scholarly journals Cross-platform Profiling of ctDNA Using ddPCR: Standardization of the Liquid Biopsy for Pediatric Diffuse Midline Glioma

2020 ◽  
Author(s):  
Daphne Li ◽  
Erin R Bonner ◽  
Kyle Wierzbicki ◽  
Eshini Panditharatna ◽  
Tina Huang ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Mutation Detection ◽  
Pediatric Brain Tumor ◽  
Circulating Tumor Dna ◽  
Allelic Frequency ◽  
Detection Sensitivity ◽  
Therapeutic Monitoring ◽  
Sample Type ◽  
Invasive Approach ◽  
Diffuse Midline Glioma

Abstract Background Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poorer prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low ctDNA concentrations is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. Methods DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n=4), CSF (n=6), plasma (n=4), and human primary pediatric glioma cells (H3.3K27M, n=2; H3WT, n=1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 AàT H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n=3). Results We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3F3A mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutionsConclusion Our study demonstrates that ctDNA is reliably and reproducibly detected in ctDNA using ddPCR, representing a clinically feasible and reproducible minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

scholarly journals Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daphne Li ◽  
Erin R. Bonner ◽  
Kyle Wierzbicki ◽  
Eshini Panditharatna ◽  
Tina Huang ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Mutation Detection ◽  
Pediatric Brain Tumor ◽  
Circulating Tumor Dna ◽  
Allelic Frequency ◽  
Detection Sensitivity ◽  
Therapeutic Monitoring ◽  
Sample Type ◽  
Invasive Approach ◽  
Diffuse Midline Glioma

AbstractDiffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

scholarly journals PDTM-42. CROSS-PLATFORM PROFILING OF ctDNA USING ddPCR: STANDARDIZATION OF THE LIQUID BIOPSY FOR PEDIATRIC DIFFUSE MIDLINE GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi196-vi196
Author(s):  
Daphne Li ◽  
Erin Bonner ◽  
Stefanie Stallard ◽  
Eshini Panditharatna ◽  
Tina Huang ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Mutation Detection ◽  
Poor Response ◽  
High Grade Glioma ◽  
Circulating Tumor Dna ◽  
Allelic Frequency ◽  
Response To Therapy ◽  
Therapeutic Monitoring ◽  
Csf Analysis ◽  
Diffuse Midline Glioma

Abstract INTRODUCTION Pediatric diffuse midline glioma (DMG) with histone H3 mutations (80%), are highly morbid tumors with poor response to therapy. We previously detected H3 mutations in circulating tumor DNA (ctDNA) from CSF derived from children with DMG and high grade glioma. Here, we describe a high-throughput, sensitive and specific approach for H3 mutation detection and quantification in plasma and CSF specimens, validated across multiple centers. METHODS DNA extracted from tissue-validated H3.3K27M specimens (4 tumor, 5 CSF, 4 plasma) and pediatric glioma cell lines (1 wild-type, 1 H3.3K27M) were used to standardize ddPCR workflows. ctDNA extracted from 500uL of CSF or plasma was pre-amplified using sequence-specific primers, then analyzed on RainDance and BioRad ddPCR systems using two sets of custom primers and fluorescent LNA probes (Assays A and B). H3 mutation allelic frequency (MAF) was determined across specimens, and validated through parallel analysis of additional matched H3.3K27M tumor tissue, CSF and plasma specimens (n=3). RESULTS ctDNA processing and detection was standardized across three institutions and two platforms with sensitive, specific and reproducible H3 mutation detection. H3K27M mutations were detected in as little as 0.3-2pg input DNA on both platforms, with comparable MAFs across instruments and assays. RainDance yielded greater overall positive droplet number detection (increased sensitivity). Differences in optimal sample input volume between platforms were circumvented by vacuum concentration or dilution to maintain equal input DNA. Assay A yielded superior specificity so was used for subsequent matched specimen analysis. Mutation detection in plasma, as in previous studies, remained challenging due to low [ctDNA], while CSF analysis yielded reliable results across assays and platforms. CONCLUSIONS We demonstrate the utility of liquid biopsy for identifying H3K27M mutations in plasma and CSF via ddPCR with low starting [DNA], representing a rapid, minimally invasive method for diagnosis and therapeutic monitoring of DMG.

