Monogenic autoinflammatory disorders (AIDs) are rare diseases caused by variants in genes regulating the innate immune system. The identification of the first four genes responsible for the prototype ...group of hereditary recurrent fevers prompted the development of genetic diagnosis, followed by external quality assessment and guidelines for the interpretation of sequence variants in these diseases. Recent changes in the diagnosis of genetic diseases, namely the implementation of next-generation sequencing (NGS), lead to discovery of the new genes associated with at least 40 novel AIDs, which revolutionized patient care and prognosis. However, these rapid advances resulted in nonstandardized molecular strategies that can influence genetic diagnosis and reporting of results. In order to assess factors, which may have an impact on performance and quality of results in the NGS era, we carried out an online survey among member laboratories of the European Molecular Genetics Quality Network, which highlighted different strategies being used and identified pitfalls that deserve discussion and improvement.
Disruptive imaging and laboratory technologies can improve clinical decision processes and outcomes in oncology. However, certain obstacles must be overcome before these technologies can be fully ...implemented as part of the standard for care. An integrative diagnostic approach represents a unique opportunity to unleash the full diagnostic potential and paves the way towards personalized cancer diagnostics. To meet this demand, an interdisciplinary Task Force of the EFLM was initiated as a consequence of an EFLM/ESR during the CELME 2019 meeting in order to evaluate the clinical value of CNAPS/CTC (circulating nucleic acids in plasma and serum/circulating tumor cells) in early detection of cancer. Here, an overview of current disruptive techniques, their clinical implications and potential value of an integrative diagnostic approach is provided. Furthermore, requirements such as the establishment of diagnostic tumor boards, development of adequate software solutions and a change of mindset towards a new generation of diagnosticians providing actionable health information are presented. This development has the potential to elevate the position and clinical recognition of diagnosticians.
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. Keywords: Lung cancer, NSCLC, cfDNA, EGFR EQA, Mutation testing
The diagnostic work-up for non-small cell lung cancer (NSCLC) requires biomarker testing to guide therapy choices. This article is the second of a two-part series. In Part 1, we summarised ...evidence-based recommendations for obtaining and processing small specimen samples (i.e. pre-analytical steps) from patients with advanced NSCLC. Here, in Part 2, we summarise evidence-based recommendations relating to analytical steps of biomarker testing (and associated reporting and quality assessment) of small specimen samples in NSCLC. As the number of biomarkers for actionable (genetic) targets and approved targeted therapies continues to increase, simultaneous testing of multiple actionable oncogenic drivers using next-generation sequencing (NGS) becomes imperative, as set forth in European Society for Medical Oncology guidelines. This is particularly relevant in advanced NSCLC, where tissue specimens are typically limited and NGS may help avoid tissue exhaustion compared with sequential biomarker testing. Despite guideline recommendations, significant discrepancies in access to NGS persist across Europe, primarily due to reimbursement constraints. The use of increasingly complex testing methods also has implications for the reporting of results. Molecular testing reports should include clinical interpretation with additional commentary on sample adequacy as appropriate. Molecular tumour boards are recommended to facilitate the interpretation of complex genetic information arising from NGS, and to collaboratively determine the optimal treatment for patients with NSCLC. Finally, whichever testing modality is employed, it is essential that adequate internal and external validation and quality control measures are implemented.
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.
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%.
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.
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.
Biomarker testing is crucial for treatment selection in advanced non-small cell lung cancer (NSCLC). However, the quantity of available tissue often presents a key constraint for patients with ...advanced disease, where minimally invasive tissue biopsy typically returns small samples. In Part 1 of this two-part series, we summarise evidence-based recommendations relating to small sample processing for patients with NSCLC. Generally, tissue biopsy techniques that deliver the greatest quantity and quality of tissue with the least risk to the patient should be selected. Rapid on-site evaluation can help to ensure sufficient sample quality and quantity. Sample processing should be managed according to biomarker testing requirements, because tissue fixation methodology influences downstream nucleic acid, protein and morphological analyses. Accordingly, 10% neutral buffered formalin is recommended as an appropriate fixative, and the duration of fixation is recommended not to exceed 24–48 h. Tissue sparing techniques, including the ‘one biopsy per block’ approach and small sample cutting protocols, can help preserve tissue. Cytological material (formalin-fixed paraffin-embedded FFPE cytology blocks and non-FFPE samples such as smears and touch preparations) can be an excellent source of nucleic acid, providing either primary or supplementary patient material to complete morphological and molecular diagnoses. Considerations on biomarker testing, reporting and quality assessment are discussed in Part 2.
