Peripheral Blood Cell-free DNA Research Articles

Plasma Cell-Free DNA in Uterine Cervical Cancer: Therapeutic Monitoring and Prognostic Values after Radical Radiotherapy.

In the present study, we aimed to establish a liquid biopsy-based monitoring method using peripheral blood cell-free DNA (cfDNA) for patients with cervical cancer who underwent radical radiotherapy (RT). Twenty-five patients with cervical cancer were prospectively recruited and treated with external beam RT and brachytherapy. In all patients, except one, chemotherapy was administered concurrently during RT. Whole peripheral blood samples were obtained at least twice from each patient. We performed next-generation sequencing (NGS) for the target-captured libraries (67 oncogenes and human papillomavirus [HPV] type 16/18) using 64 plasma cfDNA samples from the 25 participants. The ratio of HPV cfDNA and the variant allele frequency (VAF) in cfDNA was calculated, and their dynamic changes were monitored. The median follow-up duration was 25.4 months. In total, we identified 21,866 cfDNA variants. ARID1A and frameshift variants occupied the largest portion of altered genes and HIGH-grade variant types, respectively. In most cases, tumor shrinkage was followed by a decrease in the HPV ratio; however, an increase in HPV ratio indicated distant metastasis, despite the reduced tumor size. The initial HPV ratio reflecting the tumor burden was likely associated with treatment outcomes (p = 0.16). We did not determine a role for serial changes in the VAF in cfDNA. Our findings suggest that the HPV cfDNA ratio, calculated after targeted NGS, may be valuable for monitoring and predicting treatment responses. Accordingly, further validation of these findings is warranted.

Abstract OT1-18-08: Randomized phase II trial of pembrolizumab/carboplatin vs. carboplatin alone for breast cancer with chest wall recurrence: TBCRC044

Abstract Background: A significant number of patients with breast cancer may develop chest wall recurrence, which can be difficult to treat, and is associated with a poor prognosis. Given the inflammatory nature of chest wall disease, and the association of chest wall disease with lymphovascular invasion, we hypothesized that immunotherapy may be beneficial in this setting. Furthermore, combining immunotherapy and chemotherapy may have a synergistic effect; indeed, the combination of the anti-programmed cell death 1 (PD-1) antibody, pembrolizumab, with chemotherapy has been approved for metastatic triple negative breast cancer (TNBC). Consequently, in this study, we are evaluating the combination of pembrolizumab/carboplatin vs. carboplatin alone for chest wall recurrence. Trial design: This is a phase II study of patients with chest wall disease from breast cancer. Patients are randomized in a 2:1 manner to pembrolizumab/carboplatin x 6 cycles (Arm A) with the option to continue pembrolizumab +/- carboplatin after 6 cycles (Arm Ax) vs. carboplatin alone (Arm B) with the option to cross-over to pembrolizumab +/- carboplatin on progression (Arm Bx). Carboplatin is dosed at AUC 5 IV every 3 weeks, and pembrolizumab at 200 mg IV every 3 weeks. Trastuzumab may be continued with study treatment in patients with HER2 positive disease. Imaging scans (CT chest, abdomen, and pelvis, and bone scan) occur at baseline and every 3 cycles. Chest wall biopsies and peripheral blood is collected at baseline and after completing 2 cycles of treatment for correlative studies. Eligibility criteria: Patients must have chest wall disease from breast cancer. Distant metastases are allowed. TNBC, hormone receptor positive/HER2 negative disease (after 2 prior hormone therapies), and HER2 positive disease (progressed on standard HER2 directed treatment) are eligible. Any number of prior lines of chemotherapy are acceptable, including a prior platinum chemotherapy in the absence of disease progression on the platinum. Specific Aims: Primary aim is to determine disease control rate in the chest wall and other distant sites at 18 weeks of treatment with RECIST 1.1. Secondary aims including determining toxicity, progression-free survival, response based on irRECIST, and response based on tumor PD-L1 expression. Exploratory aims include studying changes in: 1) immune composition of tumor and peripheral blood, 2) peripheral blood cell-free DNA and circulating tumor cells, 3) soluble PD-L1 expression, and 4) association of MYC with PD-1 and immune markers, based on preclinical data demonstrating that MYC upregulates these markers. Statistical Methods: 84 patients (Arm A: 56, Arm B: 28) are being enrolled at 7 sites in the Translational Breast Cancer Research Consortium. The study is powered to identify a 20% difference in disease control rates between Arms A and B (HR 0.52, α= 0.10, ß=0.20). A futility analysis will occur for Arm B after 18 patients are enrolled to allow for early closure if lack of efficacy. Accrual: Present accrual is 57 patients (Arm A: 39, Arm B: 18). (NCT03095352). This trial is partly funded by grants from Merck and University of California San Francisco. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu Citation Format: Neelima Vidula, Rita Nanda, Kathy Miller, Leisha Emens, Vandana Abramson, Ben Park, Minetta C. Liu, Andrei Goga, Hope Rugo. Randomized phase II trial of pembrolizumab/carboplatin vs. carboplatin alone for breast cancer with chest wall recurrence: TBCRC044 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-18-08.