scholarly journals Clinical Value of ctDNA in Hematological Malignancies (Lymphomas, Multiple Myeloma, Myelodysplastic Syndrome, and Leukemia): A Meta-Analysis

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiangyu Tan ◽  
Han Yan ◽  
Lei Chen ◽  
Yuyang Zhang ◽  
Chunyan Sun
Keyword(s):  
Disease Progression ◽  
Sensitivity And Specificity ◽  
Hematological Malignancies ◽  
Meta Analysis ◽  
Progression Free Survival ◽  
Circulating Tumor Dna ◽  
Detection Sensitivity ◽  
Cochrane Library ◽  
Clinical Value ◽  
Invasive Approach

Background: Circulating tumor DNA (ctDNA) has offered a minimally invasive approach for the detection and measurement of cancer. However, its diagnostic and prognostic value in hematological malignancies remains unclear.Materials and methods: Pubmed, Embase, and Cochrane Library were searched for relating literature. Diagnostic accuracy variables and disease progression prediction data were pooled by the Meta-Disc version 1.4 software. Review Manager version 5.4 software was applied for prognostic data analysis.Results: A total of 11 studies met our inclusion criteria. In terms of diagnosis, the pooled sensitivity and specificity were 0.51 (95% confidence intervals (CI) 0.38–0.64) and 0.96 (95% CI 0.88–1.00), respectively. The AUSROC (area under the SROC) curve was 0.89 (95%CI 0.75–1.03). When it comes to the prediction of disease progression, the overall sensitivity and specificity was 0.83 (95% CI 0.67–0.94) and 0.98 (95% CI 0.93–1.00), respectively. Moreover, a significant association also existed between the presence of ctDNA and worse progression-free survival (HR 2.63, 95% CI 1.27–5.43, p = 0.009), as well as overall survival (HR 2.92, 95% CI 1.53–5.57, p = 0.001).Conclusions: The use of ctDNA in clinical practice for hematological malignancies is promising, as it may not only contribute to diagnosis, but could also predict the prognosis of patients so as to guide treatment. In the future, more studies are needed to realize the standardization of sequencing techniques and improve the detection sensitivity of exploration methods.

scholarly journals BIOM-47. LONGITUDINAL MONITORING OF PLASMA H3K27M IN DIFFUSE MIDLINE GLIOMA PATIENTS TREATED WITH ONC201

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii11-ii11
Author(s):  
Rohinton S Tarapore ◽  
Amanda Field ◽  
D Ashley Hill ◽  
Joshua Allen
Keyword(s):  
Radiographic Progression ◽  
Low Frequency ◽  
Circulating Tumor Dna ◽  
Allelic Frequency ◽  
Who Grade ◽  
Line Radiation ◽  
Complication Risk ◽  
Droplet Pcr ◽  
Tumor Dna ◽  
Diffuse Midline Glioma

Abstract Diffuse midline glioma, H3 K27M-mutant (DMG) is a 2016 WHO Grade IV glioma that has no established treatment beyond first-line radiation. ONC201 is an investigational small molecule that has been shown to be clinically active in recurrent DMG clinical trials. While biopsies of DMG are sometimes feasible, many patients defer secondary to complication risk. MR scans have many limitations in monitoring DMG progression, including distinguishing pseudoprogression and pseudoresponse and measuring diffuse lesions that often do not contrast enhance. Digital droplet PCR (ddPCR) is capable of sensitively detecting and quantifying the allelic frequency of circulating-tumor DNA (ctDNA) fragments against a backdrop of non-tumor DNA. Using sequence-specific probes for H3F3A (H3.3 K27M) and HIST1H3B (H3.1 K27M) ddPCR detects very low frequency variants and provides an assessment of mutational burden. A pilot cohort of 5 patients treated with ONC201 who had a range of outcomes were assessed with serial ctDNA analyses. Two patients with immediately progressive disease had a concordant H3 K27M ctDNA increase that precedes radiographic detection by 4 weeks. Two patients with >50% tumor regressions while on ONC201 had concordant H3 K27M ctDNA burden at the onset of response and subsequent radiographic progression was preceded by increases in ctDNA 8–16 weeks prior. One patient who had prolonged stable disease had decreased H3 K27M ctDNA burden over time. Upon radiographic progression, the addition of bevacizumab with ONC201 caused a radiographic pseudoresponse, however H3 K27M ctDNA remained stable. These pilot results suggest H3 K27M ctDNA may be a sensitive and accurate biomarker of disease burden. Longitudinal evaluation of H3 K27M ctDNA in a cohort of 34 recurrent contrast-enhancing H3 K27M-mutant glioma patients while on ONC201 will be reported. Primary tumor locations range across the thalamus, cerebellum, basal ganglia, temporal lobe, and midbrain; median age is 31 years old (range 20–70).

