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A growing body of research supports stromal tumour-infiltrating lymphocyte (TIL) density in breast cancer to be a robust prognostic and predicive biomarker. The gold standard for stromal TIL density quantitation in breast cancer is pathologist visual assessment using haematoxylin and eosin-stained slides. Artificial intelligence/machine-learning algorithms are in development to automate the stromal TIL scoring process, and must be validated against a reference standard such as pathologist visual assessment. Visual TIL assessment may suffer from significant interobserver variability. To improve interobserver agreement, regulatory science experts at the US Food and Drug Administration partnered with academic pathologists internationally to create a freely available online continuing medical education (CME) course to train pathologists in assessing breast cancer stromal TILs using an interactive format with expert commentary. Here we describe and provide a user guide to this CME course, whose content was designed to improve pathologist accuracy in scoring breast cancer TILs. We also suggest subsequent steps to translate knowledge into clinical practice with proficiency testing.
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Neoplasias da Mama , Humanos , Feminino , Patologistas , Linfócitos do Interstício Tumoral , Inteligência Artificial , PrognósticoRESUMO
BACKGROUND: Circulating cell free DNA (cfDNA) testing of plasma for EGFR somatic variants in lung cancer patients is being widely implemented and with any new service, external quality assessment (EQA) is required to ensure patient safety. An international consortium, International Quality Network for Pathology (IQNPath), has delivered a second round of assessment to measure the accuracy of cfDNA testing for lung cancer and the interpretation of the results. METHODS: A collaboration of five EQA provider organisations, all members of IQNPath, have delivered the assessment during 2018-19 to a total of 264 laboratories from 45 countries. Bespoke plasma reference material containing a range of EGFR mutations at varying allelic frequencies were supplied to laboratories for testing and reporting according to routine procedures. The genotyping accuracy and clinical reporting was reviewed against standardised criteria and feedback was provided to participants. RESULTS: The overall genotyping error rate in the EQA was found to be 11.1%. Low allelic frequency samples were the most challenging and were not detected by some testing methods, resulting in critical genotyping errors. This was reflected in higher false negative rates for samples with variant allele frequencies (VAF) rates less than 1.5% compared to higher frequencies. A sample with two different EGFR mutations gave inconsistent detection of both mutations. However, for one sample, where two variants were present at a VAF of less than 1% then both mutations were correctly detected in 145/263 laboratories. Reports often did not address the risk that tumour DNA may have not been tested and limitations of the methodologies provided by participants were insufficient. This was reflected in the average interpretation score for the EQA being 1.49 out of a maximum of 2. CONCLUSIONS: The variability in the standard of genotyping and reporting highlighted the need for EQA and educational guidance in this field to ensure the delivery of high-quality clinical services where testing of cfDNA is the only option for clinical management.
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Ácidos Nucleicos Livres , Neoplasias Pulmonares , Receptores ErbB/genética , Frequência do Gene , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MutaçãoRESUMO
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.
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Predisposição Genética para Doença , Variação Genética , Oncologia , Neoplasias/genética , Neoplasias/terapia , Biomarcadores Tumorais , Tomada de Decisão Clínica , Gerenciamento Clínico , Fidelidade a Diretrizes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oncologia/métodos , Oncologia/normas , Neoplasias/diagnóstico , Garantia da Qualidade dos Cuidados de Saúde , Controle de Qualidade , Reprodutibilidade dos Testes , Terminologia como AssuntoRESUMO
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.
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Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Receptores ErbB/genética , Seguimentos , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Estudos Longitudinais , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/enzimologia , Polimorfismo de Nucleotídeo Único , Controle de Qualidade , Células Tumorais CultivadasRESUMO
Objectives: The new European In Vitro Diagnostic (IVD) Regulation 2017/746 (IVDR) restricts the use of lab-developed tests (LDT) after 26th May 2022. There are no data on the impact of the IVDR on laboratories in the European Union. Methods: Laboratory tests performed in UZ Leuven were divided in four groups: core laboratory, immunology, special chemistry, and molecular microbiology testing. Each test was classified as Conformité Européenne (CE)-IVD, modified/off-label CE-IVD, commercial Research Use Only (RUO) or LDT. Each matrix was considered a separate test. Results: We found that 97.6% of the more than 11.5 million results/year were generated with a CE-IVD method. Of the 922 different laboratory tests, however, only 41.8% were CE-IVD, 10.8% modified/off-label CE-IVD, 0.3% RUO, and 47.1% LDT. Off-label CE-IVD was mainly used to test alternative matrices not covered by the claim of the manufacturer (e.g., pleural or peritoneal fluid). LDTs were mainly used for special chemistry, flow cytometry, and molecular testing. Excluding flow cytometry, the main reasons for the use of 377 LDTs were lack of a CE-IVD method (71.9%), analytical requirements (14.3%), and the fact the LDT was in use before CE-IVD available (11.9%). Conclusions: While the large majority of results (97.6%) were generated with a CE-IVD method, only 41.8% of laboratory tests were CE-IVD. There is currently no alternative on the market for 71.5% of the 537 LDTs performed in our laboratory which do not fall within the scope of the current IVD directive (IVDD). Compliance with the IVDR will require a major investment of time and effort.
