Recommendations for Tumor Mutational Burden Assay Validation and Reporting: A Joint Consensus Recommendation of the Association for Molecular Pathology, College of American Pathologists, and Society for Immunotherapy of Cancer.

Tumor mutational burden (TMB) has been recognized as a predictive biomarker for immunotherapy response in several tumor types. Several laboratories offer TMB testing, but there is significant variation in how TMB is calculated, reported, and interpreted among laboratories. TMB standardization efforts are underway, but no published guidance for TMB validation and reporting is currently available. Recognizing the current challenges of clinical TMB testing, the Association for Molecular Pathology convened a multidisciplinary collaborative working group with representation from the American Society of Clinical Oncology, the College of American Pathologists, and the Society for the Immunotherapy of Cancer to review the laboratory practices surrounding TMB and develop recommendations for the analytical validation and reporting of TMB testing based on survey data, literature review, and expert consensus. These recommendations encompass pre-analytical, analytical, and postanalytical factors of TMB analysis, and they emphasize the relevance of comprehensive methodological descriptions to allow comparability between assays.

Recommendations for Cell-Free DNA Assay Validations: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists.

Diagnosing, selecting therapy for, and monitoring cancer in patients using a minimally invasive blood test represents a significant advance in precision medicine. Wide variability exists in how circulating tumor DNA (ctDNA) assays are developed, validated, and reported in the literature, which hinders clinical adoption and may negatively impact patient care. Standardization is needed for factors affecting ctDNA assay performance and reporting, including pre-analytical variables, analytical considerations, and elements of laboratory assay reporting. The Association for Molecular Pathology Clinical Practice Committee's Liquid Biopsy Working Group (LBxWG), including organizational representation from the American Society of Clinical Oncology and the College of American Pathologists, has undertaken a full-text data extraction of 1228 ctDNA publications that describe assays performed in patients with lymphoma and solid tumor malignancies. With an emphasis on clinical assay validation, the LBxWG has developed a set of 13 best practice consensus recommendations for validating, reporting, and publishing clinical ctDNA assays. Recommendations include reporting key pre-analytical considerations and assay performance metrics; this analysis demonstrates these elements are inconsistently included in publications. The LBxWG recommendations are intended to assist clinical laboratories with validating and reporting ctDNA assays and to ensure high-quality data are included in publications. It is expected that these recommendations will need to be updated as the body of literature continues to mature.

Exploring the Utility of Multiplex Infectious Disease Panel Testing for Diagnosis of Infection in Different Body Sites: A Joint Report of the Association for Molecular Pathology, American Society for Microbiology, Infectious Diseases Society of America, and Pan American Society for Clinical Virology.

The use of clinical molecular diagnostic methods for detecting microbial pathogens continues to expand and, in some cases, supplant conventional identification methods in various scenarios. Analytical and clinical benefits of multiplex molecular panels for the detection of respiratory pathogens have been demonstrated in various studies. The use of these panels in managing different patient populations has been incorporated into clinical guidance documents. The Association for Molecular Pathology's Infectious Diseases Multiplex Working Group conducted a review of the current benefits and challenges to using multiplex PCR for the detection of pathogens from gastrointestinal tract, central nervous system, lower respiratory tract, and joint specimens. The Working Group also discusses future directions and novel approaches to detection of pathogens in alternate specimen types, and outlines challenges associated with implementation of these multiplex PCR panels.

Recommendations for Next-Generation Sequencing Germline Variant Confirmation: A Joint Report of the Association for Molecular Pathology and National Society of Genetic Counselors.

Clinical laboratory implementation of next-generation sequencing (NGS)-based constitutional genetic testing has been rapid and widespread. In the absence of widely adopted comprehensive guidance, there remains substantial variability among laboratories in the practice of NGS. One issue of sustained discussion in the field is whether and to what extent orthogonal confirmation of genetic variants identified by NGS is necessary or helpful. The Association for Molecular Pathology Clinical Practice Committee convened the NGS Germline Variant Confirmation Working Group to assess current evidence regarding orthogonal confirmation and to establish recommendations for standardizing orthogonal confirmation practices to support quality patient care. On the basis of the results of a survey of the literature, a survey of laboratory practices, and subject expert matter consensus, eight recommendations are presented, providing a common framework for clinical laboratory professionals to develop or refine individualized laboratory policies and procedures regarding orthogonal confirmation of germline variants detected by NGS.

Assessments of Somatic Variant Classification Using the Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists Guidelines: A Report from the Association for Molecular Pathology.

