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Detection of cell-free circulating tumor DNA (ctDNA) from solid tumors is a fast-evolving field with significant potential for improving patient treatment outcomes. The spectrum of applications for ctDNA assays is broad and includes very diverse intended uses that will require different strategies to demonstrate utility. On September 14-15, 2023, the National Cancer Institute held an in-person workshop in Rockville, MD titled "ctDNA in Cancer Treatment and Clinical Care." The goal of the workshop was to examine what is currently known and what needs to be determined for various ctDNA liquid biopsy use cases related to treatment and management of patients with solid tumors and to explore how the community can best assess the value of ctDNA assays and technology. Additionally, new approaches were presented that may show promise in the future. The information exchanged in this workshop will provide the community with a better understanding of this field and its potential to affect and benefit decision-making in the treatment of patients with solid tumors., (Published by Oxford University Press 2024.)

Background: We assessed the added value of incorporating carcinoembryonic antigen (CEA) to circulating tumor DNA (ctDNA) and pathological TN (pTN) stage for risk classification in stage 3 colon cancer (CC)., Patients and Methods: We retrospectively analyzed postoperative CEA values in patients with CC from the IDEA-France phase 3 trial. The relation between disease-free survival (DFS) and CEA was modeled through restricted cubic splines. Prognostic value of CEA, ctDNA, and pTN was assessed with the Kaplan-Meier method. Multivariate analysis was used to identify prognostic and predictive factors for DFS., Results: Among 696 patients (35%), CEA values were retrievable, and for 405 (20%) both CEA and ctDNA were available. An optimized CEA threshold of 2 ng/mL was identified, the 3-year DFS was 66.4% for patients above the threshold and 80.9% for those below (HR, 1.74; 95% CI, 1.33-2.28, P < .001). In multivariate analysis, CEA ≥ 2 ng/mL contributed significantly to model variability, becoming an independent prognostic factor for DFS (HR, 1.82; 95% CI,1.27-2.59), alongside ctDNA (HR, 1.88; 95% CI, 1.16-3.03) and pTN (HR, 1.78; 95% CI, 1.24-2.54). A novel integrated risk classification combining CEA, ctDNA, and pTN stage reclassified 19.8% of pT4/N2 patients as low risk and 2.5% of pT3/N1 patients as high risk. This new classification demonstrated the 3-year DFS of 80.8% for low-risk patients and 55.4% for high-risk patients (HR, 2.66, 95% CI, 1.84-3.86, P < .001)., Conclusions: Postoperative CEA value is a prognostic factor for DFS in stage 3 CC, independently of ctDNA and pTN. It advocates for systematic reporting in future adjuvant trials. Integrating both biomarkers with pTN could refine risk classification in stage 3 CC., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Despite complete resection, 20%-50% of patients with localized renal cell carcinoma (RCC) experience recurrence within 5 years. Accurate assessment of prognosis in high-risk patients would aid in improving outcomes. Here we evaluate the use of circulating tumor DNA (ctDNA) in RCC using banked samples and clinical data from a single institution., Methods: The cohort consisted of 45 RCC patients (≥pT1b) who underwent complete resection. The presence of ctDNA in plasma was determined using a personalized, tumor-informed ctDNA assay (Signatera RUO, Natera, Inc.). Relationships with outcomes and other relevant clinical variables were assessed. The median follow-up was 62 months., Results: Plasma ctDNA was detected in 18 out of 36 patients (50%) pre-operatively and was associated with increased tumor size (mean 9.3 cm vs. 7.0 cm, P < .05) and high Fuhrman grade (60% grades III-IV vs 27% grade II, P = .07). The presence of ctDNA, either pre-operatively or at any time post-operatively, was associated with inferior relapse-free survival (HR = 2.70, P = .046; HR = 3.23, P = .003, respectively). Among patients who were ctDNA positive at any time point, the sensitivity of relapse prediction was 84% with a PPV of 90%. Of note, ctDNA positivity at a post-surgical time point revealed a PPV of 100% and NPV of 64%. The lack of ctDNA detection at both time points yielded an NPV of 80%., Conclusions: Detection of plasma ctDNA using a personalized assay is prognostic of recurrence in patients with resected RCC. Herein, we describe a successful approach for its application and identify potential limitations to be addressed in future studies., (© The Author(s) 2024. Published by Oxford University Press.)

Introduction: Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC)., Methods: In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (n = 1329) from 298 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023., Results: Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, P < .0001) as well as during the surveillance period (median DFS: 11.40 months for ctDNA-positive vs NR for ctDNA-negative; HR: 12.38, P < .0001). Additionally, DFS was significantly better with KRAS wildtype status followed by KRASG12R (HR: 0.99, P = .97), KRASG12D (HR: 1.42, P = .194), and worse with KRASG12V (HR: 2.19, P = .002) status. In multivariate analysis, ctDNA detection at surveillance was found to be the most significant prognostic factor for recurrence (HR: 24.28, P < .001)., Conclusions: Perioperative tumor-informed ctDNA detection in PDAC is feasible across all stages and is associated with patient survival outcomes., (© The Author(s) 2024. Published by Oxford University Press.)

