Presence Of ctDNA Research Articles

A sensitive fluorescent sensor based on the photoinduced electron transfer mechanism for cefixime and ctDNA.

Cefixime is a third generation orally administered cephalosporin that is frequently used as a broad spectrum antibiotic against various gram-negative and gram-positive bacteria. In this study, a simple and sensitive fluorescent sensor for the determination of the cefixime and ctDNA was established based on the CdTe:Zn2+ quantum dots (QDs). The fluorescence of CdTe:Zn2+ QDs can be effectively quenched by cefixime in virtue of the surface binding of cefixime on CdTe:Zn2+ QDs and the subsequent photoinduced electron transfer process from CdTe:Zn2+ QDs to cefixime, in particular, the high sensitivity of QDs fluorescence emission to cefixime at the micromole per liter level, which render the cefixime-CdTe:Zn2+ QDs system into fluorescence "OFF" status, then turn on in the presence of ctDNA. Furthermore, the Fourier transform infrared (FTIR) spectra of characteristic bands of C-N and N-H groups of cefixime endow evidence for the interaction of cefixime with CdTe:Zn2+ QDs. The relative electrochemical behavior of the affinity of CdTe:Zn2+ QDs for cefixime and ctDNA reveals the potential molecular binding mechanism.

Detection of postoperative plasma circulating tumour DNA and lack of CDX2 expression as markers of recurrence in patients with localised colon cancer

BackgroundColon cancer (CC) is a heterogeneous disease. Novel prognostic factors beyond pathological staging are required to accurately identify patients at higher risk of relapse. Integrating these new biological factors, such as plasma circulating tumour DNA (ctDNA), CDX2 staining, inflammation-associated cytokines and transcriptomic consensus molecular subtypes (CMS) classification, into a multimodal approach may improve our accuracy in determining risk of recurrence.MethodsOne hundred and fifty patients consecutively diagnosed with localised CC were prospectively enrolled in our study. ctDNA was tracked to detect minimal residual disease by droplet digital PCR. CDX2 expression was analysed by immunostaining. Plasma levels of cytokines potentially involved in disease progression were measured using ELISAs. A 96 custom gene panel for nCounter assay was used to classify CC into colorectal cancer assigner and CMS.ResultsMost patients were classified into CMS4 (37%) and CMS2 (28%), followed by CMS1 (20%) and CMS3 (15%) groups. CDX2-negative tumours were enriched in CMS1 and CMS4 subtypes. In univariable analysis, prognosis was influenced by primary tumour location, stage, vascular and perineural invasion together with high interleukin-6 plasma levels at baseline, tumours belonging to CMS 1 vs CMS2 +CMS3, ctDNA presence in plasma and CDX2 loss. However, only positive ctDNA in plasma samples (HR 13.64; p=0.002) and lack of CDX2 expression (HR 23.12; p=0.001) were found to be independent prognostic factors for disease-free survival in the multivariable model.ConclusionsctDNA detection after surgery and lack of CDX2 expression identified patients at very high risk of recurrence in localised CC.

Significance of postoperative follow-up of patients with metastatic colorectal cancer using circulating tumor DNA.

BACKGROUNDOne 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 is a minimally invasive procedure and can be performed repeatedly at relatively short intervals (months or even weeks). The presence of the disease and the actual extent of the tumor burden (tumor mass) within the patient’s body can be monitored. This is of particular importance, especially when evaluating radicality of surgical treatment as well as for early detection of disease progression or recurrence.AIMTo confirm the radicality of surgery using ctDNA and compare available methods for detection of recurrence in metastatic colorectal cancer.METHODSA total of 47 patients with detected ctDNA and indications for resection of mCRC were enrolled in the multicenter study involving three surgical centers. Standard postoperative follow-ups using imaging techniques and the determination of tumor markers were supplemented by ctDNA sampling. In addition to the baseline ctDNA testing prior to surgery, a postoperative observation was conducted by evaluating ctDNA presence up to a week after surgery and subsequently at approximately three-month intervals. The presence of ctDNA was correlated with radicality of surgical treatment and the actual clinical status of the patient.RESULTSAmong the monitored patients, the R0 (curative) resection correlated with postoperative ctDNA negativity in 26 out of 28 cases of surgical procedures (26/28, 93%). In the remaining cases of R0 surgeries that displayed ctDNA, both patients were diagnosed with a recurrence of the disease after 6 months. In 7 patients who underwent an R1 resection, 4 ctDNA positivities (4/7, 57%) were detected after surgery and associated with the confirmation of early disease recurrence (after 3 to 7 months). All 15 patients (15/15, 100%) undergoing R2 resection remained constantly ctDNA positive during the entire follow-up period. In 22 cases of recurrence, ctDNA positivity was detected 22 times (22/22, 100%) compared to 16 positives (16/22, 73%) by imaging methods and 15 cases (15/22, 68%) of elevated tumor markers.CONCLUSIONctDNA detection in patients with mCRC is a viable tool for early detection of disease recurrence as well as for confirmation of the radicality of surgical treatment.

