Simon Patton

Guideline on the requirements of external quality assessment programs in molecular pathology

Molecular pathology is an integral part of daily diagnostic pathology and used for classification of tumors, for prediction of prognosis and response to therapy, and to support treatment decisions. For these reasons, analyses in molecular pathology must be highly reliable and hence external quality assessment (EQA) programs are called for. Several EQA programs exist to which laboratories can subscribe, but they vary in scope, number of subscribers, and execution. The guideline presented in this paper has been developed with the purpose to harmonize EQA in molecular pathology. It presents recommendations on how an EQA program should be organized, provides criteria for a reference laboratory, proposes requirements for EQA test samples, and defines the number of samples needed for an EQA program. Furthermore, a system for scoring of the results is proposed as well as measures to be taken for poorly performing laboratories. Proposals are made regarding the content requirements of an EQA report and how its results should be communicated. Finally, the need for an EQA database and a participant manual are elaborated. It is the intention of this guideline to improve EQA for molecular pathology in order to provide more reliable molecular analyses as well as optimal information regarding patient selection for treatment.

EMQN best practice guidelines for the molecular genetic diagnosis of hereditary hemochromatosis (HH)

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.

Experience and outcome of 3 years of a European EQA scheme for genetic testing of the spinocerebellar ataxias

The European Molecular Genetics Quality Network (EMQN) has been organizing an external quality assessment (EQA) scheme for molecular genetic testing of trinucleotide repeat mutations in the spinocerebellar ataxias (SCAs) since 2004. DNA samples were validated by at least two independent labs and two different methods. Together with mock clinical case descriptions and requests for specific SCA gene analyses, these were sent to registered participants each year. Laboratories were asked to use their routine procedures and protocols. A panel of assessors reviewed the final returns, including genotype results and reports, to assess the quality of (1) genotyping and (2) interpretation and reporting. A description of methods and raw data were also requested and were very useful for the final analysis. Altogether, during 3 years, 239 reports were received from the laboratories. Overall genotype error rate ranged 1.1–5.2%, a significant cause of concern. Scores for interpretation and reporting also showed that there is still much room for progress, although performance has improved over this period of assessment. The consequences of suboptimal laboratory practices, genotyping errors and misdiagnosis and of incorrect or incomplete interpretation and reporting have wide implications for patient lives, as well as for health management and counselling of relatives. EQA schemes are an important part of quality assurance in molecular genetic laboratories, and their use should become a routine part of laboratory diagnostic practice. Current evidence shows also that it is important that laboratories participate on a yearly basis and that this becomes mandatory for reference laboratories.

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.

Retaining the Confidence of the Public in Molecular Genetic Testing – Quality Assurance

The introduction of molecular genetic techniques to clinical laboratories opens the possibility of public access to a wide range of tests for many different genetic disorders. This new diagnostic area is still in a transition phase from research to service. Mechanisms for ensuring the quality of genetic testing are not yet in place but need to be developed with some urgency to retain public confidence. External quality assessment (EQA), or proficiency testing, is an independent check on a laboratory’s performance. This paper describes EQA pilot schemes and guidelines for ‘best practice’ developed in Europe and some of the problems involved in introducing quality assurance measures to service providers so that these measures are sustainable. The introduction of accreditation may provide a means of sustaining these systems. Reasons to encourage an international approach to EQA and harmonisation and mutual recognition of national and regional EQA schemes are also discussed.

Review of the implementation of plasma ctDNA testing on behalf of IQN Path ASBL: a perspective from an EQA providers’ survey

Molecular biomarker analysis for the personalised treatment of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) is becoming more common, due to the number and availability of molecular targets for predictive biomarker testing increasing [1]. Clinical laboratories must implement accurate test procedures and provide timely and reliable test results, to ensure that appropriate therapies are administered to patients [2]. The challenge for laboratories is to keep pace with molecular biomarker developments while maintaining excellence in service standards. Plasma circulating tumour DNA (ctDNA) may be found in the blood of cancer patients, alongside a larger fraction of circulating free DNA (cfDNA). Plasma ctDNA testing is becoming more common in the management of cancer patients [3]. It has several advantages: in the absence of suitable or sufficient tissue biopsy, it yields material for molecular analysis, can demonstrate molecular resistance to targeted treatment and is an alternative to invasive tissue sampling [4]. Plasma ctDNA analysis may also prove useful in cases of intra- and inter-tumour heterogeneity [5]. With formal approval from the European Medicine Agency (EMA), several clinical applications for plasma ctDNA testing are now being considered, including the detection of Epidermal Growth Factor Receptor (EGFR) mutations in the plasma of patients with advanced NSCLC [6]. The implementation of new methods such as plasma ctDNA testing can be challenging for diagnostic laboratories. Indeed, it has been shown that inexperience in specialised and complex techniques can compromise the result quality [2, 7]. To address these issues, four EQA providers came together under the umbrella organisation the International Quality Network for Pathology IQN Path (IQN Path): Association Italiana di Oncologia Medica (AIOM), European Molecular Genetics Quality Network (EMQN), European Society of Pathology (ESP) EQA and the United Kingdom National External Quality Assessment Service (UK NEQAS) for Molecular Genetics. Their aim was to survey testing methods currently in use and to pilot an EQA which assessed the standards of plasma ctDNA testing. This article summarises the results of the survey, which evaluated current laboratory practices in this field and which will subsequently inform the design of a pilot EQA scheme for plasma ctDNA testing.

Recommended practice for laboratory reporting of non‐invasive prenatal testing of trisomies 13, 18 and 21: a consensus opinion

Non‐invasive prenatal testing (NIPT) for trisomies 13, 18 and 21 is used worldwide. Laboratory reports should provide clear, concise results with test limitations indicated, yet no national or local guidelines are currently available. Here, we aim to present minimum best practice guidelines.

A comparative study of germline BRCA1 and BRCA2 mutation screening methods in use in 20 European clinical diagnostic laboratories

Background:Thousands of clinically relevant variations in BRCA1 and BRCA2 have been discovered and this poses a significant challenge with respect to the accurate detection, analysis turn-around time, characterisation and interpretation of these sequence variants.Methods:We evaluated the performance of different BRCA1/2 gene testing practices in routine diagnostic use in 20 European laboratories, with a focus on next-generation sequencing-based strategies as this is the technical approach implemented by or under adoption by most European clinical laboratories. Participant laboratories, selected on expertise and diagnostic service quality, tested 10 identical DNA samples containing a range of challenging pathogenic variants.Results:A small number of errors in the detection of pathogenic and significant variants were identified (2.6% diagnostic error rate). There was a high degree of concordance (>97%) across all laboratories for all variants detected. No systematic technical flaw was identified in the strategies employed across the participating laboratories.Conclusions:The discrepancies identified are most likely due to human error or the way the methodology has been implemented locally, for example, next-generation sequencing bioinformatics pipelines, rather than technical limitations of the methods. The choice of BRCA1/2 testing method will therefore depend on multiple factors including required throughput and turn-around times, access to equipment, expertise and budget.

Quality Assurance in Molecular Testing of Cancer

In the past 20 years, clinical laboratory science has been transformed by the use of technologies that cross the traditional boundaries between laboratory disciplines. However, during this period, issues of quality have not always been given adequate attention.