Véronique Tack

External Quality Assessment Unravels Interlaboratory Differences in Quality of RAS Testing for Anti-EGFR Therapy in Colorectal Cancer

BACKGROUND Regulations for the selection of patients with metastatic colorectal cancer for anti-EGFR treatment changed at the end of 2013. The set of mutations to be tested extended from KRAS codons 12 and 13 to KRAS and NRAS exons 2, 3, and 4. A European external quality assessment scheme monitored the performance of laboratories and evaluated the implementation of the new regulations. MATERIALS AND METHODS The 131 participating laboratories received 10 samples of formalin-fixed paraffin-embedded material, including RAS (exon 2, 3, 4) and BRAF mutations. Mock clinical data were provided for three cases. Using their routine methods, laboratories determined the genotypes and submitted three written reports. Assessors scored the results according to predefined evaluation criteria. RESULTS Half of the participants (49.3%) had completely implemented the new test requirements (codons 12, 13, 59, 61, 117, and 146 of KRAS and NRAS), and 96 laboratories (73.3%) made no genotype mistakes. Correct nomenclature, according to the Human Genome Variation Society, was used by 82 laboratories (62.6%). CONCLUSION Although regulations were effective for several months, many laboratories were not ready for full RAS testing in the context of anti-EGFR therapy. Nevertheless, in each participating country, there are laboratories that provide complete and correct testing. External quality assessments can be used to monitor implementation of new test regulations and to stimulate the laboratories to improve their testing procedures. Because the results of this program are available on the website of the European Society of Pathology, patients and clinicians can refer test samples to a reliable laboratory.

The relevance of external quality assessment for molecular testing for ALK positive non-small cell lung cancer: results from two pilot rounds show room for optimization.

Background and Purpose Molecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK rearrangement status is a critical biomarker to predict response to tyrosine kinase inhibitors such as crizotinib. To promote high quality testing in non-small cell lung cancer, the European Society of Pathology has introduced an external quality assessment scheme. This article summarizes the results of the first two pilot rounds organized in 2012–2013. Materials and Methods Tissue microarray slides consisting of cell-lines and resection specimens were distributed with the request for routine ALK testing using IHC or FISH. Participation in ALK FISH testing included the interpretation of four digital FISH images. Results Data from 173 different laboratories was obtained. Results demonstrate decreased error rates in the second round for both ALK FISH and ALK IHC, although the error rates were still high and the need for external quality assessment in laboratories performing ALK testing is evident. Error rates obtained by FISH were lower than by IHC. The lowest error rates were observed for the interpretation of digital FISH images. Conclusion There was a large variety in FISH enumeration practices. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing.

What's in a Name? A Coordinated Approach toward the Correct Use of a Uniform Nomenclature to Improve Patient Reports and Databases

The Human Genome Variation Society (HGVS) recommendations provide standardized nomenclature for reporting variants. This should be encouraged in molecular pathology—both for issuing diagnostic reports and for correct data recording in electronic databases. Many providers of external quality assessment (EQA) promote the correct use of HGVS nomenclature by scoring variant descriptions used in EQA reports. This study focuses on the type and impact of variant nomenclature errors. An assessment was made of EGFR gene variant nomenclature by four EQA providers (European Society of Pathology [ESP], European Molecular Genetics Quality Network [EMQN], United Kingdom National External Quality Assessment Service for Molecular Genetics, and the French national Gen&Tiss EQA scheme) for two EQA distributions. Laboratories testing for oncology biomarkers make different errors when describing EGFR gene variants. Significant differences were observed regarding inclusion of the correct reference sequence: EMQN participants made fewer errors compared to ESP EQA participants (P‐value = 0.015). The analysis of ESP EQA participants showed significant improvement over 2 years (P‐value = 0.016). Results demonstrate the need for improvement of variant reporting according to HGVS guidelines. Consequences of using incorrect mutation nomenclature are currently perceived as low by many laboratories, but the impact will rise with an increased reliance on databases to assist in result analysis.

A stitch in time saves nine: external quality assessment rounds demonstrate improved quality of biomarker analysis in lung cancer

Biomarker analysis has become routine practice in the treatment of non-small cell lung cancer (NSCLC). To ensure high quality testing, participation to external quality assessment (EQA) schemes is essential. This article provides a longitudinal overview of the EQA performance for EGFR, ALK, and ROS1 analyses in NSCLC between 2012 and 2015. The four scheme years were organized by the European Society of Pathology according to the ISO 17043 standard. Participants were asked to analyze the provided tissue using their routine procedures. Analysis scores improved for individual laboratories upon participation to more EQA schemes, except for ROS1 immunohistochemistry (IHC). For EGFR analysis, scheme error rates were 18.8%, 14.1% and 7.5% in 2013, 2014 and 2015 respectively. For ALK testing, error rates decreased between 2012 and 2015 by 5.2%, 3.2% and 11.8% for the fluorescence in situ hybridization (FISH), FISH digital, and IHC subschemes, respectively. In contrast, for ROS1 error rates increased between 2014 and 2015 for FISH and IHC by 3.2% and 9.3%. Technical failures decreased over the years for all three markers. Results show that EQA contributes to an ameliorated performance for most predictive biomarkers in NSCLC. Room for improvement is still present, especially for ROS1 analysis.