Circulating tumour DNA (ctDNA) as a biomarker in metachronous melanoma and colorectal cancer- a case report

BackgroundCirculating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation.Case presentationWe present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient’s tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers.ConclusionsUsing cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types.

GENE-01. THE MUTATIONAL LANDSCAPE OF PRIMARY CHORDOMAS AND THEIR SENSITIVE DETECTION IN PLASMA ctDNA BY MULTIPLE NEXT GENERATION SEQUENCING TECHNOLOGIES

Abstract CNS-associated tumors are notoriously difficult to detect in plasma. Chordomas are the most common primary spinal column malignancy, and extensive surgical procedures, along with chemotherapy and radiation are required to reduce recurrence. Currently CT, MRI, and PET are used to monitor for recurrence but are limited by surgical sequalae. In addition, needle biopsy risks tumor seeding along the biopsy track. In the largest cohort of chordomas described thus far, we characterize the mutational landscape of these tumors and show that liquid biopsy is a sensitive method for detecting cancers. 34 patients with a biopsy-confirmed diagnosis of chordoma had blood drawn before surgery, at the time of surgery, and/or at follow up appointments. Mutations in the primary tumor were identified by whole exome sequencing (WES) and droplet digital PCR (ddPCR) and/or Rapid Amplification of cDNA Ends Sequencing (RACE-Seq) was used to detect one or more of these mutations in plasma ctDNA at concurrent or later time points. The primary endpoint was detection of mutations in ctDNA in biopsy-confirmed chordoma samples. 87.9% of patients were ctDNA positive at the time of initial blood draw (p < 0.001). Follow up blood draws in twenty of the patients demonstrated that ctDNA levels reflected the clinical status of the disease. Patients with positive ctDNA levels were more likely to undergo radiotherapy (p = 0.004), and the presence of ctDNA may predict response to systemic chemotherapy and/or disease recurrence. Given the significant sequelae of biopsy and spinal surgery, liquid biopsy may be the best tool for detection and monitoring of chordomas.

A Supramolecular Interaction of a Ruthenium Complex With Calf-Thymus DNA: A Ligand Binding Approach by NMR Spectroscopy.

Lawsone itself exhibits interesting biological activities, and its complexation with a metal center can improve the potency. In this context a cytotoxic Ru-complex, [Ru(law)(dppb)(bipy)] (law = lawsone, dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2′-bipyridine), named as CBLAU, was prepared as reported. In this work, NMR binding-target studies were performed to bring to light the most accessible interaction sites of this Ru-complex toward Calf-Thymus DNA (CT-DNA, used as a model), in a similar approach used for other metallic complexes with anti-cancer activity, such as cisplatin and carboplatin. Advanced and robust NMR binding-target studies, among them Saturation Transfer Difference (STD)-NMR and longitudinal relaxometry (T1), were explored. The 1H and 31P -NMR data indicate that the structure of Ru-complex remains preserved in the presence of CT-DNA, and some linewidth broadening is also observed for all the signals, pointing out some interaction. Looking at the binding efficiency, the T1 values are highly influenced by the formation of the CBLAU-DNA adduct, decreasing from 11.4 s (without DNA) to 1.4 s (with DNA), where the difference is bigger for the lawsone protons. Besides, the STD-NMR titration experiments revealed a stronger interaction (KD = 5.9 mM) for CBLAU-DNA in comparison to non-complexed lawsone-DNA (KD = 34.0 mM). The epitope map, obtained by STD-NMR, shows that aromatic protons from the complexed lawsone exhibits higher saturation transfer, in comparison to other Ru-ligands (DPPB and bipy), suggesting the supramolecular contact with CT-DNA takes place by the lawsone face of the Ru-complex, possibly by a spatial π-π stacking involving π-bonds on nucleic acids segments of the DNA chain and the naphthoquinone group.