Circulating tumor DNA mutation as a prognostic marker in melanoma with brain metastasis.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21560-e21560
Author(s):  
Tapas Ranjan Behera ◽  
Jung Min Song ◽  
Donald Matthew Eicher ◽  
Brian Gastman ◽  
Daniel H. Farkas ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Brain Metastases ◽  
Metastatic Disease ◽  
Braf Mutation ◽  
Mutation Detection ◽  
Objective Response ◽  
Unmet Need ◽  
Circulating Tumor Dna ◽  
Metastatic Setting ◽  
Tumor Dna

e21560 Background: Prognosis in melanoma with brain metastasis is poor with a median survival of four months and a one-year survival rate of 10–20%. There is an unmet need for surveillance methods that can supplement imaging at regular intervals. Serial analysis of circulating tumor DNA (ctDNA) may aid surveillance and prognostication. A PCR-based, “specimen in/result out” testing device was employed to detect BRAF variants in plasma-derived ctDNA to evaluate the utility of rapid biomarker detection in the management of melanoma with brain metastasis. Methods: Serial blood samples from patients diagnosed with BRAF mutation-positive metastatic melanoma were collected at regular intervals. We employed a real-time PCR-based automated mutation detection system (Idylla; Biocartis, Belgium) to interrogate the plasma samples. The ctDNA mutation detection trend was analyzed relative to disease progression. Results: 39 patients with BRAF mutation positive melanoma were enrolled. 29 patients were treated in the metastatic setting, 10 in the adjuvant setting. 18 of the 29 patients with metastatic disease (62%) had brain metastases. Circulating BRAF mutation was detected in 17 of the 29 (59%) patients with metastatic disease, and was not detected in any patients treated adjuvantly. In the group with metastatic disease, this circulating biomarker changed from undetectable to detectable in eight (28%) and detectable to undetectable in three (10%). No change in circulating mutation status occurred in 18 (62%). In the eight patients who had an initial negative test that later became positive, seven (87%) had brain metastases. In three patients, ctDNA mutation detection occurred before the diagnosis of brain metastases on imaging, with a median lead time of five weeks (range, 3-12 weeks). In one patient with de novo metastatic disease admitted to the ICU, tissue was unavailable for BRAF testing but plasma was found to be positive for ctDNA BRAF detection. BRAF/MEK targeted therapy resulted in a sustained objective response. Five of six (83%) patients that had persistent ctDNA positivity had brain metastases. Among patients with brain metastases, median overall survival (mOS) of patients demonstrating >50% test positivity was numerically longer than those with <50% positivity (mOS 12.3 vs 53.5 months; p = 0.133). Conclusions: Plasma-based, rapid ctDNA testing may be useful as an aid in detecting progression and gauging prognosis in patients with melanoma treated in the metastatic setting. The dynamics of ctDNA test positivity may indicate a need for more urgent imaging, particularly of the brain. Blood-based, semi-automated ctDNA detection may serve as an attractive adjunct to scheduled imaging surveillance in melanoma.

scholarly journals Circulating Tumor Cells and Circulating Tumor DNA Detection in Potentially Resectable Metastatic Colorectal Cancer: A Prospective Ancillary Study to the Unicancer Prodige-14 Trial

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 516 ◽  
Author(s):  
François-Clément Bidard ◽  
Nicolas Kiavue ◽  
Marc Ychou ◽  
Luc Cabel ◽  
Marc-Henri Stern ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Circulating Tumor Cell ◽  
Circulating Tumor Dna ◽  
Detection Sensitivity ◽  
Clinical Validity ◽  
Mutational Status ◽  
Prospective Phase ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Tumor Dna