Molecular Characterization of Waldenström's Macroglobulinemia Patients Using Peripheral Blood Cell-Free Tumor DNA

BackgroundMutational characterization of Waldenström's macroglobulinemia (WM) currently relies on DNA from CD19-selected cells derived from bone marrow (BM) aspirates, which is an invasive technique, associated with significant patient discomfort. Peripheral blood cell-free tumour DNA (cf or ctDNA) has been recently shown to be a powerful non-invasive molecular biomarker in monitoring tumor status in several cancers. In this study, we aimed to characterize the mutational status of WM/IgM MGUS patients by using peripheral blood plasma-derived cfDNA and using matched tumor DNA (tDNA) from BM-CD19+ selected cells as a control, in order to determine whether peripheral blood , by means of cfDNA, can be used as an additional diagnostic tool in identifying the mutational profile of WM.MethodsA total of 30 consecutive patients with IgM monoclonal gammopathies were included in this study. All patients gave a written informed consent for sample collection and analysis. Peripheral blood (10-12mL) was collected in EDTA tubes and DNA was extracted using the MagMax cell free DNA isolation kit (ThermoFisher Scientific). cfDNA concentration was measured using the Qubit fluorometer. The quantities of cfDNA varied between 1.5 ng/μL and 8 ng/μL. BM aspirates were collected at the same time with peripheral blood, and were processed for CD19 enrichment, using CD19 magnetic beads (Miltenyi Biotech), followed by DNA extraction (Invitrogen). The paired tDNA and cfDNA samples from the 30 patients were analyzed for the MYD88 L265P and for CXCR4 mutations. The presence of L265P mutation was initially assessed by Allele-Specific PCR (AS-PCR) and then confirmed with direct sequencing. The presence of CXCR4 mutations was assessed with direct sequencing.ResultsThe study included patients with symptomatic WM before initiation of any therapy (N=4) , with WM under therapy (N=4) or after completion of therapy (N=12), with asymptomatic WM (N=8) and with IgM MGUS (N=2). The median age of the patients was 70 years (range 22-87). The median bone marrow infiltration of the tested samples was 28% (range 5 to 90%) and median IgM levels were 1510 mg/dL (range 80 to 7310 mg/dL).In all, but one patient, (29/30) the L265P mutation was detected in both cfDNA and tDNA (100% concordance). This mutation was initially detected by AS-PCR which was validated by direct sequencing in all patients. Assessment of CXCR4 mutation status was feasible in 29 patients; in one patient CXCR4 sequencing was unsuccessful in both cfDNA and tDNA. Mutations in CXCR4 were detected in 24% (7/29) of patients. The pathogenic mutation S338X (rs104893626) was present in 2 (7%) patients and was identified in both cfDNA and tDNA. In one patient the L50X (1/29) truncating mutation was found in tDNA but not in cfDNA. Two patients harbored the F29L (2/29) mutation and another two (2/29) the P31T mutation, both in cfDNA and in tDNA; these two mutations have not been previously reported. Synonymous mutations were also found in 10% of the tested patients in both cfDNA and tDNA. Thus, the concordance for CXCR4 mutations identified by direct sequencing of cfDNA and tDNA was 97% (in 28/29 patients).ConclusionsOur study demonstrates that in patients with IgM monoclonal gammopathies peripheral blood cell-free DNA can be used as a non-invasive method to detect the MYD88 L265P mutation with excellent concordance to BM CD19+ derived tumor DNA. Furthermore, CXCR4 mutations could also be detected with high concordance to tDNA by direct sequencing. This method could be more cost effective, as it avoids the expenses and time needed for CD19 selection, either BM or peripheral blood derived. Peripheral blood cfDNA could be used as an initial test to assess the presence of MYD88 L265P and CXCR4 mutations, so that BM testing can be limited to patients with indeterminate results. Updated results will be presented in the meeting. Further investigation is ongoing in order to assess tumor dynamics using peripheral blood. DisclosuresTerpos:Janssen: Honoraria, Research Funding; Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; Abbvie: Honoraria; Takeda: Honoraria, Other: SC member; Amgen: Honoraria, Other: SC member, Research Funding; GSK: Honoraria; BMS: Honoraria. Kastritis:Prothena: Honoraria; Janssen: Honoraria; Genesis pharma: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding. Dimopoulos:Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding.