To assess the clinical implementation of the 2017 Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists, identify content that may result in classification inconsistencies, and evaluate implementation barriers, an Association for Molecular Pathology Working Group conducted variant interpretation challenges and a guideline implementation survey. A total of 134 participants participated in the variant interpretation challenges, consisting of 11 variants in four cancer cases. Results demonstrate 86% (range, 54% to 94%) of the respondents correctly classified clinically significant variants, variants of uncertain significance, and benign/likely benign variants; however, only 59% (range, 39% to 84%) of responses agreed with the working group's consensus intended responses regarding both tiers and categories of clinical significance. In the implementation survey, 71% (157/220) of respondents have implemented the 2017 guidelines for variant classification and reporting either with or without modifications. Collectively, this study demonstrates that, although they may not yet be optimized, the 2017 guideline recommendations are being adopted for standardized somatic variant classification. The working group identified significant areas for future guideline improvement, including the need for a more granular and comprehensive classification system and education resources to meet the growing needs of both laboratory professionals and medical oncologists.

An Educational Assessment of Evidence Used for Variant Classification: A Report of the Association for Molecular Pathology.

The Association for Molecular Pathology Variant Interpretation Testing Among Laboratories (VITAL) Working Group convened to evaluate the Standards and Guidelines for the Interpretation of Sequence Variants implementation into clinical practice, identify problematic classification rules, and define implementation challenges. Variants and associated clinical information were provided to volunteer respondents. Participant variant classifications were compared with intended consensus-derived classifications of the Working Group. The 24 variant challenges received 1379 responses; 1119 agreed with the intended response (81%; 95% CI, 79% to 83%). Agreement ranged from 44% to 100%, with 16 challenges (67%; 47% to 82%) reaching consensus (≥80% agreement). Participant classifications were also compared to a calculated interpretation of the ACMG Guidelines using the participant-reported criteria as input. The 24 variant challenges had 1368 responses with specific evidence provided and 1121 (82%; 80% to 84%) agreed with the calculated interpretation. Agreement for challenges ranged from 63% to 98%; 15 (63%; 43% to 79%) reaching consensus. Among 81 individual participants, 32 (40%; 30% to 50%) reached agreement with at least 80% of the intended classifications and 42 (52%; 41% to 62%) with the calculated classifications. This study demonstrated that although variant classification remains challenging, published guidelines are being utilized and adapted to improve variant calling consensus. This study identified situations where clarifications are warranted and provides a model for competency assessment.

A memorandum of understanding has facilitated guideline development involving collaborating groups.

OBJECTIVE: Collaboration between groups can facilitate the development of high-quality guidelines. While collaboration is often desirable, misunderstandings can occur. One method to minimize misunderstandings is the pre-specification of terms of engagement in a memorandum of understanding (MOU). This study considered when an MOU may be most helpful, and which key elements should be included. STUDY DESIGN AND SETTING: An international panel of representatives from guideline groups was convened. A literature review to identify publications and other documents relevant to the establishment of MOUs between two or more guideline groups, supplemented by available source documents, was used to inform development of a draft MOU resource. This was iteratively refined until consensus was achieved. RESULTS: The level of detail in an MOU may vary based on institutional preferences and the particular collaboration. Elements within an MOU include those pertaining to: (1) scope and purpose; (2) leadership and team; (3) methods and commitment; (4) review and endorsement; and (5) publication and dissemination. CONCLUSION: Since groups may have different expectations regarding how a collaboration will unfold, an MOU may mitigate preventable misunderstandings. The result may be a higher likelihood of producing a guideline without disruption and delay.

An International Needs Assessment Survey of Guideline Developers Demonstrates Variability in Resources and Challenges to Collaboration between Organizations.

