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Contents

        Preface: The Medical Practice of Molecular Oncology Diagnostics xiii

        Fei Dong

        Panel Sequencing for Targeted Therapy Selection in Solid Tumors 309

        Nicholas Willard, Lynette Sholl, and Dara Aisner
        The genetic testing of solid tumors has evolved rapidly. With an ever-increasing number of clinically significant and/or actionable gene alterations in addition to increasing biologic, gene, or mutation-specific therapies, single-target testing is no longer suitable for many modern oncology patients. This review explores panel-based testing, including its history and evolution from prior testing modalities. We also discuss its current usefulness, as best exemplified by lung cancer, and other special considerations including a summary of the pros and cons of panel implementation and use. Lastly, we discuss the successes and challenges of panel-based testing and explore future directions.

        Molecular Diagnostic Testing for Hematopoietic Neoplasms: Linking Pathogenic Drivers to Personalized Diagnosis 325

        Christopher B. Hergott and Annette S. Kim
        Molecular diagnostics inhabit an increasingly central role in characterizing hematopoietic malignancies. This brief review summarizes the genomic targets important for many major categories of hematopoietic neoplasia by focusing on disease pathogenesis. In myeloid disease, recurrent mutations in key functional classes drive clonal hematopoiesis, on which additional variants can specify clinical presentation and accelerate progression. Lymphoblastic leukemias are frequently initiated by oncogenic fusions that block lymphoid maturation while, in concert with additional mutations, driving proliferation. The links between genetic aberrations and lymphoma patient outcomes have been clarified substantially through the clustering of genomic profiles. Finally, the addition of next-generation sequencing strategies to cytogenetics is refining risk stratification for plasma cell myeloma. In all categories, molecular diagnostics shed light on the unique mechanistic underpinnings of each individual malignancy, thereby empowering more rational, personalized care for these patients.

        Special Considerations in the Molecular Diagnostics of Pediatric Neoplasms 349

        Adam S. Fisch and Alanna J. Church
        Pediatric neoplasms have unique demands, including triaging of small biopsies for multiple testing modalities, and a pediatric cancer genome that is notably different from the adult cancer genome. Pediatric cancers are more likely to be driven by gene fusions and typically have a lower tumor mutational burden. Clinically relevant unique molecular targets exist within pediatric cancers, with important implications for diagnosis, prognosis, and treatment selection. Hence, assays and interpretation workflows must be designed thoughtfully to support molecular tumor profiling for children with cancer, including accommodation of small samples, detection of gene fusions, and consideration of potential germline associations.

        Specimen Considerations in Molecular Oncology Testing 367

        Qiong Gan and Sinchita Roy-Chowdhuri
        Proper treatment of the patient with cancer depends on an accurate diagnosis of the tumor and is further directed by prognostic and more recently therapeutic molecular signatures in the era of precision medicine. Molecular oncology testing provides diagnostic, prognostic, and therapeutic information derived from the tumor genome. The aim of this review is to provide valuable information to laboratories for choosing optimal clinical specimens for molecular oncology testing by evaluating the strengths and weaknesses of different sample types from the procurement, processing, and pre-analytic selection matching to different test platforms.

        Utility of Single-Gene Testing in Cancer Specimens 385

        Mehenaz Hanbazazh, Diana Morlote, Alexander C. Mackinnon, and Shuko Harada
        Molecular testing is now considered the standard of care to screen for disease, confirm the diagnosis, guide management, and use target therapy. Currently, several testing strategies are being used. One of the most common strategies is single-gene testing, which is often conducted for known mutations, such as BRAF in melanoma and EGFR in lung cancer. Subsequently, next-generation sequencing (NGS), which tests many genes simultaneously, was developed using targeted gene panels, whole-exome, or whole-genome sequencing. Ordering the best diagnostic tool and choosing between single-gene testing and NGS depends on several factors. In this review, we discuss different single-gene testing methodologies and the impact of using them in comparison to NGS/multigene panel.

        Analytical Principles of Cancer Next Generation Sequencing 395

        Tatyana Gindin and Susan J. Hsiao
        This article covers analytical principles of cancer next generation sequencing (NGS). Cancer samples require special considerations due to the cancer-specific applications of testing, as well as cancer sample specific issues, including low input, low tumor purity, or fixation-related artifacts. Laboratories typically use a combination of approaches around specimen processing, assay design, and bioinformatics analysis to allow for successful detection of actionable biomarkers. Examples of these approaches for cancer NGS testing are discussed and reviewed here.

