Analysis Of Cell-free DNA Research Articles

The landscape of cell-free mitochondrial DNA in liquid biopsy for cancer detection

BackgroundExisting methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection.ResultsWe survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81.ConclusionsThe mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.

GCparagon: evaluating and correcting GC biases in cell-free DNA at the fragment level.

Analyses of cell-free DNA (cfDNA) are increasingly being employed for various diagnostic and research applications. Many technologies aim to increase resolution, e.g. for detecting early-stage cancer or minimal residual disease. However, these efforts may be confounded by inherent base composition biases of cfDNA, specifically the over - and underrepresentation of guanine (G) and cytosine (C) sequences. Currently, there is no universally applicable tool to correct these effects on sequencing read-level data. Here, we present GCparagon, a two-stage algorithm for computing and correcting GC biases in cfDNA samples. In the initial step, length and GC base count parameters are determined. Here, our algorithm minimizes the inclusion of known problematic genomic regions, such as low-mappability regions, in its calculations. In the second step, GCparagon computes weights counterbalancing the distortion of cfDNA attributes (correction matrix). These fragment weights are added to a binary alignment map (BAM) file as alignment tags for individual reads. The GC correction matrix or the tagged BAM file can be used for downstream analyses. Parallel computing allows for a GC bias estimation below 1 min. We demonstrate that GCparagon vastly improves the analysis of regulatory regions, which frequently show specific GC composition patterns and will contribute to standardized cfDNA applications.

Multi-faceted attributes of salivary cell-free DNA as liquid biopsy biomarkers for gastric cancer detection

BackgroundRecent advances in circulating cell-free DNA (cfDNA) analysis from biofluids have opened new avenues for liquid biopsy (LB). However, current cfDNA LB assays are limited by the availability of existing information on established genotypes associated with tumor tissues. Certain cancers present with a limited list of established mutated cfDNA biomarkers, and thus, nonmutated cfDNA characteristics along with alternative biofluids are needed to broaden the available cfDNA targets for cancer detection. Saliva is an intriguing and accessible biofluid that has yet to be fully explored for its clinical utility for cancer detection.MethodsIn this report, we employed a low-coverage single stranded (ss) library NGS pipeline “Broad-Range cell-free DNA-Seq” (BRcfDNA-Seq) using saliva to comprehensively investigate the characteristics of salivary cfDNA (ScfDNA). The identification of cfDNA features has been made possible by applying novel cfDNA processing techniques that permit the incorporation of ultrashort, ss, and jagged DNA fragments. As a proof of concept using 10 gastric cancer (GC) and 10 noncancer samples, we examined whether ScfDNA characteristics, including fragmentomics, end motif profiles, microbial contribution, and human chromosomal mapping, could differentiate between these two groups.ResultsIndividual and integrative analysis of these ScfDNA features demonstrated significant differences between the two cohorts, suggesting that disease state may affect the ScfDNA population by altering nuclear cleavage or the profile of contributory organism cfDNA to total ScfDNA. We report that principal component analysis integration of several aspects of salivary cell-free DNA fragmentomic profiles, genomic element profiles, end-motif sequence patterns, and distinct oral microbiome populations can differentiate the two populations with a p value of < 0.0001 (PC1).ConclusionThese novel features of ScfDNA characteristics could be clinically useful for improving saliva-based LB detection and the eventual monitoring of local or systemic diseases.

1555P Cell-free DNA analysis in patients with metastatic gastroesophageal adenocarcinoma: Preliminary results of the REGIRI - PRODIGE 58 ancillary study

1555P Cell-free DNA analysis in patients with metastatic gastroesophageal adenocarcinoma: Preliminary results of the REGIRI - PRODIGE 58 ancillary study

Synergistic signal amplification of a 3D Dual-Core DNA nanomachine and PCNs@AuPdCe hybrid nanozymes for ultrasensitive electrochemical detection of Cell-free DNA

