50P Droplet digital PCR measurement of c-Met gene amplification in plasma cell free DNA in patients with advanced NSCLC

Abstract Background Babies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4α (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management. Methods Droplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies’ genotypes were ascertained postnatally by Sanger sequencing. Results Droplet digital PCR assays for GCK or HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no false-positive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample. Conclusions This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.

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Analysis of KRAS Mutation Subtype in Tissue DNA and Cell-Free DNA Using Droplet Digital PCR and the Function of Cell-Free DNA as a Recurrence Predictive Marker in Pancreatic Cancer

Biomedicines ◽

10.3390/biomedicines9111599 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1599

Author(s):

Eunsung Jun ◽

Bonhan Koo ◽

Eo Jin Kim ◽

Dae Wook Hwang ◽

Jae Hoon Lee ◽

...

Keyword(s):

Pancreatic Cancer ◽

Clinical Significance ◽

Kras Mutation ◽

Predictive Marker ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Cell Free Dna ◽

Cancer Subtypes ◽

Free Dna ◽

Mutation Burden

KRAS mutation is a major regulator in the tumor progression of pancreatic cancer. Here, we compared the frequency and mutation burden of KRAS mutation subtypes with paired tumor tissue and blood in patients and examined their clinical significance. DNA from tumor tissues and cell-free DNA (cfDNA) from preoperative blood were obtained from 70 patients with pancreatic cancer. Subtypes and mutation burdens of KRAS G12D and G12V mutations were evaluated using droplet digital PCR. Comparing the presence of mutations in tissue, accumulative and simultaneous mutations of G12D or G12V were identified of 67 (95.7%), and 48 patients (68.6%). Conversely, in blood, they were only identified in 18 (25.7%) and four (5.7%) patients; respectively. Next, comparing the mutation burden in tissue, the mutation burden varied from less than 0.1 to more than five, whereas that of cfDNA in blood was mostly between one and five, as cases with a mutation burden lower than 0.1 and higher than five were rare. Finally, the presence of the G12V mutation alone in cfDNA and the combination of the G12V mutation with elevated CA 19-9 levels were associated with poor recurrence-free survival. These fundamental data on the KRAS mutation subtypes and their clinical significance could support their potential as predictive markers for postoperative recurrence.

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Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer

Cancers ◽

10.3390/cancers12123541 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3541 ◽

Cited By ~ 2

Author(s):

Md Ismail Hosen ◽

Nathalie Forey ◽

Geoffroy Durand ◽

Catherine Voegele ◽

Selin Bilici ◽

...

Keyword(s):

Urothelial Cancer ◽

Digital Pcr ◽

Kappa Coefficient ◽

Droplet Digital Pcr ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

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