Double Drop-Off Droplet Digital PCR: A Novel, Versatile Tool for Mutation Screening and Residual Disease Monitoring in Acute Myeloid Leukemia Using Cellular or Cell-Free DNA

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Abstract OT-13-09: Translational breast cancer research consortium 044 trial: Randomized phase 2 study of pembrolizumab and carboplatin versus carboplatin alone for chest wall recurrence of breast cancer

Abstract Background: Chest wall recurrence may occur in up to 30% of patients with breast cancer, and is associated with a high morbidity and mortality. We hypothesized that immunotherapy may be beneficial in this setting because of the inflammatory nature of this disease, and the association of chest wall disease with lymphovascular invasion. Combination immunotherapy and chemotherapy may have a synergistic effect. In this study, we combined pembrolizumab, an anti-programmed cell death 1 (PD-1) antibody with carboplatin, in patients with chest wall disease. This combination has previously been shown to be effective in advanced lung cancer. Trial design: This is a 2:1 randomized phase II study of pembrolizumab and carboplatin (Arm A) versus carboplatin alone (Arm B) in patients with chest wall involvement from breast cancer. Pembrolizumab is dosed at 200 mg IV every 3 weeks, and carboplatin is dosed at AUC 5 IV every 3 weeks. Patients on Arm A have the option to continue on pembrolizumab alone after 6 cycles of combination treatment (Arm Ax). Patients on Arm B have the option to cross-over to pembrolizumab (+/- carboplatin) after 6 cycles of carboplatin alone (Arm Bx). Patients with HER2 positive disease may continue trastuzumab in addition to the study treatment. Patients undergo chest wall biopsies and peripheral blood collection for correlative studies at enrollment and after 2 cycles of treatment, and also imaging with CT chest, abdomen, and pelvis, and bone scan every 3 cycles. Eligibility criteria: Patients must have chest wall involvement from breast cancer, with or without distant metastases. Patients may have triple-negative breast cancer, hormone receptor positive, HER2- disease (after two prior lines of hormone therapy), or refractory HER2 positive disease. Patients may have received any number of lines of prior chemotherapy. A prior platinum is allowed as long as there was not disease progression on this agent. Specific Aims: 1. (Primary aim) Disease control rate in the chest wall and other distant sites at 18 weeks of treatment using RECIST 1.1 criteria, 2. progression-free survival, 3. toxicity, 4. response based on tumor PD-L1 expression, and 5. response based on irRECIST. Exploratory aims include evaluating: 1. Soluble PD-L1 expression, 2. changes in tumor and peripheral blood immune composition, 3. peripheral blood circulating tumor cells, 4. peripheral blood cell-free DNA, and 5. the association of MYC with PD-1 and TIM-3 on tumor cells, based on preclinical data to suggest that MYC may upregulate these inflammatory markers. Statistical Methods: 84 patients (56 in Arm A and 28 in Arm B) are being enrolled at 7 sites in the Translational Breast Cancer Research Consortium. The study is powered to determine a 20% difference in disease control rates between arms (HR 0.52, α= 0.10, ß=0.20). A futility analysis was originally planned to occur for Arm B after 18 patients are enrolled, but a subsequent amendment has enabled an earlier assessment after 14 patients were enrolled. Accrual: Present accrual is 40 patients. (NCT03095352). This trial is funded in part by Merck and a Development Program Grant from the University of California San Francisco. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu Citation Format: Neelima Vidula, Rita Nanda, Kathy Miller, Paula Pohlmann, Vandana Abramson, Leisha A. Emens, Ben H. Park, Minetta C. Liu, Andrei Goga, Hope S. Rugo. Translational breast cancer research consortium 044 trial: Randomized phase 2 study of pembrolizumab and carboplatin versus carboplatin alone for chest wall recurrence of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-13-09.

