Plasma Circulating Tumor DNA in Patients with Primary Central Nervous System Lymphoma

윤상은,김연정,심준호,박동현,조준훈,고영혜,박웅양,문영철,이경은,조덕,김원석,김석진 대한암학회 2022 Cancer Research and Treatment Vol.54 No.2

Purpose Analysis of circulating tumor DNA (ctDNA) in blood could allow noninvasive genetic analysis of primary tumors. Although there have been unmet needs for noninvasive methods in patients with primary central nervous system lymphoma (PCNSL), it is still not determined whether plasma ctDNA analysis could be useful for patients with PCNSL. Materials and Methods Targeted deep sequencing of 54 genes was performed in cell-free DNA isolated from plasma samples collected pretreatment, during treatment, and at the end of treatment in 42 consecutively diagnosed PCNSL patients between January 2017 and December 2018. Results Targeted sequencing of plasma cell-free DNA detected somatic mutations representing ctDNA in 11 cases (11/41, 27%). The detection of ctDNA was not related to the concentration of cell-free DNA or tumor volume. The mutation profiles of these 11 cases varied between patients. The most frequently mutated gene was PIM1 (4/11, 36.4%), whereas KMT2D, PIK3CA, and MYD88 were each observed in three patients (3/11, 27%). The mutations of 13 genes were concordantly found in primary tumor tissue and plasma ctDNA, giving a detection sensitivity of 45%. During the serial tracking of seven patients with complete response, the disappearance of ctDNA mutations was found in four patients, whereas three patients had detected ctDNA mutation at the end of treatment. Conclusion The plasma ctDNA mutation analysis still has limited value for surveillance and predicting treatment outcomes of PCNSL because the detection efficiency was lower than other systemic lymphomas. Thus, analytical platforms should be improved to overcome anatomical hurdles associated with PCNSL. PurposeAnalysis of circulating tumor DNA (ctDNA) in blood could allow noninvasive genetic analysis of primary tumors. Although there have been unmet needs for noninvasive methods in patients with primary central nervous system lymphoma (PCNSL), it is still not determined whether plasma ctDNA analysis could be useful for patients with PCNSL. Materials and MethodsTargeted deep sequencing of 54 genes was performed in cell-free DNA isolated from plasma samples collected pretreatment, during treatment, and at the end of treatment in 42 consecutively diagnosed PCNSL patients between January 2017 and December 2018.ResultsTargeted sequencing of plasma cell-free DNA detected somatic mutations representing ctDNA in 11 cases (11/41, 27%). The detection of ctDNA was not related to the concentration of cell-free DNA or tumor volume. The mutation profiles of these 11 cases varied between patients. The most frequently mutated gene was PIM1 (4/11, 36.4%), whereas KMT2D, PIK3CA, and MYD88 were each observed in three patients (3/11, 27%). The mutations of 13 genes were concordantly found in primary tumor tissue and plasma ctDNA, giving a detection sensitivity of 45%. During the serial tracking of seven patients with complete response, the disappearance of ctDNA mutations was found in four patients, whereas three patients had detected ctDNA mutation at the end of treatment. ConclusionThe plasma ctDNA mutation analysis still has limited value for surveillance and predicting treatment outcomes of PCNSL because the detection efficiency was lower than other systemic lymphomas. Thus, analytical platforms should be improved to overcome anatomical hurdles associated with PCNSL.