In the recent decade, analysis of circulating tumor DNA (ctDNA) has improved cancer care by allowing for rapid detection of actionable molecular targets. A new generation of circulating DNA tests is now becoming available commercially. These tests are characterized by a superior limit of detection of 0.01% vaF or better, allowing for the detection of radiologically occult molecular residual disease (MRD). MRD tests have the potential to revolutionize neoadjuvant and adjuvant treatment. In addition, these tests can be used as tumor markers to assess disease response. We reviewed the current evidence for the use of high-sensitivity MRD assays with particular focus on the genitourinary tumors. Multiple studies have now been reported in urothelial, renal, and recently testicular carcinoma. We find that the sensitivity varies across tumor types in the adjuvant setting and may reach a high of 100% in urothelial cancer. Specificity in tumor-informed MRD appears to be preserved across tumor types (98%-100%). Several trials are now prospectively validating MRD testing in biomarker integral studies, mainly in urothelial carcinoma., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis., Methods: We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis., Results: Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level., Conclusions: Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients., (© Association for Diagnostics & Laboratory Medicine 2024.)

Purpose: In addition to the existing biomarkers HER2 and PD-L1, FGFR2b has become an area of interest for the development of new targeted-based treatment. Given that clinical evaluation of FGFR2 targeted therapy is underway, we sought to elucidate the genomic landscape of FGFR2amp in gastroesophageal cancer (GEC) using a circulating tumor DNA (ctDNA) platform., Materials and Methods: We retrospectively evaluated the Guardant Health database from 2017 to 2022 for patients with GECs with Guardant360 ctDNA next-generation sequencing (NGS) performed. We assessed co-occurring genetic alterations for patients who harbored FGFR2amp versus FGFR2null. We also explored real-world evidence database with Guardant Health, publicly available genomic databases (MSK cohort using cBioPortal), and pooled clinical data from large-volume cancer centers for FGFR2amp GECs., Results: Less than 4% of patients with GEC in the Guardant Health database were identified to be FGFR2amp. The most commonly co-occurring gene mutations were TP53, CTNNB1, CDH1, and RHOA. Upon interrogation of the MSK cohort, these same genes were not significant on tissue NGS in the FGFR2amp cohort of GEC. In the pooled institutional cohort, we noted that FGFR2amp tumors were most commonly involving the gastroesophageal junction (GEJ). The overall survival of these patients was noted at 13.1 months., Conclusion: FGFR2 is a validated target in GECs, and the contexture of FGFR2amp will be important in defining patient subgroups with responses to FGFR2-directed therapy. Using ctDNA to provide a more detailed genomic landscape in patients with GECs will allow the advancement of targeted therapy in the near future for these aggressive cancers., (© The Author(s) 2024. Published by Oxford University Press.)

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Evidence has demonstrated that monitoring of the variable, diversity, and joining gene segments (VDJ) rearrangement of the immunoglobulin (Ig) genes in the circulating tumor DNA (ctDNA) is of value in predicting the outcomes of diffuse large B cell lymphoma (DLBCL). In this study, we investigated the role of VDJ rearrangement proportion in ctDNA for predicting DLBCL progression., Methods: Patients diagnosed with newly diagnosed DLBCL were included in this study. The VDJ sequences of IgH were detected using next-generation sequencing (NGS) in formalin-fixed paraffin-embedded tissue and/or peripheral blood. The clonotype of the highest proportion in the peripheral blood was defined as the "dominant circulating clonotype," whilst the clonotype of the highest proportion in matched tissue that is detected in peripheral blood was defined as the "dominant tissue-matched clonotype." The decision tree, a machine learning-based methodology, was used to establish a progression-predicting model through a combination of "dominant tissue-matched clonotype" proportion or "dominant circulating clonotype" proportion, and the clinicopathological information, including age, sex, cell of origin, stage, international prognostic index, lactate dehydrogenase, number of extranodal involvements and β2-microglobulin., Results: A total of 55 patients with eligible sequencing data were used for prognosis analysis, among which 36 patients had matched tissue samples. The concordance rate of "dominant circulating clonotype" and "dominant tissue-matched clonotype" was 19.44% (7/36). The decision tree model showed that the combination of extranodal involvement event and "dominant circulating clonotype" proportion (≥37%) had a clinical value in predicting the prognosis of DLBCL following combined chemotherapy (sensitivity, 0.63; specificity, 0.81; positive prediction value (PPV), 0.59; negative prediction value, 0.83; kappa value, 0.42). Noticeably, the combination of the "dominant tissue-matched clonotype" and extranodal involvement event showed a higher value in predicting the progression (sensitivity, 0.85; specificity, 0.78; PPV, 0.69; kappa value, 0.64)., Conclusion: IgH proportion detected in the ctDNA samples traced from tissue samples has a high clinical value in predicting the progression of DLBCL., (© The Author(s) 2024. Published by Oxford University Press.)

