Variation in nomenclature of somatic variants for selection of oncological therapies: Can we reach a consensus soon?

A standardized nomenclature for reporting oncology biomarker variants is key to avoid misinterpretation of results and unambiguous registration in clinical databases. External quality assessment (EQA) schemes have revealed a need for more consistent nomenclature use in clinical genetics. We evaluated the propensity of EQA for improvement of compliance with Human Genome Variation Society (HGVS) recommendations for reporting of predictive somatic variants in lung and colorectal cancer. Variant entries between 2012 and 2018 were collected from written reports and electronic results sheets. In total, 4,053 variants were assessed, of which 12.1% complied with HGVS recommendations. Compliance improved over time from 2.1% (2012) to 22.3% (2018), especially when laboratories participated in multiple EQA schemes. Compliance was better for next-generation sequencing (20.9%) compared with targeted techniques (9.8%). In the 1792 reports, HGVS recommendations for reference sequences were met for 31.9% of reports, for 36.0% of noncommercial, and 26.5% of commercial test methods. Compliance improved from 16.7% (2012) to 33.1% (2018), and after repeated EQA participation. EQA participation improves compliance with HGVS recommendations. The residual percentage of errors in the most recent schemes suggests that laboratories, companies, and EQA providers need to collaborate for additional improvement of harmonization in clinical test reporting.

Sensitive detection methods are key to identify secondary EGFR c.2369C>T p.(Thr790Met) in non-small cell lung cancer tissue samples.

BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

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

BACKGROUND: Predictive 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. METHODS: Longitudinal 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. RESULTS: Laboratory 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. CONCLUSION: The 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.

International pilot external quality assessment scheme for analysis and reporting of circulating tumour DNA.

BACKGROUND: Molecular analysis of circulating tumour DNA (ctDNA) is becoming increasingly important in clinical treatment decisions. A pilot External Quality Assessment (EQA) scheme for ctDNA analysis was organized by four European EQA providers under the umbrella organization IQN Path, in order to investigate the feasibility of delivering an EQA to assess the detection of clinically relevant variants in plasma circulating cell-free DNA (cfDNA) and to analyze reporting formats. METHODS: Thirty-two experienced laboratories received 5 samples for EGFR mutation analysis and/or 5 samples for KRAS and NRAS mutation analysis. Samples were artificially manufactured to contain 3 mL of human plasma with 20 ng/mL of fragmented ctDNA and variants at allelic frequencies of 1 and 5%. RESULTS: The scheme error rate was 20.1%. Higher error rates were observed for RAS testing when compared to EGFR analysis, for allelic frequencies of 1% compared to 5%, and for cases including 2 different variants. The reports over-interpreted wild-type results and frequently failed to comment on the amount of cfDNA extracted. CONCLUSIONS: The pilot scheme demonstrated the feasibility of delivering a ctDNA EQA scheme and the need for such a scheme due to high error rates in detecting low frequency clinically relevant variants. Recommendations to improve reporting of cfDNA are provided.

Causes behind error rates for predictive biomarker testing: the utility of sending post-EQA surveys.

External quality assessment (EQA) schemes assess the performance of predictive biomarker testing in lung and colorectal cancer and have previously demonstrated variable error rates. No information is currently available on the underlying causes of incorrect EQA results in the laboratories. Participants in EQA schemes by the European Society of Pathology between 2014 and 2018 for lung and colorectal cancer were contacted to complete a survey if they had at least one analysis error or test failure in the provided cases. Of the 791 surveys that were sent, 325 were completed including data from 185 unique laboratories on 514 incorrectly analyzed or failed cases. For the digital cases and immunohistochemistry, the majority of errors were interpretation-related. For fluorescence in situ hybridization, problems with the EQA materials were reported frequently. For variant analysis, the causes were mainly methodological for lung cancer but variable for colorectal cancer. Post-analytical (clerical and interpretation) errors were more likely detected after release of the EQA results compared to pre-analytical and analytical issues. Accredited laboratories encountered fewer reagent problems and more often responded to the survey. A recent change in test methodology resulted in method-related problems. Testing more samples annually introduced personnel errors and lead to a lower performance in future schemes. Participation to quality improvement projects is important to reduce deviating test results in laboratories, as the different error causes differently affect the test performance. EQA providers could benefit from requesting root cause analyses behind errors to offer even more tailored feedback, subschemes, and cases.

PD-L1 immunohistochemistry in non-small-cell lung cancer: unraveling differences in staining concordance and interpretation.