Concordance of circulating tumor DNA and matched formalin-fixed paraffin-embedded tumor tissue in gastric cancer as a predictor of recurrence

Seo Soo Hyun,Park Young Suk,남수경,Lee Hye Seung,Park Do Joong,Park Kyoung Un 대한종양외과학회 2023 Korean Journal of Clinical Oncology Vol.19 No.2

Purpose: Combined analysis of the variant composition of circulating tumor DNA (ctDNA) from cell-free plasma and DNA from tumor tissue could provide insight into the implications of the genetic alterations responsible for the intratumoral and intertumoral heterogeneity of gastric cancer. We aimed to evaluate the usefulness of this approach in these patients.Methods: Cell-free plasma and formalin-fixed paraffin-embedded tumor tissue samples from 46 patients with gastric cancer were examined. Targeted deep sequencing was performed using a commercially available kit.Results: The cell-free DNA (cfDNA) concentration was higher in stage II-IV versus stage I patients and in larger versus smaller tumors. Only 12 of the 36 (33.3%) alterations in the tumor tissue samples were in concordance with those in the ctDNA samples. Two variants were in concordance in stage I samples and 10 in stage II-IV samples. Actionable variants that were detected in concordance were in the stage II-IV samples. Preoperative ctDNA positivity of actionable variants was significantly associated with cfDNA concentration, lymphatic invasion, N stage, and TNM stage. Cancer recurrence was significantly associated with tumor size, lymphatic/vascular invasion, TNM stage, and ctDNA-tumor tissue variant concordance.Conclusion: Preoperative ctDNA genetic analysis using a multigene panel offers substantial clinical benefits when performed in conjunction with targeted deep sequencing of tumor tissue. Concordance between preoperative ctDNA and tumor tissue mutations may serve as a prognostic indicator in patients with gastric cancer.

A Phase II Open-Label, Multicentre Study to Assess the Anti-tumour Activity of Afatinib in Patients with Activating Epidermal Growth Factor Receptor mutation (EGFRm) from Circulating Tumor DNA (CtDNA)

이승룡,박철규,오인재,이재철,최창민,이신엽,장태원,김영철 대한결핵 및 호흡기학회 2018 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.126 No.0

About 70% of activating EGFRm can be detected using CtDNA in patients with activating EGFRm in their Tumor DNA. The treatment efficacy of afatinib was assessed in patients with lung cancer harboring EGFRm which were detected from CtDNA. Primary objective was to prove overall response rate (ORR) in response evaluable population, and the secondary endpoints were progression free survival, overall survival and safety. Ten mL of venous blood was withdrawn and plasma preparation was finished as soon as possible, no more than 4 hours after draw. Plasma samples were stored at -20℃ until delivered to Panagene. EGFRm analyses for CtDNA were performed by PANA Mutyper® EGFR kit (Panagene, Korea). EGFRm testing for tumor DNA were performed by in-house testing in each hospital with PNA clamp EGFR mutation kit or PANA Mutyper® EGFR kit. A total of 340 (331 without missing values) patients were screened for this trial from 2015 July to 2018 March. Tumor genotyping showed 24.5% (81/331), while CtDNA showed 20.5% (68/331) of positivity to detect activating EGFRm (exon 19 deletions, exon 21 and exon 18 point mutations). Among 81 subjects with tumor DNA EGFRm positive subjects, 48 showed EGFRm in their CtDNA (59% sensitivity). Types of EGFRm were completely matched between tumor DNA and CtDNA in 48 subjects. Among 21 subjects enrolled in this trial, 11 subjects had EGFRm only in CtDNA (tumor DNA EGFR wild or unknown, Group 1), and 10 subjects had same EGFRm in their CtDNA and tumor DNA (Group 2). Afatinib (40mg) was initiated in 21 (female:17, adenocarcinoma:20, NSCLC-NOS:1) subjects with mean age of 68.5 years (standard deviation 8.7). Dose modifications were made in 13 subjects (62%). Partial remission was observed in 13 subjects, stable disease in 6 subjects, and response was not evaluated in 2 subjects (ORR : 68.4%). There was no significant difference (p=0.35) in ORR between Group 1 (80%) and Group 2 (55.6%). As of July 25 2018, treatment is ongoing in 15 subjects, progression was confirmed in 3 subjects and 1 withdrew consent, 2 discontinued treatment due to serious adverse events (SAEs). A total of 5 SAEs including 2 subjects with possible drug induced lung disease were reported. In conclusion, afatinib showed favorable efficacy in subjects with NSCLC harboring EGFRm in their CtDNA. Acknowledgement: This study was funded by Boehringer Ingelheim and Panagene. ClinicalTrials.gov Identifier: NCT02629523.

