Changes In ctDNA Research Articles

Abstract 2522: Exploring the prognostic and predictive value of circulating tumor DNA in patients with advanced colorectal cancer and chemotherapy

Abstract Background: Chemotherapy improves the survival of advanced colorectal cancer (CRC) patients but recurrences do happen frequently. Early indicators of reccurence/relapse post-chemotherapy are essential for optimal clinical decision. Circulating tumor DNA (ctDNA) are cancer-specific DNA fragments in plasma. They have been considered as predictive biomarkers of early stage CRC. However, the predictive value of ctDNA in advanced CRC is still undetermined. Here, we evaluate the applicability of early changes in ctDNA as a marker that might predict clinical course in advanced CRC patients after chemotherapy. Methods: 122 patients with CRC were enrolled and their ctDNA fingerprints, designed based on individual patient's top somatic mutated genes, were determined and monitored over time (~ 3-6 months interval). The patients were classified into high- and low-ctDNA groups divided by the median concentration of ctDNA. We subsequently analyzed the correlation between overall survival (OS) and ctDNA levels in patients with or without chemotherapy. The dynamics of ctDNA were used to assess the prognosis and OS. Results: In the 122 patients, higher ctDNA levels (above median) were associated with shorter OS (HR, 2.89; CI 95% 1.45-5.79, P=0.0027). The conclusion upheld in a subset of 48 who had advanced CRC (stage IV) and received chemotherapy (HR, 1.601, CI 95% 0.61-4.17, p=0.037). Moreover, changes of ctDNA were detected early during the chemotherapy treatment course. In the same 48 patients with advanced CRC and chemotherapy, those who had undetectable and decreased ctDNA had longer OS overall, suggesting that ctDNA dynamics is predictive of clinic change in chemotherapy in advanced CRC. Finally, compared with clinical imaging diagnosis, we found that approximately 52.9% (18/34) patients belonged to a “consistent” group, in which patients with decreased/undetectable ctDNA had relieved clinical diagnosis and patients with increased ctDNA had elevated diagnosis. However, 14.7% (5/34) patients had inconsistent results between imaging diagnosis and ctDNA monitoring, and 32.4% (11/34) patients were uncertain due to unstable ctDNA levels. Conclusion: This study confirms the association between baseline levels of ctDNA and survival times of CRC patients. Additionally, it also suggests the use of early change of ctDNA as a potential biomarker of therapeutic efficacy in CRC chemotherapy. Oncologists may be able to use ctDNA fingerprints to guide treatment decisions and predict individual patient's prognosis. Citation Format: Xiu Liu, Xuan Zhang, Kai Ou, Chelong Lu, Dong Zheng, Jun Liu, Qiang Xu, Lin Yang. Exploring the prognostic and predictive value of circulating tumor DNA in patients with advanced colorectal cancer and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2522.

Abstract 28: Prognostic value of baseline and early changes of circulating cell-free (cf)DNA in the neoadjuvant setting of early stage colon cancer (CC)

Abstract Introduction Circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III CC treated with surgery +/- adjuvant chemotherapy. No study, however, has ever analysed the prognostic value of this biomarker in CC patients (pts) treated with neoadjuvant chemotherapy. We sought to evaluate the prognostic value of baseline and early, on-treatment changes of cfDNA and ctDNA in stage II-III CC pts who were treated with one cycle of neoadjuvant FOLFOX chemotherapy followed by surgery +/- adjuvant FOLFOX chemotherapy in the PePiTA trial. Methods PePITA was a multicentre, single-arm, prospective phase II trial aiming to test in vivo tumour chemosensitivity as assessd by metabolic response using 18F-FDG PET/CT scan of early stage CC and to evaluate its association with survival outcome (NCT00994864). Plasma samples were prospectively collected at baseline and 2 weeks (ie, after one cycle of neoadjuvant FOLFOX chemotherapy). cfDNA was isolated with the QIAmp circulating nucleic acid kit (Qiagen), and quantified with the Qubit fluorometer (Life-Technologies). cfDNA samples were bisulfite converted using the EZ DNA Methylation-Gold™ Kit (Zymo Research), with NPY and WIF1 being selected as universal methylation markers for ctDNA and analysed with digital droplet (dd)PCR technology. Data from ddPCR were processed with the QuantaSoft V1.6 software (BioRad). The primary outcome measure was 3-year disease-free survival (DFS). Receiver operating characteristics curve analyses, Kaplan-Meier method, cox proportional hazards models and log-rank tests were used. Statistical analyses were carried out with the SPSS for MacOS version 25.0 (SPSS Inc). Results 80 pts were included (ypStage I-II 56%, ypStage III 44%). After a median follow-up of 52.5 months, 3-year DFS was 80% (95%CI 71.2-90.8) and 5-year OS 84% (95%CI 75.2-94.9). Pts with high (≥1.2 ng/µl) baseline cfDNA level had worse 3-year DFS (48% vs 80%; HR 2.72, 95%CI 1.02-7.25; p=0.036) and 5-year OS (71% vs 90%; HR 5.36, 95%CI 1.14-25.28; p=0.017) than those with low baseline cfDNA level. In a multivariable analysis (including sex, ypStage and CEA), baseline cfDNA was the only factor showing a trend towards statistical significance (HR DFS 2.6, 95%CI 0.96-7.01; p=0.059; HR OS 4.65, 95%CI 0.97-22.32; p=0.055). Early changes of cfDNA (Δ ≥11%) after one cycle of neoadjuvant FOLFOX chemotherapy failed to predict survival (HR DFS 1.08, 95%CI 0.42-2.81; p=0.873; HR OS 0.68, 95%CI 0.19-2.39; p=0.543). ctDNA analyses are ongoing and will be presented at the meeting. Conclusions For the first time, we have shown that baseline cfDNA may predict survival outcome in early stage CC pts treated with neoadjuvant chemotherapy. Pending confirmation in larger series, testing for cfDNA at baseline could help select high-risk pts who may benefit from neoadjuvant, FOXTROT-like, treatment strategies. Citation Format: Giacomo Bregni, Elena Trevisi, Chiara Senti, Andrea Pretta, Caroline Vandeputte, Pashalina Kehagias, Elena Acedo Reina, Amélie Deleporte, Paraskevas Gkolfakis, Jean-Luc Van Laethem, Philippe Vergauwe, Marc Van den Eynde, Guido Deboever, Jos Janssens, Gauthier Demolin, Stephane Holbrechts, Marylene Clausse, Thierry De Grez, Marc Peeters, Lionel D'Hondt, Karen Geboes, Tatiana Besse-Hammer, Alexis Buggenhout, Françoise Rothé, Patrick Flamen, Alain Hendlisz, Francesco Sclafani. Prognostic value of baseline and early changes of circulating cell-free (cf)DNA in the neoadjuvant setting of early stage colon cancer (CC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 28.