Higher Incidence of Germline Mutations in DNA Damage Response Genes in Lymphoid Than in Myeloid Neoplasms without Significant Increase in the Prevalence of Clonal Hematopoiesis

Introduction:DNA damage response (DDR) genes play major role in maintaining the genomic integrity of actively dividing cells such as lymphoid and myeloid cells. Higher rate of mutagenesis and development of cancer is well established in individuals with inherited defects in one of the DDR genes. However, the prevalence of inherited abnormalities in DDR genes in patients with myeloid and lymphoid neoplasms is not well established. Furthermore, the impact of such abnormalities on the clinical course and outcomes of patients with inherited defects in DDR genes is unknown. We investigated the prevalence of germline DDR gene mutations in patients diagnosed with lymphoid neoplasms and compared findings with those in patients diagnosed with myeloid neoplasms. Method: Tissue and peripheral blood cell-free DNA (cfDNA) from 683 patients with confirmed myeloid or lymphoid neoplasms were tested using a next generation sequencing (NGS) panel of 177 genes. The germline nature of the mutation was confirmed by demonstrating that the mutations were detected in all analyzed DNA in cfDNA. Sequential patients were tested between early 2019 and 2020 without any selection, including newly diagnosed as well as relapsed/refractory patients. The DNA sequencing is based on Single Primer Extension (SPE) library preparation with unique molecular identifier (UMI) (Qiagen, Germantown, MD). Sequencing data of DNA is analyzed using the DRAGEN Platform. Sequence duplicates were removed before calculating VAF. Results:Confirmed germline mutations were detected in the following DNA repair genes: BRCA1, BRCA2, MUTYH, FANCA, FANCE, FANCG, ATM, BLM, and GEN1. In addition germline mutations in MYD88, RET, ROS1 TET2, TNFAIP3, CBL and FLT3 were also detected. Mutations were considered pathogenic only if classified as such in ClinVar database or when showed early termination. Of the 381 patients with various types of lymphoma (follicular, DLBCL, mantle, T-cell lymphoma, marginal zone, and multiple myeloma), 29 (7.6%) showed germline mutations. This is was significantly higher than the prevalence of germline mutations in myeloid neoplasms (P<0.001). In myeloid neoplasms (MDS, AML, and MPN), only 10 patients of 302 had germline mutations (3%). There was no significant difference in age at the time of testing between the two groups (median of 66 years, range 19 to 93 in lymphoid vs median of 65, range 21 to 93 in the myeloid group). We also evaluated the lymphoma group for the prevalence of clonal hematopoiesis of indeterminate potential (CHIP) as defined by the presence of isolated mutations in TET2, DNMT3A, and ASXL1 in cfDNA that were not present in the lymphoma sample. CHIP was detected in 19 of the 352 (5.39%) lymphoma patients without DDR germline abnormalities, while 3 of the 29 with germline mutation (10%) showed CHIP (P=0.2). Conclusion:The role of DDR mechanism is very significant in lymphomagenesis. Patients with germline mutations in one of the DDR genes should be monitored for the development of lymphoma. However, the presence of a defect in one of the DDR genes does not seem to significantly increase the prevalence of CHIP. The impact of the presence of DDR deficiency on therapy outcome, development of secondary neoplasm needs further investigation. Figure Disclosures Donato: Genomics Cooperative:Current equity holder in publicly-traded company.Siegel:Merck:Consultancy, Honoraria, Speakers Bureau;BMS:Consultancy, Honoraria, Speakers Bureau;Janssen:Consultancy, Honoraria, Speakers Bureau;Takeda:Consultancy, Honoraria, Speakers Bureau;Celulatiry:Consultancy;Karyopharma:Consultancy, Honoraria;Amgen:Consultancy, Honoraria, Speakers Bureau.Feldman:Rhizen:Research Funding;Corvus:Research Funding;Kite:Honoraria, Other: Travel expenses, Speakers Bureau;BMS:Consultancy, Honoraria, Research Funding;Viracta:Research Funding;Bayer:Consultancy, Honoraria;Abbvie:Honoraria;Pharmacyclics:Honoraria, Other, Speakers Bureau;Amgen:Research Funding;Cell Medica:Research Funding;Eisai:Research Funding;Kyowa Kirin:Consultancy, Research Funding;Pfizer:Research Funding;Portola:Research Funding;Janssen:Speakers Bureau;AstraZeneca:Consultancy;Trillium:Research Funding;Celgene:Honoraria, Research Funding;Takeda:Honoraria, Other: Travel expenses;Seattle Genetics, Inc.:Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau.Biran:BMS:Consultancy, Honoraria, Other: reimbursement of travel and accommodation, Research Funding, Speakers Bureau;Amgen:Consultancy, Honoraria, Other: reimbursement of travel and accomodation, Research Funding, Speakers Bureau;Janssen:Consultancy, Honoraria, Other: reimbursement of travel and accomodation, Research Funding, Speakers Bureau;Sanofi:Honoraria, Speakers Bureau;KAryopharma:Research Funding.Koprivnikar:Amgen:Speakers Bureau;Novartis:Speakers Bureau;Alexion:Speakers Bureau;BMS:Speakers Bureau.Goy:AbbVie:Research Funding;Morphosys:Research Funding;MD Anderson:Research Funding;Regional Cancer Care Associates/OMI:Current Employment;Infinity Verastem:Research Funding;Xcenda:Consultancy;COTA:Consultancy, Current equity holder in publicly-traded company, Other: leadership role;Kite, a Gilead Company:Consultancy, Current equity holder in publicly-traded company, Honoraria, Other: leadership role, Research Funding;Janssen:Consultancy, Honoraria, Other: leadership role, Research Funding;Celgene:Honoraria, Research Funding;AstraZeneca:Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: leadership role, Research Funding;Acerta:Consultancy, Honoraria, Other: leadership role, Research Funding;RCCA/OMI:Current Employment;PracticeUpdate Oncology:Consultancy;Bayer:Research Funding;CALBG:Research Funding;Constellation:Research Funding;Genentech/Roche:Research Funding;Infinity:Research Funding;Karyopharm:Research Funding;Hackensack UMC and University of Nebraska:Research Funding.