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer.

A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse. One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR. NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r = 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P = 0.0014 and HR, 11.33; log-rank P = 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5 months. Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.

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.

Presence of mEGFR ctDNA predicts a poor clinical outcome in lung adenocarcinoma.

BackgroundCirculating tumor DNA (ctDNA) is a biomarker for the selection of target agents in various malignancies. In this study, we examined the effect of ctDNA presence on the response to EGFR‐tyrosine kinase inhibitor (TKI) and on the prognosis in lung adenocarcinoma.MethodsctDNA of EGFR‐TKI sensitizing mutations (mEGFR), L858R substitution and Exon 19 deletion (E19d) mutation, was evaluated using droplet digital PCR (ddPCR) in 81 patients with lung adenocarcinoma which harbored mEGFR in the corresponding tumor tissues.ResultsThe study recruited lung cancer patients at various stages, and the sensitivity, specificity, and area under the curve (AUC) of mEGFR ctDNA detection by ddPCR were 40.0%, 88.5%, and 0.68, respectively. It showed higher sensitivity (75.0% vs. 10.0%) and AUC (0.83 vs. 0.49) in the advanced stages of lung adenocarcinoma compared with the early stages and the number of metastases and the fractional abundance of mEGFR ctDNA showed a strong correlation (σ = 0.516; P < 0.001, Spearman correlation test). There was a significantly shorter progression‐free survival and duration of disease control by EGFR‐TKIs in the ctDNA‐positive group than the negative group (14.0 vs. 41.0 months, P = 0.02 and 12.0 vs. 23.0 months, P = 0.02, log‐rank test, respectively). There was a trend for overall survival time to be shorter in patients with mEGFR ctDNA than for patients without mEGFR ctDNA (35.6 vs. 67.1 months, P = 0.06, log‐rank test).ConclusionsThese data showed that mEGFR ctDNA detection using ddPCR is useful in the advanced stages and its presence predicted distant metastasis and poor clinical outcome in lung adenocarcinoma.

Plasma clearance of RAS mutation under therapeutic pressure is a rare event in metastatic colorectal cancer.

In metastatic colorectal cancer (mCRC), circulating tumor DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. Patients with a RAS mutated tumor included in the prospective PLACOL study were monitored for ctDNA. Analyses were based on optimized targeted next-generation sequencing and/or droplet-based digital polymerase chain reaction (ddPCR). For plasma samples without detectable mutations at progression disease, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. Among the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only two out of the eight patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only 2 samples among 36 were confirmed for clearance of RAS mutation in our series. In conclusion, this study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored.

110P - Presence of circulating tumour DNA in surgically resected renal cell carcinoma is associated with advanced disease and poor patient prognosis