The ins and outs of molecular pathology reporting

The raid evolution in molecular pathology resulting in an increasing complexity requires careful reporting. The need for standardisation is clearer than ever. While synoptic reporting was first used for reporting hereditary genetic diseases, it is becoming more frequent in pathology, especially molecular pathology reports too. The narrative approach is no longer feasible with the growing amount of essential data present on the report, although narrative components are still necessary for interpretation in molecular pathology. On the way towards standardisation of reports, guidelines can be a helpful tool. There are several guidelines that focus on reporting in the field of hereditary diseases, but it is not always feasible to extrapolate these to the reporting of somatic variants in molecular pathology. The rise of multi-gene testing causes challenges for the laboratories. In order to provide a continuous optimisation of the laboratory testing process, including reporting, external quality assessment is essential and has already proven to improve the quality of reports. In general, a clear and concise report for molecular pathology can be created by including elements deemed important by different guidelines, adapting the report to the process flows of the laboratory and integrating the report with the laboratory information management system and the patient record.

Describing the Reportable Range Is Important for Reliable Treatment Decisions: A Multiple Laboratory Study for Molecular Tumor Profiling Using Next-Generation Sequencing

Because interpretation of next-generation sequencing (NGS) data remains challenging, optimization of the NGS process is needed to obtain correct sequencing results. Therefore, extensive validation and continuous monitoring of the quality is essential. NGS performance was compared with traditional detection methods and technical quality of nine NGS technologies was assessed. First, nine formalin-fixed, paraffin-embedded patient samples were analyzed by 114 laboratories by using different detection methods. No significant differences in performance were observed between analyses with NGS and traditional techniques. Second, two DNA control samples were analyzed for a selected number of variants by 26 participants with the use of nine different NGS technologies. Quality control metrics were analyzed from raw data files and a survey about routine procedures. Results showed large differences in coverages, but observed variant allele frequencies in raw data files were in line with predefined variant allele frequencies. Many false negative results were found because of low-quality regions, which were not reported as such. It is recommended to disclose the reportable range, the fraction of targeted genomic regions for which calls of acceptable quality can be generated, to avoid any errors in therapy decisions. NGS can be a reliable technique, only if essential quality control during analysis is applied and reported.

Accreditation, setting and experience as indicators to assure quality in oncology biomarker testing laboratories

BackgroundPredictive biomarkers allow clinicians to optimise cancer treatment decisions. Therefore, molecular biomarker test results need to be accurate and swiftly available. To ensure quality of oncology biomarker testing, external quality assessments (EQA) for somatic variant analyses were organised. This study hypothesised whether laboratory characteristics influence the performance of laboratories and whether these can be imposed before authorisation of biomarker testing.MethodsLongitudinal EQA data from the European Society of Pathology were available over six (metastatic colorectal cancer) and four years (non-small cell lung cancer), including the percentage of analysis errors and technical failures, and information on laboratory characteristics (accreditation status, laboratory setting, number of samples analysed and detection method). Statistical models for repeated measurements were used to analyse the association between the EQA results and the laboratory characteristics.ResultsLaboratory accreditation was associated with fewer analysis errors in early stages of biomarker introduction into the laboratory. Analysing more samples, or university and research laboratories showed better performance. Changing the detection method did not have an effect.ConclusionThe indicators support the clinicians in choosing molecular pathology laboratories by improving quality assurance and guaranteeing patient safety. Accreditation of laboratories, centralisation of biomarker testing or a university and research setting should be stimulated.

137INIMPORTANCE OF EXTERNAL QUALITY ASSESSEMENT IN IMMUNO-ONCOLOGY

ABSTRACT The important role of immunohistochemistry (IHC) is well known. IHC will recognize antigens and, consequently, identify and classify specific cells within a cell population whose morphology is heterogenous or apparently homogenous. Since many years external quality assessment (EQA) schemes for IHC are organized by NORDIQC and UK NEAQS. Since some years, IHC can be used for treatment selection in advanced NSCLC. In 2012, the European Society of Pathology (ESP) proposed an EQA scheme to promote high quality biomarker testing in NSCLC for EGFR mutation analysis and ALK rearrangement detection (IHC, FISH). From 2014 on, ROS1 testing is also included. The scheme aims to assess and improve the current status of testing in NSCLC, to provide education and remedial measures, to permit inter-laboratory comparison and to allow validation of test methods by distributing validated material harboring well-defined aberrations. In total, 173 different laboratories participated in the pilot rounds. In the first round, 29 laboratories submitted results for ALK IHC. In the second round, 58 laboratories submitted results for ALK IHC. If carefully clinically validated according to ISO 15189, ALK IHC may be considered as a screening method to select specimens for ALK FISH testing. In the context that at the time of the pilot studies no interlaboratory comparison validation studies were available, ALK IHC performance from our study shows that IHC testing is well implemented. Not surprisingly however, the error rates for IHC were greater than for FISH. Our study demonstrates improvement of ALK testing after only two rounds. It is expected that larger datasets, spanning a larger number of EQA participations will demonstrate a statistically significant improvement in performance. On scheme level, the decreased error rates in the second round demonstrated improvement for both ALK FISH and ALK IHC. Error rates for ALK FISH TMA and ALK IHC were still high (>5%), which stresses the need for continued education through EQA. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing. Disclosure: All authors have declared no conflicts of interest.