BACKGROUND: The development of rigorous, high-quality clinical guidelines increases the need for resources and skilled personnel within guideline-producing organizations. While collaboration between organizations provides a unique opportunity to pool resources and save time and effort, the collaboration presents its own unique challenges. OBJECTIVE: To assess the perceived needs and current challenges of guideline producers worldwide related to guideline development and collaboration efforts. DESIGN: Survey questions were developed by the Guidelines International Network and the US GRADE Network, pilot-tested among attendees of a guideline development workshop, and disseminated electronically using convenience and snowball sampling methods. PARTICIPANTS: A total of 171 respondents representing 30 countries and more than 112 unique organizations were included in this analysis. MAIN MEASURES: The survey included free-response, multiple-choice, and seven-point Likert-scale questions. Questions assessed respondents' perceived value of guidelines, resource availability and needs, guideline development processes, and collaboration efforts of their organization. KEY RESULTS: Time required to develop high-quality systematic reviews and guidelines was the most relevant need (median=7; IQR=5.5-7). In-house resources to conduct literature searches (median=4; IQR=3-6) and the resources to develop rigorous guidelines rapidly (median=4; IQR=2-5) were perceived as the least available resources. Difficulties reconciling differences in guideline methodology (median=6; IQR=4-7) and the time required to establish collaborative agreements (median=6; IQR=5-6) were the most relevant barriers to collaboration between organizations. Results also indicated a general need for improvement in conflict of interest (COI) disclosure policies. CONCLUSION: The survey identified organizational challenges in supporting rigorous guideline development, including the time, resources, and personnel required. Connecting guideline developers to existing databases of high-quality systematic reviews and the use of freely available online platforms may facilitate guideline development. Guideline-producing organizations may also consider allocating resources to hiring or training personnel with expertise in systematic review methodologies or utilizing resources more effectively by establishing collaborations with other organizations.

Electronic Health Records and Genomics: Perspectives from the Association for Molecular Pathology Electronic Health Record (EHR) Interoperability for Clinical Genomics Data Working Group.

The use of genomics in medicine is expanding rapidly, but information systems are lagging in their ability to support genomic workflows both from the laboratory and patient-facing provider perspective. The complexity of genomic data, the lack of needed data standards, and lack of genomic fluency and functionality as well as several other factors have contributed to the gaps between genomic data generation, interoperability, and utilization. These gaps are posing significant challenges to laboratory and pathology professionals, clinicians, and patients in the ability to generate, communicate, consume, and use genomic test results. The Association for Molecular Pathology Electronic Health Record Working Group was convened to assess the challenges and opportunities and to recommend solutions on ways to resolve current problems associated with the display and use of genomic data in electronic health records.

Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology.

To address the clinical relevance of small DNA variants in chronic myeloid neoplasms (CMNs), an Association for Molecular Pathology Working Group comprehensively reviewed published literature, summarized key findings that support clinical utility, and defined critical gene inclusions for high-throughput sequencing testing panels. This review highlights the biological complexity of CMNs [including myelodysplastic syndromes, myeloproliferative neoplasms, entities with overlapping features (myelodysplastic syndromes/myeloproliferative neoplasms), and systemic mastocytosis], the genetic heterogeneity within diagnostic categories, and similarities between apparently disparate diagnostic entities. The founding variant's hematopoietic differentiation compartment, specific genes and variants present, order of variant appearance, individual subclone dynamics, and therapeutic intervention all contribute to the clinicopathologic features of CMNs. Selection and efficacy of targeted therapies are increasingly based on DNA variant profiles present at various time points; therefore, high-throughput sequencing remains critical for patient management. The following genes are a minimum recommended list to provide relevant clinical information for the management of most CMNs: ASXL1, BCOR, BCORL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PHF6, PPM1D, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, and ZRSR2. This list is not comprehensive for all myeloid neoplasms and will evolve as insights into effects of combinations of relevant biomarkers on specific clinicopathologic characteristics of CMNs accumulate.

Standards and Guidelines for Validating Next-Generation Sequencing Bioinformatics Pipelines: A Joint Recommendation of the Association for Molecular Pathology and the College of American Pathologists.

Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.

Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists.

Next-generation sequencing (NGS) methods for cancer testing have been rapidly adopted by clinical laboratories. To establish analytical validation best practice guidelines for NGS gene panel testing of somatic variants, a working group was convened by the Association of Molecular Pathology with liaison representation from the College of American Pathologists. These joint consensus recommendations address NGS test development, optimization, and validation, including recommendations on panel content selection and rationale for optimization and familiarization phase conducted before test validation; utilization of reference cell lines and reference materials for evaluation of assay performance; determining of positive percentage agreement and positive predictive value for each variant type; and requirements for minimal depth of coverage and minimum number of samples that should be used to establish test performance characteristics. The recommendations emphasize the role of laboratory director in using an error-based approach that identifies potential sources of errors that may occur throughout the analytical process and addressing these potential errors through test design, method validation, or quality controls so that no harm comes to the patient. The recommendations contained herein are intended to assist clinical laboratories with the validation and ongoing monitoring of NGS testing for detection of somatic variants and to ensure high quality of sequencing results.

The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer: A Report of the Association for Molecular Pathology.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.