Contents

        Editorial Board,  iii
        Contributors,  v

        Preface: Molecular Pathology: What a Difference a Year Makes,  xv

        By Gregory J. Tsongalis

        Genetics

         Current Tools, Databases, and Resources for Phenotype and Variant Analysis of Clinical Exome Sequencing,  1

        By Shannon M. McNulty, Yasemin Cole, Bradford C. Powell, and Stefan Rentas
         Introduction,  1
         Phenotype association tools and databases,  2
        Virtual Gene Panels,  2
        Algorithm-Driven Gene Analysis,  3
        Capturing Phenotype Information,  3
         Variant pathogenicity: evidence codes, tools, and databases,  6
        PVS1,  6
        PS1/PM5,  7
        PS2/PM6,  7
        PS3, BS3,  7
        PM1,  7
        PM2, BA1, BS1,  7
        PM3, BP2,  8
        PP3, BP4,  8
        Automating Variant Analysis,  8
         Genomic analysis: current and future avenues to consider,  9
         Summary,  10
         Clinics care points,  11
         Disclosure,  11

        Applications of Noninvasive Prenatal Testing for Subchromosomal Copy Number Variations Using Cell-Free DNA,  17

        By Jiale Xiang and Zhiyu Peng
         Introduction,  17
        Subchromosomal Copy Number Variations,  17
        Noninvasive Prenatal Testing for Subchromosomal Copy Number Variations,  18
        Selected Copy Number Variations,  18
        Genome-Wide Copy Number Variations,  18
        Confirmatory Test for Subchromosomal Copy Number Variations,  20
        Opinions of Professional Societies,  20
         Factors affecting noninvasive prenatal testing screening subchromosomal copy number variation,  20
        Fetal Fraction,  20
        Sequencing Depth,  21
        Copy Number Variation Size,  22
        Copy Number Variation Region,  22
         Challenges,  22
         Summary,  23
         Clinics care points,  23
         Disclosure,  23

        Applications of Optical Genome Mapping in Next-Generation Cytogenetics and Genomics,  27

        By Wahab A. Khan and Diana M. Toledo
         Introduction,  27
         Significance,  28
         From massively parallel sequencing to next-generation karyo-mapping,  28
         Cytogenomic applications of optical mapping in constitutional disease,  29
        Optical Mapping Applied to Genomic Disorders Mediated by Low Copy Repeats,  29
        Optical Mapping and Single-Gene Disorders: Prototypical Example of Duchene Muscular Dystrophy,  30
        Optical Mapping in Context of Infertility and Prenatal Testing,  30
         Cytogenomic applications using optical mapping in acquired disease,  31
        Optical Mapping in Hematological Malignancies,  31
        Optical Mapping in Solid Tumor Genetics,  31
         Resolving intractable regions of the human genome: repeat expansions disorders,  31
        Facioscapulohumeral Muscular Dystrophy,  32
        Fragile X Syndrome,  32
        Amyotrophic Lateral Sclerosis,  32
         Bioinformatics workflow in optical genome mapping,  33
         Present relevance and future avenues of optical mapping in medical genetics,  34
         Summary,  34
         Disclosure,  34