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

Incidents in Molecular Pathology: Frequency and Causes During Routine Testing.

CONTEXT.­: Errors in laboratory medicine could compromise patient safety. Good laboratory practice includes identifying and managing nonconformities in the total test process. Varying error percentages have been described in other fields but are lacking for molecular oncology. OBJECTIVES.­: To gain insight into incident causes and frequency in the total test process from 8 European institutes routinely performing biomarker tests in non-small cell lung cancer and colorectal cancer. DESIGN.­: All incidents documented in 2018 were collected from all hospital services for pre-preanalytical entries before the biomarker test, as well as specific incidents for biomarker tests. RESULTS.­: There were 5185 incidents collected, of which 4363 (84.1%) occurred in the pre-preanalytical phase (all hospital services), 2796 of 4363 (64.1%) related to missing or incorrect request form information. From the other 822 specific incidents, 166 (20.2%) were recorded in the preanalytical phase, 275 (33.5%) in the analytical phase, and 194 (23.6%) in the postanalytical phase, mainly due to incorrect report content. Only 47 of 822 (5.7%) incidents were recorded in the post-postanalytical phase, and 123 (15.0%) in the complete total test process. For 17 of 822 (2.1%) incidents the time point was unknown. Pre-preanalytical incidents were resolved sooner than incidents on the complete process (mean 6 versus 60 days). For 1215 of 5168 (23.5%) incidents with known causes a specific action was undertaken besides documenting them, not limited to accredited institutes. CONCLUSIONS.­: There was a large variety in the number and extent of documented incidents. Correct and complete information on the request forms and final reports are highly error prone and require additional focus.

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

Managing Deviating EQA Results: A Survey to Assess the Corrective and Preventive Actions of Medical Laboratories Testing for Oncological Biomarkers.

Laboratories testing predictive biomarkers in lung and colorectal cancer are advised to participate in external quality assessment (EQA) schemes. This study aimed to investigate which corrective actions were taken by laboratories if predetermined performance criteria were not met, to ultimately improve current test practices. EQA participants from the European Society of Pathology between 2014 and 2018 for lung and colorectal cancer were contacted, if they had at least one analysis error or test failure in the provided cases, to complete a survey. For 72.4% of 514 deviating EQA results, an appropriate action was performed, most often including staff training (15.2%) and protocol revisions (14.6%). Main assigned persons were the molecular biologist (40.0%) and pathologist (46.5%). A change in test method or the use of complex techniques, such as next-generation sequencing, required more training and the involvement of dedicated personnel to reduce future test failures. The majority of participants adhered to ISO 15189 and implemented suitable actions by designated staff, not limited to accredited laboratories. However, for 27.6% of cases (by 20 laboratories) no corrective action was taken, especially for pre-analytic problems and complex techniques. The surveys were feasible to request information on results follow-up and further recommendations were provided.

External Quality Assessment Schemes for Biomarker Testing in Oncology: Comparison of Performance between Formalin-Fixed, Paraffin-Embedded-Tissue and Cell-Free Tumor DNA in Plasma.

Liquid biopsies have emerged as a useful addition to tissue biopsies in molecular pathology. Literature has shown lower laboratory performances when a new method of variant analysis is introduced. This study evaluated the differences in variant analysis between tissue and plasma samples after the introduction of liquid biopsy in molecular analysis. Data from a pilot external quality assessment scheme for the detection of molecular variants in plasma samples and from external quality assessment schemes for the detection of molecular variants in tissue samples were collected. Laboratory performance and error rates by sample were compared between matrices for variants present in both scheme types. Results showed lower overall performance [65.6% (n = 276) versus 89.2% (n = 1607)] and higher error rates [21.0% to 43.5% (n = 138) versus 8.7% to 16.7% (n = 234 to 689)] for the detection of variants in plasma compared to tissue, respectively. In the plasma samples, performance was decreased for variants with an allele frequency of 1% compared to 5% [56.5% (n = 138) versus 74.6% (n = 138)]. The implementation of liquid biopsy in the detection of circulating tumor DNA in plasma was associated with poor laboratory performance. It is important both to apply optimal detection methods and to extensively validate new methods for testing circulating tumor DNA before treatment decisions are made.

Staining Performance of ALK and ROS1 Immunohistochemistry and Influence on Interpretation in Non-Small-Cell Lung Cancer.

Selection of non-small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance.

IQN path ASBL report from the first European cfDNA consensus meeting: expert opinion on the minimal requirements for clinical ctDNA testing.