BackgroundCirculating tumor DNA (ctDNA) is a promising, non-invasive biomarker for preclinical detection and monitoring of various cancers. The utility of ctDNA assessment in renal cell carcinoma (RCC) in not well established. Here, we evaluate the potential of a bespoke, multiplex PCR, whole exome sequencing (WES)-based approach for ctDNA detection. MethodsThe cohort consisted of 42 patients with stage Ib-IV RCC who underwent complete surgical resection. ctDNA was measured in plasma samples drawn pre-surgery (n=34; baseline) and at post-operative time points (n=41) using the bespoke assay targeting patient-specific tumor variants. ResultsA median of 11.7ng (1.4-175ng) of cfDNA was extracted from a median plasma volume of 3.2mL (1.2-3.8mL). ctDNA was detected with a mean mutant molecules/mL of 5.3 (0.22-62). Baseline ctDNA was detected in 41% (14/34) of patients. Presence of ctDNA was significantly associated with increased tumor size (mean 9.7 vs 7.1cm, p<0.05), advanced tumor stage (stage III-IV vs I-II, p<0.05) and poorly differentiated tumors (grade III-IV vs II, p<0.0001). In the postoperative setting, 8/8 ctDNA-positive patients relapsed (positive predictive value (PPV=100%), while 16/33 ctDNA-negative patients relapsed (NPV=52%). Positive ctDNA status was associated with reduced relapse-free survival at post-surgical timepoints (HR: 2.8; 95% CI:1.2-6.6). None of the post-surgical samples from a control cohort of 17 non-relapsing patients were ctDNA-positive (specificity of 100%; median follow up of 64 months). ConclusionsPresence of presurgical ctDNA strongly correlates with advanced stage RCC. Despite low plasma volumes, the bespoke assay detected ctDNA in 41% of baseline samples. Postoperative ctDNA presence is correlated with clinical relapse. However, absence of ctDNA does not preclude recurrence as RCC is known to shed limited amounts of ctDNA. Higher sample volumes and multiregion tumor biopsies could enhance detection rates. This personalized approach has the potential to be used for ctDNA-based detection of relapse in patients with advanced stage RCC. Legal entity responsible for the studyNatera, Inc.; Fox Chase Cancer Center. FundingNatera, Inc.; Fox Chase Cancer Center. DisclosureM. Balcioglu: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. H. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. S. Dashner: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. S. Shchegrova: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. E. Kalashnikova: Full / Part-time employment: Natera, Inc. H. Pawar: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. R.G. Uzzo: Advisory / Consultancy: Janssen; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Novartis; Advisory / Consultancy: Argos. A. Aleshin: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. H. Sethi: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. R. Salari: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. M. Louie: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. B. Zimmermann: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. P. Abbosh: Advisory / Consultancy, Advisory: Janssen; Advisory / Consultancy, Advisory: AstraZeneca. All other authors have declared no conflicts of interest.

623P - Plasma clearance of RAS mutation under therapeutic pressure is a rare event in metastatic colorectal cancer

BackgroundIn metastatic colorectal cancer (mCRC), circulating tumour DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. MethodsPatients with a RAS mutated tumour included in the prospective PLACOL study, were monitored for ctDNA. Analyzes were based on optimized targeted next generation sequencing and/or droplet-based digital PCR (ddPCR). For plasma samples without detectable mutations, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. ResultsAmong the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression, and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only 2 out of the 8 patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only two samples among 36 were confirmed for clearance of RAS mutation in our series. ConclusionsThis study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored. Legal entity responsible for the studyThe authors. FundingThe Ministère de l’Enseignement Supérieur et de la Recherche, the Université Paris-Descartes, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut National du Cancer (INCA, n° 2009-1-RT-03-US-1 and 2009-RT-03-UP5-1), the Association pour la recherche contre le cancer (ARC, no. SL220100601375), the Agence Nationale de la Recherche (ANR Nanobiotechnologies; no. ANR-10-NANO-0002-09), the SIRIC CARPEM, the ligue nationale contre le cancer (LNCC, Program “Equipe labelisée LIGUE”; no. EL2016.LNCC/VaT) and Advanced Merieux Research Grant and canceropole funding (no. 2011-1-LABEL-UP5-2). DisclosureH. Blons: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck; Advisory / Consultancy: Amgen; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Pfizer. V. Taly: Advisory / Consultancy: Servier; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Raindance Technologies. W. Shu-Fang: Research grant / Funding (institution): Servier. S. Pernot: Advisory / Consultancy: Amgen; Advisory / Consultancy: Sanofi. J. Taieb: Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Advisory / Consultancy, Speaker Bureau / Expert testimony: Celgene; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sirtex; Advisory / Consultancy, Speaker Bureau / Expert testimony: Shire; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Servier; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre. P. Laurent-Puig: Advisory / Consultancy: Amgen; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Roche; Advisory / Consultancy: Lilly. A. Zaanan: Advisory / Consultancy: Amgen; Advisory / Consultancy: Baxter; Advisory / Consultancy: Lilly; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: MSD; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Roche; Advisory / Consultancy: Servier. All other authors have declared no conflicts of interest.

