Cell-free DNA In Patients Research Articles

Cell free DNA in patients with pancreatic adenocarcinoma: clinicopathologic correlations

Detection of circulating tumor DNA (ctDNA) from plasma cell free DNA (cfDNA) has shown promise for diagnosis, therapeutic targeting, and prognosis. This study explores ctDNA detection by next generation sequencing (NGS) and associated clinicopathologic factors in patients with pancreatic adenocarcinoma (PDAC). Patients undergoing surgical exploration or resection of pancreatic lesions were enrolled with informed consent. Plasma samples (4–6 ml) were collected prior to surgery and cfDNA was recovered from 95 plasma samples. Adequate cfDNA for NGS (20 ng) was obtained from 81 patients. NGS was performed using the Oncomine Lung cfDNA assay on the Ion Torrent S5 sequencing platform. Twenty-five patients (30.9%) had detectable mutations in KRAS and/or TP53 with allele frequencies ranging from 0.05 to 8.5%, while mutations in other genes were detected less frequently and always along with KRAS or TP53. Detectable ctDNA mutations were more frequent in patients with poorly differentiated tumors, and patients without detectable ctDNA mutations showed longer survival (medians of 10.5 months vs. 18 months, p = 0.019). The detection of circulating tumor DNA in pancreatic adenocarcinomas is correlated with worse survival outcomes.

Comprehensive characterization of circulating tumor cells and cell-free DNA in patients with metastatic melanoma.

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.

Cell-free DNA in patients with sepsis: long term trajectory and association with 28-day mortality and sepsis-associated acute kidney injury.

Outcome-prediction in patients with sepsis is challenging and currently relies on the serial measurement of many parameters. Standard diagnostic tools, such as serum creatinine (SCr), lack sensitivity and specificity for acute kidney injury (AKI). Circulating cell-free DNA (cfDNA), which can be obtained from liquid biopsies, can potentially contribute to the quantification of tissue damage and the prediction of sepsis mortality and sepsis-associated AKI (SA-AKI). We investigated the clinical significance of cfDNA levels as a predictor of 28-day mortality, the occurrence of SA-AKI and the initiation of renal replacement therapy (RRT) in patients with sepsis. Furthermore, we investigated the long-term course of cfDNA levels in sepsis survivors at 6 and 12 months after sepsis onset. Specifically, we measured mitochondrial DNA (mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, mt-ND1, and mitochondrially encoded cytochrome C oxidase subunit III, mt-CO3) and nuclear DNA (nuclear ribosomal protein S18, n-Rps18) in 81 healthy controls and all available samples of 150 intensive care unit patients with sepsis obtained at 3 ± 1 days, 7 ± 1 days, 6 ± 2 months and 12 ± 2 months after sepsis onset. Our analysis revealed that, at day 3, patients with sepsis had elevated levels of cfDNA (mt-ND1, and n-Rps18, all p<0.001) which decreased after the acute phase of sepsis. 28-day non-survivors of sepsis (16%) had higher levels of cfDNA (all p<0.05) compared with 28-day survivors (84%). Patients with SA-AKI had higher levels of cfDNA compared to patients without AKI (all p<0.05). Cell-free DNA was also significantly increased in patients requiring RRT (all p<0.05). All parameters improved the AUC for SCr in predicting RRT (AUC=0.88) as well as APACHE II in predicting mortality (AUC=0.86). In summary, cfDNA could potentially improve risk prediction models for mortality, SA-AKI and RRT in patients with sepsis. The predictive value of cfDNA, even with a single measurement at the onset of sepsis, could offer a significant advantage over conventional diagnostic methods that require repeated measurements or a baseline value for risk assessment. Considering that our data show that cfDNA levels decrease after the first insult, future studies could investigate cfDNA as a "memoryless" marker and thus bring further innovation to the complex field of SA-AKI diagnostics.

Evaluating the effectiveness of pre-operative diagnosis of ovarian cancer using minimally invasive liquid biopsies by combining serum human epididymis protein 4 and cell-free DNA in patients with an ovarian mass

ObjectiveTo assess the feasibility of scalable, objective, and minimally invasive liquid biopsy-derived biomarkers such as cell-free DNA copy number profiles, human epididymis protein 4 (HE4), and cancer antigen 125 (CA125)...

