Detection of Fetal Single Gene Mutations through Targeted Sequencing of Maternal cell-free DNA

Pathogenic molecular features gained specific significance in therapeutic decisions in lung carcinoma in the past decade. Initial and follow up genetic testing requres appropriate amounts and quality of tumor derived DNA, but tumor sampling, especially for disease monitoring is generally limited. Further to the peripheral blood (PB), samples from pleural fluid, accumulating in diverse lung processes might serve as an alternative source for cell-free DNA (cfDNA) for genetic profiling. In our study, cfDNA isolated from the pleural effusion and from the PB, and genomic DNA (gDNA) obtained from tissue/cellular samples were analyzed and compared from altogether 65 patients with pulmonary disease, including 36 lung adenocarcinomas. The quantity of effusion cfDNA yield appeared to be significantly higher compared to that from simultaneously collected PB plasma (23.2 vs. 4.8 ng/μl, p < 0.05). Gene mutations could be safely demonstrated from the effusion cfDNA fraction obtained from adenocarcinoma patients, 3/36 EGFR, 9/36 KRAS and 1/36 BRAF gene variants were detected. In this series, 9/13 samples showed an effusion+/plasma-mutational status, while only 1/13 samples presented with the opposite findings (effusion-/plasma+). gDNA analysis from sediment cell blocks from the identical effusion sample was surprisingly ineffective for lung adenocarcinoma profiling due to the low DNA yield. In conclusion, the cell free supernatant of pleural effusions appears to concentrate cancer derived cfDNA and seems to be particularly suitable for serial genotyping of pulmonary adenocarcinoma.

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An online tool for fetal fraction prediction based on direct size distribution analysis of maternal cell-free DNA

BioTechniques ◽

10.2144/btn-2020-0143 ◽

2020 ◽

Author(s):

Luca Bedon ◽

Josef Vuch ◽

Simeone Dal Monego ◽

Germana Meroni ◽

Vanna Pecile ◽

...

Keyword(s):

Size Distribution ◽

Prenatal Testing ◽

Distribution Analysis ◽

Blood Draw ◽

Noninvasive Prenatal Testing ◽

Cell Free Dna ◽

Maternal Cell ◽

Fetal Dna ◽

Free Dna ◽

Fetal Fraction

The discovery of circulating fetal DNA in the plasma of pregnant women has greatly promoted advances in noninvasive prenatal testing. Screening performance is enhanced with higher fetal fraction and analysis of samples whose fetal DNA fraction is lower than 4% are unreliable. Although current approaches to fetal fraction measurement are accurate, most of them are expensive and time consuming. Here we present a simple and cost-effective solution that provides a quick and reasonably accurate fetal fraction by directly evaluating the size distribution of circulating DNA fragments in the extracted maternal cell-free DNA. The presented approach could be useful in the presequencing stage of noninvasive prenatal testing to evaluate whether the sample is suitable for the test or a repeat blood draw is recommended.

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First prenatal case of 48,XXYY syndrome detected by maternal cell-free DNA testing

Journal of Obstetrics and Gynaecology ◽

10.1080/01443615.2019.1607268 ◽

2019 ◽

Vol 40 (2) ◽

pp. 270-272

Author(s):

Jian Li ◽

Li Zhen ◽

Min Pan ◽

Dong-Zhi Li

Keyword(s):

Dna Testing ◽

Cell Free Dna ◽

Maternal Cell ◽

Free Dna

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Assessing the impact of clonal hematopoiesis in disease monitoring using targeted cell-free DNA (cfDNA) sequencing technology.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14530 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14530-e14530

Author(s):

Stephanie J. Yaung ◽

Liu Xi ◽

Corinna Woestmann ◽

Christine Ju ◽

Daniel M. Klass ◽

...