Nanoplasmonic biosensors for detection of circulating tumor DNA(ctDNA) in genetic and epigenetic condition

김우현,( Nguyenhunganh ),심상준 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.1

Circulating tumor DNA (ctDNA) are promising biomarker for noninvasive cancer assay in tumor-specific mutation and methylation. However, previous methods for ctDNA detection have limitation in genetic mutations. Here we present a strategy for ultrasensitive detection of tumor-specific mutations and methylation of ctDNA based on localized surface plasmon resonance (LSPR) and the coupling plasmon mode of gold nanoparticles (AuNPs). Peptide nucleic acid (PNA) is used as a probe to capture and enrich ctDNA. The exposure of PNA-probed AuNPs to target ctDNA generates the primary response of LSPR-peak shift. Immunogold colloids are exploited as methylation detectors and plasmon coupling enhancers for secondary response. LSPR-peak shift is increased approximately 107% upon immunogold colloids binding to two methylcytosines, compared to the primary response. These results demonstrate that the sensor can simultaneously detect the hot-spot mutation and epigenetic changes on the ctDNA.

위암에서의 순환종양핵산

이현지,이선민 대한상부위장관ㆍ헬리코박터학회 2018 Korean Journal of Helicobacter Upper Gastrointesti Vol.18 No.3

Gastric cancer is still the leading cause of cancer deaths, especially in Asian countries. Recently, many studies have analyzed cell-free nucleic acids (cfNAs) circulating in the blood, for the early diagnosis of cancer and monitoring its progression. Circulating tumor nucleic acids (ctNAs) originate in a tumor and contain tumor-related genetic or epigenetic alterations. This review defines the nomenclatures of each form of cfNAs and describes the characteristics of circulating tumor DNA (ctDNA) and microRNA (miRNA), two major forms of ctNAs studied in gastric cancer research to date. We compare available studies on ctDNA, and explain trends observed in studies of miRNAs in gastric cancers. As these new blood-based biomarkers have attracted increasing attention, we have discussed several important points to be considered before the clinical translation of ctNA detection. We have also discussed the current status of research in this field, and clinical applications of specific ctNAs as tumor markers for gastric cancer diagnosis. (Korean J Helicobacter Up Gastrointest Res 2018;18:-173)

Circulating Tumor DNA–Based Genotyping and Monitoring for Predicting Disease Relapses of Patients with Peripheral T-Cell Lymphomas

김석진,김연정,윤상은,유경주,박본,박동현,조덕,김현영,조준훈,고영혜,박웅양,김원석 대한암학회 2023 Cancer Research and Treatment Vol.55 No.1

Purpose Plasma circulating tumor DNA (ctDNA) could reflect the genetic alterations present in tumor tissues. However, there is little information about the clinical relevance of cell-free DNA genotyping in peripheral T-cell lymphoma (PTCL).Materials and Methods After targeted sequencing plasma cell-free DNA of patients with various subtypes of PTCL (n=94), we analyzed the mutation profiles of plasma ctDNA samples and their predictive value of dynamic ctDNA monitoring for treatment outcomes.Results Plasma ctDNA mutations were detected in 53 patients (56%, 53/94), and the detection rate of somatic mutations was highest in angioimmunoblastic T-cell lymphoma (24/31, 77%) and PTCL, not otherwise specified (18/29, 62.1%). Somatic mutations were detected in 51 of 66 genes that were sequenced, including the following top 10 ranked genes: RHOA, CREBBP, KMT2D, TP53, IDH2, ALK, MEF2B, SOCS1, CARD11, and KRAS. In the longitudinal assessment of ctDNA mutation, the difference in ctDNA mutation volume after treatment showed a significant correlation with disease relapse or progression. Thus, a ≥ 1.5-log decrease in genome equivalent (GE) between baseline and the end of treatment showed a significant association with better survival outcomes than a < 1.5-log decrease in GE.Conclusion Our results suggest the clinical relevance of plasma ctDNA analysis in patients with PTCL. However, our findings should be validated by a subsequent study with a larger study population and using a broader gene panel.