Sequential RAS mutations evaluation in cell-free DNA of patients with tissue RAS wild-type metastatic colorectal cancer: the PERSEIDA (Cohort 2) study

PurposeRAS (KRAS/NRAS) mutational status on a tumor biopsy is mandatory to guide the best treatment in metastatic colorectal cancer (mCRC). Determining the RAS mutational status by tumor-tissue biopsy is essential in guiding the optimal treatment decision for mCRC. RAS mutations are negative predictive factors for the use of EGFR monoclonal antibodies. Cell-free DNA (cfDNA) analysis enables minimally invasive monitoring of tumor evolution.Methods/patientsPERSEIDA was an observational, prospective study assessing cfDNA RAS, BRAF and EGFR mutations (using Idylla™) in first-line mCRC, RAS wild-type (baseline tumor-tissue biopsy) patients (cohort 2). Plasma samples were collected before first-line treatment, after 20 ± 2 weeks, and at disease progression.Results117 patients were included (103 received panitumumab + chemotherapy as first-line treatment). At baseline, 7 (6.8%) patients had RAS mutations, 4 (3.9%) BRAF mutations and no EGFR mutations were detected (cfDNA, panitumumab + chemotherapy subpopulation [panitumumab + Ch]). The baseline RAS mutational status concordance between tissue and liquid biopsies was 94.0% (93.2%, panitumumab + Ch). At 20 weeks, only one patient in the study (included in the panitumumab + Ch) had an emerging cfDNA RAS mutation. No emerging BRAF or EGFR mutations were reported. At disease progression, 6 patients had emergent mutations not present at baseline (RAS conversion rate: 13.3% [6/45]; 15.0% [6/40], panitumumab + Ch).ConclusionsThe concordance rate between liquid and solid biopsies at baseline was very high, as previously reported, while our results suggest a considerable emergence of RAS mutations during disease progression. Thus, the dynamics of the genomic landscape in ctDNA may provide relevant information for the management of mCRC patients.

Abstract 6504: Exploratory analysis using serial cell-free DNA in patients treated with amivantamab in non-small cell lung cancer with EGFR exon 20 insertion mutations

Abstract Introduction: Epidermal growth factor receptor (EGFR) exon 20 insertion (ex20 ins) mutation showed limited clinical activity with conventional EGFR tyrosine kinase inhibitors (TKIs). Amivantamab is the first bispecific antibody to be approved for the treatment of metastatic non-small cell lung cancer (NSCLC) with EGFR ex20ins mutation. However, limited data is available in terms of predictive biomarkers and the resistance mechanism. In this study, we conducted an exploratory analysis to elucidate a genomic landscape using circulating tumor DNA (ctDNA) from plasma samples. Patients and Methods: This study include result from 79 blood samples, which has been prospectively collected from 27 EGFR ex20ins mutated NSCLC patients (confirmed by either tissue or plasma-based test) who were treated with amivantamab. ctDNAs were obtained from plasma, which was serially collected at baseline, after 2 cycles of treatment, 2 cycles prior to the treatment discontinuation, and at the time point of disease progression. Guardant OMNI panel (Guardant Health, CA, USA) was used for the genomic profiling. Results: The median follow-up duration was 5.8 months (range 2.7-12.7). The overall response rate was 40.7% (95% confidence interval [CI] 24.5-59.2, n=11), and median progression-free survival was 5.8 months (95% CI 3.9-15.9) with a median duration of response of 9.8 months (95% CI, 7.3-NA). Patients with EGFR ex20ins not detected from ctDNA at baseline (n=10) showed significantly longer PFS compared to patients with EGFR ex20ins identified at baseline plasma sample (n=17) (14.6 vs 3.9 months; hazard ratio [HR] 0.40, 95% CI 0.17-0.95, P&amp;lt;0.05).The most common concurrent somatic variations at baseline were TP53 (62.9%), TERT (18.5%), ATM (18.5%). Longer median PFS was observed in patients without EGFR amplification (n=18) compared to patients with concurrent EGFR amplification (n=9) (11.1 vs. 2.5 months; HR 0.30, 95% CI 0.11-0.77, P &amp;lt;0.05). The presence of plasma EGFR ex20ins at C2D15 predicted poor PFS (17.8 vs 2.7 months; HR 0.17, 95% CI 0.06-0.47, P &amp;lt;0.01), regardless of baseline its presence or absence. Based on the paired analysis using baseline and at the timepoint of disease progression, putative resistance mechanism to amivantamab was identified in 14 patients (60.8%), which were PDGFRB (28.5%), followed by PTEN (21.4%), CHEK2 (21.4%). Conclusions: This study provides a comprehensive longitudinal ctDNA analysis in patients with EGFR ex20ins mutant NSCLC who were treated with amivantamab. Our results suggested that the presence of ctDNA for EGFR ex20ins mutation and co-alteration with EGFR amplification at baseline might be related to the clinical outcomes of amivantamab. Citation Format: Geun-Ho Park, Sehhoon Park, Hyen-Ae Jung, Jong-Mu Sun, Jin Seok Ahn, Myung-Ju Ahn, Se-Hoon Lee. Exploratory analysis using serial cell-free DNA in patients treated with amivantamab in non-small cell lung cancer with EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6504.