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Hospitais Universitários/normas , Laboratórios Hospitalares/normas , Kit de Reagentes para Diagnóstico/normas , Bélgica , Técnicas de Química Analítica/normas , Técnicas de Química Analítica/estatística & dados numéricos , Hospitais Universitários/legislação & jurisprudência , Hospitais Universitários/estatística & dados numéricos , Humanos , Testes Imunológicos/normas , Testes Imunológicos/estatística & dados numéricos , Laboratórios Hospitalares/legislação & jurisprudência , Laboratórios Hospitalares/estatística & dados numéricos , Técnicas Microbiológicas/normas , Técnicas Microbiológicas/estatística & dados numéricos , Kit de Reagentes para Diagnóstico/estatística & dados numéricosRESUMO
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.
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Acreditação/métodos , Biomarcadores Tumorais/genética , Laboratórios/normas , Variação Genética , Humanos , Oncologia , Metástase Neoplásica , Controle de Qualidade , Projetos de PesquisaRESUMO
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.
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Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Neoplasias/sangue , Garantia da Qualidade dos Cuidados de Saúde , Receptores ErbB/sangue , Humanos , Mutação , Neoplasias/patologia , Proteínas Proto-Oncogênicas p21(ras)/sangueRESUMO
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.
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Bases de Dados Factuais/normas , Terminologia como Assunto , Receptores ErbB/genética , Guias como Assunto , Projeto Genoma Humano/organização & administração , Humanos , Mutação , Controle de QualidadeRESUMO
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.
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Biomarcadores Tumorais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Técnicas de Genotipagem/métodos , Laboratórios Hospitalares/normas , Proteínas Proto-Oncogênicas/genética , Garantia da Qualidade dos Cuidados de Saúde , Proteínas ras/genética , Códon , Neoplasias Colorretais/patologia , Análise Mutacional de DNA/métodos , Éxons , Genótipo , Humanos , Ensaio de Proficiência Laboratorial , Seleção de Pacientes , Proteínas Proto-Oncogênicas p21(ras)RESUMO
External quality assessment (EQA) schemes are a tool for clinical laboratories to evaluate and manage the quality of laboratory practice with the support of an independent party (i.e., an EQA provider). Depending on the context, there are different types of EQA schemes available, as well as various EQA providers, each with its own field of expertise. In this review, an overview of the general requirements for EQA schemes and EQA providers based on international guidelines is provided. The clinical and scientific value of these kinds of schemes for clinical laboratories, clinicians and patients are highlighted, in addition to the support EQA can provide to other types of laboratories, e.g., laboratories affiliated to biotech companies. Finally, recent developments and challenges in laboratory medicine and quality management, for example, the introduction of artificial intelligence in the laboratory and the shift to a more individual-approach instead of a laboratory-focused approach, are discussed. EQA schemes should represent current laboratory practice as much as possible, which poses the need for EQA providers to introduce latest laboratory innovations in their schemes and to apply up-to-date guidelines. By incorporating these state-of-the-art techniques, EQA aims to contribute to continuous learning.
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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.
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Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Colorretais/diagnóstico , Imuno-Histoquímica/normas , Hibridização in Situ Fluorescente/normas , Neoplasias Pulmonares/diagnóstico , Indicadores de Qualidade em Assistência à Saúde/normas , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/química , Neoplasias Colorretais/genética , Estudos de Viabilidade , Pesquisas sobre Atenção à Saúde , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Variações Dependentes do Observador , Valor Preditivo dos Testes , Garantia da Qualidade dos Cuidados de Saúde/normas , Melhoria de Qualidade/normas , Reprodutibilidade dos TestesRESUMO
In personalized medicine, predictive biomarker testing is the basis for an appropriate choice of therapy for patients with cancer. An important tool for laboratories to ensure accurate results is participation in external quality assurance (EQA) programs. Several providers offer predictive EQA programs for different cancer types, test methods, and sample types. In 2013, a guideline was published on the requirements for organizing high-quality EQA programs in molecular pathology. Now, after six years, steps were taken to further harmonize these EQA programs as an initiative by IQNPath ABSL, an umbrella organization founded by various EQA providers. This revision is based on current knowledge, adds recommendations for programs developed for predictive biomarkers by in situ methodologies (immunohistochemistry and in situ hybridization), and emphasized transparency and an evidence-based approach. In addition, this updated version also has the aim to give an overview of current practices from various EQA providers.
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Biomarcadores Tumorais , Testes Diagnósticos de Rotina/normas , Imuno-Histoquímica/normas , Hibridização In Situ/normas , Oncologia/normas , Neoplasias/química , Neoplasias/genética , Indicadores de Qualidade em Assistência à Saúde/normas , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Consenso , Humanos , Neoplasias/patologia , Variações Dependentes do Observador , Valor Preditivo dos Testes , Controle de Qualidade , Melhoria de Qualidade/normas , Reprodutibilidade dos TestesRESUMO
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.