Revolutionizing NSCLC Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-Term Stored Plasma

이지영,전세연,전하라,성창옥,장세진,최창민,천성민 대한암학회 2024 Cancer Research and Treatment Vol.56 No.2

Purpose Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non–small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). Materials and Methods Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. Results The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). Conclusion Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.

Establishment of External Quality Assessment Material Preparation Method for Next-Generation Sequencing-Based Liquid Biopsy Scheme

Jinyoung Hong,Joonsang Yu,Hyunjung Gu,Juhee Lee,Woochang Lee,Sail Chun,Won-Ki Min 대한임상검사정도관리협회 2022 Journal of Laboratory Medicine And Quality Assuran Vol.44 No.3

Background: Next-generation sequencing (NGS)-based liquid biopsy testing using peripheral blood is a minimally invasive technique that can identify the characteristics of tumor-derived circulating tumor DNA (ctDNA) in cellfree DNA (cfDNA). External quality assessment (EQA) should be implemented to ensure the reliability of NGS-based liquid biopsy tests. This study aims to establish a method for producing EQA materials for NGS-based liquid biopsy tests. Methods: Eight cell lines harboring clinically important somatic mutations were selected for further analysis. Genomic DNA from the cell lines was extracted and fragmented using an ultrasonicator (Covaris Inc., USA). Two EQA materials were produced by spiking fragmented DNA into fresh frozen plasma and frozen at –70℃. The manufactured EQA materials were evaluated using a cfDNA gene panel (Dxome, Korea) using NextSeq Dx (Illumina, USA). Results: After sonication, the average sizes of the fragmented DNA were 203 and 201 bp, respectively. The results of the cell-free NGS panel showed a combination of different variants between the two EQA materials, and clinically important somatic mutations were detected as intended. Conclusions: In this study, a method for manufacturing materials for an NGS-based liquid biopsy test EQA scheme is presented. EQA materials with conditions similar to ctDNA clinical specimens can be produced at a relatively low cost using cell line-derived DNA and an ultrasonicator. The distribution of adequate EQA materials can improve the reliability of NGS-based liquid biopsy tests.

Analysis of serum protein biomarkers, circulating tumor DNA, and dovitinib activity in patients with tyrosine kinase inhibitor-refractory gastrointestinal stromal tumors

Yoo, C.,Ryu, M.-H.,Na, Y. S.,Ryoo, B.-Y.,Park, S. R.,Kang, Y.-K. Oxford University Press 2014 ANNALS OF ONCOLOGY Vol.25 No.11