Abstract 5203: Identification of prognostic and predictive biomarkers for palbociclib add-on therapy using cancer panel sequencing of cell-free DNA in patients with HR-positive HER2-negative advanced and metastatic breast cancer with fulvestrant resistance

Abstract Purpose: The combination of cyclin-dependent kinase 4/6 inhibitors with endocrine therapies has shown promise for improving the survival of patients with hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative advanced and metastatic breast cancer (ABC/MBC). The FUTURE trial (UMIN000029294) demonstrated that adding palbociclib after acquiring fulvestrant resistance is potentially safe and effective. To further identify biomarkers for patient selection, this preplanned translational sub-study aims to use cancer panel sequencing of cell-free DNA (cfDNA) to discover prognostic and predictive biomarkers for additional palbociclib treatment after acquiring fulvestrant resistance in patents with HR-positive/HER2-negative ABC/MBC. Methods: Herein, we utilized 149 cfDNA samples from 61 patients with disease progression after fulvestrant monotherapy and who subsequently received fulvestrant and palbociclib. Circulating cfDNA was analyzed at the time of palbociclib addition after fulvestrant resistance, on day 15 of cycle one (D15), and at the end of treatment (EOT) using the ThruPLEX ctDNA Assay, which can identify single or multi nucleotide variants, DNA insertions, and deletions in up to 34 cancer-associated genes. Results: The genetic landscape of patients with BC undergoing palbociclib treatment was dynamic and changed over the course of treatment. Regarding progression-free survival, alterations in NRAS (Hazard Ratio (HR): 11.97, P &amp;lt; 0.005)), MAP3K1 (HR: 6.86, P &amp;lt; 0.005), and TP53 (HR: 3.6, P = 0.02) were identified in cfDNA on D15 and were linked to poor prognoses. Alterations in CBFB at baseline (HR: 5.15, P = 0.01), in BRCA2 (HR: 7.17, P &amp;lt; 0.005) and NRAS (HR: 5.32, P &amp;lt; 0.005) at D15, and ESR1 (HR: 4.37, P = 0.01) at EOT were also linked to poor prognosis, whereas alterations in WEE1 (HR: -5.84, P = 0.02) at EOT were associated with a favorable prognosis. Alterations in ESR1, WEE1, CBFB, and BRCA2 were prognostic factors at peak frequencies during treatment. Moreover, our research uncovered a novel observation that as treatment progressed, the relationship between prognostic factors focused on alterations in ESR1 and TP53 tended to weaken. Conclusion: Cancer panel testing for cfDNA identified prognostic and predictive biomarkers for palbociclib add-on therapy after acquiring fulvestrant resistance in patients with HR-positive/HER2-negative ABC/MBC. These findings have important implications for patient selection and personalized treatment strategies in such patients. Citation Format: Takashi Takeshita, Takayuki Iwamoto, Naoki Niikura, Kenichi Watanabe, Yuichiro Kikawa, Kokoro Kobayashi, Nobutaka Iwakuma, Takuho Okamura, Hiroshi Tada, Shinji Ozaki, Toshitaka Okuno, Uhi Toh, Yutaka Yamamoto, Michiko Tsuneizumi, Hiroshi Ishiguro, Norikazu Masuda, Shigehira Saji. Identification of prognostic and predictive biomarkers for palbociclib add-on therapy using cancer panel sequencing of cell-free DNA in patients with HR-positive HER2-negative advanced and metastatic breast cancer with fulvestrant resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5203.