Keyword(s):

Solid Tumors ◽

Mononuclear Cells ◽

Response To Therapy ◽

Targeted Sequencing ◽

P Value ◽

Disease Monitoring ◽

Cell Free Dna ◽

Clonal Hematopoiesis ◽

Free Dna ◽

The Impact

e14530 Background: Somatic variants found in plasma cell-free DNA (cfDNA) may derive from either solid tumors or clonal hematopoiesis (CH). Little is known about how this may impact plasma-based longitudinal disease monitoring using targeted sequencing of circulating tumor DNA (ctDNA). Methods: To assess the potential impact of CH in disease monitoring, we evaluated monitoring algorithms by targeted sequencing with and without matched peripheral blood mononuclear cells (PBMC). Samples were collected from a prospective observational study, where 62 late stage lung adenocarcinoma subjects were treated with first-line chemo or chemoradiation therapy. Pre-treatment plasma cfDNA and matched PBMC were analyzed with the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), a sequencing panel of 198 kilobases targeting cancer genes. Median input amounts of 25 ng cfDNA and 50 ng PBMC DNA were sequenced to median deduplicated depths of 4582 and 6134, respectively. Results: A median of 120 single nucleotide variants were detected per cfDNA sample, with 93.1% of these identified in matched PBMC. Most PBMC-matched cfDNA variants were germline SNPs, with allele frequency (AF) ~ 50% or 100%. A median of 1 (range 0-5) PBMC-matched cfDNA variants per sample were detected with an AF < 10%, consistent with CH. The number of these variants was positively associated with age (p-value = 0.0039) and the most frequently mutated gene was TP53. The remaining somatic variants (i.e., in cfDNA and not PBMC) had an AF range 0.03-40.9%. These PBMC-informed variants (median of 7 per sample) were used in longitudinal monitoring in the first post-treatment plasma sample to assess early response to therapy. Association between ctDNA level and progression-free survival using the same monitoring algorithm yielded nearly identical results on somatic variants derived from filtering approaches independent of matched PBMC (HR 0.32; 95% CI 0.16 - 0.65; log-rank P = 0.0009) and the PBMC-informed method (HR 0.31; 95% CI 0.14 - 0.66; log-rank P = 0.0013). Conclusions: A targeted panel focused on solid tumors by design has limited impact from CH. For disease monitoring applications in a non-MRD setting, measuring multiple variants instead of a single variant further enables robust classifiers that can moderate the impact of variants, if any, from CH.

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Targeted sequencing of plasma cell-free DNA to predict response to PD1 inhibitors in advanced non-small cell lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.8_suppl.103 ◽

2019 ◽

Vol 37 (8_suppl) ◽

pp. 103-103

Author(s):

Nicolas Guibert ◽

Greg Jones ◽

John F. Beeler ◽

Clive D. Morris ◽

Vincent Plagnol ◽

...

Keyword(s):

Clinical Outcomes ◽

Plasma Cell ◽

Progressive Disease ◽

Genetic Alterations ◽

Targeted Sequencing ◽

Cell Free Dna ◽

Invasive Approach ◽

Free Dna ◽

Poor Outcomes ◽

Immune Score

103 Background: Tumor mutational burden is an emerging biomarker of response to immune checkpoint inhibitors (ICI), whose clinical adoption is challenging, especially in liquid biopsies. We hypothesized that targeting limited but relevant genetic alterations in plasma cell-free DNA with next generation sequencing (NGS) along with early monitoring may represent a non-invasive approach to predict response to ICI. Methods: Plasma samples from responders (PFS > 6 months) and non-responders (progressive disease at first evaluation) patients collected before nivolumab (second line) initiation and at 1 month were tested using tagged amplicon sequencing of hotspots and coding regions from 36 genes, blinded to clinical outcomes and tumor genotype. Molecular profile of ctDNA, and its early kinetics (1 month) were analyzed as potential early indicators of response. Results: 98 patients were analyzed, of which 86 (39 responders, 47 non-responders) were evaluable for response. Alterations in ctDNA were detectable in 67/86 baseline samples (78%). The detection of a targetable oncogenic driver (5 EGFR, 1 ALK) was associated with progressive disease on the first CT-scan The presence of a PTEN and/or STK11 mutations (b-PS(+)) was correlated with poor outcomes (median PFS 1.5 months vs. 8 months in b-PS(-)) patients, p = 0.0007), while the presence of transversion mutations in KRAS and/or TP53 (b-KP-Tv(+)) predicted good outcomes (median PFS 11 months vs. 2 months in b-KP-Tv(-) patients, p = 0.0088). Combining these results, patients with a low “immune score” (driver and/or b-PS(+) and/or b-KP-Tv(-)) derived poor outcomes (PFS 2 months), compared with patients with a high immune score (no driver, b-PS(-) and b-KP-Tv(+), PFS 14 months, p = 0.0001, HR 2.96). Studying early changes in 65 specimens, molecular response was correlated with clinical outcomes (14 months PFS in patients with early ctDNA decrease compared to 2 months in patients with increase, p < 0.0001; HR 2.7). Using cut-off of 30% and 50% of plasma response increased the ability of ctDNA to predict response (HR 4 and 4.17, respectively). Conclusions: Targeted sequencing of plasma ctDNA and its early variations can predict response to anti-PD-1. Clinical trial information: NCT02827344.