Dynamics of circulating tumor DNA in patients with advanced solid tumors treated with cediranib and olaparib.

3035 Background: Circulating tumor DNA (ctDNA) has emerged as a potential biomarker to monitor treatment response in solid tumors. Our group previously showed that changes in ctDNA levels were predictive of radiographic response and survival in NSCLC patients receiving immunotherapy. Here we evaluated whether ctDNA dynamics could similarly be used to assess response in a PARP inhibitor-based therapy. Methods: A total of 122 patients with NSCLC, TNBC, PDAC or SCLC received cediranib (C) 30mg daily and Olaparib (O) 200mg twice daily in a phase II study NCI9881. Using a multigene NGS assay, ctDNA was measured longitudinally at baseline (T0), after 3 to 7 days of C monotherapy (T1), after 1 week of C+O combination (T2), after 4 weeks of C+O (T3), and every 4 weeks (T4+) thereafter. The first radiographic assessment was done after 8 weeks of C+O and every 8-12 weeks thereafter. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma. We defined an early ctDNA response (e-ctDNA-R) as a >10% decrease in mutant allele fraction from T0 to T2, and an early ctDNA progression (e-ctDNA-P) as a > 10% increase; otherwise, it was stable ctDNA (e-ctDNA-S). Results: In total, 493 samples were analyzed from 94 patients, and 40 unique patients had both T0 and T2 ctDNA measurements, as well as corresponding radiographic assessments. These included 10 NSCLC, 17 TNBC, 3 SCLC and 10 PDAC. Of these patients, 4, 21, and 15 patients had PR, SD, and PD as best overall radiographic response respectively. Twenty-three (57.5%) patients had either e-ctDNA-R (17, 42.5%) or e-ctDNA-S (6, 15.0%), 18 (78.3%) of whom subsequently had either radiographic partial response (PR) or stable disease (SD). Seventeen (42.5%) patients had e-ctDNA-P, 10 (58.8%) of whom then had PD. A fair agreement was observed between e-ctDNA-R/S or e-ctDNA-P and radiographic PR/SD or PD with Cohen’s k 0.38 (70% agreement). The correlation between early ctDNA changes and PFS/OS are summarized in the table. All 25 patients with PR/SD eventually progressed. Of these, 17 (68%) had >10% increase in ctDNA from the nadir prior to disease progression (median 94.6%, 95%CI 38.2%-389.1%). The time between ctDNA progression and ctDNA nadir was significantly shorter (median 21 days, 95%CI 21-28) than the time between radiographic/clinical progression and initial PR/SD (median 107 days, 95%CI 56-204, P=0.0015). Conclusions: Longitudinal ctDNA measurements could enable early assessment of treatment response, resistance, and disease progression in patients treated with PARP inhibitor-based therapy. However, in this study, tumor responses and ctDNA changes were generally not as robust as have been observed with other classes of therapy.[Table: see text]

Impact of circulating tumor DNA (ctDNA) detection on survival outcomes of patients (pts) treated with immune-checkpoint inhibitors (ICIs) in early clinical trials.

2542 Background: Detection of ctDNA is a promising tool for managing pts in oncology. Most methods require whole-genome sequencing of tumor samples followed by the design of personalized panels for tracking purposes. In this work, we evaluated the prognostic and predictive value of total ctDNA quantification, using shallow whole-genome sequencing (shWGS) exclusively from plasma samples, in a prospective cohort of pts treated with ICIs in early clinical trials. Methods: IchorCNA pipeline was used to quantify ctDNA of shWGS from plasma ctDNA samples of pts treated with ICIs in phase 1 trials, collected at baseline and prior to cycle 2 (prec2). We investigated the association and correlation of ctDNA levels with surrogate markers for tumor burden (LDH levels, summatory of target lesions (TL), liver metastasis) using Spearman and Kruskal-Wallis tests. Kaplan-Meier estimates of overall survival (OS) of pts with baseline detectable ctDNA levels versus undetectable ctDNA were calculated. A multivariate Cox proportional hazards model, including continuous classical prognostic factors (LDH, albumin, hemoglobin, derived Neutrophil-to-Lymphocyte ratio (dNLR), platelets, number of metastases sites, ECOG PS) and ctDNA was performed. An estimate of progression free survival (PFS) of pts with ctDNA increase levels in preC2 versus a non-increase group was evaluated. Results: Since January 2018, 113 pts with no standard-treatment options were included. Median (m) follow up was 14.8 months (mo). Baseline ctDNA levels correlated significantly with baseline TL (R = 0.4, p < 0.001) and LDH levels (R = 0.61, p < 0.001). Pts with liver metastasis had higher levels of ctDNA (11,68 ng/ml) versus pts with no liver disease (2,31 ng/ml) (p < 0,001). In the survival analysis pts with detectable baseline ctDNA (74 pts) had significantly shorter OS compared with pts with undetectable ctDNA (39 pts); median 9.6 m (8.4 – 16.4) and NA m (13.6-NA), respectively (HR = 2.25 [1.18-4.29] p < 0.01). In the multivariate analysis, only ctDNA and albumin levels maintained the impact in OS (HR = 1.03, p < 0.05 and HR = 0.22, p < 0.05, respectively). Pts with early increases in ctDNA had a shorter PFS compared with those with a stabilization or decrease, median 1.9 m (1.6 – 4.0) and 3.0 m (2.6 – 3.8), respectively (HR = 2.19 [1.31-3.67], p < 0.01). Conclusions: Quantification of baseline ctDNA using shWGS is a strong independent prognostic factor. Early dynamic changes of ctDNA could be a useful tool to predict PFS outcomes.

Longitudinal Relationship between Idylla Plasma ctBRAF V600 Mutation Detection and Tumor Burden in Patients with Metastatic Melanoma.

Circulating tumor DNA (ctDNA) may complement radiography for interim assessment of patients with cancer. Our objective was to explore the relationship between changes in plasma ctDNA versus radiographic imaging among patients with metastatic melanoma. Using the Idylla system, we measured B-Raf proto-oncogene (BRAF) V600 ctDNA in plasma from 15 patients with BRAF V600E/K-positive primary tumors undergoing standard-of-care monitoring, including cross-sectional computed tomography (CT) imaging. BRAF V600 mutant allele frequency (%MAF) was calculated from the Idylla Cq values and directly measured using droplet digital polymerase chain reaction (ddPCR). The Idylla ctDNA assay demonstrated 91% sensitivity, 96% specificity, 91% positive predictive value, and 96% negative predictive value for the presence of > 93 mm metastatic disease. Qualitative ctDNA results corresponded to changes in RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 status determined by CT imaging in 11 of 15 subjects (73%). Calculated %MAF results correlated with ddPCR (R2 = 0.94) and provided evidence of progressive disease 55 and 97 days in advance of CT imaging for two subjects with persistently positive qualitative results. Overall, interim ctDNA results provided evidence of partial response or progressive disease an average of 82 days before radiography. This pilot study supports the feasibility of using the Idylla plasma BRAF V600 ctDNA assay as a complement to CT scanning for routine monitoring of therapeutic response. Somatic mutation quantification based on Cq values shows promise for identifying disease progression and warrants further validation.

Combining tissue and circulating tumor DNA increases the detection rate of a CTNNB1 mutation in hepatocellular carcinoma

Background and aimsStudies suggest that mutations in the CTNNB1 gene are predictive of response to immunotherapy, an emerging therapy for advanced hepatocellular carcinoma (HCC). Analysis of circulating tumor DNA (ctDNA) offers the possibility of serial non-invasive mutational profiling of tumors. Combining tumor tissue and ctDNA analysis may increase the detection rate of mutations.This study aimed to evaluate the frequency of the CTNNB1 p.T41A mutation in ctDNA and tumor samples from HCC patients and to evaluate the concordance rates between plasma and tissue. We further evaluated changes in ctDNA after various HCC treatment modalities and the impact of the CTNNB1 p.T41A mutation on the clinical course of HCC.MethodsWe used droplet digital PCR to analyze plasma from 95 patients and the corresponding tumor samples from 37 patients during 3 years follow up.ResultsIn tumor tissue samples, the mutation rate was 8.1% (3/37). In ctDNA from HCC patients, the CTNNB1 mutation rate was 9.5% (9/95) in the pre-treatment samples. Adding results from plasma analysis to the subgroup of patients with available tissue samples, the mutation detection rate increased to 13.5% (5/37). There was no difference in overall survival according to CTNNB1 mutational status. Serial testing of ctDNA suggested a possible clonal evolution of HCC or arising multicentric tumors with separate genetic profiles in individual patients.ConclusionCombining analysis of ctDNA and tumor tissue increased the detection rate of CTNNB1 mutation in HCC patients. A liquid biopsy approach may be useful in a tailored therapy of HCC.

Early plasma circulating tumor DNA (ctDNA) changes predict response to first-line pembrolizumab-based therapy in non-small cell lung cancer (NSCLC)

BackgroundCurrently available biomarkers are imperfect in their ability to predict responses to the multiple first-line treatment options available for patients with advanced non-small cell lung cancer (NSCLC). Having an early...

P34.09 The Dynamic Changes of ctDNA and cfDNA in the Perioperative Period of Early NSCLC have the Potential to Predict the Risk of Recurrence

P34.09 The Dynamic Changes of ctDNA and cfDNA in the Perioperative Period of Early NSCLC have the Potential to Predict the Risk of Recurrence

CtDNA pathogenic variants (PVs) in homologous recombination repair (HRR) genes in patients with metastatic CRPC.

