Detecting KRAS mutations in peripheral blood of colorectal cancer patients by peptide nucleic acid clamp PCR

Abstract

We investigated the effectiveness of peptide nucleic acid (PNA) clamp PCR for detecting KRAS mutations in peripheral blood samples of colorectal cancer (CRC) patients. We compared KRAS point mutations between tumour tissue and blood samples. Forty-two patients were included in this study. We observed KRAS mutations in formalin-fixed, paraffin-embedded tissues by PCR direct sequencing and in blood samples by PNA clamp PCR. KRAS point mutations were detected in primary tumour tissue samples of 13 patients (31.0%) and in peripheral blood samples of 10 patients (23.8%). KRAS point mutations were detected in both samples for 8 patients (19.0%). The sensitivity, specificity and accuracy for detecting KRAS mutations in peripheral blood and tumour tissue samples were 61.5, 93.1 and 83.3%, respectively. The positive and negative predictive values were 80.0 and 84.4%, respectively. Five patients with mutant KRAS in their plasma preoperatively, did not exhibit KRAS mutations postoperatively. Our method detected KRAS point mutations in peripheral blood samples of CRC patients, which contained extremely small amounts of mutant cells. This method is helpful for identifying metastatic CRC patients in whom metastases will respond to EGFR-targeted monoclonal antibody therapy.

The diagnostic accuracy of circulating free DNA for the detection of KRAS mutation status in colorectal cancer: A meta‐analysis

“…A total of 5032 studies were excluded after primary screening, and 94 articles were selected for further assessment of eligibility. By rigorous evaluation, 28 studies met the inclusion criteria and were included in our present meta‐analysis. In the study reported by Taly and another study by Xu, KRAS status was detected by two different methods, and Morgan detected KRAS status both in serum and plasma, and the data from two different methods and two samples were analyzed as two independent studies.…”

Versatility of peptide nucleic acids (PNAs): role in chemical biology, drug discovery, and origins of life

“…Earlier procedure for sequencing the resistant markers from the viral isolate takes several days; however, PNA‐mediated PCR technique takes only few hours. PNA–PCR clamping technique has also been used in the detection of EGFR mutations in the circulating free DNA, BRAF V600E Mutation with Thyroid Tissue, KRAS mutations in peripheral blood of colorectal cancer patients, and T790M Epidermal Growth Factor Receptor Mutation . The natural diets of larval marine animals and Japanese eel leptocephali were also investigated using PNA‐directed PCR clamping …”

WspółCzesne Metody Wykrywania Mutacji Genu EGFR Jako Czynnika Predykcyjnego Dla Terapii Ukierunkowanej Molekularnie Chorych NA Niedrobnokomórkowego Raka PłUca—Czy Istnieje “ZłOty Standard” Diagnostyczny?

“…Cel ten może zostać osiągnięty różnymi metodami, na przykład poprzez pozytywną selekcję zmutowanej sekwencji DNA z wykorzystaniem starterów PCR specyficznych dla poszukiwanego allelu (ASP-PCR, allele-specific primer PCR), hybrydowych sond fluorescencyjnych selektywnie amplifikujących i wykrywających zmutowany allel (Scorpion-ARMS, Scorpion Amplified Refractory Mutation System), a także poprzez wykorzystanie różnic w temperaturze topnienia homo-i heterodupleksów DNA (COLD-PCR, CO-amplification at Lower Denaturation temperature-Polymerase Chain Reaction) [21][22][23]. Selekcja zmutowanego allelu bywa też negatywna i dokonuje się poprzez blokowanie amplifikacji DNA typu natywnego, na przykład przez użycie antysensownych cząsteczek kwasu peptydonukleinowego (PNA, peptide nucleic acid) w metodach PNA-LNA PCR clamp, PNA clamp oraz PNA-SmartAmp2 czy też eliminację DNA o sekwencji natywnej za pomocą enzymu restrykcyjnego (ME-PCR, mutant-enriched PCR) [24][25][26][27].…”