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Noninvasive Cell-Free DNA-Based Prenatal Detection of Microdeletions Using Single Nucleotide Polymorphism–Targeted Sequencing

Obstetrics and Gynecology ◽

10.1097/01.aog.0000447172.98639.e5 ◽

2014 ◽

Vol 123 ◽

pp. 167S

Author(s):

Matthew Rabinowitz ◽

Melissa Savage ◽

Barbara Pettersen ◽

Styrmir Sigurjonsson ◽

Matthew Hill ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Targeted Sequencing ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Cell Free Dna ◽

Prenatal Detection ◽

Free Dna

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Identification of Somatic Mutations in Papanicolaou Smear DNA and Plasma Circulating Cell-Free DNA for Detection of Endometrial and Epithelial Ovarian Cancers: A Pilot Study

Frontiers in Oncology ◽

10.3389/fonc.2020.582546 ◽

2020 ◽

Vol 10 ◽

Author(s):

Xuan Jiang ◽

Weihua Li ◽

Jiaxin Yang ◽

Shuzhen Wang ◽

Dongyan Cao ◽

...

Keyword(s):

Gene Mutation ◽

Somatic Mutations ◽

Pap Smear ◽

Hot Spot ◽

Gene Mutations ◽

Gene Panel ◽

Pap Smears ◽

Cell Free Dna ◽

Free Dna ◽

Circulating Cell Free Dna

ObjectivesThe aim of this study was to identify tumor-derived DNA from Papanicolaou (Pap) smear and plasma specimens collected from patients with endometrial cancer or atypical hyperplasia (EC/AH) or epithelial ovarian cancer (OC).MethodsTumor tissues, peripheral blood, and Pap smear samples were collected from patients with EC/AH and patients with epithelial OC. Somatic mutations of tumor specimens in EC/AH and OC were examined by whole-exome sequencing using a 127-driver gene panel from The Cancer Genome Atlas (TCGA). A nine-gene EC/AH panel and an eight-gene OC panel were established based on the identified significantly mutated genes in the EC/AH and OC tumor specimens. Circulating single-molecule amplification and resequencing technology (cSMART) was applied to evaluate somatic mutations in Pap smear DNA and plasma circulating cell-free DNA (ccfDNA) using the EC/AH and OC gene panels.ResultsIn EC/AH group, there existed 22 tumors and 14 of the 22 tumors contributed hot spot mutations for the EC/AH nine-gene panel. In the Pap smear subgroup, all 21 Pap smears tested positive. Nine out of 11 (81.8%) identified the same gene mutations with their matched tumors and the remaining 10 Pap smears all tested positive. In the plasma subgroup, 10 out of 26 (38.5%) plasmas tested positive. One out of 13 (7.7%) identified the same gene mutation with its matched tumor and 5 out of the remaining 13 plasmas (38.5%) tested positive. In OC group, there existed 17 tumors and 16 of the 17 tumors contributed hot spot mutations for the OC eight-gene panel. In the Pap smear subgroup, all 11 Pap smears tested positive. Five out of 10 (50.0%) identified the same gene mutations with their matched tumors and the remaining one Pap smear also tested positive. In the plasma subgroup, all 22 plasmas tested positive. Ten out of 14 (71.4%) identified the same gene mutation with their matched tumors and the remaining 4 plasmas all tested positive.ConclusionsTumor-derived DNA can be detected in Pap smears and plasmas from patients with EC/AH or epithelial OC. Using a small gene-panel, early detection of EC/AH and OC might be promising. However, the value of plasma ccfDNA for EC/AH requires further investigation.

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