Real‐Time <scp>PCR</scp> and Droplet Digital <scp>PCR</scp>: two techniques for detection of the <scp>JAK</scp>2V617F mutation in Philadelphia‐negative chronic myeloproliferative neoplasms

Fontanelli, Giulia; Baratè, Claudia; Ciabatti, Elena; Guerrini, Francesca; Grassi, Susanna; Re, Marzia Del; Morganti, Riccardo; Petrini, Iacopo; Arici, Roberta; Barsotti, Simone; Metelli, Maria Rita; Danesi, Romano; Galimberti, Sara

doi:10.1111/ijlh.12404

Cited by 29 publications

(27 citation statements)

References 37 publications

(49 reference statements)

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“…5,12,24 Limit of mutant detection with ddPCR depends on the amount of DNA input and has been reported to be of 0.01%-0.1% 5,12,24 Limit of mutant detection with ddPCR depends on the amount of DNA input and has been reported to be of 0.01%-0.1%…”

Section: Discussionmentioning

confidence: 99%

“…[4][5][6][7][8][9] Droplet digital PCR (ddPCR) is an end-point measurement using limiting dilution from partition of target DNA into droplets and Poisson statistics, allowing detection and quantification of the mutated allele burden in 1 run, without the need of a standard curve or a reference gene. [4][5][6][7][8][9] Droplet digital PCR (ddPCR) is an end-point measurement using limiting dilution from partition of target DNA into droplets and Poisson statistics, allowing detection and quantification of the mutated allele burden in 1 run, without the need of a standard curve or a reference gene.…”

Section: Introductionmentioning

confidence: 99%

“…[4][5][6][7][8][9] Droplet digital PCR (ddPCR) is an end-point measurement using limiting dilution from partition of target DNA into droplets and Poisson statistics, allowing detection and quantification of the mutated allele burden in 1 run, without the need of a standard curve or a reference gene. 5,12 Liquid biopsies, also called cell-free DNA (cfDNA), are now widely used for identification of many physiological and pathological conditions. 5,12 Liquid biopsies, also called cell-free DNA (cfDNA), are now widely used for identification of many physiological and pathological conditions.…”

Section: Introductionmentioning

confidence: 99%

“…[1][2][3] Detection of JAK2 V617F in DNA extracted from peripheral blood (PB) is hence included as part of the diagnostics workup of MPNs. [4][5][6][7][8][9] Droplet digital PCR (ddPCR) is an end-point measurement using limiting dilution from partition of target DNA into droplets and Poisson statistics, allowing detection and quantification of the mutated allele burden in 1 run, without the need of a standard curve or a reference gene. 10,11 Several studies have demonstrated the concordance between real-time quantitative PCR (qPCR) and ddPCR in identifying JAK2 mutations in patients with MPN using DNA extracted from PB.…”

Section: Introductionmentioning

confidence: 99%

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JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

Nystrand

Waage

Jonassen

2017

Int J Lab Hematology

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…[4][5][6][7][8][9] Droplet digital PCR (ddPCR) is an end-point measurement using limiting dilution from partition of target DNA into droplets and Poisson statistics, allowing detection and quantification of the mutated allele burden in 1 run, without the need of a standard curve or a reference gene. 5,12 Liquid biopsies, also called cell-free DNA (cfDNA), are now widely used for identification of many physiological and pathological conditions. 5,12 Liquid biopsies, also called cell-free DNA (cfDNA), are now widely used for identification of many physiological and pathological conditions.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

Nystrand

Waage

Jonassen

2017

Int J Lab Hematology

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“…Even if the lower detection limit (LOD) of the assay was 0.01% (See Supplemental File), we identified the 0.1% as cut-off of positivity as also suggested by several studies [23–24, 27–29]. All samples were tested in duplicate with both qPCR and also with digital PCR (ddPCR) to confirm the evaluation [51–52] (See Supplemental File). In addition to JAK2 V617F mutation, CALR and MPL mutations were screened for all patients to obtain a comprehensive molecular characterization and to exclude the co-existence of additional mutations [44–46, 53–54].…”

Section: Methodsmentioning

confidence: 95%

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study

et al. 2017

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Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%.Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (‘’Histology-based’’ diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (‘’Clinical-based’’ or ‘’Molecular-based’’ diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process.Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion.

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Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

Biron

Kostiuk

Isaac

et al. 2016

Cancer

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BACKGROUND:The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultrasensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC. METHODS: Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care. RESULTS: Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was 1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7. CONCLUSIONS: The ddPCR of HPV E6/E7 is a novel and highly specific method of detecting HPV-16 in OPSCC.

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Real‐Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2^V617F mutation in Philadelphia‐negative chronic myeloproliferative neoplasms

Abstract: In conclusion, our study showed that DD-PCR could represent a new and promising technological evolution for detection of JAK2 mutation in MPNs.

Cited by 29 publications

References 37 publications

JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

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Real‐Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2V617F mutation in Philadelphia‐negative chronic myeloproliferative neoplasms

Abstract: In conclusion, our study showed that DD-PCR could represent a new and promising technological evolution for detection of JAK2 mutation in MPNs.

Cited by 29 publications

References 37 publications

JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

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Real‐Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2^V617F mutation in Philadelphia‐negative chronic myeloproliferative neoplasms