment, indicating a potential utility of this approach in the clinical management of NSCLC.

Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care.

BackgroundIn women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control.MethodsA multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed.DiscussionThe FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT.Trial registrationClinicalTrials.gov Identifier: NCT02694497.

Abstract 483: Detection of circulating tumor DNA in oral rinses and plasma from head and neck cancer cases in Argentina

Abstract In 2012, almost 700,000 new head and neck cancer (HNSCC) cases were estimated to occur worldwide with two thirds of those cases occurring in developing countries. Within the geographic areas in Latin America, characterized by high incidence rates of head and neck cancer, the region comprising Argentina, southern Brazil and Uruguay has the highest levels of incidence. Despite current therapy approaches, the survival rate is around 50%, and the main reason for treatment failure is the frequent development of regional recurrences, affecting around 30% of patients. In this context the use of ctDNA (specific biomarker for tumors) using techniques of mass sequencing (high sensitivity and specificity) is presented as a promising tool for noninvasive diagnosis and monitoring of HNSCC cases. Our primary goal was to provide a comprehensive evaluation of the presence of ctDNA in both plasma and oral rinses from a series of HNSCC cases without previous knowledge of tumor mutational status. 43 cases diagnosed as head and neck squamous cell carcinoma were selected from a multi center case-control study conducted in Argentina between 1998 and 2002. Subsite and stage distribution was as follows: 14 oral cavity, 6 oropharynx, 1 hypopharynx, 15 larynx and 7 overlapping lesions. 6 cases were stage III and 17 stage IV. Additionally, 50 controls matched by center, age, sex, smoking and alcohol status were included to assess a threshold of the background noise after sequencing. Targeted sequencing was performed independently in duplicate on plasma, oral rinses, germline DNA and the tumor material using the Ion Proton™ System. Sequencing included the entire coding region of TP53 as the most frequently mutated gene in HNSCC. Both Plasma and oral rinses samples were sequenced at an average depth of 10000X. For somatic variant calling we used a recently developed new statistical model and a bioinformatics pipeline based on Poisson distribution for estimation of sequencing errors distribution to accurately identify variants above the sequencing background. Statistical tests were carried out in R statistical programming environment. 74% of tumor samples sequenced harboured at least one TP53 somatic mutation. 10 TP53 variants were identified in the corresponding plasma samples and 1 of these variants matched the tumor mutation, possibly reflecting high tumor heterogeneity. In contrast, 90% of the variants detected in the tumor were also found in in the oral rinses with allelic frequencies ranging from 0.002 to 0.05 showing higher specificity of oral rinses as a surrogate sample for mutation detection. The detection and analysis of ctDNA in noninvasive samples might be included as a potential biomarker to improve early diagnosis and patient monitoring in HNSCC cases. Citation Format: Sandra Perdomo, Patrice Avogbe, Marta Vilensky, James Mckay, Paul Brennan. Detection of circulating tumor DNA in oral rinses and plasma from head and neck cancer cases in Argentina. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 483.

Abstract 3139: Liquid biopsy (ctDNA) testing in clinical management of solid cancers: 5-years of experience