166P - Analysis of circulating tumour DNA for early relapse detection in stage III colorectal cancer after adjuvant chemotherapy

BackgroundCirculating tumor DNA (ctDNA) has emerged as a promising biomarker for early prediction of relapse across different tumor types. In patients with colorectal cancer (CRC), multiple studies have analyzed ctDNA to monitor tumor burden using fixed gene panels and droplet digital PCR. Here, we use a highly sensitive and specific, bespoke, whole exome-based NGS approach (Signatera™) for ctDNA monitoring. MethodsA cohort of 33 patients with stage III CRC who underwent surgery and were treated with at least 4 months of adjuvant chemotherapy was analyzed. Mutational profiles derived from primary tumor tissue and germline DNA whole exome were used to design assays targeting tumor-specific somatic variants. The bespoke assays were used for ctDNA detection in plasma samples. Relapse-free survival (RFS) was calculated for patients stratified by ctDNA status. ResultsPlasma samples (n=44; average volume=1.8mL) from patients (N=33) were analysed for the presence of ctDNA. Of the five ctDNA-positive patients, clinical follow-up was available for three patients, all of whom relapsed (100%; 3/3); three of 27 ctDNA-negative patients (11%) also clinically relapsed. Molecular relapse through ctDNA analysis was detected up to 668 days ahead of radiological imaging with an average lead time of 305 days. The majority of relapses in ctDNA-positive patients (67%; 2/3) occurred within a year of follow-up, while no relapses were observed in ctDNA-negative patients during the one-year time frame. All plasma samples (n=34) from 24 non-relapsing patients were ctDNA negative, corresponding to a specificity of 100%. The presence of ctDNA was associated with a markedly reduced RFS compared to ctDNA-negative patients (HR: 5.6; 95% CI: 0.6-52.1; p<0.01). ConclusionsThe study results indicate that ctDNA status is associated with high relapse risk in patients with CRC and can serve as a predictor of patient outcome. Despite low plasma volumes (<5mL) and lack of longitudinal samples for analysis, ctDNA was detected in 50% of relapse cases. Legal entity responsible for the studyNatera, Inc. and NSABP Foundation. FundingNatera, Inc., Bayer, NSABP Foundation. DisclosureH. Sethi: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. T.J. George: Research grant / Funding (institution): Merck; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Pharmacyclics. S. Shchegrova: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. A.S. Tin: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. A. Olson: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. D. Renner: Full / Part-time employment: Natera, Inc. E. Kalashnikova: Full / Part-time employment: Natera, Inc. M. Louie: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. R. Salari: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. B. Zimmermann: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. A. Aleshin: Shareholder / Stockholder / Stock options, Full / Part-time employment, I am an employee of Natera and own stock/options to stock.: Natera, Inc. All other authors have declared no conflicts of interest.

CtDNA As a Potential Prognostic Marker for Locally Advanced Rectal Cancer Patients Receiving Neoadjuvant Chemo-Radiation Therapy on Disease-Free Survival (DFS)