Discovery of novel DNA methylation biomarker panels for the diagnosis and differentiation between common adenocarcinomas and their liver metastases

Differentiation between adenocarcinomas is sometimes challenging. The promising avenue for discovering new biomarkers lies in bioinformatics using DNA methylation analysis. Utilizing a 2853-sample identification dataset and a 782-sample independent verification dataset, we have identified diagnostic DNA methylation biomarkers that are hypermethylated in cancer and differentiate between breast invasive carcinoma, cholangiocarcinoma, colorectal cancer, hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma and stomach adenocarcinoma. The best panels for cancer type exhibit sensitivity of 77.8–95.9%, a specificity of 92.7–97.5% for tumors, a specificity of 91.5–97.7% for tumors and normal tissues and a diagnostic accuracy of 85.3–96.4%. We have shown that the results can be extended from the primary cancers to their liver metastases, as the best panels diagnose and differentiate between pancreatic adenocarcinoma liver metastases and breast invasive carcinoma liver metastases with a sensitivity and specificity of 83.3–100% and a diagnostic accuracy of 86.8–91.9%. Moreover, the panels could detect hypermethylation of selected regions in the cell-free DNA of patients with liver metastases. At the same time, these were unmethylated in the cell-free DNA of healthy donors, confirming their applicability for liquid biopsies.

Detection of Copy-Number Variation in Circulating Cell-Free DNA in Patients With Uveal Melanoma.

Somatic chromosomal alterations, particularly monosomy 3 and 8q gains, have been associated with metastatic risk in uveal melanoma (UM). Whole genome-scale evaluation of detectable alterations in cell-free DNA (cfDNA) in UM could provide valuable prognostic information. Our pilot study evaluates the correlation between genomic information using ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA in UM and associated clinical outcomes. ULP-WGS of cfDNA was performed on 29 plasma samples from 16 patients, 14 metastatic UM (mUM) and two non-metastatic, including pre- and post-treatment mUM samples from 10 patients treated with immunotherapy and one with liver-directed therapy. We estimated tumor fraction (TFx) and detected copy-number alterations (CNAs) using ichorCNA. Presence of 8q amplification was further analyzed using the likelihood ratio test (LRT). Eleven patients with mUM (17 samples) of 14 had detectable circulating tumor DNA (ctDNA). 8q gain was detected in all 17, whereas monosomy 3 was detectable in 10 of 17 samples. TFx generally correlated with disease status, showing an increase at the time of disease progression (PD). 8q gain detection sensitivity appeared greater with the LRT than with ichorCNA at lower TFxs. The only patient with mUM with partial response on treatment had a high pretreatment TFx and undetectable on-treatment ctDNA, correlating with her profound response and durable survival. ctDNA can be detected in mUM using ULP-WGS, and the TFx correlates with DS. 8q gain was consistently detectable in mUM, in line with previous studies indicating 8q gains early in primary UM and higher amplification with PD. Our work suggests that detection of CNAs by ULP-WGS, particularly focusing on 8q gain, could be a valuable blood biomarker to monitor PD in UM.

Detection of the JAK2 V617F Mutation in Urinary Cell-free DNA in Patients with Myeloproliferative Neoplasms.