Abstract Background: Detection and profiling of circulating tumor DNA (ctDNA) is an attractive tool for management of cancer patients, in particular for early detection of the relapse after surgery or monitoring of response to systemic therapy. The main advantage is minimal invasivity and applicability to a wide range of solid cancers. Methodologies are based on digital PCR with the limit of detection (LOD) below 0.01% mutated alleles, however, these often require significant amounts of input DNA (10s to 100s of ng). In the present work we demonstrate routine detection and clinical utility of ctDNA in a cohort of 423 patients covering a range of 5 different solid tumors. Methods: ctDNA is detected by somatic mutations found in primary tumor tissue by applying a specific mutation panel targeted to the tumor tape. Detection was done by denaturing capillary electrophoresis (input DNA at concentrations of 5 pg, LOD 0.03 - 1%). The cohort included samples from 257 colorectal cancer patients (CRC), 97 patients with ductal adenocarcinoma of the pancreas (PDAC), 32 patients with non-small cell lung cancer (NSCLC), 12 patients with gastric adenocarcinoma (GA) and 6 patients with head and neck cancers (HNC). A longitudinal monitoring of ctDNA levels prior to surgery, a week after surgery and at three-month follow-up intervals, was performed in 16 CRC patients. Overall ctDNA detection rate, radicality of resection, disease recurrence, tumor dynamics and survival prognosis were evaluated. Results: ctDNA rates were at 32% for NSCLC, 31% for CRC, 30% for GA, 25% for PDAC and 25% for HNC. When looking at a subgroup of patients in Stage IV of the disease the rates increased to 53% for NSCLC, 77% for CRC, 50% for GA, 46% for PDAC and 50% for HNC. 14 out 16 CRC patients with R0 resection remained ctDNA negative (88%), two patients dropped out of the study. Follow-up monitoring lead to detection of progression in 9 out of 13 patients (69%). In 3 patients (23%), ctDNA positivity preceded standard detection by CT scan. In 5 patients with follow-up exceeding 1 year (5 to 10 sample acquisitions over 15 to 28 month period) the ctDNA levels correlated with the clinical course of the disease (progression/stabilization/remission). There was a borderline statistically significance for prognostic role of ctDNA presence in PDAC patients with 140 days vs. 200 days (P = 0,0519, long-rank test) for ctDNA positive and negative, respectively. Conclusion: We have introduced a ctDNA method to routine management of 5 different solid cancers. Our results indicate clinical utility for resection radicality confirmation as well as early detection of disease progression and tumor dynamics in CRC patients. The same is applicable to approx. 50% of patients with advanced GA, NSCLC and HNC. CtDNA positivity may also indicate a negative prognosis for PDAC patients. Supported by IGA MZ grant no. NT 13660. Citation Format: Lucie Benesova, Barbora Belsanova, Tereza Halkova, Jiri Pudil, Bohus Bunganic, Milos Pesek, Bretislav Gal, Miroslav Zavoral, Miroslav Ryska, Marek Minarik. Liquid biopsy (ctDNA) testing in clinical management of solid cancers: 5-years of experience. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3139.

Prognostic value of circulating tumour DNA in patients undergoing curative resection for pancreatic cancer.

Background:Pancreatic ductal adenocarcinoma (PDAC) is frequently diagnosed at an advanced stage, leading to a poor prognosis. Therefore, interest in the development of non-invasive biomarkers for prognostic prediction has grown rapidly. Here, we assessed the clinical implications of v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated circulating tumour DNA (ctDNA) as a useful surrogate biomarker in patients with resectable PDAC.Methods:We used droplet digital polymerase chain reaction to detect rare mutant tumour-derived KRAS genes in plasma cell-free DNA (cfDNA) as ctDNA. Samples were collected from 105 patients who underwent pancreatoduodenectomy for PDAC at a single institution. Overall survival (OS) was analysed according to the presence of ctDNA.Results:Among the 105 cases, ctDNA was detected in 33 (31%) plasma samples. The median OS durations were 13.6 months for patients with ctDNA (ctDNA+) and 27.6 months for patients without ctDNA. Patients who were ctDNA+ had a significantly poorer prognosis with respect to OS (P<0.0001).Conclusions:Our findings suggested that the presence of ctDNA in plasma samples could be an important and powerful predictor of poor survival in patients with PDAC. Accordingly, ctDNA detection might be a promising approach with respect to PDAC treatment.

Development and Validation of A Spectrofluorimetric Determination of Calf Thymus DNA Using a Terbium-Danofloxacin Probe

