Paired cfDNA Research Articles

Utility of circulating plasma cell-free DNA for detection and quantification of brain arteriovenous malformations

Brain arteriovenous malformations (BAVMs) are primarily associated with somatic activating pathogenic KRAS variants. KRAS-targeting therapy is emerging as a potential treatment for BAVMs. However, current molecular diagnosis relies on surgically obtained tissue samples. Here, we investigated the feasibility of using cell-free DNA (cfDNA) from the blood for molecular diagnosis in sporadic BAVM patients. We included 31 BAVM patients and extracted genomic DNA from BAVM tissues, while paired cfDNA was isolated from plasma. Additionally, fifty plasma cfDNA samples from unaffected individuals served as controls. By utilizing droplet digital polymerase chain reaction (ddPCR), we tested KRAS c.35 G>A p.Gly12Asp (p.G12D) and c.35 G>T p.Gly12Val (p.G12V) variants in all samples. Among the 31 BAVM samples, KRAS somatic mutations were identified in 24 patients (77 %), comprising 79 % (19 out of 24) p.G12D and 21 % (5 out of 24) p.G12V variants. The variant frequencies (VFs) ranged from 0.227 % to 8.327 %, with positive droplets ranging from 17 to 1025. 63 % (15 out of 24) of patients with KRAS mutations had≥2 positive droplets in their cfDNA samples. In contrast, in none of the 50 control samples more than two positive droplets were detected. Specifically, 13 plasma samples (68 %) were positive for p.G12D mutation. The VFs in plasma cfDNA samples ranged from 0.042 % to 5.172 %. Furthermore, ddPCR demonstrated a sensitivity of 63 %, specificity of 100 %, positive predictive value of 100 %, and negative predictive value of 81 % for detecting plasma cfDNA. The VFs in mutant tissues had an inverse trend with the largest nidus sizes, volumes, and patient age, while an opposite trend was observed in plasma cfDNA. Taken together, we successfully detected pathogenic somatic activating KRAS variants in cfDNA obtained from the plasma of BAVM patients. The diagnostic utility of liquid biopsy for BAVMs will facilitate the development of personalized therapeutic approaches and offer opportunities for novel strategies to halt, slow, or delay disease progression.

MSK-ACCESS powered with SOPHiA DDM: Performance analysis of a decentralized MSK-ACCESS solution.

e15063 Background: Blood plasma cell-free DNA (cfDNA) testing of cancer patients allows for genomic profiling when tissue sampling is too invasive or of insufficient quality. This approach has demonstrated clinical utility to identify treatment targets, screen for resistance and monitor disease burden. MSK-ACCESS (Memorial Sloan Kettering – Analysis of Circulating cfDNA to Evaluate Somatic Status) is a validated, high sensitivity cfDNA assay employed at MSK to identify biologically actionable tumor alterations in clinical care. SOPHiA GENETICS is developing a decentralized solution of this assay that incorporates major features of MSK-ACCESS such as molecular barcoding, duplex-based variant calling, the inclusion of tumor-informed prior knowledge and identification of somatic alterations through the removal of both germline and clonal hematopoiesis (CH) variants. Here, we present the results of a performance study obtained during product development. Methods: A cohort of 24 plasma cfDNA and matched white blood cell (WBC) DNA samples were collected from patients with solid tumors and processed at MSK according to the validated standard operating procedures and bioinformatic analyses. In parallel, for the same set of materials libraries were constructed at SOPHiA GENETICS using the proprietary CUMIN molecular identifiers, captured with probes targeting the updated MSK-ACCESS v2 set of 146 genes and sequenced on an Illumina NovaSeq 6000. Variants were called using a duplex-aware approach by SOPHiA GENETICS and germline and CH variants were identified and filtered by paired cfDNA – WBC DNA analysis. Accuracy of the decentralized “MSK-ACCESS powered with SOPHiA DDM” solution was assessed through concordance to the orthogonal results obtained by MSK. Results: A total of 104 somatic SNV/Indels were detected with variant allele fractions (VAF) ranging from 0.1% to 97.0% with a median of 5 variants per sample. The positive percent agreement (PPA) for all variants above the limit of detection of the original MSK-ACCESS assay was 98.9% with 99.98% specificity. For the subset of prior-knowledge variants PPA was 95.6% down to 0.1% VAF. A total of additional 59 variants with ≤ 15% VAF were found both in cfDNA and WBC and assigned as CH-derived. Importantly, these occurred not only in CH-associated genes but also in common oncogenes such as BRCA1/2 and ERBB2, highlighting the utility of matched WBC analysis to correctly identify somatic variants. Conclusions: The results demonstrate that the decentralized “MSK-ACCESS powered with SOPHiA DDM” solution exhibits a high degree of agreement to the results achieved by MSK. Further studies will be conducted to expand the analytical performance evaluation.