        Principles and Validation of Bioinformatics Pipeline for Cancer Next-Generation Sequencing 409

        Somak Roy
        Clinical bioinformatics plays a key role in the implementation of clinical next-generation sequencing (NGS) testing infrastructure. Bioinformatics workflows in a clinical laboratory are complex and therefore need to be validated as part of an end-to-end NGS assay validation before clinical use. The validation cohort should be representative of the types of samples, types of variants, lower limits of detection of the assay, as well as sequence context of the panel. When validating an NGS bioinformatics pipeline, the pipeline validation lifecycle should be adhered to. Software containers and modern software automation tools can allow the building of a scalable and reliable clinical bioinformatics infrastructure and can be implemented in clinical bioinformatics operations in a phased way depending on the size and skillset of the bioinformatics team.

        Best Practice for Clinical Somatic Variant Interpretation and Reporting 423

        Jeffrey Schubert, Jinhua Wu, Marilyn M. Li, and Kajia Cao
        Because the clinical impact of cancer genomics is being increasingly recognized, tumor sequencing will likely continue to expand in breadth and scope. Therefore, it is vital for laboratory professionals to adopt the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists guidelines and create a standardized system of classification and nomenclature for somatic variants. Combining robust bioinformatics pipelines with thorough data analysis is necessary to efficiently and reproducibly identify and assess the impact of clinically relevant variants.

        Molecular Detection of Oncogenic Gene Rearrangements 435

        Zehra Ordulu and Valentina Nardi
        Oncogenic gene rearrangements have been exponentially significant for clinical management of cancer, from diagnosis to therapy and disease monitoring. Testing algorithms should be created with caution, and sample type, accessibility to testing method, turnaround time, and economic aspects should be taken into consideration. Herein, different molecular technologies for detecting these gene rearrangements are discussed and the benefits and limitations of each method are highlighted.

        Copy Number Analysis in Cancer Diagnostic Testing 451

        Tara Spence and Adrian M. Dubuc
        Accurate detection of copy number alterations (CNAs) has become increasingly important in clinical oncology for the purpose of diagnosis, prognostication, and disease management. Cytogenetic approaches for the detection of CNAs, including karyotype, fluorescence in situ hybridization (FISH), and chromosomal microarray, remain mainstays in clinical laboratories. Yet, with rapidly decreasing costs and improved accuracy of CNA detection using emerging technologies such as next-generation sequencing and optical genome mapping, we are approaching a new era of cytogenomics and molecular oncology. The aim of this review is to describe the benefits and limitations associated with the routine clinical application of available classic, emerging, and projected future technologies for the detection of CNAs in oncology.

        Molecular Biomarkers of Response to Cancer Immunotherapy 469

        Lauren L. Ritterhouse and Tasos Gogakos
        Harnessing the immune system to advance cancer therapy has offered a new weapon in the quiver of clinical oncology. The lack of uniform, robust, or durable responses in many patients has necessitated the development of approaches for the accurate prediction of subgroups that are most likely to benefit from immunotherapy. This has led to the development and regulatory approval of predictive biomarkers, as well as associated companion diagnostics. Despite these strides, there still exists great heterogeneity in the choice of biomarkers, the laboratory assays that generate them, and their overall clinical utility. This article surveys broadly the predictive biomarkers of response to cancer immunotherapy, focusing on the biomarkers with current Food and Drug Administration (FDA) approval, and raising awareness of issues that may affect their broad applicability.

        When Tissue Is the Issue: Expanding Cell-Free DNA “Liquid Biopsies” to Supernatants and Nonplasma Biofluids 485

        Vera Paulson, Eric Q. Konnick, and Christina H. Lockwood
        While tissue biopsy remains the gold standard for tumor biomarker testing, assays using plasma-derived cfDNA, aka circulating-tumor DNA (ctDNA), have recently demonstrated validity in the setting of limited tissue or recurrent disease. Tumor-derived cfDNA is also present in nonplasma biofluids and supernatants procured through interventional procedures. Evaluation of cfDNA extracted from these fluids may have benefits at nearly every stage of cancer patient management, from diagnosis and prognosis to monitoring disease progression and predicting therapeutic response. This review will focus on preanalytical, analytical, and postanalytical variables that must be considered when analyzing “liquid biopsies” outside the plasma compartment.

        Germline Testing for the Evaluation of Hereditary Cancer Predisposition 497

        Ozge Ceyhan-Birsoy
        Germline testing for hereditary cancer predisposition has become increasingly important in the management of patients with cancer. Recent studies have demonstrated that hereditary cancer predisposition is more common than previously recognized and germline pathogenic variants may be actionable for patient treatment strategies. This article reviews the significance of hereditary cancer predisposition assessment and highlights the current practices in germline genetic testing approaches.