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.

A risk-management approach for effective integration of biomarkers in clinical trials: perspectives of an NCI, NCRI, and EORTC working group.

Clinical cancer research today often includes testing the value of biomarkers to direct treatment and for drug development. However, the practical challenges of integration of molecular information into clinical trial protocols are increasingly appreciated. Inherent difficulties include evidence gaps in available biomarker data, a paucity of robust assay methods, and the design of appropriate studies within the constraints of feasible trial operations, and finite resources. Scalable and proportionate approaches are needed to systematically cope with these challenges. Therefore, we assembled international experts from three clinical trials organisations to identify the common challenges and common solutions. We present a practical risk-assessment framework allowing targeting of scarce resources to crucial issues coupled with a library of useful resources and a simple actionable checklist of recommendations. We hope that these practical methods will be useful for running biomarker-driven trials and ultimately help to develop biomarkers that are ready for integration in routine practice.

Developing translational research infrastructure and capabilities associated with cancer clinical trials.

The integration of molecular information in clinical decision making is becoming a reality. These changes are shaping the way clinical research is conducted, and as reality sets in, the challenges in conducting, managing and organising multi-disciplinary research become apparent. Clinical trials provide a platform to conduct translational research (TR) within the context of high quality clinical data accrual. Integrating TR objectives in trials allows the execution of pivotal studies that provide clinical evidence for biomarker-driven treatment strategies, targeting early drug development trials to a homogeneous and well defined patient population, supports the development of companion diagnostics and provides an opportunity for deepening our understanding of cancer biology and mechanisms of drug action. To achieve these goals within a clinical trial, developing translational research infrastructure and capabilities (TRIC) plays a critical catalytic role for translating preclinical data into successful clinical research and development. TRIC represents a technical platform, dedicated resources and access to expertise promoting high quality standards, logistical and operational support and unified streamlined procedures under an appropriate governance framework. TRIC promotes integration of multiple disciplines including biobanking, laboratory analysis, molecular data, informatics, statistical analysis and dissemination of results which are all required for successful TR projects and scientific progress. Such a supporting infrastructure is absolutely essential in order to promote high quality robust research, avoid duplication and coordinate resources. Lack of such infrastructure, we would argue, is one reason for the limited effect of TR in clinical practice beyond clinical trials.

Using lessons learnt from key stakeholders to increase support for scaling the Reach Up Early Childhood Parenting program.

Introduction: Sustainable implementation of early childhood programs requires resources, materials and methods that are adaptable, scalable and feasible for delivery through multiple sectors. Additional or modified program resources may be required to meet emerging needs, as programs go to scale. An active and effective monitoring, evaluation and learning (MEL) process may enable programs to be responsive to demands. The Reach Up: Early Childhood Parenting program, is designed primarily for disadvantaged children under 4 years of age in low- and middle-income countries (LMICs) to promote their development through playful caregiver interactions. The curriculum, training manuals and other materials and resources support implementers in the adaptation of the intervention, implementation, workforce training, monitoring and evaluation. This paper reports on how data collected from key informants drove modifications to program processes, materials and resources. Methods: We conducted in-depth interviews with 14 key informants (including program managers, lead trainers, academics, consultants and workforce personnel) on their experiences with Reach Up across 15 LMICs where the program has been implemented. We also reviewed written records generated from (i) structured small group discussions at a Knowledge Exchange meeting of 31 Reach Up partners and (ii) notes from working groups formed at the meeting and tasked to continue working post-meeting to find solutions to support ongoing implementation. The transcripts from the in-depth interviews and the meeting records were analysed using thematic analysis with a mixture of pre-defined categories and data-driven sub-themes. Results: The main findings indicated that there was a need to: (i) develop advocacy and communication resources and materials to aid prospective implementers and other stakeholders, to make decisions for implementation, (ii) revise and/or add to the content and format of the curriculum and add content in the training and other supporting manuals and (iii) enhance the training process. Conclusion: The feedback from the key global partners informed the development of new knowledge materials, resources and processes and modifications to existing program materials and resources. These will help to support advocacy, ongoing implementations, and the process of transitioning the Reach Up early childhood intervention to scale.

The Reach Up Parenting Program, Child Development, and Maternal Depression: A Meta-analysis.

