The Italian external quality assessment for RAS testing in colorectal carcinoma identifies methods-related inter-laboratory differences.
BACKGROUND
In 2014 the European Medicines Agency included exon 2, 3 and 4 KRAS and NRAS testing for the selection of metastatic colorectal cancer (mCRC) patients eligible for the therapy with anti-EGFR monoclonal antibodies. The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology (SIAPEC) organized an external quality assessment (EQA) scheme for CRC to evaluate inter-laboratory consistency and to ensure standardization of the results in the transition from KRAS to all-RAS testing.


METHODS
Ten formalin fixed paraffin embedded specimens including KRAS/NRAS (exons 2, 3, 4) and BRAF (codon 600) mutations were validated by three referral laboratories and sent to 88 participant centers. Molecular pathology sample reports were also requested to each laboratory. A board of assessors from AIOM and SIAPEC evaluated the results according to a predefined scoring system. The scheme was composed of two rounds.


RESULTS
In the first round 36% of the 88 participants failed, with 23 centers having at least one false positive or false negative while 9 centers did not meet the deadline. The genotyping error rate was higher when Sanger sequencing was employed for testing as compared with pyrosequencing (3 vs 1.3%; p = 0.01; Pearson Chi Square test). In the second round, the laboratories improved their performance, with 23/32 laboratories passing the round. Overall, 79/88 participants passed the RAS EQA scheme. Standardized Human Genome Variation Society nomenclature was incorrectly used to describe the mutations identified and relevant variations were noticed in the genotype specification.


CONCLUSIONS
The results of the Italian RAS EQA scheme indicate that the mutational analyses are performed with good quality in many Italian centers, although significant differences in the methods used were highlighted. The relatively high number of centers failing the first round underlines the fundamental role in continued education covered by EQA schemes.
Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"-IRCCS, Via M. Semmola, 80131, Naples, Italy. nicnorm@yahoo.com.|Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale Tumori "Fondazione Giovanni Pascale" IRCCS, Naples, Italy. nicnorm@yahoo.com.
Nicola
N
Normanno
Medical Oncology, S. Maria Hospital - IRCCS, Reggio Emilia, Italy. cpinto@ao.pr.it.
Carmine
C
Pinto
Department of Human Pathology and Oncology, University of Florence, Florence, Italy. francesca.castiglione@unifi.it.
Francesca
F
Castiglione
Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale Tumori "Fondazione Giovanni Pascale" IRCCS, Naples, Italy. francesca.fenizia@hotmail.it.
Francesca
F
Fenizia
Department of Pathology and Laboratory Medicine, European Institute of Oncology, Milan, Italy. massimo.barberis@ieo.it.
Massimo
M
Barberis
Center of Predictive Molecular Medicine, University-Foundation, CeSI Biotech, Chieti, Italy. amarchetti@unich.it.
Antonio
A
Marchetti
Division of Pathological Anatomy, Department of Surgical, Medical, Molecular Pathology and Critical Care, University of Pisa, Pisa, Italy. gabriella.fontanini@med.unipi.it.
Gabriella
G
Fontanini
Pathology Section, Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy. gaderosa@unina.it.
Gaetano
G
De Rosa
Department of Human Pathology and Oncology, University of Florence, Florence, Italy. gl.taddei@unifi.it.
Gian Luigi
GL
Taddei
D003062:Codon; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D066246:ErbB Receptors / Q000378:metabolism; D005060:Europe; D005091:Exons; D005189:False Positive Reactions; D005557:Formaldehyde / Q000737:chemistry; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D007558:Italy; D059021:Laboratory Proficiency Testing; D015233:Models, Statistical; D009154:Mutation; D009362:Neoplasm Metastasis; D010232:Paraffin / Q000737:chemistry; D011786:Quality Control; D015203:Reproducibility of Results; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; D005557:Formaldehyde; D010232:Paraffin; D066246:ErbB Receptors; D018631:ras Proteins