<P>To identify biomarkers of dovitinib, a novel antiangiogenic tyrosine kinase inhibitor, soluble serum protein markers involved in the VEGF and FGF pathways and mutations in circulating tumor (ct) DNA were examined in patients with gastrointestinal stromal tumors. Changes in sVEGFR-2 levels were associated with dovitinib activity and secondary mutations in ctDNA were related with poor prognosis .</P><P><B>Background</B></P><P>An exploratory translational analysis was conducted as part of a phase II study of dovitinib to assess the relevance of soluble serum proteins and circulating tumor (ct) DNA (ctDNA) as biomarkers in patients with tyrosine kinase inhibitor (TKI)-refractory gastrointestinal stromal tumors (GISTs).</P><P><B>Patients and methods</B></P><P>Predose serum samples were collected from 30 patients on day 1 of cycle 1 and cycle 2. Serum levels of angiogenesis-related proteins were assessed by enzyme-linked immunosorbent assay, and Beads, emulsions, amplification, and magnetics (BEAMing) assays were carried out to detect mutations in serum ctDNA.</P><P><B>Results</B></P><P>Dovitinib increased vascular endothelial growth factor (VEGF)<SUB>165</SUB> (1.26-fold, <I>P</I> = 0.006), VEGF-A (1.27-fold, <I>P</I> = 0.004), placental growth factor (6.0-fold, <I>P</I> = 0.002), fibroblast growth factor 23 (1.45-fold, <I>P</I> = 0.02), and interleukin 8 (1.75-fold, <I>P</I> = 0.04) levels, and decreased soluble vascular endothelial growth factor receptor (sVEGFR)-2 levels (0.8-fold, <I>P</I> = 0.001). The changes in sVEGFR-2 were significantly associated with metabolic response determined by positron emission tomography (<I>P</I> = 0.02) and progression-free survival (PFS; <I>P</I> = 0.02). Secondary kinase mutations were identified in the ctDNA of 11 patients (41%), and these patients all had mutations involving <I>KIT</I> exon 17. Patients with secondary <I>KIT</I> mutations had significantly worse overall survival {median, 5.5 months [95% confidence interval (CI) 3.8–7.2 months]} than those with no detectable secondary mutations [9.8 months (95% CI 9.6–10.0 months); hazard ratio = 2.7 (95% CI 1.0–7.3); <I>P</I> = 0.047].</P><P><B>Conclusions</B></P><P>Changes in sVEGFR-2 levels were associated with dovitinib-mediated antitumor activity. Genotyping of serum ctDNA with BEAMing is useful for the identification of resistant mutations potentially associated with poor prognosis in patients with GISTs.</P>

A Phase II Trial of Osimertinib as the First-Line Treatment of Non–Small Cell Lung Cancer Harboring Activating EGFR Mutations in Circulating Tumor DNA: LiquidLung-O-Cohort 1

박철규,조현주,최유덕,오인재,김영철 대한암학회 2021 Cancer Research and Treatment Vol.53 No.1

Purpose Osimertinib is a potent, irreversible third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for both EGFR-activating and T790M resistant mutation. The treatment efficacy of osimertinib was assessed in previously untreated patients with metastatic non–small cell lung carcinoma (NSCLC) harboring activating EGFR mutations in circulating tumor DNA (ctDNA) as well as tumor DNA. Materials and Methods Patients with activating EGFR mutations in their tumor DNA underwent screening with ctDNA analysis using Mutyper and Cobas v2 assays. Enrolled subjects received osimertinib 80 mg, once daily. Primary endpoint was objective response rate (ORR) and secondary endpoints were ctDNA test sensitivity, progression-free survival (PFS), duration of response (DoR), and safety. Results Among 39 screened patients, 29 were ctDNA positive for activating EGFR mutations and 19 were enrolled (ex19del, n=11; L858R/L861Q, n=7; G719A, n=1). Median age was 70 and most patients had brain metastases (15/19, 79%). ctDNA test sensitivity for activating EGFR mutations was 74% using both methods and 62% (Mutyper) or 64% (Cobas v2) for individual methods. ORR was 68% (13/19), median PFS was 11.1 months (95% confidence interval [CI], 0.0 to 26.7), and median DoR was 17.6 months (95% CI, 3.5 to 31.7). ORR and median PFS were significantly superior with ex19del (91%; 21.9 months; 95% CI, 5.5 to 38.3) than with L858R/L861Q (43%; 5.1 months; 95% CI, 2.3 to 7.9). One patient discontinued the drug because of drug-related interstitial pneumonitis. Conclusion Osimertinib had favorable efficacy in the first-line treatment of metastatic NSCLC harboring activating EGFR mutations in ctDNA as well as tumor DNA.