BACKGROUND AND OBJECTIVES: Evidence is needed on effective approaches to build parents' ability to promote child development feasible in low- and middle-income countries. Our objective was to synthesize impact of the Reach Up early childhood parenting program in several low- and middle-income countries and examine moderation by family and implementation characteristics. METHODS: Systematic search using PubMed and Academic Search Elite/EBSCO Host. Randomized controlled trials of the Reach Up program from 1985 to February 2022 were selected. Data were extracted by 2 independent researchers. Primary outcomes were child cognitive, language, and motor development. Secondary outcomes were home stimulation and maternal depressive symptoms. We synthesized pooled effect sizes using random effect inverse-variance weighting and effect modification by testing pooled subgroup effect estimates using the χ2 test for heterogeneity. RESULTS: Average effect size across 18 studies ranged from 0.49 (95% confidence interval [CI] 0.32 to 0.66) for cognition, 0.38 (CI 0.24 to 0.51) for language, 0.27 (CI 0.13 to 0.40) for motor development, 0.37 (CI 0.21 to 0.54) for home stimulation, and -0.09 (CI -0.19 to 0.01) for maternal depressive symptoms. Impacts were larger in studies targeted to undernourished children, with mean enrollment older than age 12 months and intervention duration 6 to 12 months. Quality of evidence assessed with the Cochrane Assessment of Risk of Bias and GRADE system was moderate. Instruments used to assess child development varied. In moderator analyses, some subgroups included few studies. CONCLUSIONS: Reach Up benefits child development and home stimulation and is adaptable across cultures and delivery methods. Child and implementation characteristics modified the effects, with implications for scaling.

Paradigm changes in health lead to paradigm changes in pathology.

For the sake of safety and quality of care as well as efficiency of care processes, health systems undergo a paradigm change towards personalized, ubiquitous, health services. This change includes preventive and predictive medicine based on advanced translational medicine. Here we introduce domain-specific, organizational, and technical paradigms, requirements and solutions for personalized, ubiquitous, care. Emphasizing the formal aspects of modeling and implementing Telehealth and personal health (pHealth) interoperability and the entailed multidisciplinary integration, and illustrate the drivers behind and benefits of personalized medicine with a specific focus on the changing trends and impact on pathology, especially emphasizing Telepathology.

Prognostic significance of the neutrophil/lymphocyte ratio in patients undergoing treatment with nivolumab for recurrent non-small-cell lung cancer.

AIM: We investigated the prognostic potential of pretherapy measurement of the neutrophil/lymphocyte ratio (NLR) in patients (n = 56) with non-small-cell lung cancer deemed suitable for treatment with nivolumab. MATERIALS & METHODS: This was a multicenter, noninterventional, retrospective data analysis, involving five oncology centers. RESULTS: Patients with prenivolumab NLR values of <5 and ≥5 had respective median overall survival (OS) values of 14.5 and 7.02 months (p = 0.0026). Patients with ≤2 and >2 metastatic sites had median OS values of 11.4 and 6.1 months, respectively (p = 0.0174). A Cox multiple regression model revealed baseline NLR ≥5 as the only variable significantly associated with decreased OS (p < 0.0447). CONCLUSION: Pretreatment elevated NLR values are associated with poor outcomes in patients with recurrent metastatic non-small-cell lung cancer treated with nivolumab.

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials.

Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting.

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

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.

A framework for the development of effective anti-metastatic agents.

Most cancer-related deaths are a result of metastasis, and thus the importance of this process as a target of therapy cannot be understated. By asking 'how can we effectively treat cancer?', we do not capture the complexity of a disease encompassing >200 different cancer types - many consisting of multiple subtypes - with considerable intratumoural heterogeneity, which can result in variable responses to a specific therapy. Moreover, we have much less information on the pathophysiological characteristics of metastases than is available for the primary tumour. Most disseminated tumour cells that arrive in distant tissues, surrounded by unfamiliar cells and a foreign microenvironment, are likely to die; however, those that survive can generate metastatic tumours with a markedly different biology from that of the primary tumour. To treat metastasis effectively, we must inhibit fundamental metastatic processes and develop specific preclinical and clinical strategies that do not rely on primary tumour responses. To address this crucial issue, Cancer Research UK and Cancer Therapeutics CRC Australia formed a Metastasis Working Group with representatives from not-for-profit, academic, government, industry and regulatory bodies in order to develop recommendations on how to tackle the challenges associated with treating (micro)metastatic disease. Herein, we describe the challenges identified as well as the proposed approaches for discovering and developing anticancer agents designed specifically to prevent or delay the metastatic outgrowth of cancer.

Roles enacted by Clinical Nurse Leaders across the healthcare spectrum: A systematic literature review.