Prediction of blood-based biomarkers and subsequent design of bisulfite PCR-LDR-qPCR assay for breast cancer detection

BackgroundInterrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR.MethodsIn this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood.ResultsOur results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27).ConclusionThis report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.

Eliminating the Need for Pancreas Biopsy Using Peripheral Blood Cell-free DNA

Eliminating the Need for Pancreas Biopsy Using Peripheral Blood Cell-free DNA

Using plasma cell-free DNA to monitor the chemoradiotherapy course of cervical cancer.

The liquid biopsy is being integrated into cancer diagnostics and surveillance. However, critical questions still remain, such as how to precisely evaluate cancer mutation burden and interpret the corresponding clinical implications. Herein, we evaluated the role of peripheral blood cell-free DNA (cfDNA) in characterizing the dynamic mutation alterations of 48 cancer driver genes from cervical cancer patients. We performed targeted deep sequencing on 93 plasma cfDNA from 57 cervical cancer patients and from this developed an algorithm, allele fraction deviation (AFD), to monitor in an unbiased manner the dynamic changes of genomic aberrations. Differing treatments, including chemotherapy (n = 22), radiotherapy (n = 14) and surgery (n = 15), led to a significant decrease in AFD values (Wilcoxon, p = 0.029). The decrease of cfDNA AFD values was accompanied by shrinkage in the size of the tumor in most patients. However, in a subgroup of patients where cfDNA AFD values did not reflect a reduction in tumor size, there was a detection of progressive disease (metastasis). Furthermore, a low AFD value at diagnosis followed a later increase of AFD value also successfully predicted relapse. These results show that plasma cfDNA, together with targeted deep sequencing, may help predict treatment response and disease development in cervical cancer.