Modern diagnostic methods (next-generation sequencing) are one of the current hopes with regard to a personalised medicine. By applying detailed genetic analysis, it is possible to not only improve ...the prediction of potential risks (as, e.g., concerning hereditary breast cancer) but also the precision of therapy by targeting it to a specific genetic variant. However, there is no international standard for creating, structuring and/or transferring the results of a genetic test report. This type of test report often contains large amounts of complex information, and a standardised and consistent structure would offer potential benefits to all. These include reduced expenditure of time (due to the elimination of information-conversion steps), improved safety (due to a reduction in the occurrence of transmission errors, misunderstanding or misinterpretation of content) and improved clinical information gathering (by the respective linkage to scientific data and literature). Especially in regard to secondary use, a standardised (electronic) format would improve the suitability of these data in retrospective studies and basic research. In this study, we analysed the format and content of 96 genetic testing reports (germline and somatic) from Germany, Switzerland and Austria. Based on these results, we summarised and discussed potentially critical data that were demonstrated to be reported inconsistently, and propose a baseline structure for reporting that would also ease future electronic conversion.
Tumor mutational burden (TMB) has recently been approved as an agnostic biomarker for immune checkpoint inhibitors. However, methods for TMB testing have not yet been standardized. The International ...Quality Network for Pathology (IQNPath) organized a pilot external quality assessment (EQA) scheme for TMB testing. The aim of this program was the validation of the materials and the procedures for the EQA of this complex biomarker. Five formalin-fixed paraffin-embedded (FFPE) cell lines were selected to mimic the various TMB values observed in clinical practice. The FFPE samples were tested with the FoundationOne CDx (F1CDx) assay as the reference test and three commercially available targeted sequencing panels. Following this internal validation, the five cell lines were sent to 29 laboratories selected on the basis of a previous survey. Nineteen of the 23 laboratories that submitted results (82.6%) used targeted sequencing for TMB estimation. Only two laboratories performed whole exome sequencing (WES) and two assessed TMB by clinical exome. A high variability in the reported TMB values was observed. The variability was higher for samples with the highest TMB value according to the F1CDx test. However, good reproducibility of the TMB score was shown by laboratories using the same panel. The majority of laboratories did not indicate a TMB cut-off value for clinical interpretation. In conclusion, this pilot EQA scheme suggests that it is feasible to run such an EQA program for TMB assessment. However, the results of our pilot highlight the numerous challenges for the standardization of this test.
Molecular genetic testing for hereditary hemochromatosis (HH) is recognized as a reference test to confirm the diagnosis of suspected HH or to predict its risk. The vast majority (typically >90%) of ...patients with clinically characterized HH are homozygous for the p.C282Y variant in the HFE gene, referred to as HFE-related HH. Since 1996, HFE genotyping was implemented in diagnostic algorithms for suspected HH, allowing its early diagnosis and prevention. However, the penetrance of disease in p.C282Y homozygotes is incomplete. Hence, homozygosity for p.C282Y is not sufficient to diagnose HH. Neither is p.C282Y homozygosity required for diagnosis as other rare forms of HH exist, generally referred to as non-HFE-related HH. These pose significant challenges when defining criteria for referral, testing protocols, interpretation of test results and reporting practices. We present best practice guidelines for the molecular genetic diagnosis of HH where recommendations are classified, as far as possible, according to the level and strength of evidence. For clarification, the guidelines' recommendations are preceded by a detailed description of the methodology and results obtained with a series of actions taken in order to achieve a wide expert consensus, namely: (i) a survey on the current practices followed by laboratories offering molecular diagnosis of HH; (ii) a systematic literature search focused on some identified controversial topics; (iii) an expert Best Practice Workshop convened to achieve consensus on the practical recommendations included in the guidelines.
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