Zandra C. Deans

Guidance for laboratories performing molecular pathology for cancer patients

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.

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.

Simultaneous mutation scanning for gross deletions, duplications and point mutations in the DMD gene

We have developed a technique to screen for gross deletions/duplications and point mutations using one streamlined approach. Fluorescent multiplex quantitative PCR is used to determine the copy number of each exon, followed by conformation sensitive capillary electrophoresis (CSCE) of the same PCR products on a multi-capillary genetic analyser. We have developed this technique to screen all 79 exons of one of the largest human genes currently known (dystrophin) using 12 multiplex PCR assays. A blind trial of 50 male and 50 female samples, in which 84 mutations had previously been found and characterized by other techniques, showed 100% sensitivity and specificity. We then applied this method to screen over 100 patient samples previously screened for deletions and duplications of 28 exons from the two hotspot regions. Our data show that combining a full deletion/duplication screen with CSCE will detect a mutation in 98% of Duchenne muscular dystrophy patients and 93% of Becker muscular dystrophy patients where the clinical diagnosis is certain. This technique is applicable to any gene and is particularly suited to mutation screening of large genes, decreasing the time taken for a complete gene screen for nearly all mutation types.

Improvement in the quality of molecular analysis of EGFR in non-small-cell lung cancer detected by three rounds of external quality assessment

Background The clinical need to determine the presence of epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancers (NSCLC) in order to make informed decisions for patient treatment has seen the widespread introduction of EGFR molecular testing in many laboratories. To ensure high-quality molecular testing and allow laboratories to externally measure the standard of the service, an external quality assessment (EQA) scheme was provided to assess the whole testing process. Methods Formalin-fixed paraffin-embedded NSCLC tumour sections were distributed to laboratories for routine EGFR molecular testing, and the genotyping accuracy, interpretation of the result and clerical accuracy of the report were independently assessed. Results Three rounds of assessment have identified many genotyping errors and have highlighted the need for external assessment and education in many testing laboratories. The main issues raised were the importance of accurate genotyping, including the use of common mutation nomenclature, clear unambiguous interpretation of the result, the impact of tumour sample assessment regarding amount of tumour being analysed and the heterogeneity of the sample on the molecular test result. Conclusions Improvements in all these areas were observed during the progression of the three EQA rounds, however, continuous unacceptably high genotyping error rates demonstrate the clear need for continual external assessment and education in this field.

Next generation diagnostic molecular pathology: Critical appraisal of quality assurance in Europe

Tumor evaluation in pathology is more and more based on a combination of traditional histopathology and molecular analysis. Due to the rapid development of new cancer treatments that specifically target aberrant proteins present in tumor cells, treatment decisions are increasingly based on the molecular features of the tumor. Not only the number of patients eligible for targeted precision medicine, but also the number of molecular targets per patient and tumor type is rising. Diagnostic molecular pathology, the discipline that determines the molecular aberrations present in tumors for diagnostic, prognostic or predictive purposes, is faced with true challenges. The laboratories have to meet the need of comprehensive molecular testing using only limited amount of tumor tissue, mostly fixed in formalin and embedded in paraffin (FFPE), in short turnaround time. Choices must be made for analytical methods that provide accurate, reliable and cost‐effective results. Validation of the test procedures and results is essential. In addition, participation and good performance in internal (IQA) and external quality assurance (EQA) schemes is mandatory. In this review, we critically evaluate the validation procedure for comprehensive molecular tests as well as the organization of quality assurance and assessment of competence of diagnostic molecular pathology laboratories within Europe.

The UK NEQAS for Molecular Genetics scheme for gastrointestinal stromal tumour: findings and recommendations following four rounds of circulation

Aims To describe the UK NEQAS for Molecular Genetics scheme for gastrointestinal stromal tumour (GIST) and to report and interpret the findings of four rounds of circulation of this quality assurance programme for KIT/PDGFRA mutation analyses. Methods Samples of GISTs from formalin-fixed paraffin-embedded tissue blocks were circulated to registered participants of the UK NEQAS for Molecular Genetics scheme for GIST. Three samples were provided per annual circulation from 2008 to 2011 inclusive. The participants were required to analyse the samples for KIT and/or PDGFRA mutations using their routine protocols, and the anonymised participants reports were assessed and an annual scheme report issued. Results The genotyping error rates for the 2008, 2009, 2010 and 2011 circulations were 13%, 33%, 19% and 4%, respectively. These errors were either missed or incorrectly described mutations. There was an overall false negative rate of 2% and false positive rate of 0%. The main recommendations that arose from these circulations were: (1) inclusion of reference accession numbers in reports; (2) avoidance of the term ‘heterozygous’ when analysing DNA from tumour tissue unless there was certainty that only neoplastic DNA was studied; and (3) the need to screen KIT exons 9, 11, 13 and 17 and PDGFRA exons 12, 14 and 18 before classifying a GIST as ‘wild type’. Conclusions The UK NEQAS for Molecular Genetics scheme emphasises the potential complexities of KIT/PDGFRA mutation analyses for GISTs and provides recommendations to help optimise such genotyping and reporting. The scheme has also demonstrated its educational value among participating laboratories.

Molecular Genetics External Quality Assessment Pilot Scheme for KRAS Analysis in Metastatic Colorectal Cancer

Laboratories are increasingly required to perform molecular tests for the detection of mutations in the KRAS gene in metastatic colorectal cancers to allow better clinical management and more effective treatment for these patients. KRAS mutation status predicts a patients likely response to the monoclonal antibody cetuximab. To provide a high standard of service, these laboratories require external quality assessment (EQA) to monitor the level of laboratory output and measure the performance of the laboratory against other service providers. National External Quality Assurance Services for Molecular Genetics provided a pilot EQA scheme for KRAS molecular analysis in metastatic colorectal cancers during 2009. Very few genotyping errors were reported by participating laboratories; however, the reporting nomenclature of the genotyping results varied considerably between laboratories. The pilot EQA scheme highlighted the need for continuing EQA in this field which will assess the laboratories ability not only to obtain accurate, reliable results but also to interpret them safely and correctly ensuring that the referring clinician has the correct information to make the best clinical therapeutic decision for their patient.

Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.

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.

External quality assessment of BRAF molecular analysis in melanoma

The availability of BRAF inhibitors has given metastatic melanoma patients an effective new treatment choice and molecular testing to determine the presence or absence of a BRAF codon 600 mutation is pivotal in the clinical management of these patients. This molecular test must be performed accurately and appropriately to ensure that the patient receives the most suitable treatment in a timely manner. Laboratories have introduced such testing; however, some experience low sample throughput making it critical that an external quality assurance programme is available to help promote a high standard of testing, reporting and provide an educational aspect for BRAF molecular testing. Laboratories took part in three rounds of external quality assessment (EQA) during a 12-month period giving participants a measure of the accuracy of genotyping, clinical interpretation of the result and experience in testing a range of different samples. Formalin fixed paraffin embedded tissue sections from malignant melanoma patients were distributed to participants for BRAF molecular testing. The standard of testing was generally high but distribution of a mutation other than the most common, p.(Val600Glu), highlighted concerns with detection or reporting of the presence of rarer mutations. The main issues raised in the interpretation of the results were the importance of clear unambiguous interpretation of the result tailored to the patient and the understanding that the treatment is different from that given to other stratified medicine programmes. The variability in reporting and wide range of methodologies used indicate a continuing need for EQA in this field.