Adult medulloblastoma (MB) is a rare disease affecting 0.6 persons per million adults over 19 years of age. The SHH-activated/TP53-wild type is the most common subtype, accounting for 60% of adult MBs, being characterized by mutations in PTCH1, SMO, or the TERT promoter. Several small studies demonstrate objective but short-lived responses to SMO inhibitors such as vismodegib or sonidegib. Like other oncogene-addicted solid tumors, detection of the corresponding drivers through liquid biopsy could aid in the molecular diagnosis and monitoring of the disease through less invasive procedures. However, most studies have only evaluated cerebrospinal fluid as the ctDNA reservoir, and very limited evidence exists on the role of liquid biopsy in plasma in patients with primary central nervous system tumors, including MB. We present the case of a 26-year-old patient with a recurrent MB, in which next-generation sequencing (FoundationOne CDx) revealed a mutation in PTCH1, allowing the patient to be treated with vismodegib in second line, resulting in a durable benefit lasting for 1 year. Using an in-house digital PCR probe, the PTCH1 mutation could be tracked in ctDNA during treatment with first-line chemotherapy and while on treatment with vismodegib, demonstrating a precise correlation with the radiological and clinical behavior of the disease., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands)., Methods: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance., Results: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752&#95;I759del) (69%), EGFR p.(N771&#95;H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately., Conclusions: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine., (© Association for Diagnostics & Laboratory Medicine 2024.)

Motivation: Microsatellite instability (MSI) is a promising biomarker for cancer prognosis and chemosensitivity. Techniques are rapidly evolving for the detection of MSI from tumor-normal paired or tumor-only sequencing data. However, tumor tissues are often insufficient, unavailable, or otherwise difficult to procure. Increasing clinical evidence indicates the enormous potential of plasma circulating cell-free DNA (cfNDA) technology as a noninvasive MSI detection approach., Results: We developed MSIsensor-ct, a bioinformatics tool based on a machine learning protocol, dedicated to detecting MSI status using cfDNA sequencing data with a potential stable MSIscore threshold of 20%. Evaluation of MSIsensor-ct on independent testing datasets with various levels of circulating tumor DNA (ctDNA) and sequencing depth showed 100% accuracy within the limit of detection (LOD) of 0.05% ctDNA content. MSIsensor-ct requires only BAM files as input, rendering it user-friendly and readily integrated into next generation sequencing (NGS) analysis pipelines., Availability: MSIsensor-ct is freely available at https://github.com/niu-lab/MSIsensor-ct., Supplementary Information: Supplementary data are available at Briefings in Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)

Despite The Central Dogma states the destiny of gene as 'DNA makes RNA and RNA makes protein', the nucleic acids not only store and transmit genetic information but also, surprisingly, join in intracellular vital movement as a regulator of gene expression. Bioinformatics has contributed to knowledge for a series of emerging novel nucleic acids molecules. For typical cases, microRNA (miRNA), long noncoding RNA (lncRNA) and circular RNA (circRNA) exert crucial role in regulating vital biological processes, especially in malignant diseases. Due to extraordinarily heterogeneity among all malignancies, hepatocellular carcinoma (HCC) has emerged enormous limitation in diagnosis and therapy. Mechanistic, diagnostic and therapeutic nucleic acids for HCC emerging in past score years have been systematically reviewed. Particularly, we have organized recent advances on nucleic acids of HCC into three facets: (i) summarizing diverse nucleic acids and their modification (miRNA, lncRNA, circRNA, circulating tumor DNA and DNA methylation) acting as potential biomarkers in HCC diagnosis; (ii) concluding different patterns of three key noncoding RNAs (miRNA, lncRNA and circRNA) in gene regulation and (iii) outlining the progress of these novel nucleic acids for HCC diagnosis and therapy in clinical trials, and discuss their possibility for clinical applications. All in all, this review takes a detailed look at the advances of novel nucleic acids from potential of biomarkers and elaboration of mechanism to early clinical application in past 20 years., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: There are no established molecular biomarkers for patients with breast cancer receiving combination endocrine and CDK4/6 inhibitor (CDK4/6i). We aimed to determine whether genomic markers in circulating tumor DNA (ctDNA) can identify patients at higher risk of early progression on fulvestrant therapy with or without palbociclib, a CDK4/6i., Methods: PALOMA-3 was a phase III, multicenter, double-blind randomized controlled trial of palbociclib plus fulvestrant (n = 347) vs placebo plus fulvestrant (n = 174) in patients with endocrine-pretreated estrogen receptor-positive (ER+) breast cancer. Pretreatment plasma samples from 459 patients were analyzed for mutations in 17 genes, copy number in 14 genes, and circulating tumor fraction. Progression-free survival (PFS) was compared in patients with circulating tumor fraction above or below a prespecified cutoff of 10% and with or without a specific genomic alteration. All statistical tests were 2-sided., Results: Patients with high ctDNA fraction had worse PFS on both palbociclib plus fulvestrant (hazard ratio [HR] = 1.62, 95% confidence interval [CI] = 1.17 to 2.24; P = .004) and placebo plus fulvestrant (HR = 1.77, 95% CI = 1.21 to 2.59; P = .004). In multivariable analysis, high-circulating tumor fraction was associated with worse PFS (HR = 1.20 per 10% increase in tumor fraction, 95% CI = 1.09 to 1.32; P < .001), as was TP53 mutation (HR = 1.84, 95% CI = 1.27 to 2.65; P = .001) and FGFR1 amplification (HR = 2.91, 95% CI = 1.61 to 5.25; P < .001). No interaction with treatment randomization was observed., Conclusions: Pretreatment ctDNA identified a group of high-risk patients with poor clinical outcome despite the addition of CDK4/6 inhibition. These patients might benefit from inclusion in future trials of escalating treatment, with therapies that may be active in these genomic contexts., (© The Author(s) 2020. Published by Oxford University Press.)