Background: Analysis of biomolecules is required in many biomedical research areas. A spectrofluorimetric method is proposed for determination of calf thymus DNA (ctDNA) based on the fluorescence enhancement of terbium-danofloxacin (Tb3+-Dano) in the presence of ctDNA. Methods: A probe with maximum excitation and emission wavelengths of 347 nm and 545 nm, respectively, was developed. The enhanced fluorescence intensity of Tb3+-Dano system was proportional to the concentration of ctDNA. The effective factors and the optimum conditions for the determination of ctDNA were studied. Under the optimum conditions of [Tris buffer]= 0.01 mol L-1 (pH 7.8), [ Tb3+]= 1×10-5 mol L-1 and [Dano]= 5×10-5 mol L-1, the maximum response was achieved. The developed method was evaluated in terms of accuracy, precision and limit of detection. Results: The linear concentration range for quantification of ctDNA was 36-3289 ng mL-1 and the detection limit (S/N=3) was 8 ng mL-1. The concentration of DNA extracted from Escherichia coli as an extracted sample was also determined using the developed probe. The concentration of DNA in extracted sample was determined using UV assay and developed method, the results were satisfactory. Conclusion: The proposed method is a simple, practical and relatively interference free method to follow up the concentrations of ctDNA.

MP03-09 CIRCULATING TUMOR DNA AS A BIOMARKER IN ADVANCED RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Advanced (including Drug Therapy) I1 Apr 2016MP03-09 CIRCULATING TUMOR DNA AS A BIOMARKER IN ADVANCED RENAL CELL CARCINOMA Mark Ball, Michael Gorin, Gunes Gunters, Phillip Pierorazio, George Netto, Channing Paller, Hans Hammers, Luis Diaz, and Mohamad Allaf Mark BallMark Ball More articles by this author , Michael GorinMichael Gorin More articles by this author , Gunes GuntersGunes Gunters More articles by this author , Phillip PierorazioPhillip Pierorazio More articles by this author , George NettoGeorge Netto More articles by this author , Channing PallerChanning Paller More articles by this author , Hans HammersHans Hammers More articles by this author , Luis DiazLuis Diaz More articles by this author , and Mohamad AllafMohamad Allaf More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1902AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There are currently no serum biomarkers for renal cell carcinoma (RCC). Circulating tumor DNA (ctDNA) has been shown to correlate with advanced disease and response to therapy in several solid malignancies; however little is known about the presence of ctDNA in patients with RCC. We sough to characterize the presence of ctDNA in patients with locally advanced and metastatic RCC. METHODS Pre-operative serum was collected from 4 patients with locally advanced (>T3b) (n=2) or metastatic (n=2) clear cell RCC. DNA from fresh-frozen paraffin-embedded (FFPE) nephrectomy specimens was isolated, and a next-generation sequencing assay to identify somatic mutations in 300 cancer-associated genes was used to determine tissue mutations. One high allele frequency mutation was selected for each sample. Primers for these mutations were constructed for digital PCR of matched serum specimens. Patients with positive pretreatment ctDNA were followed longitudinally to assess changes in ctDNA levels with treatment. RESULTS Next generation sequencing identified known mutations in all 4 tumors specimens. Candidate mutations selected were from the following genes: VHL, BAP1, PBRM1, NF. Serum PCR failed to detect mutant alleles in either locally advanced case, but ctDNA was detected in 1 metastatic patient with substitution mutation resulting in a splice site donor of VHL. In this patient, ctDNA burden decreased after nephrectomy and initiation of systemic therapy, but increased with disease progression (Figure 1). CONCLUSIONS In this pilot study, ctDNA was detected in 1 of 4 patients. In patients with detectable ctDNA at baseline, ctDNA dynamics may correlate with tumor burden. Future directions will work to optimize the detection of ctDNA in patients with metastatic RCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e22 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Mark Ball More articles by this author Michael Gorin More articles by this author Gunes Gunters More articles by this author Phillip Pierorazio More articles by this author George Netto More articles by this author Channing Paller More articles by this author Hans Hammers More articles by this author Luis Diaz More articles by this author Mohamad Allaf More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Circulating DNA and Survival in Solid Tumors.

The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes. A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan-Meier curves for OS at 1 and 3 years. Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02-3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20-7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59-2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93-4.13; P < 0.001, respectively). High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors. Circulating DNA is associated with worse outcome in solid tumors.

Hypermethylated circulating DNA detection using picoliter droplet-based PCR in colorectal cancer.