Neoadjuvant chemo-radiation therapy (nCRT) plus surgery is the standard treatment for patients with locally advanced rectal cancer (LARC). However, how to predict and select patients who may achieve clinical complete response is still an unsolved issue. We conducted a pilot study to evaluate the potential role of ctDNA as a biomarker to predict treatment outcome. We found that ctDNA clearance was related to TRG level in previous studies. Now we try to explore using ctDNA as a prognostic factor and improve risk stratification in locally advanced rectal cancer (LARC). In this study, we recruited 119 patients with LARC receiving nCRT. 595 serial plasma samples were collected at d0, d15, d25 of radiotherapy as well before and 7 days post-surgery. The level of ctDNA was calculated by dynamic monitoring the mutant allele frequency of somatic mutations in plasma. Plasma and tissue samples were subjected to targeted-NGS using a 422 cancer-related genes panel. We followed up patients with concomitant CT until disease progression or death. Serial samples’ data analysis showed detection of pre-treatment mutations after completion of nCRT was significantly associated with worse disease-free survival (DFS) (P<0.05). Patients with ctDNA mutations clearance during nCRT significantly have a better DFS compare with patients with ctDNA non-clearance (P=0.02). The presence of TP53 mutations is predictive of treatment outcome and shows a statistically significant worse DFS (2 years) (54.3% VS 87.0%, P=0.0005). Predictive model based on support vector machine was developed for prediction of pCR achieving a mean AUC of 0.85 assessed by repeated cross validation. Through tracking clonal extinction, persistence and emergence, patients were grouped into four evolutionary subtypes with distinct TRG and DFS. Our data showed the prognostic value of ctDNA on DFS. In patients treated with nCRT, the presence of ctDNA after completion of nCRT was associated with an inferior DFS (P=0.02). Dynamic monitoring of ctDNA can be used to predict TRG and prognosis in LARC patients receiving nCRT. ctDNA sequencing depicts the evolutionary trajectories of sensitive and resistant clones during nCRT in LARC. CtDNA could potentially be used to guide patient selection for nCRT.

Effect of gemini surfactants on binding interactions of Coumarin 485 with calf thymus deoxyribonucleic acid in presence of nanotubes of β-cyclodextrin

Present study is focused on the binding of a dye, Coumarin 485 (C-485) with Calf thymus deoxyribonucleic acid (ctDNA), explored using the spectroscopic tools such as UV–visible absorption, steady-state fluorescence and fluorescence anisotropy, and time-resolved fluorescence spectroscopy and dynamic light scattering measurements. The hyperchromic shifts in UV–visible absorption and fluorescence bands depict that the C-485 binds with the ctDNA. Absorption spectrum of C-485 in presence of ctDNA, ethidium bromide displacement assay and iodide ion quenching experiment confirm that C-485 binds with ctDNA through the groove binding mode. β-Cyclodextrin (β-CD) molecules (host) in presence of C-485 (guest) form the nanotubes and secondary aggregates of nanotubes which has been explored as the carriers of C-485 for ctDNA. The binding efficiency of C-485 with β-CD is reduced in presence of ctDNA. Gemini surfactants, m-4-m (m = 12, 14 and 16) induce the release of C-485 from the cavities of nanotubes of β-CD. After getting released from the cavities, C-485 molecules bind with ctDNA in the form of inclusion complexes with β-CD/un-complexed state at a low concentration range of a gemini surfactant. A gemini surfactant with comparatively longer hydrocarbon tails pushes the guest molecules (C-485) out of the nanotubular cavities of β-CD more efficiently due to greater extent of hydrophobic interactions between the cavities and tails.

Monitoring circulating tumor DNA by analyzing personalized cancer-specific rearrangements to detect recurrence in gastric cancer

Circulating tumor DNA (ctDNA) has emerged as a candidate biomarker for cancer screening. However, studies on the usefulness of ctDNA for postoperative recurrence monitoring are limited. The present study monitored ctDNA in postoperative blood by employing cancer-specific rearrangements. Personalized cancer-specific rearrangements in 25 gastric cancers were analyzed by whole-genome sequencing (WGS) and were employed for ctDNA monitoring with blood up to 12 months after surgery. Personalized cancer-specific rearrangements were identified in 19 samples. The median lead time, which is the median duration between a positive ctDNA detection and recurrence, was 4.05 months. The presence of postoperative ctDNA prior to clinical recurrence was significantly correlated with cancer recurrence within 12 months of surgery (P = 0.029); in contrast, no correlation was found between cancer recurrence and the presence of preoperative ctDNA, suggesting the clinical usefulness of postoperative ctDNA monitoring for cancer recurrence in gastric cancer patients. However, the clinical application of ctDNA can be limited by the presence of ctDNA non-shedders (42.1%, 8/19) and by inconsistent postoperative ctDNA positivity.

