De este modo, “ los Oncólogos podrán conocer previamente si dicha terapia va a tener efecto en sus pacientes o hay que recurrir a otras alternativas terapéuticas ” .
Tanto la European Society for Medical Oncology (ESMO) como la American Society of Clinical Oncology (ASCO) han publicado sendas directrices que obligan a la detección de dichas alteraciones genéticas antes de la implementación del tratamiento.
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ASCO , 3 Junio 2014 .
Author(s): Maria Luisa Villahermosa, Juan Moscoso del Prado, Nuria Manjon, Ana Isabel Moraga, Maria Jesus Sanz, Javier Hernandez-Losa, Javier Gomez-Roman, Jesper Bonde, Jose Luis Rodriguez-Peralto, Rosario Cospedal; Genomica SAU (Zeltia Group), Madrid, Spain; Pathology Department, Vall d'Hebron University Hospital, Barcelona, Spain; Hospital Universitario Marqués de Valdecilla, Santander, Spain; Molecular Pathology Lab, Department of Pathology, Hvidovre Hospital, Copenhagen, Denmark; Pathological Anatomy Service. Hospital Doce de Octubre, Madrid, Spain .
Abstract :Background:
RAS family mutation status is an important potential clinical variable for treatment visitation in metastatic colon cancer (mCRC). Around 40% of colorectal tumors carry mutations in KRAS exon 2 (codons 12 and 13) which is a negative predictor of response to anti-EGFR therapies. Recent studies extend the range of mutations to include KRAS exons 3 and 4 or NRAS exons 2, 3 as negative response outcome predictors to anti-EGFR-containing therapy. Here, we present a novel diagnostic assay based upon multiplex ARMS-PCR and microarray detection system (CLART technology) to identify infrequent KRAS and NRAS mutations in formalin fixed paraffin embedded CRC specimens.
Methods:
We designed specific ARMS-primers and hybridization probes for detecting Q61H (183 A>C), K117N (351 A>C), K117N (351 A>T), A146T and A146V mutations in KRAS; and G12D, Q61H (183 A>T), Q61L, Q61N and Q61R mutations in NRAS. Clinical testing was performed using 33 clinical samples which contained the targets of this assay. Moreover, clinical specimens with wild type alleles and with known mutations in KRAS (codons 12, 13) and BRAF were tested to show that no cross reactivity to these targets were detected. The results were cross checked by comparison to Sanger sequencing methodology.
Results:
Analytical sensitivity was done using recombinant plasmids, results ranged between 100 - 1000 copies/µl for all point mutations excepting three of them (KRAS Q61H -183 A>C-, KRAS K117N -351 A>T- and NRAS Q61L) that were 10000 copies /µl. NRAS Q61R, Q61L, Q61H, Q61L and KRAS A146T mutations were analyzed using specific cell lines, the sensitivity was between 1-5ng. Diagnostic Sensitivity and Specificity data was over 90% and 98% for all mutations respectively. Concordance between present assay and Sanger sequencing were ≥95%.
Conclusions:
CLART NRAS iKRAS simultaneously detects the most prevalent mutations of NRAS and infrequent KRAS mutations with sensitivity and reproducibility ≥95%, being capable of detecting mutations in samples with less than 5% mutated alleles. Our data supports the use of this technology for clinical testing prior to anti-EGFR treatment, for both KRAS and NRAS status in human colorectal cancer samples.