Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer

Azuara, Daniel; Ginestà, Mireia M.; Gausachs, Mireia; Rodríguez-Moranta, Francisco; Fabregat, Joan; Busquets, Juli; Peláez, Núria; Boadas, Jaume; Galter, Sara; Moreno, Vı́ctor; Costa, José; de, Javier; Capellà, Gabriel

doi:10.1373/clinchem.2012.186577

Cited by 53 publications

(41 citation statements)

References 42 publications

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“…Mutations in KRAS (exons 2/3/4), NRAS (exons 2/3/4), and BRAF (V600E) were detected by conventional qPCR (sensitivity of about 1%-10%) and by nanofluidic dPCR (sensitivity of about 0.05%-0.1%) of genomic DNA as previously described (23).…”

Section: Dna Extraction and Primary Pcr Reactionsmentioning

confidence: 99%

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Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

Azuara

Santos

López-Dóriga

et al. 2016

Molecular Cancer Therapeutics

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The clinical significance of low-frequent RAS pathwaymutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy.

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Section: Dna Extraction and Primary Pcr Reactionsmentioning

confidence: 99%

“…Several highly sensitive techniques are currently being evaluated. Among them, digital PCR (dPCR) is an increasingly applied technique that offers a number of advantages for both detecting and quantifying nucleic acids (22,23).…”

Section: Introductionmentioning

confidence: 99%

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

Azuara

Santos

López-Dóriga

et al. 2016

Molecular Cancer Therapeutics

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“…The Light Cycler ® 480 software version 1.5 (Roche Applied Science) was used to determine the genotype of the sample by measuring the intensity distribution of the used dyes following PCR amplification. This technique has an analytical sensitivity of 5% in a background of wild-type DNA (11).…”

Section: Patients and Samples Between January 2004 Andmentioning

confidence: 99%

“…This may well reflect a field effect within the pancreas or, when the tumor is already evident, the possibility that exfoliated cells only represent part of a tumor that is intrinsically heterogeneous (11). Aberrant promoter methylation of tumor suppressor genes is a frequent and early event in multiple tumors, including carcinomas of the pancreas and the periampullary area (12,13).…”

Section: Introductionmentioning

confidence: 99%

APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

Ginestà¹,

Díaz‐Riascos²,

Busquets³

et al. 2016

Oncology Letters

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Abstract. Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71-80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms.

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“…KRAS mutant alleles have been detected in gastrointestinal malignancies, including colorectal carcinoma, using nanofluidic digital PCR which showed a better performance in detection of mutation KRAS in colorectal adenomas compared to conventional PCR. [145] Nanoparticles have been coupled with short cancer specific oligonucleotides (aptamers) for targeted binding to prostate specific membrane antigen positive cells in prostate cancer cell lines. [146] Finally, serum detection of colorectal cancer has been achieved with gold nanoparticles using SERS spectroscopy in a study which exemplified the use of this approach as a viable, minimally invasive screening method for colorectal cancer.…”

Section: Cancer Nanotechnologymentioning

confidence: 99%

Prospects of ‘Omics Based Molecular Approaches in Colorectal Cancer Diagnosis and Treatment in the Developing World: A Case Study in Cape Town, South Africa

Adeola¹,

Goosen²,

Goldberg³

et al. 2014

Colorectal Cancer - Surgery, Diagnostics and Treatment

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Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer

Cited by 53 publications

References 42 publications

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

Prospects of ‘Omics Based Molecular Approaches in Colorectal Cancer Diagnosis and Treatment in the Developing World: A Case Study in Cape Town, South Africa

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