Objective Testing for the Janus activating kinase 2 (JAK2) V617F mutation is important for diagnosing and treating myeloproliferative neoplasms (MPNs). Recently, urine cell-free DNA (ucfDNA) was reported to be useful for detecting tumor-specific gene mutations in several solid tumors. However, its utility in detecting such mutations in hematological malignancies has not yet been assessed. In this study, we assessed whether or not the JAK2 V617F mutation could be detected in ucfDNA and whether or not its positivity rate in ucfDNA was associated with the JAK2 V617F allele ratio of peripheral blood cells in patients with MPN. Methods The JAK2 V617F allele ratio of genomic DNA from peripheral blood cells was determined using quantitative polymerase chain reaction (qPCR) or droplet digital PCR (ddPCR). ucfDNA was subjected to ddPCR. The correlation between the JAK2 V617F mutation positivity rates of blood-derived DNA and those of ucfDNA was assessed. Materials Twelve patients with polycythemia vera and 12 patients with essential thrombocythemia were enrolled. Ethylenediaminetetraacetic acid-treated peripheral blood (100 mL) and 15-30 mL of fresh urine were used. Results The JAK2 V617F mutation was detected in the ucfDNA from all 20 JAK2 V617F mutation-positive patients. In addition, the JAK2 V617F mutation positivity rate of ucfDNA was correlated with the JAK2 V617F allele ratio of blood-derived DNA, including in both estimated glomerular filtration rate (eGFR) groups (patients with an eGFR ≥50 or <50 mL/min/1.73 m2). Conclusion Our results indicate that ucfDNA is a valuable tool for diagnosing and monitoring MPN. Given these findings, other disease-specific gene mutations in hematological malignancies may also be detectable in ucfDNA.

Liquid biopsy in H3K27M diffuse midline glioma.

Diffuse midline glioma (DMG) with H3K27M mutation is an aggressive and difficult to treat pediatric brain tumor. Recurrent gain of function mutations in H3.3 (H3.3A) and H3.1 (H3C2) at the 27th lysine to methionine (H3K27M) are seen in over 2/3 of DMGs, and are associated with a worse prognosis. Due to the anatomical location of DMG, traditional biopsy carries risk for neurologic injury as it requires penetration of vital midline structures. Further, radiographic (MRI) monitoring of DMG often shows nonspecific changes, which makes therapeutic monitoring difficult. This indicates a critical need for more minimally invasive methods, such as liquid biopsy, to understand, diagnose, and monitor H3K27M DMG. Here, we review the use of all modalities to date to detect biomarkers of H3K27M in cerebrospinal fluid (CSF), blood, and urine, and compare their effectiveness in detection, diagnosis, and monitoring treatment response. We provide specific detail of recent efforts to monitor CSF and plasma H3K27M cell-free DNA in patients undergoing therapy with the imipridone ONC201. Lastly, we discuss the future of therapeutic monitoring of H3K27M-DMG, including biomarkers such as mitochondrial DNA, mutant and modified histones, and novel sequencing-based approaches for improved detection methods.

Plasma cell-free DNA in patients with acute promyelocytic leukaemia treated with arsenic trioxide.

Cell-free DNA (cfDNA) is free DNA found in circulating blood that originates from apoptosis or necrosis, and elevated cfDNA concentrations have been reported in cancers and other diseases. In this study, the concentrations and fragment distributions of plasma cfDNA were preliminary investigated in elderly (n = 1) and paediatric (n = 1) patients with acute promyelocytic leukaemia (APL) treated with arsenic trioxide (ATO). A slight increase in cfDNA concentrations was observed in the APL patients compared with healthy controls. The change in plasma cfDNA concentrations corresponded to the change in plasma arsenic concentrations during ATO treatment. The fragment distribution pattern did not differ before and during treatment. Three ladder fragments were observed in part of the cfDNA in the second consolidation therapy in an elderly APL patient and the first consolidation therapy of a paediatric APL patient, while two fragments were observed in all other treatment periods. Moreover, APL-related gene mutations were successfully genotyped from plasma cfDNA by using polymerase chain reaction-based methods and these results are consistent with those from leukocytes. This study is the first to report the concentrations and fragment patterns of cfDNA from APL patients treated with ATO. The results suggested that plasma cfDNA concentration in APL patients increased with ATO treatment and that cfDNA is released mainly via neutrophil extracellular traps (and/or necrosis) in addition to apoptosis. To confirm whether cfDNA concentrations and fragment patterns can be used as a biomarker for APL treated with ATO, further accumulative data are needed.

Targeted Molecular Profiling of Circulating Cell-Free DNA in Patients With Advanced Hepatocellular Carcinoma.