Circulating extracellular DNA levels are acutely elevated in ischaemic stroke and associated with innate immune system activation

ABSTRACTObjective: The objective of this work was to assess the ability of peripheral blood cell-free DNA (cfDNA) levels to identify ischaemic stroke early in the acute phase of care, as well as to examine the relationship between peripheral blood cfDNA levels and stroke-induced innate immune system activation. Methods: Upon emergency department admission, peripheral blood samples were obtained from 43 patients experiencing acute ischaemic stroke and 20 patients identified as stroke mimics. Plasma cfDNA levels were measured using quantitative polymerase chain reaction (qPCR), infarct volume and NIH stroke scale (NIHSS) were used to assess injury severity, and peripheral blood neutrophil count was used as a measure of innate immune system status.Results: Peripheral blood cfDNA levels were significantly elevated in patients suffering stroke relative to those diagnosed as stroke mimics, and could differentiate between groups with 86% (95% CI = 72–95%) sensitivity and 75% (95% CI = 51–91%) specificity. Furthermore, cfDNA levels displayed significant positive associations between both infarct volume and peripheral blood neutrophil count within the stroke group. Conclusions: These findings suggest that assessment of peripheral blood cfDNA levels may be useful for the identification of ischaemic stroke in the acute care setting, and provide associative evidence that cfDNA is a potential activator of the peripheral innate immune system in response to cerebral ischaemia.

Peripheral Blood Cell Free DNA to Monitor Mutational Response in Epidermal Growth Factor Receptor–Mutant Non–Small Cell Lung Cancer

Peripheral Blood Cell Free DNA to Monitor Mutational Response in Epidermal Growth Factor Receptor–Mutant Non–Small Cell Lung Cancer

Higher Mutation Rate in Patients with Aplastic Anemia Using Peripheral Blood cfDNA As Compared with Bone Marrow Cells

Background:We have reported that peripheral blood cell-free DNA (cfDNA) is reliable for detecting bone marrow molecular abnormalities in patients with hematologic neoplasms. However, not clear is whether cfDNA is sufficient to detect mutations present at low variant allele frequency (VAF). Since patients with aplastic anemia (AA usually have relatively small clones in blood and bone marrow (BM), we compared mutations detected in BM cells with those detected in peripheral blood cfDNA from patientswith this disease.Methods: A total of 120 paired BM aspirate and PB plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. We used NucliSenS EasyMAG automated platform for extracting total nucleic acid from PB plasma collected in EDTA. All paired BM and plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA), which covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X.Results: One hundred twenty paired BM and cfDNA samples from 96 patients with aplastic anemia were tested. Of the 96 patients, 33 (34%; equivalent to 48 samples, 40%) had one or more mutations. We identified 54 different somatic mutations in these patients, of which 45 were unique. There was no significant difference (P=0.71, Sign test) in allele frequency between cfDNA and BM. The median mutant allele frequency was 10.9% in cfDNA and 12.6% in BM cells, and 40 of the 54 mutations had allele frequency ≤20% in BM cells, while 45 samples had allele frequency ≤20 in cfDNA. Six of the 33 patients with somatic mutations (18%) showed mutations in plasma cfDNA but not in BM. In contrast, 2 patients (6%) showed mutations in BM cells and not in cfDNA. One of these two patients had a mutation in ASXL1 gene detected in BM cells but not in cfDNA and a subsequent sample showed the same ASXL1mutation in BM cells and not in cfDNA, and a second clone with a different ASXL1 mutation detected in both BM cells and cfDNA. Overall concordance between BM cells and cfDNA in the 120 samples was 92% and there was no statistically significant difference between the two sample types (P=0.6).Summary and Conclusions: Seven samples (from 7 patients) of the 120 tested samples showed mutations in cfDNA and not in BM cells while 3 samples (from 2 patients) showed mutations in BM and not in cfDNA. VAF of mutations in cfDNA were similar to those in BM cells. Therefore, peripheral blood cfDNA should be tested in addition to BM cells for detecting mutations in patients with AA. Peripheral blood cfDNA can be used as a reliable means for monitoring patients with AA. cfDNA testing can be used as an alternative testing to bone marrow even when mutant allele frequency in bone marrow is <20%. cfDNA may be an especially valuable source of mutation detection in marrow failure, in which marrow aspirates may not contain sufficient cells for accurate mutation analysis. DisclosuresAlbitar:Neogenomics Laboratories: Employment. Townsley:Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Young:GSK/Novartis: Research Funding. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Peripheral blood cell-free DNA is an alternative tumor DNA source reflecting disease status in myelodysplastic syndromes.