Background: The correlation between blood-based tumor mutation burden (bTMB) and tissue-based tumor mutation burden(tTMB) has not been broadly tested in a multicancer cohort. Here, we assess the correlation between bTMB with tTMB in phase I trial patients treated with immunotherapy. As an exploratory analysis, we evaluated circulating tumor DNA (ctDNA) dynamics in responders., Methods: Patients treated with immunotherapy at the Princess Margaret phase I trials unit were enrolled. Pretreatment plasma ctDNA and matched normal blood controls were collected. Available archival tissue formalin-fixed paraffin-embedded (FFPE) samples were analyzed. A 425-gene panel was used to sequence both ctDNA and FFPE samples. Samples with TMB within the highest tertile were considered as high TMB., Results: Thirty-eight patients were accrued from 25 different trials, 86.8% of which involved an anti-PD-1/PD-L1 agent. Thirty patients (78.9%) had detectable mutations in ctDNA, of which the median (range) bTMB was 5 (1-53) mutations per megabase (mut/Mb). Of the 22 patients with available FFPE samples, mutations were detected in 21 (95.4%); the median (range) tTMB was 6 (2-124) mut/Mb. Among the 16 patients with detectable mutations in both FFPE and ctDNA, a statistically significant correlation between bTMB and tTMB was observed ( ρ  = 0.71; P  = .002). High TMB was not associated with better survival. All 3 responders had a decrease in the variant allele frequency of mutations detected in ctDNA at a second timepoint relative to baseline, indicating a potential early marker of response., Conclusions: In this small series, bTMB correlated with tTMB. An on-treatment decrease in VAF of mutations detected in ctDNA at baseline was observed in responders. Larger studies to verify our findings are warranted., (© The Author(s) 2021. Published by Oxford University Press.)

DNA methylation is an important epigenetic regulator in gene expression and has several roles in cancer and disease progression. MethHC version 2.0 (MethHC 2.0) is an integrated and web-based resource focusing on the aberrant methylomes of human diseases, specifically cancer. This paper presents an updated implementation of MethHC 2.0 by incorporating additional DNA methylomes and transcriptomes from several public repositories, including 33 human cancers, over 50 118 microarray and RNA sequencing data from TCGA and GEO, and accumulating up to 3586 manually curated data from >7000 collected published literature with experimental evidence. MethHC 2.0 has also been equipped with enhanced data annotation functionality and a user-friendly web interface for data presentation, search, and visualization. Provided features include clinical-pathological data, mutation and copy number variation, multiplicity of information (gene regions, enhancer regions, and CGI regions), and circulating tumor DNA methylation profiles, available for research such as biomarker panel design, cancer comparison, diagnosis, prognosis, therapy study and identifying potential epigenetic biomarkers. MethHC 2.0 is now available at http://awi.cuhk.edu.cn/∼MethHC., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Numerous publications have reported the incidental detection of occult malignancies upon routine noninvasive prenatal testing (NIPT). However, these studies were not designed to evaluate the NIPT performance for cancer detection., Methods: We investigated the sensitivity of a genome-wide NIPT pipeline, called GIPSeq, for detecting cancer-specific copy number alterations (CNAs) in plasma tumor DNA (ctDNA) of patients with breast cancer. To assess whether a pregnancy itself, with fetal cell-free DNA (cfDNA) in the maternal circulation, might influence the detection of ctDNA, results were compared in pregnant (n = 25) and nonpregnant (n = 25) cancer patients. Furthermore, the ability of GIPSeq to monitor treatment response was assessed., Results: Overall GIPSeq sensitivity for detecting cancer-specific CNAs in plasma cfDNA was 26%. Fifteen percent of detected cases were asymptomatic at the time of blood sampling. GIPSeq sensitivity mainly depended on the tumor stage. Also, triple negative breast cancers (TNBC) were more frequently identified compared to hormone-positive or HER2-enriched tumors. This might be due to the presence of high-level gains and losses of cfDNA or high ctDNA loads in plasma of TNBC. Although higher GIPSeq sensitivity was noted in pregnant (36%) than in nonpregnant women (16%), the limited sample size prohibits a definite conclusion. Finally, GIPSeq profiling of cfDNA during therapy allowed monitoring of early treatment response., Conclusions: The results underscore the potential of NIPT-based tests, analyzing CNAs in plasma cfDNA in a genome-wide and unbiased fashion for breast cancer detection, cancer subtyping and treatment monitoring in a pregnant and nonpregnant target population., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC's Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors' presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency., (© American Association for Clinical Chemistry 2020.)

Background: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion., Methods: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls., Results: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats., Conclusion: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays., (© American Association for Clinical Chemistry 2020.)

Background: Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential., Methods: We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis., Results: Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples., Conclusion: The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool., Methods: We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy., Results: The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response., Conclusions: This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making., Methods: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites., Results: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT., Conclusions: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows., (© 2019 American Association for Clinical Chemistry.)

Uterine leiomyomas (LM) and leiomyosarcomas (LMS) are considered biologically unrelated tumors due to their cytogenetic and molecular disparity. Yet, these tumors share morphological and molecular characteristics that cannot be differentiated through current clinical diagnostic tests, and thus cannot be definitively classified as benign or malignant until surgery. Newer approaches are needed for the identification of these tumors, as has been done for other tissues. The application of next generation sequencing enables the detection of new mutations that, when coupled to machine learning bioinformatic tools, advances our understanding of chromosomal instability. These approaches in the context of LM and LMS could allow the discovery of genetic variants and possible genomic markers. Additionally, the potential clinical utility of circulating cell-free tumor DNA could revolutionize the noninvasive detection and monitoring of these tumors. Here, we seek to provide a perspective on the molecular background of LM and LMS, recognizing their distinct molecular features that may lead to improved diagnosis and personalized treatments, which would have a measurable impact on women's reproductive health., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Circulating tumor DNA (ctDNA) assays are increasingly used for clinical decision-making, but it is unknown how well different assays agree. We aimed to assess the agreement in ctDNA mutation calling between BEAMing (beads, emulsion, amplification, and magnetics) and droplet digital PCR (ddPCR), 2 of the most commonly used digital PCR techniques for detecting mutations in ctDNA., Methods: Baseline plasma samples from patients with advanced breast cancer enrolled in the phase 3 PALOMA-3 trial were assessed for ESR1 and PIK3CA mutations in ctDNA with both BEAMing and ddPCR. Concordance between the 2 approaches was assessed, with exploratory analyses to estimate the importance of sampling effects., Results: Of the 521 patients enrolled, 363 had paired baseline ctDNA analysis. ESR1 mutation detection was 24.2% (88/363) for BEAMing and 25.3% (92/363) for ddPCR, with good agreement between the 2 techniques (κ = 0.9l; 95% CI, 0.85-0.95). PIK3CA mutation detection rates were 26.2% (95/363) for BEAMing and 22.9% (83/363) for ddPCR, with good agreement (κ = 0.87; 95% CI, 0.81-0.93). Discordancy was observed for 3.9% patients with ESR1 mutations and 5.0% with PIK3CA mutations. Assessment of individual mutations suggested higher rates of discordancy for less common mutations ( P = 0.019). The majority of discordant calls occurred at allele frequency <1%, predominantly resulting from stochastic sampling effects., Conclusions: This large, clinically relevant comparison showed good agreement between BEAMing and ddPCR, suggesting sufficient reproducibility for clinical use. Much of the observed discordancy may be related to sampling effects, potentially explaining many of the differences in the currently available ctDNA literature., (© 2019 American Association for Clinical Chemistry.)

Background: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) is commercially available and increasingly adopted in clinical practice despite a paucity of prospective data to support its use., Methods: Patients with advanced lung cancers who had no known oncogenic driver or developed resistance to current targeted therapy (n = 210) underwent plasma NGS, targeting 21 genes. A subset of patients had concurrent tissue NGS testing using a 468-gene panel (n = 106). Oncogenic driver detection, test turnaround time (TAT), concordance, and treatment response guided by plasma NGS were measured. All statistical tests were two-sided., Results: Somatic mutations were detected in 64.3% (135/210) of patients. ctDNA detection was lower in patients who were on systemic therapy at the time of plasma collection compared with those who were not (30/70, 42.9% vs 105/140, 75.0%; OR = 0.26, 95% CI = 0.1 to 0.5, P < .001). The median TAT of plasma NGS was shorter than tissue NGS (9 vs 20 days; P < .001). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 56.6% (60/106, 95% CI = 46.6% to 66.2%). Among patients who tested plasma NGS positive, 89.6% (60/67; 95% CI = 79.7% to 95.7%) were also concordant on tissue NGS and 60.6% (60/99; 95% CI = 50.3% to 70.3%) vice versa. Patients who tested plasma NGS positive for oncogenic drivers had tissue NGS concordance of 96.1% (49/51, 95% CI = 86.5% to 99.5%), and directly led to matched targeted therapy in 21.9% (46/210) with clinical response., Conclusions: Plasma ctDNA NGS detected a variety of oncogenic drivers with a shorter TAT compared with tissue NGS and matched patients to targeted therapy with clinical response. Positive findings on plasma NGS were highly concordant with tissue NGS and can guide immediate therapy; however, a negative finding in plasma requires further testing. Our findings support the potential incorporation of plasma NGS into practice guidelines., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Osimertinib, a third generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), is active against EGFR-mutant non-small cell lung cancer (NSCLC) resistant to first-/second-generation EGFR-TKIs with the T790M mutation. T790M monitoring in plasma circulating tumor DNA (ctDNA) in patients receiving EGFR-TKIs is less invasive than re-biopsy and could provide valuable clinical information., Methods: Patients with advanced or postoperative recurrent NSCLC with sensitizing EGFR mutations who were planned to receive or were receiving first-/second-generation EGFR-TKI treatment without disease progression were eligible for enrollment. Plasma samples at baseline and every 1-2 months thereafter were analyzed for EGFR mutation status using the cobas®EGFR Mutation Test v2., Results: Between September 2016 and March 2017, 122 patients at 15 Japanese institutions were enrolled. In August 2018, 1291 plasma samples from 121 patients were analyzed for EGFR mutation status. At baseline, a sensitizing EGFR mutation was detected in 29 (23.9%) of 121 patients and T790M mutation was detected in three (2.5%). At follow-up, 66 (54.5%) patients experienced disease progression and 64 (52.9%) discontinued first-line EGFR-TKI treatment. Twenty-two (18.2%) patients showed T790M in plasma ctDNA, of which 15(68.2%) received osimertinib. Although 31 patients received re-biopsy to examine EGFR status at disease progression, T790M was detected in only nine (22.0%) patients, of which 7 (77.8%) received osimertinib., Conclusions: ctDNA monitoring during EGFR-TKI treatment is useful for detecting T790M mutation. The efficacy of osimertinib treatment based on T790M status in plasma ctDNA remains to be established, warranting further research., (© The Author(s) 2019. Published by Oxford University Press.)