Liquid biopsy testing is a new laboratory-based method that detects tumour mutations in circulating free DNA (cfDNA) derived from minimally invasive blood sampling techniques. Recognising the significance for clinical testing, in 2017, IQN Path provided external quality assessment for liquid biopsy testing. Representatives of those participating laboratories were invited to attend a workshop to discuss the findings and how to achieve quality implementation of cfDNA testing in the clinical setting, the discussion and outcomes of this consensus meeting are described below. Predictive molecular profiling using tumour tissue in order to select cancer patients eligible for targeted therapy is now routine in diagnostic pathology. If insufficient tumour tissue material is available, in some circumstances, recent European Medicines Agency (EMA) guidance recommends mutation testing with plasma cfDNA. Clinical applications of cfDNA include treatment selection based on clinically relevant mutations derived from pre-treatment samples and the detection of resistant mutations upon progression of the disease. In order to identify tumour-related mutations in amongst other nucleic acid material found in plasma samples, highly sensitive laboratory methods are needed. In the workshop, we discussed the variable approaches taken with regard to cfDNA extraction methods, the tests, and considered the impact of false-negative test results. We explored the lack of standardisation of complex testing procedures ranging from plasma collection, transport, processing and storage, cfDNA extraction, and mutation analysis, to interpretation and reporting of results. We will also address the current status of clinical validation and clinical utility, and its use in current diagnosis. This workshop revealed a need for guidelines on with standardised procedures for clinical cfDNA testing and reporting, and a requirement for cfDNA-based external quality assessment programs.

European follow-up of incorrect biomarker results for colorectal cancer demonstrates the importance of quality improvement projects.

Biomarker analysis for colorectal cancer has been shown to be reliable in Europe with 97% of samples tested by EQA participants to be correctly classified. This study focuses on errors during the annual EQA assessment. The aim was to explore the causes and actions related to the observed errors and to provide feedback and assess any improvement between 2016 and 2017. An electronic survey was sent to all laboratories with minimum one genotyping error or technical failure on ten tumor samples. A workshop was organized based on 2016 survey responses. Improvement of performance in 2017 was assessed for returning participants (n = 76), survey respondents (n = 13) and workshop participants (n = 4). Survey respondents and workshop participants improved in terms of (maximum) analysis score, successful participation, and genotyping errors compared to all returning participants. In 2016, mostly pre- and post-analytical errors (both 25%) were observed caused by unsuitability of the tumor tissue for molecular analysis. In 2017, most errors were due to analytical problems (50.0%) caused by methodological problems. The most common actions taken (n = 58) were protocol revisions (34.5%) and staff training (15.5%). In 24.1% of issues identified no action was performed. Corrective actions were linked to an improved performance, especially if performed by the pathologist. Although biomarker testing has improved over time, error occurrence at different phases stresses the need for quality improvement throughout the test process. Participation to quality improvement projects and a close collaboration with the pathologist can have a positive influence on performance.

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.

External Quality Assessment Identifies Training Needs to Determine the Neoplastic Cell Content for Biomarker Testing.

Neoplastic cell content determination is crucial for biomarker testing. It is known that interobserver variation exists, but largescale data are missing about variation in tumor delineation and cell content determination. Results were obtained from the external quality assessment program for metastatic colorectal cancer from the European Society of Pathology (N = 5776 observations). The study included three parts: current practices were surveyed, neoplastic cell content estimations and delineations were retrieved from stained slides, and clinical reports were analyzed. Seventeen of 43 pathologists determined the neoplastic cell content in a tumor-rich area for DNA extraction and took immune cells (n = 37), tumor cell distribution (n = 33), desmoplastic stroma (n = 30), necrosis (n = 29), and mucus (n = 23) into account. The selected area was highly variable, and the average difference between the highest and lowest estimation ranged between 51% and 78% (2011 to 2017). The number of overestimations was alarmingly high in samples containing <30% tumor cells. Of concern is that 33 of 105 laboratories reported a wild-type result in a sample without tumor in 2017. Standardization of neoplastic cell content determination is needed for test outcome interpretation. The authors' data show variation in estimation practices, tumor delineations and estimations, and interpretation problems (n = 226 reports). Further training for selecting the most suitable block and creating clear reports is urgently needed.

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.

Detection of EGFR Variants in Plasma: A Multilaboratory Comparison of a Real-Time PCR EGFR Mutation Test in Europe.

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

Three Rounds of External Quality Assessment in France to Evaluate the Performance of 28 Platforms for Multiparametric Molecular Testing in Metastatic Colorectal and Non-Small Cell Lung Cancer.

Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education.