The Clinical Nurse Leader (CNL) is a master's prepared nurse envisioned to provide clinical leadership at the microsystem level to ensure safe, high quality patient-centered care. The American Association of Colleges of Nurses defined ten 'fundamental aspects' of CNL practice, but as the certified CNL population grows, data suggest they are filling a variety of positions besides formally designated CNL roles. This article reports the results of a systematic review of CNL literature to better understand what roles and activities certified CNLs are enacting when not hired into formally designated CNL roles. Sixty-nine articles met inclusion criteria. Roles identified include: faculty, 62%; clinical management/executive, 12%; specialty clinician, 11%; and staff nurse, 9%. In these roles, certified CNLs are reviewing literature, conducting research, and/or writing commentaries on CNL education and practice and other health foci such as alarm fatigue, insulin practices, and physical exercise for cancer-related fatigue. Results indicate that despite a lack of formal tracking of certified CNLs over time, the available information identifies a variety of roles and job titles used by this group of professional nurses. The study findings add to the body of knowledge informing overall understanding of the CNL initiative.

Results of the UK NEQAS for Molecular Genetics reference sample analysis.

AIMS: In addition to providing external quality assessment (EQA) schemes, United Kingdom National External Quality Assessment service (UK NEQAS) for Molecular Genetics also supports the education of laboratories. As an enhancement to the Molecular Pathology EQA scheme, a human cell-line reference sample, manufactured by Thermo Fisher Scientific (AcroMetrix), was provided for analysis. This contained many variants, present at frequencies between 1% and 17.9%. METHODS: One hundred and one laboratories submitted results, with a total of 2889 test results on 53 genes being reported. Known polymorphisms, 46/2889 (1.59%) results, were excluded. Variants detected in the seven most commonly reported (and clinically relevant) genes, KRAS, NRAS, BRAF, EGFR, PIK3CA, KIT and PDGFRA, are reported here, as these genes fall within the scope of UK NEQAS EQA schemes. RESULTS: Next generation sequencing (NGS) was the most commonly performed testing platform. There were between 5 and 27 validated variants in the seven genes reported here. Eight laboratories correctly reported all five NRAS variants, and two correctly reported all eight BRAF variants. The validated mean variant frequency was lower than that determined by participating laboratories, with single-gene testing methodologies showing less variation in estimated frequencies than NGS platforms. Laboratories were more likely to correctly identify clinically relevant variants. CONCLUSIONS: Over 100 laboratories took the opportunity to test the 'educational reference sample', showing a willingness to further validate their testing platforms. While it was encouraging to see that the most widely reported variants were those which should be included in routine testing panels, reporting of variants was potentially open to interpretation, thus clarity is still required on whether laboratories selectively reported variants, by either clinical relevance or variant frequency.

An exploration into study design for biomarker identification: issues and recommendations.

Genomic profiling produces large amounts of data and a challenge remains in identifying relevant biological processes associated with clinical outcome. Many candidate biomarkers have been identified but few have been successfully validated and make an impact clinically. This review focuses on some of the study design issues encountered in data mining for biomarker identification with illustrations of how study design may influence the final results. This includes issues of clinical endpoint use and selection, power, statistical, biological and clinical significance. We give particular attention to study design for the application of supervised clustering methods for identification of gene networks associated with clinical outcome and provide recommendations for future work to increase the success of identification of clinically relevant biomarkers.

CpG island methylation of DNA damage response genes in advanced ovarian cancer.

We have determined the methylation frequencies of 24 CpG islands of genes associated with DNA damage responses or with ovarian cancer in 106 stage III/IV epithelial ovarian tumors. We have analyzed this data for whether there is evidence of a CpG island methylator phenotype or associations of CpG island methylation with response to chemotherapy in advanced ovarian cancer. Frequent methylation was observed for OPCML, DCR1, RASSF1A, HIC1, BRCA1, and MINT25 (33.3%, 30.7%, 26.4%, 17.3%, 12.3%, and 12.0%, respectively), whereas no methylation was observed for APAF-1, DAPK, FANCF, FAS, P14, P21, P73, SOCS-3, and SURVIVIN. The remaining genes showed only a low frequency of methylation, <10%. Unsupervised gene shaving identified a nonrandom pattern of methylation for OPCML, DCR1, RASSF1A, MINT25, HIC1, and SFRP1, supporting the concept of concordant methylation of these genes in ovarian cancer. Methylation of at least one of the group of genes involved in DNA repair/drug detoxification (BRCA1, GSTP1, and MGMT) was associated with improved response to chemotherapy (P = 0.013). We have examined the frequency of a polymorphism in the DNA methyltransferase gene DNMT3b6, which has been previously reported to affect gene transcription and cancer risk. The genetic polymorphism in the DNMT3b6 gene promoter (at position -149) is not significantly associated with the concordant methylation observed, but is weakly associated with the overall frequency of methylation at the genes examined (P = 0.04, n = 56). This supports the hypothesis that genetic factors affecting function of DNMT genes may underlie the propensity of tumors to acquire aberrant CpG island methylation.