Abstract 2293: Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck cancer

Abstract Purpose: Liquid biopsy approaches are revolutionizing cancer monitoring and have been considered potential mediators for therapeutic efficacy in multiple cancers. The investigation of different liquid biopsy biomarkers may lead to a better understanding of the cancers they are designed to detect and facilitate optimal management. This study aimed to identify and compare the epigenomic and genomic profiles of tumor biomarkers in circulation using cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in plasma and saliva samples collected from patients with head and neck squamous cell carcinoma (HNSCC). Methods: In a prospective trial, we performed whole-genome bisulfite sequencing on paired cfDNA and exoDNA from saliva versus plasma of 16 HNSCC patients. cfDNA was directly extracted from plasma and saliva, while exoDNA was extracted from purified exosomes after characterization. DNAs were then converted by Zymo EZ DNA Methylation-Direct kit and libraries were constructed with xGen™ Methyl-Seq Kit and sequenced on the Illumina NovaSeq platform. Sequencing data analyses were performed using Bismark, GATK, and methylKit for methylation pattern and cfTools for Tumor Fraction Estimation (TFE). Results: Methylation data demonstrated heterogeneities across sample types. Comparisons of ctDNA as a reference for exoDNA showed 7147 hypermethylated and 9984 hypomethylated regions for plasma samples but showed 131 hypermethylated and 123 hypomethylated regions for saliva samples. Comparisons of plasma as a reference for saliva, 37 hypermethylated and 95 hypomethylated regions were noted in exoDNA, while 117 hypermethylated and 262 hypomethylated regions in ctDNA. The mean level of tumor-derived DNA was above 3%, the lower limit of sensitivity to indicate the presence of tumor cutoff as suggested by the cfTools. There were 3.24%, 3.14%, and 3.14% tumor-derived DNA in plasma cfDNA, plasma exoDNA, and saliva exoDNA, respectively. However, the tumor fraction in saliva cfDNA was below this cutoff (2.88%). A statistically significant (P < 0.001) difference in TFE was observed between combined cfDNA and exoDNA data from plasma and saliva, but the difference between combined sample types cfDNA and exoDNA did not reach statistical significance (P = 0.306). The linear mixed-effects model for TFE influence between the different groups revealed a significant interaction between sample sources (plasma and saliva) and sample types (cfDNA and exoDNA) (P = 0.005), as well as a significant effect of sample source on TFE (P < 0.001). Conclusions: This pilot investigation provides evidence that exoDNA and cfDNA have different molecular profiles in plasma and saliva samples from the same HNSCC patient, and indicates that saliva carries additional tumor markers for HNSCC. These results help to advance our understanding of the potential impact of different liquid biopsy sample types in cancer management. Citation Format: Mouadh Barbirou, Amanda Miller, David Baek, Allison Hameier, Ashley Wetzel, Alban Linnenbach, Kathryn Nunes, Pablo Llerena, Voichita Bar-Ad, Adam Luginbuhl, Ubaldo Martinez Outschoorn, Ronald Myers, Joseph Curry, Chun Wang, Hushan Yang. Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck cancer [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 2293.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: TFH lymphomas (TFHL) commonly harbor mutations in TET2, RHOA, DNMT3A and IDH2. While RHOA and IDH2 appear restricted to the neoplastic cells, TET2 and DNMT3A mutations occur in a significant proportion of cases in a hematopoietic progenitor cell and can also be detected in B or myeloid cells. Cell-free DNA (cfDNA) sequencing allows the detection of circulating tumor DNA in solid cancers or B-cell lymphomas, with predictive value, but few data exist on cfDNA sequencing in TFHL. Methods: In the frame of the ORACLE study, a phase 3 trial comparing oral azacitidine to investigator choice treatment in relapsed or refractory TFHL patients, we collected tumor biopsies and plasma in streck tube at inclusion, after 3 cycles, and at progression. Tumour and cfDNA were sequenced by NGS using 9 genes, amplicon-based libraries. Median sequencing depth was 2386X in tumor and 3519X in cfDNA. Results: Among patients with confirmed TFHL treated in LYSA centers, we collected 45 samples at inclusion, 16 after cycle 3, and 14 at progression. The median cfDNA concentration at each time point was respectively 7507 (IQR 3418–19414), 5182 (3438–12429), and 15750 (7316–26953) hEG/mL. Results of cfDNA at inclusion and tumor sequencing were compared in 43 patients. Common TET2 mutations were detected in the tumor of 36/43 (84%) and in cfDNA of 33/43 (77%) patients, with a median variant allele frequency (VAF) of 17.75% vs. 9.8% respectively. RHOAG17V was detected in 29/43 (67%, VAF 10%) and in 24/43 (59%, VAF 4.4%), DNMT3A in 13/43 (30%, VAF 17.9%) and 15/73 (35%, VAF 17.9%), and IDH2 in 12/43 (28%, VAF 5.9%) and 8/43 (19%, VAF 5.4%) of tumor biopsies and cfDNA samples respectively. Only one patient had detectable TET2, DNMT3A, IDH2, and RHOA mutations in the cfDNA and not in the tumor biopsy, corresponding to a tumor with low neoplastic cell content. By contrast, 3 patients had DNMT3A mutations, not affecting the R882 residue, detected in the cfDNA but not in the tumor likely corresponding to clonal hematopoiesis not related to the TFHL. We also compared 25 paired cfDNA samples collected at inclusion and after cycle 3 (including progression sample if occurring at cycle 3 or before). In all but one patient, we observed the persistence of TET2 and DNMT3A mutations, even in responding patients, confirming that these mutations are not restricted to neoplastic cells, and suggesting that treatment, especially with 5-azacitidine, does not affect the clonal hematopoiesis. By contrast, among the 14/25 patients with a detectable RHOA mutation in cfDNA at inclusion, progression-free survival was longer in the 4 patients with the disappearance of the RHOA mutations in cfDNA at cycle 3 than in the 10 others (median 29 vs. 3 months, p = 0.01). The research was funded by: BMS Force Hémato Keywords: Aggressive T-cell non-Hodgkin lymphoma, Liquid biopsy, Minimal residual disease No conflicts of interests pertinent to the abstract.