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Rearranjo Gênico , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Transtornos Linfoproliferativos/genética , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase , Humanos , Ensaio de Proficiência Laboratorial , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Variações Dependentes do Observador , Valor Preditivo dos Testes , Garantia da Qualidade dos Cuidados de Saúde , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
If genome sequencing is performed in health care, in theory the opportunity arises to take a further look at the data: opportunistic genomic screening (OGS). The European Society of Human Genetics (ESHG) in 2013 recommended that genome analysis should be restricted to the original health problem at least for the time being. Other organizations have argued that 'actionable' genetic variants should or could be reported (including American College of Medical Genetics and Genomics, French Society of Predictive and Personalized Medicine, Genomics England). They argue that the opportunity should be used to routinely and systematically look for secondary findings-so-called opportunistic screening. From a normative perspective, the distinguishing characteristic of screening is not so much its context (whether public health or health care), but the lack of an indication for having this specific test or investigation in those to whom screening is offered. Screening entails a more precarious benefits-to-risks balance. The ESHG continues to recommend a cautious approach to opportunistic screening. Proportionality and autonomy must be guaranteed, and in collectively funded health-care systems the potential benefits must be balanced against health care expenditures. With regard to genome sequencing in pediatrics, ESHG argues that it is premature to look for later-onset conditions in children. Counseling should be offered and informed consent is and should be a central ethical norm. Depending on developing evidence on penetrance, actionability, and available resources, OGS pilots may be justified to generate data for a future, informed, comparative analysis of OGS and its main alternatives, such as cascade testing.
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Testes Genéticos/normas , Genética Humana/normas , Guias de Prática Clínica como Assunto , Sociedades Médicas/normas , Europa (Continente) , Testes Genéticos/ética , Genética Humana/ética , Genética Humana/organização & administração , HumanosRESUMO
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.
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DNA Tumoral Circulante/sangue , Fixadores/farmacologia , Formaldeído/farmacologia , Neoplasias/sangue , Neoplasias/diagnóstico , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/genética , Frequência do Gene , Humanos , Biópsia Líquida , Oncologia/métodos , Mutação , Neoplasias/patologia , Dados PreliminaresRESUMO
BACKGROUND: The clinical spectrum associated with cystic fibrosis transmembrane conductance regulator (CFTR) variant p.Arg117His is highly variable, ranging from full-blown cystic fibrosis (CF) in a small number of cases to CFTR-related disorders (CFTR-RDs) or no symptoms at all. Therefore, taking into account phenotype variability is essential for interpretation. External quality assessment (EQA) schemes can help laboratories to objectively assess the quality of genotyping and reporting by the laboratory. METHODS: We performed a retrospective longitudinal data analysis on laboratory performance regarding the interpretation of p.Arg117His during CF EQA scheme participation. Completeness and accuracy of reporting on two mock clinical cases were each compared over time (case 1: 2005, 2007 and 2012; case 2: 2015 and 2018). These cases concerned subjects compound heterozygous for p.Phe508del and p.Arg117His in cis with 7T, but with different clinical backgrounds (family planning (case 1) versus diagnostic testing for a child (case 2)). Furthermore, we analyzed the influence of previous participations, annual test volume, accreditation status and laboratory setting on overall performance. RESULTS: Overall performance improved over time, except during the 2007 CF EQA scheme. In addition, previous participations had a beneficial effect on laboratory performance. Accreditation status, annual test volume and laboratory setting did not significantly influence total interpretation scores. CONCLUSIONS: In general, laboratories performed well on both cases, although reporting on the variable clinical spectrum of p.Arg117His in cis with 7T and on the disease liability of individual CFTR variants can still improve. Moreover, this study underlined the educational role of CF EQA schemes.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Testes Genéticos/métodos , Feminino , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Estudos Longitudinais , Masculino , Mutação , Fenótipo , Estudos RetrospectivosRESUMO
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.
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Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Hibridização in Situ Fluorescente/métodos , Laboratórios Hospitalares , Patologistas , Proto-Oncogene Mas , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Ácidos Nucleicos Livres/análise , DNA Tumoral Circulante/análise , Biópsia Líquida , Neoplasias/patologia , Análise Mutacional de DNA/métodos , Prova Pericial/métodos , Humanos , Biópsia Líquida/métodos , Mutação/genética , Neoplasias/diagnósticoRESUMO
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.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Erros de Diagnóstico , Ensaio de Proficiência Laboratorial/normas , Técnicas de Diagnóstico Molecular/normas , Melhoria de Qualidade/normas , Indicadores de Qualidade em Assistência à Saúde/normas , Neoplasias Colorretais/patologia , Congressos como Assunto , Europa (Continente) , Seguimentos , Feedback Formativo , Predisposição Genética para Doença , Pesquisas sobre Atenção à Saúde , Humanos , Variações Dependentes do Observador , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
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.