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell‐free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma‐PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher‐risk groups than that in lower‐risk groups (P = 0.034). The concentration of plasma‐/serum‐PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160‐bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM‐DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB‐2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.

Utilization of Peripheral Blood Cell-Free DNA in Myelodysplastic Syndromes: Clinical and Molecular Characteristics and Utilization for Genetic Analyses Using Conventional and Next-Generation Strategies

Abstract Background: Genetic mutations are detected in over 90% of patients with myelodysplastic syndromes (MDS). Some mutations may be critical not only for the pathogenesis of MDS, but also for disease progression to acute myelocytic leukemia and the acquirement of drug resistance; however, the detailed functions of these mutations remain unclear. To determine the roles of these mutations, genetic analyses using serially harvested tumor DNA are required. To date, genomic DNA from bone marrow (BM) cells has been utilized for genetic analyses of MDS patients, but bone marrow aspiration cannot be performed repeatedly because it causes physical pain in patients. Recently, peripheral blood cell-free DNA (PB-cfDNA) obtained from plasma and/or serum has received much attention as an alternative tumor DNA source, especially for solid tumors. Here, we demonstrate that PB-cfDNA may be used as an alternative DNA source instead of BM cells, that it reflects MDS disease status, and that the mutations in MDS can be successfully detected from PB-cfDNA by Sanger sequencing and next-generation targeted sequencing analyses. Aims: 1) Confirmation of the molecular and clinical basis of PB-cfDNA in MDS; 2) detection of genetic mutations using PB-cfDNA with conventional and next-generation sequencing strategies; and 3) optimization of sequencing conditions using PB-cfDNA. Methods: PB-cfDNA from patients with MDS and other related diseases (N = 33) and normal volunteer donors (N = 19) was analyzed. The concentration of PB-cfDNA was measured and correlations between the PB-cfDNAconcentration and clinical data were analyzed. DNA sequencing was performed using Sanger sequencing and targeted sequencing was performed using a TruSight Myeloid Sequencing Panel (Ilumina). Results: The plasma PB-cfDNA concentration was significantly higher in MDS patients than in normal donors (p = 0.0405), and was significantly higher in those with a higher International Prognostic Scoring System (IPSS) score than in those with a lower score (p = 0.0339). The concentration of plasma and serum PB-cfDNA was significantly correlated with the serum lactate dehydrogenase (LDH)level (both p &lt; 0.0001) and the blast cell count in PB (plasma, p = 0.0373; serum, p = 0.0274). Since PB-cfDNA showed a fragmented pattern reflecting its oligonucleosomal structure, amplification using primers for PCR-based sequencing analyses were optimized when the size of the PCR products were approximately 160 bp. For Sanger and targeted sequencing, 1 and 50 ng of PB-cfDNA, respectively, were required for each assay. Almost all genetic mutations detected by Sanger and targeted sequencing using BM cells from MDS patients (TET2, IDH2, SETBP1, U2AF1, SRSF2, NRAS, TP53) were also detected using the PB-cfDNA, but they were not detected in the germline controls. Serially harvested PB-cfDNA samples from a patient with refractory anemia with excess blasts-1 (RAEB-1) who underwent cord blood transplantation (CBT) after 5-azacytidine treatment were utilized for genetic analyses; they showed a correlation between PB-cfDNA concentration and the clinical course, and that the U2AF1 and SETBP1 mutations detected before CBT were no longer detectable 150 days after CBT. Discussion: These data suggest that PB-cfDNA likely originates from MDS clones, which reflect their disease status, and that they can be utilized as a biomarker and an alternative promising source of tumor DNA instead of BM cells for genetic analyses; including not only conventional Sanger sequencing, but also next-generation targeted sequencing. However, since the PB-cfDNA concentration of some MDS patients was too low (less than 10 ng/1 mL plasma) to be used for targeted sequencing and/or whole exome sequencing, optimization of the sensitivity of sequencing analyses is still required. Disclosures Tomita: Janssen Pharmaceutical K.K.: Consultancy. Ishikawa:GlaxoSmithKline K.K.: Research Funding. Kiyoi:Alexion Pharmaceuticals: Research Funding; Eisai Co., Ltd.: Research Funding; Japan Blood Products Organization: Research Funding; FUJIFILM RI Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Bristol-Myers Squibb: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents &amp; Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding.

