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The Italian external quality assessment for RAS testing in colorectal carcinoma identifies methods-related
Nicola Normanno1,2, Carmine Pinto3, Francesca Castiglione4
1Cell 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.
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.
Conclusion:
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.
Insights
The Italian RAS External Quality Assessment (EQA) scheme showed that while many centers perform quality mutational analyses for metastatic colorectal cancer (mCRC) patients, differences in methods and initial failure rates highlight the need for continued education and standardization in RAS testing.
Area of Science:
- Oncology
- Molecular Pathology
- Genetics
Background:
- European Medicines Agency mandated KRAS/NRAS testing for anti-EGFR therapy selection in mCRC patients.
- External Quality Assessment (EQA) schemes are crucial for standardizing RAS testing in Italy.
- The study transitioned from KRAS to comprehensive RAS (including NRAS) testing assessment.
Purpose of the Study:
- To evaluate inter-laboratory consistency in RAS mutation testing for mCRC.
- To ensure standardization of results during the shift to all-RAS testing.
- To assess the performance of Italian laboratories in molecular pathology for cancer diagnostics.
Main Methods:
- An EQA scheme involving 88 participant centers and 10 validated specimens with known KRAS/NRAS/BRAF mutations.
- Two rounds of testing with molecular pathology sample reports evaluated by a board of assessors.
- Genotyping methods compared included Sanger sequencing and pyrosequencing.
Main Results:
- 36% of participants failed the first round, with genotyping errors higher in Sanger sequencing compared to pyrosequencing (3% vs 1.3%).
- Performance improved in the second round, with 79/88 participants passing the overall RAS EQA scheme.
- Inconsistent use of Human Genome Variation Society nomenclature for mutation description was observed.
Conclusions:
- RAS mutational analyses are generally performed with good quality in many Italian centers.
- Significant differences in testing methodologies were identified.
- EQA schemes play a vital role in continuous education and improving laboratory performance in molecular diagnostics.
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