In patients with a suspected malignancy, standard-of care management currently includes histopathologic examination and analysis of tumor-specific molecular abnormalities. Herein, we present a 77-year-old patient with an abdominal mass suspected to be a gastrointestinal stromal tumor (GIST) but without the possibility to collect a tumor biopsy. Cell-free DNA extracted from a blood sample was analyzed for the presence of mutations in GIST-specific genes using next generation sequencing. Furthermore, liquid biopsies were used to monitor the levels of mutant DNA copies during treatment with a tumor-specific mutation droplet digital PCR assay that correlated with the clinical and radiological response. Blood-based testing is a good alternative for biopsy-based testing. However, it should only be applied when biopsies are not available or possible to obtain because overall, in only 50%-85% of the cell-free plasma samples is the known tumor mutation detected., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2019.)

Background: Tissue tumor mutational burden (TMB) has emerged as a potential biomarker predicting response to anti-programmed cell death-1 protein receptor (PD-1)/programmed cell death-1 protein ligand (PD-L1) therapy, but few studies have explored using circulating tumor DNA (ctDNA) TMB in non-small cell lung cancer (NSCLC)., Materials and Methods: A total of 136 patients with NSCLC with ctDNA testing were retrospectively evaluated from a single institution, along with a validation cohort from a second institution. ctDNA TMB was derived using the number of detected mutations over the DNA sequencing length., Results: Higher ctDNA TMB was significantly correlated with smoking history ( p  < .05, chi-squared test). Among patients treated with immune checkpoint inhibitors ( n  = 20), higher ctDNA TMB was significantly correlated with shorter progressive free survival (PFS) and overall survival (OS; 45 vs. 355 days; hazard ratio [HR], 5.6; 95% confidence interval [CI], 1.3-24.6; p  < .01, and OS 106 days vs. not reached; HR, 6.0; 95% CI, 1.3-27.1; p  < .01, respectively). In a small independent validation cohort ( n  = 12), there was a nonsignificant numerical difference for higher ctDNA TMB predicting shorter OS but not PFS. ctDNA TMB was not correlated with RECIST tumor burden estimation in the subset of patients treated with immune checkpoint blockade., Conclusion: The findings indicate that higher ctDNA TMB, at the current commercial sequencing length, reflects worse clinical outcomes., Implications for Practice: Biomarkers to identify patients who will respond to immune checkpoint blockade are critical. Tissue tumor mutational burden (TMB) has emerged as a viable biomarker to predict response to anti-PD-1/PD-L1 therapy, but few studies have explored the meaning and potential clinical significance of noninvasive, blood-based TMB. Here, we investigated circulating tumor DNA (ctDNA) TMB and present data demonstrating that current ctDNA TMB may reflect tumor burden and that ctDNA panels with a greater number of mutations may be necessary to more accurately reflect tissue TMB., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2019.)

Motivation: Cancer is a complex disease that involves rapidly evolving cells, often forming multiple distinct clones. In order to effectively understand progression of a patient-specific tumor, one needs to comprehensively sample tumor DNA at multiple time points, ideally obtained through inexpensive and minimally invasive techniques. Current sequencing technologies make the 'liquid biopsy' possible, which involves sampling a patient's blood or urine and sequencing the circulating cell free DNA (cfDNA). A certain percentage of this DNA originates from the tumor, known as circulating tumor DNA (ctDNA). The ratio of ctDNA may be extremely low in the sample, and the ctDNA may originate from multiple tumors or clones. These factors present unique challenges for applying existing tools and workflows to the analysis of ctDNA, especially in the detection of structural variations which rely on sufficient read coverage to be detectable., Results: Here we introduce SViCT , a structural variation (SV) detection tool designed to handle the challenges associated with cfDNA analysis. SViCT can detect breakpoints and sequences of various structural variations including deletions, insertions, inversions, duplications and translocations. SViCT extracts discordant read pairs, one-end anchors and soft-clipped/split reads, assembles them into contigs, and re-maps contig intervals to a reference genome using an efficient k-mer indexing approach. The intervals are then joined using a combination of graph and greedy algorithms to identify specific structural variant signatures. We assessed the performance of SViCT and compared it to state-of-the-art tools using simulated cfDNA datasets with properties matching those of real cfDNA samples. The positive predictive value and sensitivity of our tool was superior to all the tested tools and reasonable performance was maintained down to the lowest dilution of 0.01% tumor DNA in simulated datasets. Additionally, SViCT was able to detect all known SVs in two real cfDNA reference datasets (at 0.6-5% ctDNA) and predict a novel structural variant in a prostate cancer cohort., Availability: SViCT is available at https://github.com/vpc-ccg/svict. Contact:faraz.hach@ubc.ca., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies., Methods: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction., Results: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS , parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing., Conclusions: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution., Clinicaltrialsgov Identifier: NCT02112357., (© 2018 American Association for Clinical Chemistry.)

Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500-1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1-16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection.

Background: Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring., Subjects, Materials, and Methods: Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival., Results: Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival., Conclusion: Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring., Implications for Practice: Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers, suggest that ctDNA can be an effective marker for disease monitoring and that ctDNA level over time is a better predictor of survival than the dynamics of the commonly used biomarker CA19-9. Therefore, ctDNA analysis can be a useful tool for monitoring PDAC treatment response. These results should be further validated in larger sample numbers., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

Background: The current standard for determining eligibility of patients with metastatic melanoma for BRAF-targeted therapy is tissue-based testing of BRAF mutations. As patients are rarely rebiopsied, detection in blood might be advantageous by enabling a comprehensive assessment of tumor mutational status in real time and thereby representing a noninvasive biomarker for monitoring BRAF therapy., Methods: In all, 634 stage I to IV melanoma patients were enrolled at 2 centers, and 1406 plasma samples were prospectively collected. Patients were assigned to 3 separate study cohorts: study 1 for assessment of circulating tumor DNA (ctDNA) as part of companion diagnostics, study 2 for assessment of ctDNA for patients with low tumor burden and for follow-up, and study 3 for monitoring of resistance to BRAF inhibitor (BRAFi) or mitogen-activated protein kinase inhibitor therapy., Results: Overall, a high degree of concordance between plasma and tissue testing results was observed at 90.9% (study 1) and 90.1% (study 2), respectively. Interestingly, discrepant results were in some cases associated with nonresponse to BRAFi (n = 3) or a secondary BRAF -mutant malignancy (n = 5). Importantly, ctDNA results correlated with the clinical course of disease in 95.7% and with response to treatment. Significantly, the detection of BRAF mutant ctDNA preceded relapse assessed by Response Evaluation Criteria in Solid Tumors, and was more specific than serum S100 and lactate dehydrogenase., Conclusions: Blood-based testing compares favorably with standard-of-care tissue-based BRAF mutation testing. Importantly, blood-based BRAF testing correlates with the clinical course, even for early-stage patients, and may be used to predict response to treatment, recurrence, and resistance before radioimaging under BRAFi therapy, thereby enabling considerable improvements in patient treatment., (© 2018 American Association for Clinical Chemistry.)

Sensitive detection of the single nucleotide variants in cell-free DNA (cfDNA) may provide great opportunity for minimally invasive diagnosis and prognosis of cancer and other related diseases. Here, we demonstrate a facile new strategy for quantitative measurement of cfDNA mutations at low abundance in the cancer patients' plasma samples. The method takes advantage of a novel property of lambda exonuclease which effectively digests a 5'-fluorophore modified dsDNA with a 2-nt overhang structure and sensitively responds to the presence of mismatched base pairs in the duplex. It achieves a limit of detection as low as 0.02% (percentage of the mutant type) for BRAFV600E mutation, NRASQ61R mutation and three types of EGFR mutations (G719S, T790M and L858R). The method enabled identification of BRAFV600E and EGFRL858R mutations in the plasma of different cancer patients within only 3.5 h. Moreover, the terminal structure-dependent reaction greatly simplifies the probe design and reduces the cost, and the assay only requires a regular real-time PCR machine. This new method may serve as a practical tool for quantitative measurement of low-abundance mutations in clinical samples for providing genetic mutation information with prognostic or therapeutic implications.

Background: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited., Methods: We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan ® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events., Results: The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations., Conclusions: This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy., (© 2017 American Association for Clinical Chemistry.)

Background: Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy., Methods: NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples., Results: In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification., Conclusions: In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer., (© 2017 American Association for Clinical Chemistry.)

Background: Real-time knowledge of the somatic genome can influence management of patients with metastatic castration-resistant prostate cancer (mCRPC). While routine metastatic tissue biopsy is challenging in mCRPC, plasma circulating tumor DNA (ctDNA) has emerged as a minimally invasive tool to sample the tumor genome. However, no systematic comparisons of matched "liquid" and "solid" biopsies have been performed that would enable ctDNA profiling to replace the need for direct tissue sampling., Methods: We performed targeted sequencing across 72 clinically relevant genes in 45 plasma cell-free DNA (cfDNA) samples collected at time of metastatic tissue biopsy. We compared ctDNA alterations with exome sequencing data generated from matched tissue and quantified the concordance of mutations and copy number alterations using the Fisher exact test and Pearson correlations., Results: Seventy-five point six percent of cfDNA samples had a ctDNA proportion greater than 2% of total cfDNA. In these patients, all somatic mutations identified in matched metastatic tissue biopsies were concurrently present in ctDNA. Furthermore, the hierarchy of variant allele fractions for shared mutations was remarkably similar between ctDNA and tissue. Copy number profiles between matched liquid and solid biopsy were highly correlated, and individual copy number calls in clinically actionable genes were 88.9% concordant. Detected alterations included AR amplifications in 22 (64.7%) samples, SPOP mutations in three (8.8%) samples, and inactivating alterations in tumor suppressors TP53 , PTEN , RB1 , APC , CDKN1B , BRCA2 , and PIK3R1 . In several patients, ctDNA sequencing revealed robust changes not present in paired solid biopsy, including clinically relevant alterations in the AR, WNT, and PI3K pathways., Conclusions: Our study shows that, in the majority of patients, a ctDNA assay is sufficient to identify all driver DNA alterations present in matched metastatic tissue and supports development of DNA biomarkers to guide mCRPC patient management based on ctDNA alone., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor-specific genetic alterations. The clinical utility has been intensively investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor-specific mutations, whereas the use of total cell-free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta-analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection., Materials and Methods: A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed, and meta-analysis applied on both aggregate data extraction and individual patients' data., Results: Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing [beads, emulsification, amplification, and magnetics] technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta-analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03-2.82, p  < .0001)., Conclusion: The total cfDNA levels are high in patients with mCRC and bear strong prognostic information, which should be tested prospectively by using a predefined cut-off value based on normal values in healthy cohorts. Finally, the potential use of cfDNA for detection of tumor-specific mutations was emphasized in a large individual patients' data meta-analysis., Implications for Practice: Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2017.)