Clinical and Molecular Significance of Peripheral Blood Cell-Free DNA in B-Cell Lymphomas for Detection of Genetic Mutations and Correlation with Disease Status

[Backgrounds]Genetic abnormalities in B-cell lymphoma (BCL) have been analyzed by using tumor cell-derived DNA (tDNA) obtained from biopsy samples at diagnosis, while the amount of available samples is often limited due to the advance in diagnostic technique. Furthermore since repetitive biopsies during the clinical course are not generally acceptable, sequential analysis of tumor samples is difficult. The peripheral blood circulating cell-free DNA (PB-cfDNA), which is detected in human plasma or serum, has been shown to be a possible alternative source for DNA mutation analysis and an indicator for monitoring disease status especially in solid tumors. PB-cfDNA, if available, should be quite beneficial for lymphoma practice in terms of invasiveness and accessibility compared to the conventional biopsies, however the role of PB-cfDNA in BCL remains unknown. We thus investigate the feasibility of PB-cfDNA in diagnosing and monitoring disease status of BCLs.[Aims]To uncover the role of PB-cfDNA in BCLs, we evaluated the clinical and molecular features and the association with lymphoma status, and further investigated the usefulness for global genetic analyses.[Patients and Methods]The study population included 38 BCLs patients (diffuse large B-cell lymphoma (DLBCL), n=27; follicular lymphoma (FL), n=10; nodal marginal zone B-cell lymphoma (NMZL), n=1) diagnosed at Nagoya University Hospital and affiliated hospitals. Plasma separated from PB and tumor cells from biopsy samples were collected at diagnosis and during their clinical course with written informed consent. PB-cfDNA and tDNA were extracted from plasma (n=107) and tumor samples, respectively. The concentration and quality of PB-cfDNA were analyzed by the gel electrophoresis and BioAnalyzer (Agilent Technologies, Inc.). For genetic analyses, the Sanger sequencing and whole-exome sequencing (WES) using HiSeq 2000 (Illumina) were performed. PB mononuclear cells from each patient were used as the matched germline control. The profiles of genetic mutations of PB-cfDNA and tDNA were analyzed.[Results]In 31 of 38 (81%) patients (DLBCL, n=24; FL, n=6; NZML, n=1), PB-cfDNA was successfully obtained from plasma samples at diagnosis. The median PB-cfDNA concentrations in DLBCL and FL patients were 58.3 ng/mL (range, 1.4-8754.5) and 15.5 ng/mL (0.1-164.9), respectively (p=0.2127). Intriguingly, the concentration of PB-cfDNA in a specific subtype of DLBCL was much higher (median, 631.9 ng/mL; range, 70.4-1109.5). LDH and CRP were significantly correlated with the PB-cfDNA concentration (correlation coefficient, r = 0.9582 and 0.8023, respectively, n=38). The PB-cfDNA concentration in patients with B-symptom was significantly higher than that in patients without B-symptom (p=0.011) and tended to be higher in patients with advanced clinical stages and bone marrow invasion, although not statistically significant. In the time course analyses, the concentration of PB-cfDNA increased immediately after the commencement of chemotherapy and rapidly decreased within a week after the beginning of treatment, probably reflecting the release of PB-cfDNA after the collapse of tumor cells due to chemotherapy. When analyzed by WES, PB-cfDNA and tDNA were shown to have discrete sets of mutations; a set of mutations were shared by both samples and tended to have higher variant allele frequencies, whereas others were private to an either sample, suggestive of the presence of regional tumor heterogeneity. In a specific BCL subtype, WES easily detected mutations in PB-cfDNA, even though mutations were hardly detected in tDNA because of a relatively lower tumor content.[Discussion and conclusions]PB-cfDNA could be detected in high proportion of BCL patients. Considering the correlation coefficient with clinical indicators and detection of mutations, PB-cfDNA in BCL should be mainly originated from lymphoma cells and closely associated with tumor aggressiveness and disease progression. Moreover, we could successfully perform WES analyses using PB-cfDNA, indicating that PB-cfDNA was an alternate source for detecting lymphoma specific genetic abnormalities. PB-cfDNA might be a novel diagnostic tool for patients in whom sufficient tumor samples are not obtained by conventional diagnostic procedure. DisclosuresKiyoi:Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Bristol-Myers Squibb: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding.