The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

2017 ◽  
Vol 36 (2) ◽  
pp. 429-435 ◽  
Author(s):  
Laura S. Hiemcke-Jiwa ◽  
Monique C. Minnema ◽  
Joyce H. Radersma-van Loon ◽  
N. Mehdi Jiwa ◽  
Mirthe de Boer ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Liquid Biopsies ◽  
Sensitive Technique ◽  
Highly Sensitive

scholarly journals Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239819
Author(s):  
Matilda Holm ◽  
Emma Andersson ◽  
Emerik Osterlund ◽  
Ali Ovissi ◽  
Leena-Maija Soveri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Metastatic Colorectal Cancer ◽  
Disease Progression ◽  
Primary Tumor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Plasma Samples ◽  
Liquid Biopsies ◽  
Generation Sequencing

Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45–63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

scholarly journals Detection of AR-V7 in Liquid Biopsies of Castrate Resistant Prostate Cancer Patients: A Comparison of AR-V7 Analysis in Circulating Tumor Cells, Circulating Tumor RNA and Exosomes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 688 ◽  
Author(s):  
Mohammed Nimir ◽  
Yafeng Ma ◽  
Sarah A. Jeffreys ◽  
Thomas Opperman ◽  
Francis Young ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Digital Pcr ◽  
Liquid Biopsies ◽  
Tumor Biopsy ◽  
Highly Sensitive ◽  
Castrate Resistant Prostate Cancer ◽  
Castrate Resistant

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker.

Highly Sensitive Droplet Digital PCR for MYD88L265P Mutation Detection and Minimal Residual Disease Monitoring in Waldenström Macroglobulinemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2645-2645
Author(s):  
Daniela Drandi ◽  
Elisa Genuardi ◽  
Paola Ghione ◽  
Daniele Grimaldi ◽  
Barbara Mantoan ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Mutation Detection ◽  
Residual Disease ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Advisory Committees ◽  
Highly Sensitive ◽  
Sensitive Tool ◽  
Minimal Residual

Abstract Background. Recently, the somatic MYD88L265P mutation has been found as the hallmark of Waldenström Macroglobulinemia (WM), being detectable in nearly 90% of cases, as well as in up to 50% of IgM MGUS, rarely in other non-Hodgkin lymphomas and never in multiple myeloma (MM). Beyond its potential diagnostic role, this mutation has been associated with tumor growth and therapy resistance. Moreover, MYD88L265P might represent an ideal marker for minimal residual disease (MRD) monitoring in a disease whose therapeutic scenario has been rapidly changing, with many new available and highly effective drugs (nucleoside analogues, proteasome and BTK-inhibitors). However, the current MYD88L265P allele-specific quantitative PCR (ASqPCR) diagnostic tool lacks sensitivity (1.00E-03) and thus is not suitable for MRD. Moreover, is not useful to test peripheral blood (PB), that harbors low concentrations of circulating tumor cells (especially after immunochemotherapy), neither to assess cell-free DNA (cfDNA), usually present at very low amount in plasma. Therefore, our study aims: 1) to assess whether a highly sensitive tool as droplet digital PCR (ddPCR) might be helpful in MYD88L265P screening; 2) to evaluate whether MYD88L265P might be a suitable marker for MRD monitoring in WM. Methods. Bone marrow (BM) and PB samples were collected at diagnosis and during follow-up from a local series of patients affected by WM, IgM MGUS and IgG-secreting lymphoplasmacytic lymphoma (LPL), as well as samples from healthy subjects and MM were used as negative controls. Genomic (gDNA) and cell-free DNA (cfDNA) were extracted as recommended (Qiagen). MYD88L265P was assessed on 100 ng of gDNA by ASqPCR as previously described [Xu 2013] and by ddPCR, using a custom dual labelled probe assay (Bio-Rad). When available, 50 ng of cfDNA were tested for MYD88L265P, only by ddPCR. ddPCR was performed on 20 µl of reaction at 55°C for 40 cycles, run on QX100 droplet reader and analyzed by QuantaSoft v1.6.6 (Bio-Rad). MYD88L265P ASqPCR level was estimated as described [Treon 2012]. ΔCT<8.4 identified a MYD88L265P positive sample. Similarly, MYD88L265P ddPCR cut-off was settled on the highest healthy samples level. IGH rearrangements identification and IGH-based MRD analysis were performed as previously described [van der Velden 2007]. Results. Once the ddPCR assay was optimized, the sensitivity of MYD88L265P ddPCR was compared to ASqPCR on a ten-fold serial dilution standard curves built with a 70% MYD88L265P mutated WM sample, previously identified by Sanger sequencing [Treon 2012]. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Thereafter, overall 105 samples (48 BM, 57 PB, 52 diagnosis and 53 follow up) from 58 patients (49 WM, 5 IgM MGUS and 4 LPL) as well as 20 controls (15 healthy subjects and 5 MM) were tested by both methods. 32/33 (97%) diagnostic BM scored positive for MYD88L265P by both ddPCR and ASqPCR (being the only one negative a WM), while ddPCR, was able to detect more mutated cases, than ASqPCR, among diagnostic PB samples: 15/19 (79%) vs 9/19 (47%) (Table1). Moreover, to investigate whether the MYD88L265P ddPCR tool could be used for MRD detection we compared it to the standardized IGH-based MRD. An IGH-based MRD marker was found in 40/53 (75%) patients (37 WM and 3 LPL). Five Patients, so far analyzed, with baseline and follow up samples (18 BM, 5 PB) showed highly superimposable results between the two methods. Finally, pivotal results on cfDNA from 10 patients showed higher median levels of MYD88L265P mutation in plasma if compared to PB. Conclusions. We developed a new tool for diagnosis and MRD monitoring in WM, showing that: 1) ddPCR is a highly sensitive tool for MYD88L265P detection, especially useful in low infiltrated samples, like PB; 2) MYD88L265P can be effectively and easily used for MRD monitoring in WM, achieving similar results to standardized IGH-based MRD; 3) cfDNA recovered from plasma might be an attractive alternative for MYD88L265P detection, deserving further investigation. Methodological validation against IgH-based MRD detection and Flow cytometry and correlations with clinical impact are currently ongoing on external samples series. Table 1.PATIENTSWM (45)LPL (2)IgM MGUS (5)TISSUEBMPBBMPBBMPBSAMPLES31141114MYD88L265P ddPCR/ASqPCR30/3011/71/10/01/14/2 TABLE 1. MYD88L265P mutation detection in diagnostic samples: ddPCR vs ASqPCR Disclosures Boccadoro: Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies

2018 ◽  
Vol 64 (12) ◽  
pp. 1762-1771 ◽  
Author(s):  
Mariana Fitarelli-Kiehl ◽  
Fangyan Yu ◽  
Ravina Ashtaputre ◽  
Ka Wai Leong ◽  
Ioannis Ladas ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Gene Copy Number ◽  
Fold Increase ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Patients With Cancer ◽  
Free Circulating Dna

Abstract BACKGROUND Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a “glass ceiling” in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations. METHODS We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9–2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6–1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9–2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.

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