The management of patients with colorectal cancer (CRC) and potentially resectable liver metastases (LM) requires quick assessment of mutational status and of response to pre-operative systemic therapy. In a prospective phase II trial (NCT01442935), we investigated the clinical validity of circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) detection. CRC patients with potentially resectable LM were treated with first-line triplet or doublet chemotherapy combined with targeted therapy. CTC (Cellsearch®) and Kirsten RAt Sarcoma (KRAS) ctDNA (droplet digital polymerase chain reaction (PCR)) levels were assessed at inclusion, after 4 weeks of therapy and before LM surgery. 153 patients were enrolled. The proportion of patients with high CTC counts (≥3 CTC/7.5mL) decreased during therapy: 19% (25/132) at baseline, 3% (3/108) at week 4 and 0/57 before surgery. ctDNA detection sensitivity at baseline was 91% (N=42/46) and also decreased during treatment. Interestingly, persistently detectable KRAS ctDNA (p = 0.01) at 4 weeks was associated with a lower R0/R1 LM resection rate. Among patients who had a R0/R1 LM resection, those with detectable ctDNA levels before liver surgery had a shorter overall survival (p < 0.001). In CRC patients with limited metastatic spread, ctDNA could be used as liquid biopsy tool. Therefore, ctDNA detection could help to select patients eligible for LM resection.

scholarly journals Cerebrospinal Fluid Access Devices in Pediatric Diffuse Midline Glioma Patients: Literature Review and Evaluation of Institutional Practices

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Daphne Li ◽  
Wendy Stellpflug ◽  
Amanda Muhs Saratsis
Keyword(s):  
Cerebrospinal Fluid ◽  
Liquid Biopsy ◽  
Tumor Biology ◽  
Significant Proportion ◽  
Circulating Tumor Dna ◽  
Response To Treatment ◽  
Institutional Practices ◽  
Medline Search ◽  
Tumor Dna ◽  
Diffuse Midline Glioma

Abstract INTRODUCTION Diffuse midline gliomas (DMG) are the number one cause of cancer death in children. H3K27M mutations occur in 80% of DMG, with distinct tumor biology and poorer response to treatment. H3K27M is detectable in cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA), depending on CSF tumor proximity, and correlates with tumor volume and treatment response. Ventricular access devices (VAD) for serial CSF sampling (liquid biopsy) could therefore play a significant role in DMG management. Here, we set to characterize VAD placement practices in pediatric DMG. METHODS A retrospective review of patients <21 yr treated for DMG at our institution was performed (1984-2019). A MEDLINE search was conducted to identify reports of VAD placement in DMG. Full-text English reports of patients = 21 yr with VAD outcomes were analyzed. RESULTS A total of 106 DMG patients at our institution were identified. In total 49% had brainstem disease (n = 52). A total of 46.23% (n = 49) had VADs: 32.65% transient (ETV n = 5, EVD n = 11), 67.35% permanent (reservoir n = 7, shunt n = 26). A total of 17 had ETV at biopsy, 7 with concurrent reservoir placement. Of 10 ETV patients without initial reservoir, 5 ultimately underwent permanent VAD placement (reservoir n = 1, shunt n = 4). A total of 9 patients received EVDs at tumor surgery, 8 required EVD for acute hydrocephalus (HCP), with 6 converted to shunts. A total of 15 shunts were placed at tumor diagnosis: 4 required revision (27%). A total of 14 articles describing 240 DMG patients cited HCP in 22%-100%, with VAD placement in 22%-63%, and shunt-induced extraneural metastases in 7. Ventricular chemotherapy via indwelling reservoirs (481 patients) was associated with 29 infectious and 50 noninfectious complications. Standardized reservoir access procedures decreased infection rates. CONCLUSION VAD placement is clinically indicated in a significant proportion of pediatric DMG patients, with low morbidity. Ventricular CSF is superior to lumbar for ctDNA sequencing and quantification. VAD placement should therefore be considered to facilitate liquid biopsy in DMG.

Sign in / Sign up

Export Citation Format

Share Document