Next-generation sequencing (NGS) of tumor-derived, circulating cell-free DNA (cfDNA) may aid in diagnosis, prognostication, and treatment of patients with hepatocellular carcinoma (HCC). The operating characteristics of cfDNA mutational profiling must be determined before routine clinical implementation. This was a single-center, retrospective study with the primary objective of defining genomic alterations in circulating cfDNA along with plasma-tissue genotype agreement between NGS of matched tumor samples in patients with advanced HCC. cfDNA was analyzed using a clinically validated 129-gene NGS assay; matched tissue-based NGS was analyzed with a US Food and Drug Administration-authorized NGS tumor assay. Fifty-three plasma samples from 51 patients with histologically confirmed HCC underwent NGS-based cfDNA analysis. Genomic alterations were detected in 92.2% of patients, with the most commonly mutated genes including TERT promoter (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%), and TSC2 (14%). In total, 37 (73%) patients underwent paired tumor NGS, and concordance was high for mutations observed in patient-matched plasma samples: TERT (83%), TP53 (94%), CTNNB1 (92%), ARID1A (100%), and TSC2 (71%). In 10 (27%) of 37 tumor-plasma samples, alterations were detected by cfDNA analysis that were not detected in the patient-matched tumors. Potentially actionable mutations were identified in 37% of all cases including oncogenic/likely oncogenic alterations in TSC1/2 (18%), BRCA1/2 (8%), and PIK3CA (8%). Higher average variant allele fraction was associated with elevated alpha-fetoprotein, increased tumor volume, and no previous systemic therapy, but did not correlate with overall survival in treatment-naïve patients. Tumor mutation profiling of cfDNA in HCC represents an alternative to tissue-based genomic profiling, given the high degree of tumor-plasma NGS concordance; however, genotyping of both blood and tumor may be required to detect all clinically actionable genomic alterations.

The mutational landscape of circulating cell free DNA in patients with gastric cancer.

e16055 Background: Gastric cancer (GC) is a malignant tumor with a high lethality rate, with the highest incidence occurring in China. Gastric cancer is phenotypically heterogeneous, therefore the identification of genomic alteration is crucial for guiding clinical treatment. In this study, we aimed to use liquid biopsies to detect somatic mutations of GC patients and describe the mutational landscape of circulating tumor DNA. Methods: The blood samples of 157 Chinese patients with gastric malignancies were collected between 2018 to 2022. Somatic alterations were detected by either whole exome sequencing or high-depth targeted gene panel sequencing of ctDNA, using white blood cells as germline DNA control. Results: In our cohort, 1002 alterations of 323 genes were detected from plasma samples. We used MutSigCV tool to define the significantly mutated genes (SMG) and identified 16 SMG. Overall, the five most common mutated genes were TP53 (52.6%), LRP1B (15.4%), ARID1A(14.7%), SYNE1(12.2%), CDH1(10.9%). Recurrent mutations were identified in TP53 genes including c.524G &gt; A, c.713G &gt; T, which lead to loss of function of P53 protein according to evidence of clinical actionability from OncoKB (http://oncokb.org/). Genetic amplification of ERBB2 was detected in 8 patients (5.1%). 6 out of 95 samples were microsatellite instability (MSI), 3 were MSI-H and 3 were MSI-L.The median blood tumor mutation burden (bTMB) of all samples was 1.97 (range 0–143.51) mutations/Mb, 2 patients (1.27%) to high bTMB (≥ 20 mutations/Mb), 37 patients (23.57%) to intermediate bTMB (5-19 mutations/Mb), 118 patients (75.16%) to low bTMB ( &lt; 5 mutations/Mb). The Co-occurrence and mutual exclusivity of gastric cancer variations was investigated. Mutually exclusive mutation patterns were observed in TP53-KRAS (p &lt; 0.05), TP53-ARID1A (p &lt; 0.05), TP53-CDH1 (p &lt; 0.05) gene pairs. In addition, Gene Ontology (GO) and KEGG analysis were performed to analyze enrichment of somatic mutated genes and understand their biological function. The altered signaling pathway included PI3K−Akt signaling, MAPK signaling, Ras signaling, Rap1 signaling etc. 61 genes with somatic mutations were enriched to PI3K–Akt signaling pathway. Conclusions: In this study, we characterized the somatic mutational landscape of gastric cancer in 157 Chinese patients utilizing ctDNA which might guide clinical treatment.