A genome-wide cell-free DNA methylation analysis identifies an episignature associated with metastatic luminal B breast cancer.

Breast cancers of the luminal B subtype are frequent tumors with high proliferation and poor prognosis. Epigenetic alterations have been found in breast tumors and in biological fluids. We aimed to profile the cell-free DNA (cfDNA) methylome of metastatic luminal B breast cancer (LBBC) patients using an epigenomic approach to discover potential noninvasive biomarkers. Plasma cfDNA was analyzed using the Infinium MethylationEpic array in a cohort of 14 women, including metastatic LBBC patients and nontumor controls. The methylation levels of cfDNA and tissue samples were validated with droplet digital PCR. The methylation and gene expression data of 582 primary luminal breast tumors and 79 nontumor tissues were obtained from The Cancer Genome Atlas (TCGA). We found an episignature of 1,467 differentially methylated CpGs that clearly identified patients with LBBC. Among the genes identified, the promoter hypermethylation of WNT1 was validated in cfDNA, showing an area under the ROC curve (AUC) of 0.86 for the noninvasive detection of metastatic LBBC. Both paired cfDNA and primary/metastatic breast tumor samples showed hypermethylation of WNT1. TCGA analysis revealed significant WNT1 hypermethylation in the primary tumors of luminal breast cancer patients, with a negative association between WNT1 methylation and gene expression. In this proof-of-principle study, we discovered an episignature associated with metastatic LBBC using a genome-wide cfDNA methylation approach. We also identified the promoter hypermethylation of WNT1 in cfDNA as a potential noninvasive biomarker for luminal breast cancer. Our results support the use of EPIC arrays to identify new epigenetic noninvasive biomarkers in breast cancer.

Abstract 3396: Identification of small cell lung cancer stage-specific DNA methylation in patients using liquid biopsies

Abstract Introduction: Small cell lung cancer (SCLC) is a deadly disease and patients often suffer from recurrent disease. Biologic mechanisms of recurrence are unclear. Epigenetic mechanisms, like DNA methylation, may be operant. SCLC is rarely resected; therefore, the SCLC methylome is understudied due to scarce tumor tissue. Methods: In this study, we examined the pre-treatment methylome of 72 SCLC patients at our institution through cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) on plasma cell-free DNA (cfDNA) and on sheared genomic DNA from paired peripheral blood leukocytes (PBLs) (n = 72) to increase tumor signal specificity. cfMeDIP-seq was also performed on an independent cohort of healthy non-cancer controls (n = 20) and on tumors from circulating tumor cell-derived xenograft (CDX) models (n = 12 CDXs of 72 patients). cfMeDIP-seq libraries were sequenced at a depth of 100 million reads, paired-end, on the NovaSeq 6000. For all bioinformatic analyses, chromosomes 1-22 were binned into 300-bp windows (n = 9.6e6 genome-wide windows); reads from cfMeDIP-seq were tallied per bin. To filter out noise from non-tumor cells, ENCODE blacklist regions (n = 1e6 windows) were removed and CG-rich (>5 CGs per window), PBL-associated windows with MeDEStrand-converted beta-values < 0.2 were kept (n = 190,769 windows). Subsequent cfDNA analyses were performed using these 190,769 windows. Results: 33 (45.8%) and 39 (54.2%) of the 72 patients had limited-stage (LS) and extensive-stage (ES) SCLC, respectively. Most were current or former smokers (65/72, 90.3%). Using filtered windows (n = 190,769), methylated cfDNA from SCLC were distinguished from healthy controls: SCLC cfDNA was enriched for hypermethylated CpG islands and shore regions, whereas controls had more methylated open-sea regions. SCLC cohort by consensus clustering of the top 5000 most variant windows revealed two distinct cfDNA methylation patterns. Cluster A (9 ES, 22 LS) and Cluster B (33 ES, 11 LS) were significantly different by stage (X2(1) = 13.79, p < 0.001). There was no significant difference in sex (p = 0.81). Patients in Cluster A had a lower concentration of cfDNA (median = 7.2ng/ml) compared to Cluster B (median = 15.9ng/ml) but not statistically significant (p = 0.33). High concordance between genome-wide methylome profiles was found between paired patient cfDNA and CDX tumor, demonstrating that cfDNA is representative of tumor methylation (Pearson's correlation median r = 0.86). Conclusion: We identified stage-specific methylation patterns in the plasma of SCLC patients using the cfMeDIP-seq assay that may reveal novel epigenetic and biologic mechanisms of SCLC disease progression. Moreover, CDXs recapitulate the methylome of SCLC cell-free patient samples highlighting their utility in future work as representative models for methylome analysis. Citation Format: Sami Ul Haq, Sabine Schmid, Mansi K. Aparnathi, Katrina Hueniken, Luna J. Zhan, Danielle Sacdalan, Janice J.N. Li, Devalben Patel, Dangxiao Cheng, Vivek Philip, Geoffrey Liu, Scott V. Bratman, Benjamin H. Lok. Identification of small cell lung cancer stage-specific DNA methylation in patients using liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3396.

Genetic Characteristics Associated With Drug Resistance in Lung Cancer and Colorectal Cancer Using Whole Exome Sequencing of Cell-Free DNA.

Circulating cell-free DNA (cfDNA) can be used to characterize tumor genomes through next-generation sequencing (NGS)-based approaches. We aim to identify novel genetic alterations associated with drug resistance in lung cancer and colorectal cancer patients who were treated with EGFR-targeted therapy and cytotoxic chemotherapy through whole exome sequencing (WES) of cfDNA. A cohort of 18 lung cancer patients was treated with EGFR TKI or cytotoxic chemotherapy, and a cohort of 37 colorectal cancer patients was treated with EGFR monoclonal antibody or cytotoxic chemotherapy alone. Serum samples were drawn before and after development of drug resistance, and the genetic mutational profile was analyzed with WES data. For 110 paired cfDNA and matched germline DNA WES samples, mean coverage of 138x (range, 52–208.4x) and 47x (range, 30.5–125.1x) was achieved, respectively. After excluding synonymous variants, mutants identified in more than two patients at the time of acquired resistance were selected. Seven genes in lung cancer and 16 genes in colorectal cancer were found, namely, APC, TP53, KRAS, SMAD4, and EGFR. In addition, the GPR155 I357S mutation in lung cancer and ADAMTS20 S1597P and TTN R7415H mutations in colorectal cancer were frequently detected at the time of acquired resistance, indicating that these mutations have an important function in acquired resistance to chemotherapy. Our data suggest that novel genetic variants associated with drug resistance can be identified using cfDNA WES. Further validation is necessary, but these candidate genes are promising therapeutic targets for overcoming drug resistance in lung cancer and colorectal cancer.

Detection of Locoregional Minimal Residual Disease Immediately After Surgery Through Multi-Omic Analysis of Tumor-Associated Cell-Free DNA in Surgical Drain Fluid

<h3>Purpose/Objective(s)</h3> Liquid biopsy markers of minimal residual disease (MRD) after cancer treatment have been investigated for detection of recurrence, but detection of locoregional MRD using plasma remains a challenge. Our laboratory examined the use of liquid biopsy technology in surgical drain fluid (SDF) immediately after surgery. Here, we: 1) validate the detection of tumor-associated cell-free DNA (cfDNA) in SDF and 2) demonstrate the utility of SDF cfDNA for clinical decision-making in two cancer types. We hypothesize that tumor-associated cfDNA in SDF provides an early, objective measure of locoregional MRD that can guide adjuvant therapy. <h3>Materials/Methods</h3> We collected tumor, SDF, and plasma samples from patients undergoing surgery for HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) and thyroid cancer. TaqMan qPCR assays were designed for detection of HPV DNA and BRAF V600 mutations in HPV (+) OPSCC and thyroid cancer, respectively. HPV(+) OPSCC PCR results were validated using next-generation HPV capture sequencing in a subset of patients. <h3>Results</h3> 62 HPV (+) OPSCC and 13 thyroid carcinoma cases were included. We correlated SDF HPV cfDNA with pathology and showed a dose-dependence between HPV copy number, node positivity, and extranodal extension (ENE). In patients with ENE, locoregional MRD detection rate was 93% in SDF versus 53% in plasma (p=0.035, Fisher's). We validated results using HPV capture sequencing in a subset (n=6) and noted 100% concordance between PCR data and HPV subtyping in SDF. We then measured actionable mutations in SDF and associations with locoregional recurrence. We determined BRAF V600 mutant status in 13 papillary and anaplastic thyroid carcinomas and in paired cfDNA from SDF. In three known BRAF V600 cases, two patients with extrathyroidal extension were SDF mutant positive; one recurred locoregionally while the other only recently had surgery. The SDF mutant negative patient is disease-free at 9 months. <h3>Conclusion</h3> We present the use of a novel multi-omic platform to quantify tumor-associated cfDNA in surgical drain fluid as an immediate postoperative marker of locoregional MRD. Our data suggest tumor-associated cfDNA in surgical drain fluid is highly correlated with aggressive pathologic features in two cancer types, and could enable more accurate determination of adjuvant therapy and prognosis. When combined with plasma tumor-associated cfDNA, this may enable locoregional and distant molecular restaging after surgery. Future clinical applications may include identifying candidates for adjuvant therapy de-escalation after surgery in HPV+ OPSCC; in thyroid cancer, this assay could assist in radioactive iodine therapy planning or introduce targeted therapies into the adjuvant setting.

Personalized Circulating Tumor DNA Dynamically Predicts Response and/or Relapse in Hematological Malignancies

Background: With the recent advent of molecular techniques such as droplet digital PCR (ddPCR), tumor specific genomic component in the plasma or serum, known as circulating tumor DNA (ctDNA), represents one of the most sensitive and noninvasive biomarkers in solid tumors. In the field of hematological malignancies, it remains to be elucidated whether ctDNA monitoring enables accurate prediction of clinical relapse in individual patients. To address this issue, we retrospectively investigated 17 patients with hematological malignancies who had achieved complete or partial remission after first-line induction therapy.Methods: Our cohort consisted of 8 patients with acute myeloid leukemia, 3 with multiple myeloma (MM), 2 with B cell non-Hodgkin's lymphoma, 2 with myelodysplastic syndromes, 1 with B cell acute lymphoblastic leukemia, and 1 with T cell non-Hodgkin's lymphoma. Serial biopsy and matched serum samples were collected at the time of diagnosis (termed “baseline”) and then throughout the treatment course. Cell-free DNA (cfDNA) was extracted from serum samples. We subjected DNA extracted from baseline tumor DNA and buccal swab to next generation sequencing (NGS), identifying one or two somatic driver mutations. We designed personalized ddPCR assays for each somatic mutation identified (n=16). Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. The first day of 1st line induction therapy was set to day 0 for estimation of the disease-free survival (DFS). For subset analysis of patients who relapsed during follow-up, the day of re-induction therapy initiation for relapse is re-set to day 0. Thereafter, the cumulative incidence of relapse, as assessed by ctDNA or by clinical parameters, was estimated and compared.Results: The amount of cfDNA at diagnosis was significantly higher compared with paired cfDNA at remission (p=0.037). NGS screening identified somatic driver mutations in all cases, with 2 mutations found in 2 individuals. Overall, we could construct ddPCR assay for all patients; these mutations included NRAS, KRAS, TP53, GATA2, STAG2, JAK3, MYD88, B2M, NPM1, DNMT3A, U2AF1, and SF3B1 . NGS and ddPCR assays had a good concordance in assessing the same tumor rich samples with regard to the variant allele frequency (VAF) (r2=0.96; p< 0.01). CtDNA could be detected in matched cfDNA from baseline serum. Moreover, the dynamic longitudinal changes of ctDNA VAF reflected the observed overall clinical response, as determined by clinical biomarker or imaging. 8 patients clinically relapsed at a median of 297 days post 1st line therapy (termed “relapsed group”), with the remaining 9 patients disease free at a median of 332 days post 1st line therapy (termed “remission group”). Notably, all of the patients in relapsed group became ctDNA detectable or had ctDNA increased at two consecutive points (termed “ctDNA-increase”). In marked contrast, all of the patients in remission group remained ctDNA undetectable or stable (termed “ctDNA-stable”). Patients with ctDNA-increased had a significantly inferior DFS: 2-year DFS 12.5% in patients with ctDNA-increased vs. 100% in ctDNA-stable, p=0.0115 (Figure 1A). Finally, we compared the timing of molecular relapse and clinical relapse, as assessed by ctDNA and by conventional biomarkers (flowcytometry in leukemia or serum free light chain in MM), respectively, in relapsed group. As a result, molecular relapse as assessed by ctDNA had a median of 30 days lead time (range, 4 to 119 days) over clinical relapse as assessed by conventional clinical parameters (Figure 1B).Conclusions: In summary, personalized ctDNA monitoring could identify patients with impending relapse in advance of established clinical parameters in hematological malignancies. This might allow for clinicians to initiating earlier therapeutic interventions for impending relapse. Further prospective studies in a much larger cohort of hematological malignancies are needed to confirm these findings. [Display omitted] DisclosuresNagamura-Inoue:Tsubakimoto Chain CO.: Research Funding; Rohto Pharmaceutical Co., Ltd.: Research Funding; AMED grant: Research Funding. Tojo:AMED grant: Research Funding.

Liquid Biopsy for the Identification of Intravascular Large B-Cell Lymphoma

[Backgrounds] Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).[Patients and Methods] Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.[Results] All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.[Conclusion] Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL. [Display omitted] DisclosuresChiba:Nippon Shinyaku: Honoraria, Research Funding.

Personalized Circulating Tumor DNA Predicts Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Relapse is a main event of therapy failure for patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) undergoing allogeneic hematopoietic stem cell transplantation (alloSCT). Hence, a novel strategy to detect minimal residual disease (MRD) is required for earlier therapeutic intervention in impending post alloSCT relapse. Recent evidence indicated that screening of driver mutations by next generation sequencing (NGS) followed by detection of circulating tumor DNA (ctDNA) via droplet digital PCR (ddPCR) is a promising approach for MRD monitoring in patients with MDS (Paul Y, et al. Blood. 2017). Here, in this study, we investigated whether serial ctDNA monitoring could identify patients who are most likely to relapse after alloSCT.Methods: We performed a retrospective study in 31 patients with AML and MDS undergoing alloSCT. Cell-free DNA (cfDNA) was extracted from serum samples. We subjected primary tumor DNA, extracted from a diagnostic peripheral blood or bone marrow (BM), and buccal swab DNA, to NGS, identifying one or two somatic driver mutations. We designed unique ddPCR assays for each somatic mutation identified (n=25). The primary endpoint was cumulative incidence of relapse rate (CIR) and the secondary endpoint was overall survival (OS). CIR and OS were calculated by the Kaplan-Meier method. The log-rank test was used for group comparison.Results: 31 patients were enrolled in our study: our cohort consisted of 10 patients with de novo AML, 15 with AML with myelodysplasia related changes, and 6 with MDS. The median age was 52 years (17-68 years) and 5 patients (16%) are in complete remission at alloSCT. Stem cell source included related bone marrow (n=2) and cord blood (n=29). The preparative regimen included myeloablative conditioning (n=17) and reduced intensity conditioning (n=14). NGS screening identified 26 somatic driver mutations in 30 of 31 (97%) cases, with 2 mutations found in 5 individuals. Overall, we could construct ddPCR assays for 29 of 31 (94%) patients: these mutations included NRAS, TP53, NPM1, DNMT3A, GATA2, etc. NGS and ddPCR assays had a good concordance in assessing the same tumor rich samples with regard to the variant allele frequency (VAF).The amount of cfDNA at diagnosis was significantly higher compared with paired cfDNA at remission (n=23; p=0.0133, paired t-test). Of 37 matched serial time points, VAF between BM and serum ctDNA indicates apparent correlation (r2=0.92, p<0.0001). CtDNA was detected in 10 of 25 (40%) samples at 1 month after alloSCT and 7 of 21 (33%) samples at 3 months (termed “ctDNA1-positive” and “ctDNA3-positive”, respectively).We next evaluated whether the positivity of ctDNA at 1 month and 3 months after alloSCT was associated with higher CIR and lower OS. As expected, patients with ctDNA1-positive and ctDNA3-positive had significantly superior CIR and inferior OS: 2-year CIR 51% in ctDNA1-positive patients vs. 9% in negative patients, p=0.0005 (Figure 1A); 2-year OS 48% in ctDNA1-positive patients vs. 83% in negative patients, p=0.0152 (Figure 1B); 2-year CIR 57% in ctDNA3-positive patients vs. 23% in negative patients, p=0.0055 (Figure 1C); 2-year OS 50% in ctDNA3-positive patients vs. 88% in negative patients, p=0.0344 (Figure 1D), respectively.Conclusions: In summary, personalized ctDNA monitoring could detect MRD, which alloSCT has failed to eradicate, and thus identify patients with high-risk of relapse. This might allow for clinicians to initiating earlier therapeutic interventions for impending relapse. Further prospective studies in a much larger cohort of AML and MDS undergoing alloSCT are required to confirm these findings.Figure 1. CIR and OS based on ctDNA status at 1 month and 3 months after alloSCT [Display omitted] DisclosuresNagamura-Inoue:Tsubakimoto Chain CO.: Research Funding; AMED grant: Research Funding; Rohto Pharmaceutical Co., Ltd.: Research Funding. Tojo:AMED grant: Research Funding.

Clinical Experience of Cerebrospinal Fluid–Based Liquid Biopsy Demonstrates Superiority of Cell-Free DNA over Cell Pellet Genomic DNA for Molecular Profiling

Cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) offers unique opportunities for genomic profiling of tumors involving the central nervous system but remains uncommonly used in clinical practice. We describe our clinical experience using cfDNA from CSF for routine molecular testing using Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (targeting 468 cancer-related genes). In all, 148 cfDNA samples were assessed, comparing results of cfDNA versus genomic DNA (gDNA; gDNA from cell pellets) derived from the same CSF sample and the primary tumor. Of these, 71.6% (106/148) were successfully sequenced. Somatic alterations (mutations and fusions) were observed in 70.8% (75/106) of the samples; 97.3% (73/75) comprised variants confirming central nervous system involvement by a previously diagnosed tumor, 14.7% (11/75) had additional variants consistent with a therapy-related resistance mechanism, and 2.7% (2/75) had variants that independently diagnosed a new primary. Among samples with paired cfDNA and gDNA sequencing results, cfDNA was more frequently positive for at least one mutation [43.6% (55/126) versus 19.8% (25/126)] and harbored 1.6× more mutations (6.94 versus 4.65; P=0.005), with higher mean variant allele fractions (41.1% versus 13.0%; P<0.0001). Among mutation-positive cfDNAs, the corresponding gDNA was frequently negative (44.6%; 25/55) or failed sequencing (17.8%; 9/55). Routine molecular profiling of cfDNA is superior to gDNA from CSF, facilitating the capture of mutations at high variant allele frequency, even in the context of a negative cytology.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq) is a new ultra-sensitive UMI-based NGS library preparation technology for use with cfDNA and cfRNA

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20–25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

Distinct profile of cell-free DNA in malignant pleural effusion of non-small cell lung cancer and its impact on clinical genetic testing.

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result.Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes.Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167.Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA.

Clinical Validation of a NGS Capture Panel to Identify Mutations, Copy Number Variations and Translocations in Patients with Multiple Myeloma

Clinical Validation of a NGS Capture Panel to Identify Mutations, Copy Number Variations and Translocations in Patients with Multiple Myeloma

Abstract PR19: Monitoring of melanoma progression utilizing multiplatform biomarkers of blood cell-free DNA

Abstract Blood biopsies utilizing cell-free DNA (cfDNA) represent an alternative, minimally invasive source that permits repeated sampling to enable melanoma patient monitoring during treatment and follow-up. However, the sensitivity of cfDNA biomarker detection is limited to tumor sampling availability, which can vary significantly. This encourages the use of multiplatform biomarkers to improve sensitivity in assessing melanoma progression. Here we explored the detection of different cfDNA biomarker types, DNA mutations and copy number amplification (CNA), in blood of melanoma patients. To assess the utility of these cfDNA biomarker types, we utilized a highly sensitive multigene (54-gene cancer panel) digital next-generation sequencing (NGS) assay containing 0.1% sensitivity or multiplex droplet digital PCR ddPCR. cfDNA mutations were assessed by a multigene digital NGS assay in a cohort of preoperative stage III/IV patients rendered disease-free (n=44), whereby 75% of patients had detectable cfDNA genomic aberrations. Patients with high tumor mutation burden (TMB) (≥3 mutations) were associated with worse overall survival (OS) (p=0.048) compared to those with &amp;lt;3 mutations. Next, we assessed 98 serial bleeds obtained from a cohort of preoperative stage IIIA-C melanoma patients rendered disease-free followed by adjuvant immunotherapy with follow-up until stage IV distant metastasis (n=12), where cfDNA mutation burden (CMB) levels correlated with TMB (p=0.019). This enabled earlier detection of stage IV disease recurrence compared to imaging (p&amp;lt;0.01), and captured tumor heterogeneity, and rising CMB levels prerecurrence clinical detection. An expanded 70-gene digital NGS panel was used to assess melanoma brain metastasis (MBM) in 26 preoperative MBM patients who had paired resected MBM tumors-cfDNA. Interestingly, tumor-identified MBM mutations were detected in the paired cfDNA at a concordance of 48%, 54%, and 58% at mutant allele frequency cut-offs of 0%, 0.5%, and 1%, respectively. Encouragingly, cfDNA-tumor SNV concordance was 88% in known melanoma driver genes (BRAF/NRAS). We then assessed a novel 1q21.3 CNA during melanoma progression in patients receiving checkpoint inhibitor immunotherapy (CII). Using a multiplex ddPCR assay targeting four 1q21.3 gene targets, we identified 77% (n=31) of patients at the time of progressive disease while on CII had 1q21.3 CNA in plasma whereby patients with stable disease, partial response, or disease free had low or no 1q21.3 CNA cfDNA. These results demonstrate the ability to use different multiplatform cfDNA biomarkers to monitor melanoma disease progression for recurrence and treatment response. The study demonstrates that cfDNA as mutation detection can be improved in melanoma using other genomic aberrations in monitoring immunotherapy in melanoma patients. Citation Format: David S. Hoon, Selena Y. Lin, Rebecca Gentry, Steve O’Day. Monitoring of melanoma progression utilizing multiplatform biomarkers of blood cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR19.

Abstract A11: Serum cell-free DNA methylome-based signatures distinguish pituitary tumor from other neoplasias and by clinicopathologic features

Abstract Background: Several reports have indicated that distinct epigenomic patterns of pituitary tumor (PT) tissue, specifically DNA methylation, distinguish these tumors according to their functionality and could be involved in their pathogenesis. Thus far, molecular diagnosis and classification systems that guide clinical management of these tumors rely on the tissue profiling obtained by invasive surgical approaches (e.g., excision). However, increasing evidence showed that CNS tumors release cell material into the circulation, despite the existence of the blood-brain barrier, creating an opportunity for molecular profiling using a liquid biopsy. Although the pituitary portal system and the invasion of the cavernous system by PT may facilitate the spillage of tumor cell material into the bloodstream, until now liquid biopsy has not been described in pituitary tumors. Considering the stability, the cell specificity, and the reported role of DNA methylation in PT, we investigated the feasibility to detect and define PT-specific methylation-based signatures in the serum of patients harboring these tumors. Methods and Findings: In order to identify PT-specific methylation-based signatures in the serum of patients with PT, we conducted unsupervised and supervised analysis of the methylome profile of paired serum cfDNA (EPIC array) and/or tissue from 13 patients with pituitary macroadenomas (9 males; median age: 62; 9 nonfunctioning/4 functioning, 6 invasive/7 noninvasive), 4 controls serum (nontumor and healthy), and patients with other CNS tumors or conditions (114 gliomas, 6 meningiomas, 1 brain metastasis, 1 colloid cyst, 6 radiation necrosis). Serum PT-tissue-specific methylation was also used as input into a machine learning algorithm to generate a PT score, which was tested in an independent cohort. Unsupervised and supervised analysis indicated that the serum methylome from patients harboring PT was distinct from controls and other CNS diseases. A generated PT score using the 37 identified PT tissue-specific methylation probes was able to distinguish serum from patients harboring PT from other CNS tumors. Serum-derived PT function-specific DMP also distinguished PT according to functional status. Serum-derived invasive-specific DMPs were less prominent to define invasive status in an independent cohort of PT, most probably due to the limited number of cases. Conclusion: This is the first study to show the feasibility of profiling methylome in the serum of patients with PT using cfDNA. In addition, we identified unique methylation signatures that distinguished PT from other CNS tumors and according to distinct PT subtypes. These results underpin the potential role of methylation profile and liquid biopsy as a noninvasive approach to assess clinically relevant molecular features that can be used as diagnostic, prognostic, and surveillance in patients with PT. Citation Format: Michael Wells, Karam Asmaro, Jack Rock, Thais Sabedot, Maritza Mosella, Tathiane Malta, Kevin Nelson, James Snyder, Steven Kalkanis, Ana Valeria Castro, Houtan Noushmehr. Serum cell-free DNA methylome-based signatures distinguish pituitary tumor from other neoplasias and by clinicopathologic features [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A11.

Cell-free DNA and exoDNA analysis in metastatic colorectal cancer patients (mCRC).

e16093 Background: Liquid biopsy is a growing field in translational cancer research. Two of the most studied liquid biopsy biomarkers are cell-free DNA (cfDNA) and exosomes, nano-sized vesicles that transport protein and nucleic acids including DNA (exoDNA). Therefore, both cfDNA and exoDNA are potentially useful to investigate the molecular landscape of tumor with a minimally invasive approach. Here we investigate the prognostic and predictive role of both cfDNA and exoDNA in mCRC using Next Generation Sequencing (NGS) analysis. Methods: From July 2017 to September 2018, samples of 40 mCRC patients were collected at the Medical Oncology of the AOUP Paolo Giaccone of Palermo. Blood samples were collected in EDTA-tubes before chemotherapy infusion (T1), at the first instrumental evaluation (T2) and every 2-3 months (T3). Plasma was used to isolate cfDNA and exosomes using the QIAamp Circulating Nucleic Acid and ExoEasy Maxi Kit respectively. ExoDNA was isolated through the QIAamp DNA micro kit. NGS analysis was conducted on IonS5 with AmpliSeq Cancer Hotspot Panel v2, which includes hotspot regions of 50 cancer-related genes. Results: Patients with cfDNA concentration &gt; 0.47ng/μl have worse PFS compared with those with &lt; 0.47 ng/μl cfDNA level. NGS analysis from 16 cfDNA samples showed a total of 292 mutations, with a median of 5 non-synonymous mutations in T1 vs. 8 non-synonymous mutations in T2 sample. We report an inverse and significant correlation between non-synonymous mutations count and disease-specific survival (DSS). Only 2 cfDNA samples carried KRAS mutations, despite it were previously reported in 5 paired tissue samples. An interesting result emerged from exoDNA sequencing of CRC20. KRAS mutation was identified in T1 exoDNA but not in the paired cfDNA samples, suggesting that exoDNA is able to intercept KRAS mutation earlier than cfDNA. Conclusions: cfDNA analysis could be a useful tool for better patients’ stratification. exoDNA could allow an early interception of disease recurrence that could be explained by the ability of exosomes to protect their cargo from degradation.

Genetic Variants Detected Using Cell-Free DNA from Blood and Tumor Samples in Patients with Inflammatory Breast Cancer.

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

RAS/RAF mutations in tumor samples and cell-free DNA from plasma and bone marrow aspirates in multiple myeloma patients.

Purpose: To evaluate the detection of gene mutations in bone marrow biopsy and circulating free DNA (cfDNA) from plasma in multiple myeloma (MM).Experimental design: We used cell-free DNA from plasma and bone marrow to test BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (ddPCR), and evaluated results with clinical outcomes.Results: We found of 83 patients, the detectable mutation frequencies for the above four genes were 4 (5%), 13 (16%), 3 (4%) and 14 (17%) in bone marrow, respectively. The median variant allelic frequency (VAF) in most mutations were 1.595%. In 17 paired cfDNA samples, the detectable mutation frequencies for the above four genes were 5 (30%), 1 (6%), 0 (0%) and 3 (18%) respectively, and the median VAF rate was 2.9%. Agreement between bone marrow DNA and plasma cfDNA were 76%, 100%, 100% and 100% compared to the tissue detections, respectively. In 17 patients with paired bone marrow and plasma samples, the above four mutations were 3 (18%), 1 (6%), 0 (0%) and 2 (12%) respectively, with the agreement rates of 88%, 88%, 100% and 100% compared to tissue detections. Of 57 patients with available outcome data, high mutation VAF had a shorter median survival than patients with low mutation VAF (P=0.0322).Conclusions: Oncogenic mutations in BRAF, KRAS and NRAS genes can be detected in the bone marrow and plasma cfDNA with ddPCR in patients with MM patients and high VAF is associated with short survival.