The potential for early cancer detection through cell-free DNA (cfDNA) analysis is hindered by the low abundance and susceptibility to chemical damage of cfDNA, presenting a formidable challenge for precision analysis. In this study, we introduce an innovative electrochemical biosensor that leverages a three-dimensional (3D) dual-core DNA nanomachine driven by exonuclease III (EXO III) and trimetallic-deposited porous coordination network-224 hybrid nanozymes (PCNs@AuPdCe). The DNA nanomachine is designed with target cfDNA and 8-legged polyamidoamine-ssDNA (octopus-like) as dual dynamic cores, while magnetic beads serve as the 3D orbit, enabling highly stable cascade amplification from 1 to 8 to N. The newly synthesized PCNs@AuPdCe exhibits remarkable superoxide dismutase-, catalase-, and peroxidase-like activity and serves as a signal tag. The synergistic amplification resulting from the dual-core DNA nanomachine and PCNs@AuPdCe enables the biosensor to detect cfDNA levels as low as “a” M with a broad detection range from 1 fM to 1 nM. Analysis of real plasma samples demonstrates the robust anti-interference ability and promising clinical applications of the biosensor. Therefore, the construction of this electrochemical biosensor is of great significance to promote the development of in vitro diagnostic technology for early cancer screening.

Cell-Free DNA Fragmentomics for Early Detection of Malignant Transformation in NF1 Patients Harboring Plexiform Neurofibromas

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that are resistant to radiotherapy and chemotherapy, and are associated with poor survival. MPNST typically arise from benign or atypical plexiform neurofibromas (PN) in the setting of neurofibromatosis type 1 (NF1). Unfortunately, early detection by imaging and clinical examination remains very challenging. We hypothesize that cell-free DNA fragmentomic analysis can be used to precisely detect the risk for developing MPNST in NF1 patients, which could significantly improve clinical outcomes in the future through early cancer detection. We performed low-pass whole genome sequencing of plasma cell-free DNA samples from healthy controls (n = 21), patients with PN (n = 113), AN (n = 39) and MPNST (n = 71). Cell-free DNA was analyzed at a per-fragment level by tracking 4-nucleotide end motifs, and using a bin-wise fragmentomic machine learning approach called DELFI, which integrates across ratios of short (100-150 bp) versus long (151-220 bp) fragments in 5 megabase bins across the genome. End motif frequencies were compared between MPNST and PN patients, and receiver operating characteristic (ROC) analysis was used to classify disease states using DELFI fragmentomic scores. Using DELFI for genome-wide fragmentomics, followed by ROC analysis, we obtained the following accuracies: MPNST vs. AN (acc 0.62), MPNST vs. PN (acc 0.83), MPNST vs. healthy (acc 0.86), AN vs. PN (acc 0.87), AN vs. healthy (acc 0.72) and PN vs. healthy (acc 0.88). DELFI-based cell-free DNA fragmentomics was superior to the ichor CNA-based copy number alteration classifier that we published previously (Szymanski et al. PLOS Medicine, 2021) for all the aforementioned group comparisons (p < 0.001 by McNemar's test). Strikingly, the two AN cases with DELFI scores most closely resembling MPNST were separately identified by independent clinical care teams to have very high-risk features and recent history warranting short-interval follow up. Specifically, one patient had a paraspinal AN exhibiting new onset pain and rapid growth on imaging, while the other had increased FDG uptake of multiple AN lesions in a supraclavicular field treated years earlier with radiotherapy. We also measured fragment end motifs across different patient groups, and found that the CTCA end motif was more commonly present in cell-free DNA from MPNST and PN patients compared to healthy donors (Bonferroni-corrected p-value < 0.001). This study suggests that fragmentomic analysis of cell-free DNA can accurately risk-stratify NF1 patients with plexiform neurofibromas. Cell-free DNA fragmentomics outperformed our previously published copy number alteration method for distinguishing closely related patient subgroups with different malignancy risks. Atypical neurofibroma patients with cell-free DNA fragmentomic scores suggestive of MPNST were deemed clinically very high-risk for malignant transformation.

Efficacy of cell-free DNA as a diagnostic biomarker in breast cancer patients

Breast cancer is the most prevalent and leading cause of mortality worldwide among women. Cell-free DNA (cfDNA) analysis is an alternative quantitative approach to conventional methods for cancer diagnosis. The current research project aimed to determine the efficacy of cfDNA as a diagnostic biomarker in breast cancer patients in Pakistan. Eighty-four female breast cancer patients were selected as cases, and 152 healthy females as controls. Immunohistochemistry was performed to identify tumor biomarkers along with clinical profiling. cfDNA was extracted from serum using the phenol–chloroform method. The cfDNA level in the serum was estimated using Agarose Gel Electrophoresis and Nanodrop. SPPS version 25.0 was used to perform statistical analyses. The results showed that the cancer biomarkers were significantly associated with breast cancer. The changes in hematological parameters were insignificant, whereas the biochemical parameter variations between the cases and controls were statistically significant. A significant association of cfDNA level with breast cancer was observed. Further cfDNA levels and cancer biomarkers were not statistically significant. A significant correlation was observed between cfDNA and biochemical parameters, except for creatinine, whereas hematological parameters showed no significant correlation.ROC analysis declared cfDNA as an authentic diagnostic marker for breast cancer. It was concluded that the level of cfDNA is significantly increased in breast cancer patients and can be utilized as a diagnostic biomarker.

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (One Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

P204 Use of cell-free DNA analysis to determine need for renal transplant biopsy after detection of circulating donor-specific anti-HLA antibodies in renal transplant recipients

P204 Use of cell-free DNA analysis to determine need for renal transplant biopsy after detection of circulating donor-specific anti-HLA antibodies in renal transplant recipients

A highly efficient scheme for library preparation from single-stranded DNA

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.

Redefining precision radiotherapy through liquid biopsy.

Precision radiotherapy refers to the ability to deliver radiation doses with sub-millimetre accuracy. It does not however consider individual variation in tumour or normal tissue response, failing to maximise tumour control and minimise toxicity. Combining precise delivery with personalised dosing, through analysis of cell-free DNA, would redefine precision in radiotherapy.

Noninvasive Evaluation of Fetal Zygosity in Twin Pregnancies Involving a Binary Analysis of Single-Nucleotide Polymorphisms

Twin pregnancy constitutes significant risks for maternal and fetal health, which is usually detected by ultrasound examination at early gestation. However, the imaging-based approach may not accurately identify all twins confounded by practical or clinical variables. The analysis of fetal cell-free DNA in noninvasive prenatal screening assays can completement the ultrasound method for twin detection, which differentiates fraternal or identical twins based on their distinct genotypes. Here, a new noninvasive prenatal screening employing high-coverage next-generation sequencing for targeted nucleotide polymorphisms was developed for detection of zygosity and determination of fetal fraction in twin pregnancies. This method utilizes a binary analysis of both the number and allelic fraction of fetus-specific single-nucleotide polymorphisms to infer the zygosity. In 323 samples collected from 215 singleton, 90 dizygotic, and 18 monozygotic twin pregnancies, all 90 dizygotic twins were correctly detected, with a 100% sensitivity and a 100% specificity. In addition, this method can detect complex pregnancies, such as egg donors, contamination, and twins with complete hydatidiform mole. The fetus-specific fetal fraction change was monitored in nine dizygotic twin pregnancies, which demonstrated highly variable dynamics of fetal cell-free DNA turnover up to 7 weeks after twin reduction. Overall, this study provides a new noninvasive prenatal screening strategy for the accurate identification of twin zygosity and quantification of fetal fraction, which has important clinical implications for the management of twin pregnancies.

TIPS-10 FEASIBILITY OF A MULTI-MODALITY CENTRAL NERVOUS SYSTEM SCREENING EVALUATION IN HER2+ BREASTCANCER PATIENTS

Abstract BACKGROUND Breast cancer is the most common cancer in the world and the second most common to metastasize to the central nervous system (CNS). CNS-metastases are the most common type of brain tumor and have a rising incidence. They are associated with neurologic morbidity and mortality and remain a major clinical challenge. Survival withCNS-metastases is short with a median survival in the order of months. The incidence of CNS-metastases is 5.1% in breast cancer however true prevalence is not known; at autopsy this incidence ranges between 20-40%. One contributing factor towards the development of CNS-metastases in breast cancer is HER2-positivity. Between 25%-48% of patients with HER2-positive metastatic breast cancer develop CNS-metastases. Currently, routine CNS surveillance imaging is not recommended, however guidelines suggest having a low threshold for evaluation with imaging and/or cerebrospinal fluid (CSF) analysis because of the high rate of CNS-metastases. The clinical relevance of occult CNS-metastases, survival after identification of occult CNS-metastases and the importance of earlier initiation of CNS therapy are unknown. Existing treatment strategies for CNS-metastases rely on a combinatorial approach of chemotherapy, radiotherapy and/or surgery which typically affords limited disease control. This trial seeks to study if surveillance imaging and CSF analysis are feasible as early disease detection mechanisms. METHODS This is a two-cohort study to establish feasibility of multi-modality CNS surveillance. Patients with HER2+ metastatic breast cancer patients (with no known CNS-metastases) who are either Stage IV (Cohort A) or Stage II/III are eligible (Cohort B). On study, patients will undergo MRI Brain and CSF analysis at 6 monthly intervals. CSF analysis includes cytology, circulating tumor cells and cell-free DNA analysis. We plan to enroll 20 patients (10 onto each cohort). 1 of 20 patients have enrolled. This is the first multi-modality screening feasibility study for patients HER2-positive breast cancer.

Label-Free Human Disease Characterization through Circulating Cell-Free DNA Analysis Using Raman Spectroscopy.

Circulating cell-free DNA (ccfDNA) is a liquid biopsy biomaterial attracting significant attention for the implementation of precision medicine diagnostics. Deeper knowledge related to its structure and biology would enable the development of such applications. In this study, we employed Raman spectroscopy to unravel the biomolecular profile of human ccfDNA in health and disease. We established reference Raman spectra of ccfDNA samples from healthy males and females with different conditions, including cancer and diabetes, extracting information about their chemical composition. Comparative observations showed a distinct spectral pattern in ccfDNA from breast cancer patients taking neoadjuvant therapy. Raman analysis of ccfDNA from healthy, prediabetic, and diabetic males uncovered some differences in their biomolecular fingerprints. We also studied ccfDNA released from human benign and cancer cell lines and compared it to their respective gDNA, confirming it mirrors its cellular origin. Overall, we explored for the first time Raman spectroscopy in the study of ccfDNA and provided spectra of samples from different sources. Our findings introduce Raman spectroscopy as a new approach to implementing liquid biopsy diagnostics worthy of further elaboration.

Non-Invasive Prenatal Testing (NIPT): Reliability, Challenges, and Future Directions.

Non-invasive prenatal testing was first discovered in 1988; it was primarily thought to be able to detect common aneuploidies, such as Patau syndrome (T13), Edward Syndrome (T18), and Down syndrome (T21). It comprises a simple technique involving the analysis of cell-free foetal DNA (cffDNA) obtained through maternal serum, using advances in next-generation sequencing. NIPT has shown promise as a simple and low-risk screening test, leading various governments and private organizations worldwide to dedicate significant resources towards its integration into national healthcare initiatives as well as the formation of consortia and research studies aimed at standardizing its implementation. This article aims to review the reliability of NIPT while discussing the current challenges prevalent among different communities worldwide.

A Systematic Review and Meta-analysis of Cell-Free DNA Testing for Detection of Fetal Sex Chromosome Aneuploidy

ABSTRACT Noninvasive prenatal screening has become a valuable tool in detecting a variety of disorders and abnormalities, including sex chromosome aneuploidies (SCAs). The test uses cell-free DNA (cfDNA) analysis to identify risk for chromosomal abnormalities, which can then be more fully investigated using other tests. Much of previous research focused on cfDNA has assessed autosomal aneuploidies, but few studies have specifically focused on SCAs. The aim of this systematic review and meta-analysis was to assess the accuracy of cfDNA testing in identifying SCAs in singleton pregnancies. Studies were identified by keywords associated with cfDNA, SCA, and noninvasive prenatal testing, and studies were included based on relevance. Inclusion criteria were limited to singleton pregnancies that underwent cfDNA testing with confirmation by diagnostic genetic testing between January 1, 2010 and December 1, 2021. A total of 21 articles were selected for the final meta-analysis. All included studies reported results for monosomy X (45,X), representing nearly 13,000 tests with 254 confirmed positive results, 133 false-positives, and 18 false-negatives. Estimated sensitivity based on this sample was 98.8%, with specificity of 99.4%. Positive and negative predictive values were 14.5% and 100%, respectively. Of the 21 included studies, 16 also reported results for Klinefelter syndrome (47,XXY), representing just over 11,000 tests with 62 confirmed positive results, 10 false-positives, and 11 false-negatives. Estimated sensitivity was 100% and specificity was 100%, with positive predictive value of 97.7% and negative predictive value of 100%. Thirteen of 21 studies included reported results for triple X syndrome (47,XXX), representing roughly 10,000 tests with 38 confirmed positive results, 18 false-positives, and 1 false-negative. This showed an estimated sensitivity of 100% and specificity of 99.9%, with positive predictive value of 61.6% and negative predictive value of 100%. Finally, 9 of the studies included in this meta-analysis reported results for Jacobs syndrome (47,XYY), representing approximately 8500 tests with 22 confirmed positive results, 2 false-positives, and no false-negatives. This led to an estimated sensitivity and specificity of 100%, with positive and negative predictive values also 100%. These results provide evidence that cfDNA testing is effective in identification of SCAs in pregnancies at high risk. This particular population limits the generalizability of the findings but adds to the literature surrounding the effectiveness and accuracy of this method. The analysis used strict criteria to define the studies that were included, yet still included a large sample of tests (12,000), which adds to the statistical and practical significance of the analysis. Generalizing the findings of this study is difficult based on both the fact that the sample did not include low-risk individuals and that positive predictive values varied greatly with prevalence, impacting clinical significance. Overall, cfDNA testing for SCAs has both high sensitivity and specificity, along with a high negative predictive value of 99.99% or above for each SCA examined. Further research is needed to confirm these findings and extend knowledge to a more diverse population.

Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.

Continuous centrifugal microfluidics identifies the marker and size heterogeneity of circulating trophoblasts for accurate non-invasive prenatal diagnosis

As the prevalence of newborns with congenital anomalies continues to increase, the importance of prenatal diagnosis has been emphasized. Since the commonly used methods for prenatal diagnosis, such as amniocentesis/chorionic villi sampling, are invasive and increase the risk of miscarriage, cell-free fetal DNA (cffDNA) analysis has been widely used in clinic. However, it still has critical drawbacks, such as experimental failure at low fetal fraction of cffDNA and limited accuracy due to the need of statistical prediction, reducing the reliability of results. Therefore, there is an urgent need for non-invasive prenatal diagnosis (NIPD) with circulating fetal cells, such as circulating trophoblasts (cTBs), containing complete fetal genetic information. However, current technologies are biased (marker- and size-based) isolation approaches and are limited in practical utility in clinic. Here we introduce a fully automated, unbiased isolation platform for maximal yield and heterogeneity study of cTBs, using a continuous centrifugal microfluidics-based circulating trophoblast disc (CCM-CTBD). The CCM-CTBD achieved consistently high recovery rates using three different trophoblast cell lines with heterogeneous sizes and marker expressions. Next, we successfully isolated cTBs from maternal peripheral blood, and identified their surface marker and size heterogeneity. In addition, we found unreported cells with double-positive staining (<CD45> and <EpCAM/HLA-G/Pan-CK>) and confirmed their fetal origin by identifying the Y chromosome. Moreover, the platform could isolate fetal cells as early as 8 gestational weeks. This unbiased isolation platform can potentially be a powerful tool for accurate, safe, and early prenatal diagnosis with clinical utility.

Individualized dynamic methylation-based analysis of cell-free DNA in postoperative monitoring of lung cancer

BackgroundThe feasibility of DNA methylation-based assays in detecting minimal residual disease (MRD) and postoperative monitoring remains unestablished. We aim to investigate the dynamic characteristics of cancer-related methylation signals and the feasibility of methylation-based MRD detection in surgical lung cancer patients.MethodsMatched tumor, tumor-adjacent tissues, and longitudinal blood samples from a cohort (MEDAL) were analyzed by ultra-deep targeted sequencing and bisulfite sequencing. A tumor-informed methylation-based MRD (timMRD) was employed to evaluate the methylation status of each blood sample. Survival analysis was performed in the MEDAL cohort (n = 195) and validated in an independent cohort (DYNAMIC, n = 36).ResultsTumor-informed methylation status enabled an accurate recurrence risk assessment better than the tumor-naïve methylation approach. Baseline timMRD-scores were positively correlated with tumor burden, invasiveness, and the existence and abundance of somatic mutations. Patients with higher timMRD-scores at postoperative time-points demonstrated significantly shorter disease-free survival in the MEDAL cohort (HR: 3.08, 95% CI: 1.48–6.42; P = 0.002) and the independent DYNAMIC cohort (HR: 2.80, 95% CI: 0.96–8.20; P = 0.041). Multivariable regression analysis identified postoperative timMRD-score as an independent prognostic factor for lung cancer. Compared to tumor-informed somatic mutation status, timMRD-scores yielded better performance in identifying the relapsed patients during postoperative follow-up, including subgroups with lower tumor burden like stage I, and was more accurate among relapsed patients with baseline ctDNA-negative status. Comparing to the average lead time of ctDNA mutation, timMRD-score yielded a negative predictive value of 97.2% at 120 days prior to relapse.ConclusionsThe dynamic methylation-based analysis of peripheral blood provides a promising strategy for postoperative cancer surveillance.Trial registrationThis study (MEDAL, MEthylation based Dynamic Analysis for Lung cancer) was registered on ClinicalTrials.gov on 08/05/2018 (NCT03634826). https://clinicaltrials.gov/ct2/show/NCT03634826.

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.