138 Background: HRR PVs can serve as predictive biomarkers and two PARP inhibitors are approved for metastasic CRPC (mCRPC) pts. Published data are predominantly focused on tissue-based assays, but obtaining tissue from mCRPC pts is problematic. In a large tissue based series (PROfound), 4047 mCRPC pts had tumor samples submitted for genomic testing but only 69% had interpretable results. No data were published from PROfound enumerating pts without available tissue to submit. Herein we assess frequency of PVs from selected HRR genes using a ctDNA assay. Methods: 292 mCRPC pts at Tulane Cancer Center were assessed for detectable HRR ctDNA changes using the Guardant 360 assay (which assesses the HRR genes BRCA1, BRCA2, and ATM). Results: 20/292 (6.8%) pts had a PV in ATM. However only 4/292 (1.4%) had > 1% mutant allelic fraction. Germline testing occurred in 18/20 of the ctDNA ATM PV pts and 0/18 had a germline PV. The PROfound series had 6.3% somatic PVs in ATM. 18/292 pts (6.2%) had a PV in BRCA2 and 12/292 (4.1%) had a mutant allelic fraction of > 1%. Germline testing was performed in 17/18 with BRCA2 ctDNA PVs and 9/17 had germline PVs. The PROfound series had 9.7% somatic BRCA2 PVs. BRCA1 PVs were detected in 6/292 (2.1%) pts and 3/292 (1%) had a mutant allelic fraction > 1%. 6/6 of the ctDNA PVs has germline testing and 1/6 had a BRCA1 PV. The PROfound series had 1.3% somatic PVs in BRCA1. Conclusions: Using ctDNA essay, it is feasible to measure PVs in only a small subset of HRR genes in mCRPC pts. These assays fail to detect deep deletions, a known and important mechanism of HRR gene loss. The ctDNA mutant allelic fractions are often low. The ability of ctDNA PVs using this assay to predict treatment effects are unknown.

Abstract PS5-02: Assessment of early ctDNA dynamics to predict efficacy of targeted therapies in metastatic breast cancer: Results from plasmaMATCH trial

Abstract Background: Early changes in circulating tumour DNA (ctDNA) levels may identify which patients respond to therapy earlier than imaging, with ctDNA levels falling rapidly in patients who respond to therapy. The plasmaMATCH trial assessed the utility of ctDNA testing with an error-corrected 73-gene targeted panel (Guardant360, Guardant Health) to allocate patients to four mutation matched therapy cohorts. ESR1-extended fulvestrant (A), HER2-neratinib +/- fulvestrant (B), AKT1-capivasertib + fulvestrant (C), AKT basket-capivasertib (D). Here, we report paired baseline and early on treatment ctDNA analysis from plasmaMATCH, to establish the optimal criteria for predicting progression free survival (PFS). Methods: In plasmaMATCH treatment cohorts, plasma samples were collected for ctDNA analysis pre-treatment at cycle 1-day 1 (C1D1) and cycle 2-day 1 (C2D1) timepoints, and sequenced with the Guardant 360 assay. Patients were included if they had a minimum of 14 days of treatment in the first cycle. Multiple different methods were investigated to integrate variant allele fractions (VAF) of mutations identified at each timepoint to estimate the level of ctDNA, including maximum VAF, mean VAF and weighted mean VAF, and weighted mean VAF of clonal mutations at C1D1. Variants with a VAF <0.3%, set as the limit of detection, in C1D1 were excluded. Genes frequently mutated in CHIP were excluded (GNAS, JAK2, IDH1, IDH2 and ATM) from the weighted mean VAF of clonal mutations method. The circulating DNA ratio (CDR) was calculated as the ratio of C2D1 level relative to C1D1 level. The optimal cut-point for predicting PFS was assessed by fitting a range of cutpoints for each VAF integration method, identifying the cut-point with the highest Harrell’s C-index. Results: A total of 142 patients were enrolled into plasmaMATCH cohorts A-D, 79 patients had samples sent for paired C1D1-C2D1 plasma ctDNA sequencing, 1 failed sequencing and 1 insufficient treatment, and 77 (54%) patients had assessable C1D1-C2D1 plasma ctDNA sequencing results (45 cohort A, 12 cohort B, 12 cohort C, 8 cohort D). A weighted mean of clonal mutations in C1D1 ctDNA sequencing was the optimal method for integrating VAF, with peak C-Index 0.67. At the optimal C-index cutoff of 0.132, median PFS with high ctDNA CDR was 2.4 months (95% CI 2.0-3.7) and with suppressed ctDNA CDR was 9.9 months (95% CI 7.0-13.7) (HR 4.3, 95% CI 2.4-7.6, p<0.0001). Early changes in ctDNA level were also predictive in cohorts A extended dose fulvestrant alone (HR 5.8, 95% CI 2.2-16, p=0.0001) and cohorts B-D of targeted therapy (HR 3.8, 95% CI 1.7-8.6, p=0.00063). In analysis that was not pre-planned, patients with undetectable ctDNA at C2D1 had a particularly good outcome (p<0.0001, table 1). Conclusions: We identify an optimal methodology for assessing early dynamic changes in ctDNA that predicts treatment efficacy in patients with metastatic breast cancer. This methodology will require validation in independent data-sets, and if validated would allow trials of adapting therapy on the basis of early ctDNA dynamics. Table 1ctDNA dynamics categoryMedian PFS months (95%CI)6-month PFSORRUndetectable (N=11) CDR=018.2 (10.2-NA)91%9/11 (82%)Suppressed (N=14) CDR <0.132 and >05.4 (4.6-NA)48%6/14 (43%)High (N=52) CDR >=0.1322.4 (2.0-3.7)8%4/52 (8%) Citation Format: Javier Pascual, Rosalind J Cutts, Belinda Kingston, Sarah Hrebien, Lucy S Kilburn, Sarah Kernaghan, Laura Moretti, Katie Wilkinson, Andrew M Wardley, Iain R Macpherson, Richard D Baird, Rebecca Roylance, Michael Hubank, Giselle Walsh, Iris Faull, Kimberly C Banks, Richard B Lanman, Isaac Garcia-Murillas, Judith M Bliss, Alistair Ring, Nicholas C Turner. Assessment of early ctDNA dynamics to predict efficacy of targeted therapies in metastatic breast cancer: Results from plasmaMATCH trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-02.

Abstract OT-03-07: Solti-1804 HER2-PREDICT: A biomarker research study of DS8201-A-U301 -U302 and -U303 trials

Abstract Background: Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in 15-20% of breast cancers (BC). At the same time, HER2-low tumors (immunohistochemistry 1+ or 2+ with no gene amplification) comprise ~50% of all BC. DS-8201a is an anti-HER2 antibody drug conjugate that has shown very promising response rates both in HER2+ and HER2low BC. However, not all patients respond or benefit to the same extend. Thus, there is a need to identify predictive biomarkers. Here, we hypothesize that by performing several molecular studies in both tissue and plasma samples of those patients participating in the pivotal DESTINY-Breast trials, we will shed more light about the molecular features of HER2+ BC and better characterized the patient population according to their benefit from this promising new anti-HER2 agent. Methods: HER2-PREDICT is a multi-center, observational study within the biomarker program of SOLTI group, which will include patients who will participate, are participating or previously participated in the Daiichi Sankyo INC sponsored phase III trials: DS8201-A-U301 (NCT03523585), -U302 (NCT03529110) and -U303 (NCT03734029). Patients with HER2-positive or HER2-low unresectable and/or metastatic breast cancer may be included in SOLTI-1804 HER2-PREDICT study if randomized to the DS-8201a arm. All patients need to consent for obtaining a fresh tumor biopsy or donating an archival metastatic biopsy. Primary tumors are allowed under SOLTI acceptance. Additionally, patients included before initiating DS-8201a therapy will provide blood samples for biomarker analyses on Cycle 1 Day 1 (C1D1), C2D1 and end of treatment. The primary objectives are (1) to identify an optimal ERBB2 mRNA cut-off point predictive of Ds-8201a response and (2) to evaluate the correlation of baseline ERBB2 mRNA levels (as a continuous variable) with overall response rate (ORR) in the Ds-8201a-treated cohorts. Secondary objectives includes: to evaluate the association of ERBB2 mRNA levels, PAM50 intrinsic subtypes and immune-related genes with ORR, progression-free survival and overall survival; to design a new gene expression signature predictive of Ds-8201a benefit; to correlate early changes in ctDNA with Ds-8201a benefit and to identify acquired somatic mutations of resistance to DS8201a upon progression in plasma samples. Collection of tumor biopsies is an essential part of this study. Pathological analysis includes hematoxylin and eosin (H&E) staining, identification of areas with greater amount of tumor cells and determination of their tumor cell percentage. RNA will be isolated and analyzed at the nCounter (Nanostring Technologies). Molecular intrinsic subtypes will be identified by a research-based version of PAM50. Furthermore, we aim to evaluate 771 additional genes (+5 housekeeping genes) that encompass important genomic signatures and individual genes of importance for breast cancer by means of the nCounter®Breast Cancer 360 Panel. Somatic mutations in PIK3CA, TP53, and additional genes (e.g., GATA3 and ERBB2) will be identified using next-generation sequencing (NGS). Also, a comprehensive NGS gene panel will be performed under the Ion Torrent or Illumina platforms to DNA extracted from FFPE tumor blocks and to circulating tumor DNA (ctDNA) in plasma samples. Current status: Since December 13th, 2019, a total of 10 patients have been included, 5 of them with blood samples. As of today, 13 out of 15 Spanish sites are recruiting patients. Clinical trial identification: NCT04257162 Citation Format: Aleix Prat, Joaquin Gavilá, Sonia Pernas, Josefina Cruz, Cristina Saura, Esteban Nogales, Maria Bermejo, Javier Salvador Bofill, Vanesa Quiroga, Laura Garcia-Estévez, Serafin Morales, Sonia Servitja, Rubén del Toro, Patricia Galván, Núria Chic, Débora Martinez, Fara Brasó-Maristany, Jordi Canes, Laia Paré, Juan M Ferrero-Cafiero, Patricia Villagrasa. Solti-1804 HER2-PREDICT: A biomarker research study of DS8201-A-U301 -U302 and -U303 trials [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-03-07.

Abstract OT-13-04: Solti-1716. Targeting non-Luminal disease by PAM50 with pembrolizumab + paclitaxel in Hormone Receptor-positive/HER2-negative advanced/metastatic breast cancer patients who have progressed on or after CDK 4/6 inhibitor treatment (TATEN trial)

Abstract Background. Patients with metastatic Hormone Receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) are usually treated with CDK4/6 inhibitors combined with endocrine therapy (ET) as first line treatment. The addition of CDK4/6 inhibitors to endocrine therapy has demonstrated improved progression free survival (PFS), overall response rate (ORR) and more recently overall survival (OS). However, there is a 20% of patients who do not benefit from these drugs and those who respond eventually progress to the treatment. Notably, there is no standard treatment for patients progressing to CDK4/6 inhibitors. The information provided by the intrinsic subtypes (PAM50) highlight the potential value of BC molecular classification as a prognosis and predictive marker. Within HR+/HER2- disease, patients with non-luminal subtypes (HER2-enriched and Basal-like) present poorer prognosis than those with luminal subtypes, may be more sensitive to chemotherapy, and have higher expression of immune-related genes and tumor infiltrating lymphocytes (TILs). Recently, immunotherapy has been approved for treating metastatic triple negative breast cancer and several trials evaluating the action of immune checkpoint inhibitors are ongoing, including HR+/HER2- BC patients. TATEN study aims to evaluate the combination of pembrolizumab and chemotherapy in metastatic HR+/HER2-, PAM50 non-luminal BC. Study design. TATEN is an open-label, single arm, multicenter phase II study evaluating treatment with pembrolizumab in combination with paclitaxel in patients with locally advanced or metastatic non-luminal HR+/HER2- BC who had recurrence or progression while receiving previous therapy with a CDK4/6 inhibitor plus endocrine therapy in the adjuvant and/or metastatic setting. Tumor samples collected during advanced/metastatic disease are mandatory. No prior chemotherapy for inoperable locally advanced or metastatic BC is permitted. Eligible patients will receive pembrolizumab 200 mg every 3 weeks (on day 1 of each 21-day cycle, beginning in Cycle 1) in combination with paclitaxel 80 mg/m2 administered at days 1, 8, 15 of each 21-day cycle beginning at cycle 2. The primary endpoint of the study is to evaluate ORR according to RECIST V1.1. The study will use a Simon’s 2-stage design and will include up to 46 patients. If 6 or more responses are observed in up to 15 patients in the first stage, the trial will continue to the second stage and 31 additional patients may be evaluated for a maximum total of 46 evaluable patients. The null hypothesis will be rejected if 19 or more responses are observed. Tumor assessments will be performed every 9 weeks. Secondary endpoints include clinical benefit rate (CBR), PFS, duration of response (DoR), time to response (TtR), OS, as well as to assess the correlation of clinical benefit (ORR, PFS) with PD1 mRNA expression and early dynamic changes in ctDNA after 1 cycle of pembrolizumab (collection of blood samples at Cycle 1 Day 1, Cycle 2 Day 1 and end of treatment are mandatory). Safety and tolerability of the combination will also be assessed. Exploratory objectives include to determine ORR, PFS, DoR and TtR based on iRECIST and to identify predictive biomarkers of response to pembrolizumab plus paclitaxel. Patients will be enrolled in 7 sites in Spain. Recruitment started in July 2020. This study is financially supported by MSD. NCT04251169. Citation Format: Eva Ciruelos, Tomás Pascual, Nuria Chic, Montserrat Muñoz, Begoña Bermejo, Juan Antonio Virizuela, Mafalda Oliveira, Silvia Vázquez, Salvador Blanch, Laia Paré, Fernando Salvador, Patricia Villagrasa, Aleix Prat. Solti-1716. Targeting non-Luminal disease by PAM50 with pembrolizumab + paclitaxel in Hormone Receptor-positive/HER2-negative advanced/metastatic breast cancer patients who have progressed on or after CDK 4/6 inhibitor treatment (TATEN trial) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-13-04.

Insights into the binding properties of calf thymus DNA with lopinavir from spectroscopic and computational studies

Lopinavir (LPV), an important antiviral drug, can inhibit effectively wild type and mutant HIV protease. In this work, molecular interaction mechanism between LPV and calf thymus deoxyribonucleic acid (ct-DNA) was characterized by spectroscopic, viscosity and molecular docking approaches to attain some important information about the LPV-ct-DNA interaction including binding affinity, binding mode, main driving-forces and structural change of ct-DNA. UV–vis titration data demonstrated that the LPV-ct-DNA complex was formed, and the binding affinity under the studied conditions was weak due to the binding constant at the level of 103 M−1. The negative values of ΔH0 (−26.63 kJ∙mol−1) and ΔS0 (−24.11 J∙mol−1∙K−1) revealed that hydrogen bonding and Van der Waals forces played an indispensable part in the LPV-ct-DNA complexation process. The outcomes from viscosity measurements excluded the possibility of intercalation mode between LPV and ct-DNA. The results from UV–vis titration experiments, displacement experiments and influence of ionic strength indicated that LPV bound to the minor groove of ct-DNA, which was further confirmed by docking studies. Besides, a minor change in the conformation of ct-DNA can be observed from CD spectral analysis. Our studies scientifically elucidated the binding behavior between LPV and ct-DNA for the first time and were beneficial to assessing the pharmacological properties of this antiviral drug.

Circulating tumour DNA as a biomarker in resectable and irresectable stage IV colorectal cancer; a systematic review and meta-analysis.

For patients with metastatic colorectal cancer, stratification for treatment (surgery or chemotherapy) is often based on crude clinicopathological characteristics like tumour size and number of lesions. Circulating tumour DNA (ctDNA) acts as a potential biomarker of disease trajectory and biology, allowing better stratification. This study aims to systematically review ctDNA in stage IV colorectal cancer to assess its potential role as a prospective biomarker to guide management decisions. A literature search was performed to identify studies where the measurement of ctDNA in stage IV colorectal cancer was correlated with a clinical outcome (radiological response, secondary resection rate, PFS, DFS or OS). Twenty-eight studies were included, reporting on 2823 patients. Circulating tumour DNA was detectable in between 80% and 90% of patients prior to treatment. Meta-analysis identified a strong correlation between detectable ctDNA after treatment (surgery or chemotherapy) and overall survival (HR 2.2, 95% CI 1.79-2.69, p<0.00001), as well as progression-free survival (HR 3.15, 95% CI 2.10-4.73, p<0.00001). ctDNA consistently offered an early marker of long-term prognosis in irresectable disease, with changes after one cycle of systemic therapy demonstrating prognostic value. In resectable disease treated with curative intent, detection of ctDNA offered a lead time over radiological recurrence of 10 months. Circulating tumour DNA is detectable in the majority of resectable and irresectable patients. The presence of ctDNA is clearly associated with shorter overall survival, with changes in ctDNA an early biomarker of adverse disease behaviour. Prospective trials are essential to test its clinical efficacy.

The Prognostic Impact of Circulating Tumour DNA in Melanoma Patients Treated with Systemic Therapies-Beyond BRAF Mutant Detection.

In this study, we evaluated the predictive value of circulating tumour DNA (ctDNA) to inform therapeutic outcomes in metastatic melanoma patients receiving systemic therapies. We analysed 142 plasma samples from metastatic melanoma patients prior to commencement of systemic therapy: 70 were treated with BRAF/MEK inhibitors and 72 with immunotherapies. Patient-specific droplet digital polymerase chain reaction assays were designed for ctDNA detection. Plasma ctDNA was detected in 56% of patients prior to first-line anti-PD1 and/or anti-CTLA-4 treatment. The detection rate in the immunotherapy cohort was comparably lower than those with BRAF inhibitors (76%, p = 0.0149). Decreasing ctDNA levels within 12 weeks of treatment was strongly concordant with treatment response (Cohen's k = 0.798, p < 0.001) and predictive of longer progression free survival. Notably, a slower kinetic of ctDNA decline was observed in patients treated with immunotherapy compared to those on BRAF/MEK inhibitors. Whole exome sequencing of ctDNA was also conducted in 9 patients commencing anti-PD-1 therapy to derive tumour mutational burden (TMB) and neoepitope load measurements. The results showed a trend of high TMB and neoepitope load in responders compared to non-responders. Overall, our data suggest that changes in ctDNA can serve as an early indicator of outcomes in metastatic melanoma patients treated with systemic therapies and therefore may serve as a tool to guide treatment decisions.

CtDNA as a predictor of outcome in patients treated with neoadjuvant atezolizumab in muscle invasive urothelial cancer

To explore the clinical utility of circulating tumor DNA (ctDNA) with neoadjuvant atezolizumab and radical cystectomy (RC) in muscle invasive urothelial bladder cancer (MIUC). Forty patients from a phase II of neoadjuvant atezolizumab (2 cycles) and RC study in MIUC were included (NCT02662309). Whole-exome sequencing was performed on tumor and matched normal DNA to identify tumor-specific mutations and design a personalized multiplexed PCR Next Generation Sequencing (NGS) assay (bespoke, Signatera TM ) for ctDNA detection in plasma. Samples were taken at baseline, post-atezolizumab but pre-surgery, and 1-6 months post-surgery. Response (pathological complete response (pCR) and major pathological response (mPR)) and recurrence free survival (RFS) were correlated with ctDNA status (Pos/Neg) and level. Responders are pCR and mPR patients, whereas non-responders are SD and relapse patients. The pCR rate and 2 years RFS was 21.4% and 73.5%, respectively. ctDNA positivity at baseline, post-neoadjuvant pre-surgery (PreCx), and post-surgery (PostCx) were 25/40 (63%), 14/30 (47%), and 5/36 (14%), respectively. ctDNA status was highly prognostic at all timepoints, where PostCx had an HR of 78.11 (p<0.001), and zero events were observed for ctDNA negative patients at baseline and PreCx. An association between the change in ctDNA during neoadjuvant atezolizumab treatment and patient outcome was observed (mean of -90% ctDNA change in responders versus +27% in non-responders (p=0.04)). Patients who were PDL1 positive at baseline were more likely to be ctDNA positive at baseline (p=0.01), which was not the case for post-treatment time points. For patients who were ctDNA positive at baseline, PDL1-positive patients had better response rates than PDL1-negative (35% vs. 0%), as well as superior RFS (HR=0.11, p=0.0085). Pre-treatment ctDNA positivity was enriched in SCCL (Lund) and basal squamous (TCGA) patients. Tumors from ctDNA positive patients at baseline were enriched for immune signatures and the epithelial to mesenchymal transition (EMT) signature. ctDNA dynamics across timepoints correlates with clinical outcomes, including response rates and RFS in patients treated with neoadjuvant atezolizumab in MIUC. Immune biomarkers may be relevant in these responses. ctDNA may help the development of personalized therapy in this setting in the future.

Prognostic and Predictive Impact of Circulating Tumor DNA in Patients with Advanced Cancers Treated with Immune Checkpoint Blockade.

The utility of circulating tumor DNA (ctDNA) as a biomarker in patients with advanced cancers receiving immunotherapy is uncertain. We therefore analyzed pretreatment (n = 978) and on-treatment (n = 171) ctDNA samples across 16 advanced-stage tumor types from three phase I/II trials of durvalumab (± the anti-CTLA4 therapy tremelimumab). Higher pretreatment variant allele frequencies (VAF) were associated with poorer overall survival (OS) and other known prognostic factors, but not objective response, suggesting a prognostic role for patient outcomes. On-treatment reductions in VAF and lower on-treatment VAF were independently associated with longer progression-free survival and OS and increased objective response rate, but not prognostic variables, suggesting that on-treatment ctDNA dynamics are predictive of benefit from immune checkpoint blockade. Accordingly, we propose a concept of "molecular response" using ctDNA, incorporating both pretreatment and on-treatment VAF, that predicted long-term survival similarly to initial radiologic response while also permitting early differentiation of responders among patients with initially radiologically stable disease. SIGNIFICANCE: In a pan-cancer analysis of immune checkpoint blockade, pretreatment ctDNA levels appeared prognostic and on-treatment dynamics predictive. A "molecular response" metric identified long-term responders and adjudicated benefit among patients with initially radiologically stable disease. Changes in ctDNA may be more dynamic than radiographic changes and could complement existing trial endpoints.This article is highlighted in the In This Issue feature, p. 1775.

DNA binding and antiradical potential of ethyl pyruvate: Key to the DNA radioprotection

DNA is the store house of all necessary hereditary information for growth of cells and tissues. Physiological functionality of DNA depends on its 3D helical structure and any distortion in a structure may lead to mutation and genomic instability that may translate into disease like cancer. In order to prevent DNA damage, an exogenous compound is required that can either scavenge the excess free radicals or enhance the structural integrity of DNA through binding. In the present study, the binding mechanism of ethyl pyruvate (EP) with DNA models using different spectroscopic techniques was investigated for their structural integrity. Besides, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays were performed to determine the antioxidant scavenging of EP. Plasmid DNA relaxation assay was performed to assess the radioprotection efficacy of EP in the plasmid DNA. Circular dichroism (CD) and UV–Vis absorbance spectroscopic data confirmed the conformation change in ctDNA upon binding with EP. The molecular docking visualized that EP stacks between the DNA bases with a glide score of −2.117 kcalmol while EP binds in the minor groove region of DNA with the glide score of −1.414 kcalmol . DPPH and FRAP data confirmed that EP scavenges significantly radicals at higher concentrations. In vitro radioprotection study in plasmid DNA pBR322 showed that EP retained the supercoiled form of plasmid DNA at 50 Gy radiation dose.

Interim Cell-Free Circulating Lymphoma DNA Analysis of the Phase 2 Accept Trial

Introduction Plasma cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) genotyping in Diffuse Large B-cell Lymphoma (DLBCL) holds great promise for early detection and response prediction. DLBCL is a highly heterogenous disease with diverse tumor behaviour and outcomes, yet the delineation of poor- risk groups remains challenging. Genomically driven molecular risk stratification, using ultradeep next generation sequencing (NGS) provides a comprehensive view of the mutational landscape of lymphoma and enables ultrasensitive disease detection. Here, we present an interim analysis of ctDNA kinetics from ACCEPT, a phase Ib/II study of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with previously untreated DLBCL. Methods Plasma samples were collected at four time points, at baseline and before the second, third and final cycles of chemotherapy. Extracted cell-free DNA (cfDNA) was quantified by fluorometry, prior to undergoing library preparation and next-generation sequencing using the CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) methodology. The targeted sequencing panel covered 799 regions over 50 genes (&amp;gt;200Kb) known to be involved in lymphomagenesis. Analysis was carried out with a custom bioinformatics pipeline using error-corrected deduplication and a database of known artefacts to reduce background noise and improve sensitivity for low-level somatic variant detection. Patients were closely monitored through the study period by clinical examination and PET-CT scanning to provide objective endpoints against which the ctDNA response could be correlated. Results Fifteen patients had samples analysed for all four time points at the time of the interim analysis and were included in the study. Ten (66%) had late stage disease, nine (60%) poor risk disease (IPI &amp;gt; 2). Early vs late stage of disease at presentation showed a strong correlation with baseline levels of both total cfDNA (3,946 HGE/ml vs 21,613 HGE/ml) and ctDNA (842HGE/ml vs 6,380 HGE/ml). Twelve patients (80%) had trackable variants detected at baseline. The majority of these cleared after the first cycle of chemotherapy (Figure A; representative patient). Two cases (Figures B and C) had variants persistently detectable at high variant allele frequency (VAF) (0.059 and 0.068). These patients both had primary refractory disease. One patient from the first acalabrutinib dose escalation cohort and the second patient was found to be ineligible and withdrawn from the primary analysis because of continuous co-administration of omeprazole during therapy which inhibits acalabrutinib absorption. All others achieved a metabolic complete metabolic remission (mCR) after the final cycle of therapy. A third pattern of response noted was (Figure D) showing disappearance of detectable variants at baseline but progressive emergence of an acquired TP53 variant throughout follow-up that was not detected in the patient's germline sample. This high-risk patient with IPI of 5 and triple-hit DLBCL achieved a mCR and remains in remission in follow-up. Conclusion The clearance of ctDNA from the plasma of the responding patients in our study is consistent with the prior literature, as is the poor prognostic significance of persisting variants after the first cycle of chemotherapy. The rapid kinetics of the changes in ctDNA is noted and is potentially of advantage over other modalities of monitoring, such as PET-CT scan. Data on the entire cohort (n=30) and longer follow-up will be available at the time of the meeting. Figure 1 Disclosures Davies: Roche: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte: Consultancy; Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead: Research Funding; Celegene, Roche, Kite Pharma, Celegene: Honoraria. Johnson:Oncimmune: Consultancy; Bristol-Myers: Honoraria; Celgene: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Kite Pharma: Honoraria; Kymera: Honoraria; MorphoSys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Boehringer Ingelheim: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy; Oncimmune: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy. Schuh:Illumina: Other: Consulting fees; Roche: Other: Consulting fees; Gilead: Other: Consulting fees; Abbvie: Other: Consulting fees.

Circulating Tumor DNA Allows Early Treatment Monitoring in BRAF- and NRAS-Mutant Malignant Melanoma.

We evaluated circulating tumor DNA (ctDNA) for detecting tumor burden in melanoma and examined whether early changes in the number of ctDNA copies predict response to treatment. We included 12 patients with stage III and 50 patients with stage IV melanoma with BRAF exon 15 or NRAS exon 3 mutations in tumor tissue. We used droplet digital polymerase chain reaction to retrospectively analyze serial plasma samples for mutation-positive ctDNA. Matched plasma and serum samples were positive for ctDNA, lactate dehydrogenase, and S100 in 113 (45.8%), 108 (43.7%; not significant), and 58 (23.5%; P < .0001) of 247 samples from 50 patients with stage IV melanoma, and in 17 (63%), eight (29.6%; P = .014), and five (18.5%; P < .0001) of 27 samples from 12 patients with stage III melanoma. The number of mutant ctDNA copies correlated with concentrations of lactate dehydrogenase (r = 0.50) and S100 (r = 0.64), tumor volume (r2 = 0.58), and tumor metabolic activity (r2 = 0.83). Within 30 days before surgery, initiation of treatment, or change in treatment, ctDNA, LDH, and S100 were positive in 76.8%, 53.6% (P = .01), and 46.4% (P < .001) of patients, respectively. In patients with stage III or IV melanoma, early changes in ctDNA within 1 month after initiation of treatment correctly predicted RECIST response categories in 19 of 20 patients. Detectable ctDNA within 30 days after surgery or initiation of systemic treatment predicted inferior progression-free survival in patients with stage III disease (P = .018). In patients with stage IV disease, 10 or more copies of ctDNA per mL at first follow-up indicated shorter progression-free survival (3.8 v 9 months; hazard ratio, 4.05; 95% CI, 1.56 to 10.53). ctDNA indicated active tumor and was an adverse prognostic marker for tumor progression. Dynamic changes in ctDNA allowed prediction of response early after initiation of treatment. These data support the use of ctDNA to guide treatment in melanoma.