Elevated Donor Derived Cell-free DNA In Patients Without Clinical Rejection

Elevated Donor Derived Cell-free DNA In Patients Without Clinical Rejection

Serological, fragmentomic, and epigenetic characteristics of cell-free DNA in patients with lupus nephritis.

The biological characteristics of plasma circulating cell-free DNA (cfDNA) are related to the pathogenesis of lupus nephritis (LN). The aim of this study was to explore the biological characteristics of cfDNA in patients with LN in terms of serology, fragment omics, and epigenetics, and to discuss the possibility of liquid biopsy for cfDNA as an alternative to conventional tissue biopsy. cfDNA was extracted from plasma samples of 127 patients with systemic lupus erythematosus (64 with LN, 63 without LN). The cfDNA concentration was determined using the Qubit method. Next-generation sequencing cfDNA methylation profiling was performed for three LN patients and six non-LN patients. The methylation panel was designed based on data from The Cancer Genome Atlas cohort. The fragmentation index, motif score, and DELFI score were calculated to explore the fragmentation profile of cfDNA in patients with LN. Statistical and machine learning methods were used to select features to calculate the methylation scores of the samples. Patients with LN had significantly lower cfDNA concentrations (P = 0.0347) than those without LN. This may be associated with the presence of anti-double-stranded DNA antibodies (r = -0.4189; P = 0.0296). The mean DELFI score (proportion of short fragments of cfDNA) in patients with LN was significantly higher than that in patients without LN (P = 0.0238). Based on the pan-cancer data, 73, 66, 8, and 10 features were selected and used to calculate the methylation scores. The mean methylation scores of these features in patients with LN differed significantly from those in patients without LN (P = 0.0238). The specificity of cfDNA in patients with LN was identified using serological, fragmentomic, and epigenetic analyses. The findings may have implications for the development of new molecular markers of LN.

Evolution of RAS Mutations in Cell-Free DNA of Patients with Tissue RAS Wild-Type Metastatic Colorectal Cancer Receiving First-Line Treatment: The PERSEIDA Study.

The serial analysis of cell-free DNA (cfDNA) enables minimally invasive monitoring of tumor evolution, providing continuous genetic information. PERSEIDA was an observational, prospective study assessing the cfDNA RAS (KRAS/NRAS) mutational status evolution in first-line, metastatic CRC, RAS wild-type (according to baseline tumor tissue biopsy) patients. Plasma samples were collected before first-line treatment, after 20 ± 2 weeks, and at disease progression. One hundred and nineteen patients were included (102 received panitumumab and chemotherapy as first-line treatment-panitumumab subpopulation). Fifteen (12.6%) patients presented baseline cfDNA RAS mutations (n = 14 [13.7%], panitumumab subpopulation) (mutant allele fraction ≥0.02 for all results). No patients presented emergent mutations (cfDNA RAS mutations not present at baseline) at 20 weeks. At disease progression, 11 patients (n = 9; panitumumab subpopulation) presented emergent mutations (RAS conversion rate: 19.0% [11/58]; 17.7% [9/51], panitumumab subpopulation). In contrast, three (5.2%) patients presenting baseline cfDNA RAS mutations were RAS wild-type at disease progression. No significant associations were observed between overall response rate or progression-free survival and cfDNA RAS mutational status in the total panitumumab subpopulation. Although, in patients with left-sided tumors, a significantly longer progression-free survival was observed in cfDNA RAS wild-type patients compared to those presenting cfDNA RAS mutations at any time. Continuous evaluation of RAS mutations may provide valuable insights on tumor molecular dynamics that can help clinical practice.

Detection of BRCA1, and BRCA2 Alterations in Matched Tumor Tissue and Circulating Cell-Free DNA in Patients with Prostate Cancer in a Real-World Setting.

Poly (ADP-ribose) polymerase (PARP) inhibitors are approved for patients with metastatic castration-resistant prostate cancer harboring deleterious or suspected deleterious BRCA1 and/or 2 mutations. Identifying patients with prostate cancer harboring these mutations may be challenging. Circulating cell-free DNA (cfDNA) provides an avenue for an easier detection of these mutations. Herein, we aimed to evaluate the concordance of BRCA mutations in the tumor tissue and cfDNA in patients with metastatic prostate cancer in the real-world setting. Somatic genomic profiling results were obtained from a clinical cohort of patients at our institution who had at least two samples tested. One of the samples needed to be from either primary or metastatic tissue. Concordance was adjusted to not include mutation types that the cfDNA platforms were not designed to detect. The presence or absence of mutations in the BRCA gene was assessed in a total of 589 samples, including 327 cfDNA samples, from 260 patients with metastatic prostate cancer. The median time between the first test and any subsequent test was 22.8 (0.0-232) months. BRCA mutation was present in the patient's original prostate tissue in 23 samples (3.9%) of patients. The adjusted concordance between prostate tumor tissue and cfDNA was 97.9% [95% CI, 95.3-99.1%]. The adjusted concordance between metastatic samples and cfDNA was 93.5% [95% CI, 86.4-97.3%]. Of the patients who had a BRCA mutation detected in their prostate tissue, there was a 70% probability of detecting a BRCA mutation in the patient's cfDNA as well. For patients who did not have a detectable BRCA mutation in their primary prostate tissue, the probability of detecting a subsequent one later in the disease course was less than 0.9%. There is a high level of concordance between tissue and blood for BRCA mutations. Testing cfDNA can provide reliable information on BRCA mutational status and is a viable alternative to solid tissue sequencing when unavailable. The development of a new BRCA mutation later in the disease course is a rare event.

The level and integrity of plasma circulating cell-free DNA in patients with primary multiple myeloma.

To evaluate the clinical research related to the level and integrity of circulating free DNA (cfDNA) in the plasma of patients with multiple myeloma (MM). The plasma samples of 56 patients with newly diagnosed MM and 60 healthy volunteers were collected. ALU247 fragment and ALU115 fragment were used as target genes, and quantitative polymerase chain reaction (qPCR) was used to assess the plasma of the patient and healthy control groups. The cfDNA level in MM was analyzed, and the ALU247/ALU115 ratio was used to calculate the integrity of cfDNA. The correlation between the cfDNA level and integrity and the clinical characteristics of patients with primary MM was analyzed, and their value in efficacy monitoring and prognostic evaluation was evaluated. The plasma concentrations of ALU247 and ALU115 and the integrity of cfDNA in patients with primary MM were significantly higher than those in the healthy controls (P<0.05). The ALU247 fragment concentration was markedly correlated with the Durie-Salmon (D-S), International Staging System (ISS), and Revised-International Staging System (R-ISS) stages (P<0.05). After three courses of induction chemotherapy, the levels of ALU247, ALU115, and cfDNA integrity in both groups were lower than those before chemotherapy (P<0.05). Patients with curative effects of CR, sCR, and VGPR were classified into the ≥ very good partial response (VGPR) group (n=38), while those with curative effects of PR and SD were allocated into the <VGPR group (n=18). In addition, after chemotherapy, the levels of ALU247, ALU115, and cfDNA integrity of patients in the ≥ VGPR group were significantly lower than those in the < VGPR group (P<0.05). The follow-up results showed that the progression-free survival (PFS) of MM patients with low ALU247 expression was considerably longer than that of MM patients with high ALU247 expression (33.59±1.15 vs. 27.31±2.16, P<0.05). CfDNA levels were significantly elevated in MM patients, and the ALU247 fragment concentration was remarkably correlated with multiple clinical features and had important clinical value for efficacy monitoring and prognostic assessment.

241P Genomic analysis of plasma cell-free DNA in patients with heavily pretreated HER-2+ metastatic breast cancer (MBC)

We explored the accumulated genomic alterations and their clinical significance through circulating tumor DNA (ctDNA) analysis in patients who were enrolled in the KCSG BR18-14/KM10B trial. This trial investigated the efficacies of trastuzumab biosimilar with cytotoxic chemotherapy in HER2+ MBC patients who had failed 2 or more HER2 directed chemotherapies.