Neuroendocrine carcinoma of the cervix is an ultra-rare malignancy with a poor prognosis and limited treatment options. Checkpoint blockade immunotherapy has rapidly developed into an emerging standard of care for several common disease types. Interestingly, in preclinical and retrospective clinical data, radiation therapy has been demonstrated to synergize with checkpoint inhibitors. Here we report a patient with metastatic, chemotherapy-refractory neuroendocrine carcinoma who presented with partial bowel obstruction due to a large tumor burden. Genomic analysis demonstrated a high number of alterations on liquid biopsy (circulating tumor DNA [ctDNA]), which prompted treatment with stereotactic body radiation therapy (SBRT) combined with anti-programmed cell death protein 1 antibody. Tissue rebiopsy and comprehensive genomic profiling confirmed high tumor mutational burden and a mismatch repair gene defect. The patient manifested near-complete systemic resolution of disease, ongoing at 10+ months. We discuss the novel treatment modality of SBRT combined with a checkpoint inhibitor and the implications of molecular profiling and tumor mutational burden as potential predictors of response., Key Points: High-grade, large-cell neuroendocrine carcinoma of the cervix is an ultra-rare malignancy that carries a grim prognosis.Next-generation sequencing may reveal key mutations in MSH2 genes amongst others. MSH2 mutations target the DNA mismatch repair process and can predispose patients to malignancies with high mutational burdens.Immunotherapy combined with radiation therapy can elicit a significant response, both within and outside the field of radiation. The latter is termed the "abscopal" effect, perhaps mediated by radiation-induced cross presentation of tumor antigens resulting in immune activation.Sequencing of blood-derived ctDNA showed a high number of alterations, and tissue sequencing confirmed a high tumor mutational burden as a consequence of a mismatch repair gene defect. This observation led to a therapeutic "match" with an anti- programmed cell death protein 1 antibody combined with SBRT, resulting in a durable (10+ months), near-complete remission in a patient with advanced chemotherapy-refractory disease., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2017.)

Breast cancer (BC) is the most common malignancy in women worldwide, with approximately two-thirds having hormone receptor-positive (HR+) tumors. New endocrine therapy (ET) strategies include combining ET agents as well as adding inhibitors targeting growth factors, angiogenesis, the mechanistic target of rapamycin, phosphoinositide 3-kinase (PI3K), or cyclin-dependent kinase 4/6 to ET. Level 1 evidence supports use of fulvestrant plus anastrozole or palbociclib plus letrozole as first-line therapy for HR+/HER- advanced BC with special consideration for the former in ET-naïve patients, as well as everolimus plus exemestane or palbociclib plus fulvestrant as second-line therapy with special consideration in select first-line patients. Although the safety profiles of these combinations are generally predictable and manageable, both everolimus and palbociclib are associated with an increased risk of potentially serious or early-onset toxicities requiring individualized a priori adverse event risk stratification, earlier and more rigorous agent-specific monitoring, and patient education. Although each of these combinations improves progression-free survival, none with the exception of anastrazole plus fulvestrant have demonstrated improved overall survival. PI3K catalytic-α mutations assessed from circulating tumor DNA represent the first potentially viable serum biomarker for the selection of ET combinations, and new data demonstrate the feasibility of this minimally invasive technique as an alternative to traditional tissue analysis. Therapeutic ratios of select ET combinations support their use in first- and second-line settings, but optimal sequencing has yet to be determined., The Oncologist: 2017;22:12-24 IMPLICATIONS FOR PRACTICE: Emerging data show that new endocrine therapy (ET) combinations can improve progression-free and overall survival outcomes in patients with hormone receptor-positive, HER2-negative (HR+/HER-) advanced breast cancer. Level 1 evidence supports consideration of dual ET regimens, particularly in ET-naïve patients, or palbociclib plus letrozole as first-line therapy, as well as the addition of mTOR or CDK4/6 inhibitors to established ET in the second-line setting and in select first-line patients. Some combinations are associated with increased risk of class-specific toxicities that will require individualized risk stratification, earlier and more rigorous agent-specific monitoring, and patient education. Recent data on a noninvasive biomarker assay that predicts response to a phosphoinositide 3-kinase inhibitor demonstrates the feasibility of this minimally invasive technique as an alternative to traditional tissue analysis., (© AlphaMed Press 2016.)