Abstract 2281: The role of baseline and early dynamics of ctDNA in predicting response and prognosis of early and advanced gastroesophageal adenocarcinomas

Abstract Background: Circulating tumor DNA (ctDNA) is emerging as a valuable less-invasive adjunct to tissue biopsy for real time monitoring and personalization of cancer treatment. This study aimed to determine the prognostic value of baseline ctDNA in both early and advanced gastroesophageal adenocarcinoma (GEA) and establish whether dynamic changes in ctDNA provides useful response and prognosis information. Patients and Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue DNA and serial plasma cell-free DNA (cfDNA) were obtained from 36 patients (23 early stage (63.9%), 13 advanced stage (36.1%)) undergoing treatment for GEA. Tumor DNA was analyzed by targeted next generation sequencing (NGS) (custom ampliseq six gene panel) and Nanostring™ nCounter® technology (87 gene panel). In each patient, selected mutations and gene amplifications were profiled in serial cfDNA samples using a combination of NGS, droplet digital PCR and real-time quantitative PCR. Results: Mutations and/or gene amplifications were identified in tumor DNA of 33/36 patients (91.7%). Patient specific profiling detected ctDNA in 19/33 patients (57.6%) at baseline: 9/22 with early stage disease (40.9%) and 10/11 with advanced stage disease (90.9%). Objective Response Rate (ORR) by RECIST 1.1 criteria was 71.4% (10/14) for patients who were ctDNA negative at baseline (group A) and 52.9% (9/17) for patients with detectable ctDNA at baseline (group B). Multivariate Cox regression analysis, adjusted for stage of disease and patient performance status, showed presence of ctDNA at baseline was associated with both reduced progression free survival (PFS) and overall survival (OS) [hazard ratio (HR) of 6.2 (95% CI 1.9-19.8, P = 0.002) and 7.3 (CI 1.9-28.2, P = 0.004), respectively]. The median PFS was 34.7 months and 12.1 months and median OS was not reached and 14.5 months for group A and B, respectively (Mantel-Cox Log Rank P = 0.008 and P &amp;lt; 0.001, respectively). ctDNA was detected before relapse in 3/22 (13.6%) early stage patients and before progression in 5/11 (45.5%) advanced stage patients, with an overall median lead-time of 6.3 weeks before radiological evidence of relapse or progression. Conclusion: This pilot study suggests that presence of ctDNA at baseline and recurrence during treatment are poor prognostic biomarkers both in early and advanced GEA. Citation Format: Ali Abdulnabi Suwaidan, Mark R. Openshaw, Barbara Ottolini, David Guttery, Daniel Fernandez Garcia, Cathy J. Richards, Jacqui A. Shaw, Anne L. Thomas. The role of baseline and early dynamics of ctDNA in predicting response and prognosis of early and advanced gastroesophageal adenocarcinomas [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 2281.

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.

Mutant KRAS and TP53 with high mutation allelic frequency in ctDNA as poor outcome predictors in metastatic pancreatic cancer.