622 Background: Circulating tumor DNA (ctDNA) is thoroughly investigated as a surrogate biomarker of tumor follow-up, in different cancer types, such as colorectal cancer (CRC). Droplet-based digital PCR (ddPCR) is a highly sensitive and also quantitative method for detection of very low amount of ctDNA. Since many different genes can be mutated within a specific tumor type and also wide mutation spectrum can occur within a specific gene, procedures for ctDNA monitoring can be time consuming and need to be improved for a routinely use. To overcome these drawbacks, we characterized the methylation status of 3 genes frequently hypermethylated in CRC to identify universal markers for tumor follow-up. Methods: The characterization of the methylated status of the WIF, NPY and PENK genes in the tumor DNA was performed in 56 CRC of different stages and 45 corresponding plasma samples using droplet-based dPCR, after DNA bisulfite conversion. A two-panels assay (with albumin as a reference) was developed. Methylation level of these 3 genes in tumor tissues was compared to corresponding normal tissues (n = 22) and plasma samples (MetctDNA). To validate, plasma samples of additional 91 patients were analyzed for the presence of ctDNA both by the characterization of KRAS, BRAF, TP53 and PIK3CA mutations (MutctDNA) and of MetctDNA, at various stages of their follow-up, and 9 of them had MetctDNA assessment during treatment follow-up. Results: All tumor samples were positive for WIF1 and/or NPY markers. Hypermethylation of these two genes was significantly higher in tumor tissue compared to normal, independently of the tumor stage (p &lt; 0.0001). MetctDNA could be detected in 75% of metastatic CRC patients and 24% of localized CRC patients (stage 1 to 3). MetctDNA and MutctDNA fractions were strongly correlated (R2 &gt; 0.9, p &lt; 0.0001). During follow-up, MetctDNA levels changes allowed monitoring of tumor evolution in different stages CRC patients. Conclusions: These results indicate that determination of MetctDNA by droplet-based dPCR can reach same efficiency than MutctDNA for ctDNA assessment, using only 2 markers, and thus could be considered as a universal surrogate marker of tumor follow-up in CRC patients.

Cyclodextrin induced controlled delivery of a biological photosensitizer from a nanocarrier to DNA.

In this article, we have addressed to a demanding physicochemical aspect of therapeutic and drug research. We have reported a simple yet prospective technique that can be exploited for the controlled delivery of drugs and/or bioactive small molecules to the most relevant biomolecular target DNA. Exploiting various steady state and time resolved spectroscopic techniques together with the DNA helix melting study, we have shown that a biologically significant photosensitizer, namely, phenosafranin (PSF), can be quantitatively transferred to the DNA from the micellar nanocarrier made up of sodium tetradecyl sulfate (STS) using the external stimulant β-cyclodextrin (β-CD). The complexation property of β-CD with the nanocarrier (STS) has been utilized for the controlled release of the probe from the micelle to the DNA. Non-toxicity of the stimulant and the noninvasive nature of the carrier towards the target are expected to add to the suitability of this approach from a clinical perspective.

Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer

We used KRAS mutations to investigate the clinical relevance of circulating tumor DNA (ctDNA) measurements in patients with advanced pancreatic cancer. Fifty-three blood samples were collected from 14 prospectively recruited patients prior to chemotherapy (gemcitabine or FOLFIRINOX) and subsequently every month during treatment. Samples were processed by density centrifugation and plasma DNA isolation. A Peptide–nucleic acid–clamp PCR was then used to detect KRAS mutations (present in >90% of pancreatic cancers) as a surrogate marker for ctDNA. Plasma samples from 29 healthy individuals were analyzed as a reference group. Results were compared to conventional monitoring measures and survival data. Median follow-up time was 3.7 months (range 0.6–12.9 months).Ten (71%) patients had a positive KRAS status in the plasma samples obtained prior to chemotherapy, indicating the presence of ctDNA. Among the patients who were ctDNA-positive before chemotherapy, nine (90%) experienced disease progression during follow-up, compared to one (25%) of four ctDNA-negative patients (P = 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P = 0.014 and 0.010, respectively). Of the 14 patients, ten had ≥2 follow-up samples; in several of these patients, the ctDNA level changed substantially during the course of chemotherapy. Changes in ctDNA levels corresponded both with radiological follow-up data and CA19-9 levels for several patients.This pilot study supports the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. Recruitment of more patients is ongoing to corroborate these findings.