Results of a worldwide external quality assessment of cfDNA testing in lung Cancer.

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.

Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

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.

HGVS Nomenclature in Practice: An Example from the United Kingdom National External Quality Assessment Scheme.

The recommendations for the description of sequence variants from the Human Genome Variation Society (HGVS) were published in 2000. Over the years, the recommendations became widely adopted, especially in human clinical genetics and DNA laboratory reporting. As part of a testing scheme performed by the United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Molecular Genetics, we assessed the current variability in the use and interpretation of the guidelines by diagnostic laboratories based across the globe. Twenty-six participating laboratories gave 21 different descriptions. Six laboratories gave fully compliant HGVS descriptions, 12 laboratories reported the correct variant, although not using the recommended format, whilst eight laboratory reports (31%) were not correct. The results indicate that available tools to check variant descriptions were not used. We conclude that education appears to be the way forward to eliminate the observed variability in data reporting.

External quality assessment (EQA) for tumor mutational burden: results of an international IQN path feasibility pilot scheme.

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.

Implementation of circulating tumour DNA multi-target mutation testing in plasma: a perspective from an external quality assessment providers' survey.

Demand for large-scale tumour profiling across cancer types has increased in recent years, driven by the emergence of targeted drug therapies. Analysing alternations in plasma circulating tumour DNA (ctDNA) for cancer detection can improve survival; ctDNA testing is recommended when tumour tissue is unavailable. An online survey of molecular pathology testing was circulated by six external quality assessment members of IQN Path to registered laboratories and all IQN Path collaborative corporate members. Data from 275 laboratories across 45 countries were collected; 245 (89%) perform molecular pathology testing, including 177 (64%) which perform plasma ctDNA diagnostic service testing. The most common tests were next-generation sequencing-based (n = 113). Genes with known stratified treatment options, including KRAS (n = 97), NRAS (n = 84), and EGFR (n = 130), were common targets. The uptake of ctDNA plasma testing and plans to implement further testing demonstrates the importance of support from a well-designed EQA scheme.

Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path).

While tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

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.

Core promoter-dependent TFIIB conformation and a role for TFIIB conformation in transcription start site selection.

The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5' and 3' parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus.

RNA polymerase (pol) III transcription is a major determinant of biosynthetic capacity, providing essential products such as tRNA and 5S rRNA. It is controlled directly by the tumour suppressors RB and p53. High-risk types of human papillomavirus (HPV), such as HPV16, express the oncoproteins E6 and E7 that can inactivate p53 and RB, respectively. Accordingly, both E6 and E7 stimulate pol III transcription in cultured cells. HPV16-positive cervical biopsies express elevated levels of tRNA and 5S rRNA when compared to biopsies that test negative for HPV or are infected with the lower risk HPV11. Integration of viral DNA into the host cell genome stimulates expression of E6 and E7 and correlates with induction of tRNA and 5S rRNA. Expression of mRNA encoding the pol III-specific transcription factor Brf1 also correlates with the presence of integrated HPV16. Brf1 levels are limiting for tRNA and 5S rRNA synthesis in cervical cells. Furthermore, pol III-transcribed genes that do not use Brf1 are not induced in HPV16-positive biopsies. Three complementary mechanisms may therefore allow high-risk HPV to stimulate production of tRNA and 5S rRNA: E6-mediated removal of p53; E7-mediated neutralization of RB; and induction of Brf1. The resultant increase in biosynthetic capacity may contribute to deregulated cell growth.

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.

Making the most of pathological specimens: molecular diagnosis in formalin-fixed, paraffin embedded tissue.

The development of commercial reagents designed specifically for use with formalin-fixed paraffin-embedded (FFPE) tissue has unlocked the diagnostic potential of this prolific resource. The availability of archival FFPE tissue and tissue from current patients make it an ideal resource for molecular testing. Despite its stability and ability to preserve morphological information, FFPE provides a number of technical challenges to the study of biomolecules. In particular, the cross-linking and processing present problems in the extraction and isolation of DNA, RNA and protein and affect their use in downstream analysis. Here we will discuss some of the problems of FFPE tissue, how they can be overcome and how FFPE material can be used within clinical molecular diagnostics.

Phosphorylation of TFIIB links transcription initiation and termination.

The general transcription factor TFIIB plays a central role in preinitiation complex (PIC) assembly and the recruitment of RNA polymerase II (RNA pol II) to the promoter. Recent studies have revealed that TFIIB engages in contact with the transcription termination region and also with complexes that are involved in 3' end processing and/or termination. Here we report that TFIIB can be phosphorylated within the N terminus at serine 65 in vivo and that the phosphorylated form of TFIIB is present within (PICs). Surprisingly, TFIIB serine 65 phosphorylation is required after the phosphorylation of serine 5 of RNA pol II C-terminal domain (CTD) has occurred, but before productive transcription initiation begins. We show that phosphorylation of TFIIB at serine 65 regulates the interaction between TFIIB and the CstF-64 component of the CstF 3' cleavage and polyadenylation complex. This directs the recruitment of CstF (cleavage stimulatory factor) to the terminator and also the recruitment of the CstF and CPSF (cleavage and polyadenylation specific factor) complexes to the promoter. Our results reveal that phosphorylation of TFIIB is a critical event in transcription that links the gene promoter and terminator and triggers initiation by RNA pol II.

Epstein-Barr virus induces cellular transcription factors to allow active expression of EBER genes by RNA polymerase III.

The EBER genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These EBER transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of EBER RNA. These include phosphorylation of ATF2, which interacts with EBER promoters. A second is induction of TFIIIC, a pol III-specific factor that activates EBER genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate EBER expression in vivo, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential.

Human La is found at RNA polymerase III-transcribed genes in vivo.

The human La autoantigen can bind to nascent RNA transcripts and has also been postulated to act as an RNA polymerase III (pol III) transcription initiation and termination factor. Here, we show by chromatin immunoprecipitation (ChIP) that La is associated with pol III-transcribed genes in vivo. In contrast, the Ro autoantigen, which can also bind pol III transcripts, is not found at these genes. The putative pol III transcription factors NF1 and TFIIA are also not detected at class III genes. Binding of La remains when transcription is repressed at mitosis and does not correlate with the presence of polymerase at the gene. However, gene occupancy depends on the phosphorylation status of La, with the less prevalent, unphosphorylated form being found selectively on pol III templates.

TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo.

Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation-mediated inactivation of TFIIIB, an essential complex comprising the TATA-binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2-cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase-arrested cells, but remains associated with promoters in condensed chromosomes, along with TBP. In contrast, Bdp1 is selectively released. Repression can be reversed by raising the concentration of Brf1 or Bdp1. The data support a model in which hyperphosphorylation disrupts TFIIIB during mitosis, compromising its ability to support transcription.

The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB.

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNA(Leu) genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.