e15763 Background: This study was to investigate the feasibility and the prognostic value of circulating tumor DNA (ctDNA) in metastatic pancreatic cancer (MPC). Methods: From 2015 to 2018 in our center, 40 MPC patients treated with nab-paclitaxel based first-line chemotherapy were prospectively collected both tumor tissue and blood samples, in which the genomic profiling of 425 genes was identified by next-generation sequencing. High mutation allelic frequency (MAF) was defined &gt; 30% and &gt; 5% in tumor tissue and blood, respectively. Kappa statistics were used to compare mutant (mt) genes in tissue and ctDNA. Progression-free and overall survival (PFS, OS) were assessed with Kaplan-Meier and Cox methods. Results: Among 40 MPC patients, tumor tissue and blood samples were available in 34 and 38 patients for somatic and germline alternation test, respectively. The most commonly mutant gene were KRAS (31/34 in tissue with a median MAF of 29.4%, 29/38 in ctDNA with a mMAF of 8.2%), and TP53 (28/34 in tissue with a mMAF of 31.1%, 25/38 in ctDNA with a mMAF of 7.4%). Moderate agreement was seen between ctDNA and tumor tissue (mt KRAS: κ = 0.54, P = 0.001; mt TP53: κ = 0.74, P &lt; 0.001). Mutation in CDNK2A and SMAD4 genes were detected in 8 and 6 patients in tissue and ctDNA, respectively. Germline alternation was found in 7 genes in 9 patients (9/40). High MAF of mt KRAS (r = 0.51, P = 0.005) or mt TP53 (r = 0.50, P = 0.005) in ctDNA was correlated with high CA199 levels (&gt; 5000 u/ml) at baseline. MT KRAS in tissue with high MAF was associated with poor OS (high 7.5m vs low 10.1m, P = 0.001) in univariate and multivariate analyses (HR 3.87, 95%CI 1.47 to 10.19). Univariate analyses showed mt KRAS and mt TP53 in ctDNA with high MAF were associated with poor PFS ( KRAS and TP53: high 3.4m and 3.0m vs low 6.1m and 5.7m, P = 0.001 and P = 0.004, respectively) and OS ( KRAS and TP53: high 5.3m and 5.3m vs low 12.6m and 10.1m, P &lt; 0.001, respectively). The presence of ctDNA in any above four mt driver gene with high MAF was associated with poor PFS (HR 3.79, 95%CI 1.71 to 8.42) and OS (HR 7.21, 95%CI 2.69 to 19.34) in multivariate COX model, when adjusted by age, sex, tumor differentiation, and baseline CA199 level. Conclusions: The presence of mt KRAS and mt TP53 with high MAF in ctDNA was associated with worse PFS and OS in MPC patients. Peripheral ctDNA testing demonstrated an alternative promising prognostic biomarker for MPC patients before treatment.

Neoadjuvant nivolumab in resectable non-small cell lung cancer: Extended follow-up and molecular markers of response.

8524 Background: Improved therapy is needed for patients (pts) with early-stage non-small cell lung cancer (NSCLC), as the majority relapse after curative resection. Our group reported the first trial of neoadjuvant PD-1 blockade in resectable NSCLC, finding therapy to be safe and feasible. Here we report extended clinical follow-up and long-term molecular response data from this trial. Methods: IV nivolumab 3 mg/kg was given every 2 weeks for 2 doses prior to surgery in 20 pts with resectable NSCLC at Johns Hopkins and MSKCC. Blood for correlative studies was taken prior to each dose of nivolumab, prior to surgery, 2-4 weeks post-surgery, and during long-term follow up. In a subgroup of pts, longitudinal molecular data was assessed in peripheral blood for circulating tumor DNA (ctDNA) and dynamics of tumor-infiltrating T-cell clonotypes. Results: At median follow up of 30 months (m), 15 of 20 pts are disease-free and alive. Two pts have died (one from relapsed disease). Median recurrence free survival (RFS) has not been reached. The 24m RFS rate is 69% (95% CI: 51-93). Thus far, presence of ctDNA at diagnosis and major pathologic response (MPR - ≤10% viable tumor in resected specimen) do not associate with RFS. One long-term immune-related adverse event has occurred (skin, G3). All pts who on pathologic review had ≥30% reduction in viable tumor in response to nivolumab demonstrated clearance of detectable ctDNA from blood prior to surgery. Pts with MPR experienced expansion of neoantigen-specific T-cells in peripheral blood. In one patient with ongoing disease free status, expansion of tumor-associated T-cells has persisted in peripheral blood beyond 15m from surgery. By contrast, in a patient who had detectable peri-operative ctDNA and 75% residual disease at surgery, minimal T-cell expansion was observed in peripheral blood, with a decreasing frequency of expanded T-cell clones over time that correlated with eventual cancer relapse. Conclusions: Long-term follow up reinforces the safety of neoadjuvant nivolumab in resectable NSCLC. Analysis of ctDNA and peripheral T-cell expansion in responders compared with non-responders suggests potential biomarkers for response and surveillance. While RFS data is encouraging, phase 3 trials are ongoing to evaluate efficacy of PD-(L)1 blockade in early-stage NSCLC. Clinical trial information: NCT02259621.