Transparency of genetic testing services for 'health, wellness and lifestyle': analysis of online prepurchase information for UK consumers.

The declining cost of DNA sequencing has been accompanied by a proliferation of companies selling 'direct-to-consumer genetic testing' (DTC-GT) services. Many of these are marketed online as tools for enabling citizens to make more informed decisions about their health, wellness and lifestyle. We assessed the 'information for consumers' provided by these companies at the prepurchase stage, which could influence initial decisions to part with money, data or tissue samples. A scoping exercise revealed 65 DTC-GT companies advertising their services online to consumers in the United Kingdom, of which 15 met our inclusion criteria. We benchmarked their consumer information against the good practice principles developed by the UK Human Genetics Commission (HGC). No provider complied with all the HGC principles and overall levels of compliance varied considerably. Although consent for testing was discussed by all but one company, information about data reuse for research or other purposes was often sparse and consent options limited or unclear. Most did not provide supplementary support services to help users better understand or cope with the implications of test results. We provide recommendations for updating the preconsumer transparency aspects of the HGC guidelines to ensure their fitness-for-purpose in this rapidly changing market. We also recommend improving coordination between relevant governance bodies to ensure minimum standards of transparency, quality and accountability. Although DTC-GT has many potential benefits, close partnership between consumers, industry and government, along with interdisciplinary science input, are essential to ensure that these innovations are used ethically and responsibly.

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine - Part 2: Immunohistochemistry Test Performance Characteristics.

All laboratory tests have test performance characteristics (TPCs), whether or not they are explicitly known to the laboratorian or the pathologist. TPCs are thus also an integral characteristic of immunohistochemistry (IHC) tests and other in situ, cell-based molecular assays such as DNA or RNA in situ hybridization or aptamer-based testing. Because of their descriptive, in situ, cell-based nature, IHC tests have a limited repertoire of appropriate TPCs. Although only a few TPCs are relevant to IHC, proper selection of informative TPCs is nonetheless essential for the development of and adherence to appropriate quality assurance measures in the IHC laboratory. This paper describes the TPCs that are relevant to IHC testing and emphasizes the role of TPCs in the validation of IHC tests. This is part 2 of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 4: Tissue Tools for Quality Assurance in Immunohistochemistry.

The numbers of diagnostic, prognostic, and predictive immunohistochemistry (IHC) tests are increasing; the implementation and validation of new IHC tests, revalidation of existing tests, as well as the on-going need for daily quality assurance monitoring present significant challenges to clinical laboratories. There is a need for proper quality tools, specifically tissue tools that will enable laboratories to successfully carry out these processes. This paper clarifies, through the lens of laboratory tissue tools, how validation, verification, and revalidation of IHC tests can be performed in order to develop and maintain high quality "fit-for-purpose" IHC testing in the era of precision medicine. This is the final part of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 1: Fit-for-Purpose Approach to Classification of Clinical Immunohistochemistry Biomarkers.

Technical progress in immunohistochemistry (IHC) as well as the increased utility of IHC for biomarker testing in precision medicine avails us of the opportunity to reassess clinical IHC as a laboratory test and its proper characterization as a special type of immunoassay. IHC, as used in current clinical applications, is a descriptive, qualitative, cell-based, usually nonlinear, in situ protein immunoassay, for which the readout of the results is principally performed by pathologists rather than by the instruments on which the immunoassay is performed. This modus operandi is in contrast to other assays where the instrument also performs the readout of the test result (eg, nephelometry readers, mass spectrometry readers, etc.). The readouts (results) of IHC tests are used either by pathologists for diagnostic purposes or by treating physicians (eg, oncologists) for patient management decisions, the need for further testing, or follow-up. This paper highlights the distinction between the original purpose for which an IHC test is developed and its subsequent clinical uses, as well as the role of pathologists in the analytical and postanalytical phases of IHC testing. This paper is the first of a 4-part series, under the general title of "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

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.