        Hematopathology

         Diagnosis of Variant Translocations in Acute Promyelocytic Leukemia,  37

        By Brittany B. Coffman and Devon Chabot-Richards
         Introduction,  37
         Clinical features,  37
         Microscopy,  38
         Immunophenotype and special stains,  38
         Genetic profile,  38
         Variant translocations,  41
        ZBTB16-RARA t(11;17)(q23;q21),  41
        IRF2BP2-RARA t(1;17)(q42;q21),  42
        NPM1-RARA t(5;17)(q35;q21),  42
        STAT5B-RARA t(17;17)(q21;q21),  43
        BCOR-RARA t(X;17)(p11;q21),  43
        FIP1L1-RARA t(4;17)(q12;q21),  43
        FNDC3B-RARA t(3;17)(q26;q21),  43
        GTF2I-RARA t(7;17)(q11;q21),  44
        NABP1-RARA t(2;17)(q32;q21),  44
        NUMA1-RARA t(11;17)(q13;q21),  44
        PRKAR1A-RARA t(17;17)(q21;q24),  44
        STAT3-RARA t(17;17)(q21;q21),  44
        TBL1XR1-RARA t(3;17)(q26;q21),  45
        TFG-RARA t(3;14;17)(q12;q11;21),  45
         Molecular prognostic markers,  45
         Treatment,  45
         Prognosis,  46
         Summary,  46
         Disclosure,  46

        Next-Generation Sequencing for Measurable Residual Disease Assessment in Acute Leukemia,  49

        By Alexandra E. Kovach, Gordana Raca, Deepa Bhojwani, and Brent L. Wood
         Introduction,  49
         Next generation sequencing-based measurable residual disease assessment: performance characteristics and comparison with other assessment methodologies,  50
         Disease-specific applications of next generation sequencing-based measurable residual disease assessment,  54
        Acute lymphoblastic leukemia (B-ALL and T-ALL),  54
        Acute myeloid leukemia,  55
         Additional methodologies and considerations,  57
         Summary,  57
         Clinics care points,  57
         Disclosure,  58

        Molecular Profile of BCR-ABL1 Negative Myeloproliferative Neoplasms and Its Impact on Prognosis and Management,  65

        By Ayman Qasrawi and Ranjana Arora
         Introduction,  65
         Driver mutations in myeloproliferative neoplasms,  66
        Janus Kinase 2 Structure and Function,  66
        JAK2 V617F Mutation,  67
        Other JAK2 Mutations,  68
        CALR,  68
        MPL,  68
         Additional mutations in myeloproliferative neoplasms,  68
        Genes Involved in Epigenetic Regulation by DNA Methylation,  69
        Genes Involved in Histone Modification,  69
        Genes Involved in Messenger RNA Splicing,  70
        Genes Involved in Signaling Pathways,  71
        Genes Involved in Transcription Regulation,  71
         Clinical applications of next-generation sequencing in myeloproliferative neoplasms,  71
        Mutational Allele Burden in Myeloproliferative Neoplasms,  71
        Incorporation of Next-generation Sequencing in Prognostic Scores,  72
        Therapeutic Targets,  73
         Summary,  74
         Disclosure,  74

        Infectious Disease

         Engineering Consideration for Emerging Essential Nucleic Acid Tests for Point-of-Care Diagnostics,  81

        By Ethan P.M. LaRochelle and Amogha Tadimety
         Introduction,  81
         Significance,  85
         Three stages of nucleic acid test,  85
        Sample Preparation,  85
        Amplification,  85
        Detection,  85
        Engineering Considerations,  85
        Microfluidics,  86
        Lateral Flow,  87
        Optical Sensing,  88
         Discussion and summary,  89
         Summary and future directions,  89

        Emerging Molecular Diagnostic Methods for Prosthetic Joint Infections,  93

        By Robert Hamilton and Samantha Stephen
         Introduction,  93
         Discussion,  95
         Summary,  99
         Clinics care points,  100
         Disclosure,  100

        Pharmacogenomics

         The Role of the Human Gutome on Chronic Disease: A Review of the Microbiome and Nutrigenomics,  103

        By Carrie C. Hoefer, Leah K. Hollon, and Jennifer A. Campbell
         Introduction,  103
         Significance,  104
        Gut Microbiome,  104
        Nutrigenomics and Disease,  104
        Diet and the Gutome,  105
         Discussion and future perspectives,  109
        Metabolic Syndromes and the Gutome,  109
        The Gutome and Cancer,  112
         Summary,  112
         Disclosure,  112

        The Importance of Use of Genetics to Guide Hypertension Therapy: Using β-Blockade as an Example,  117

        By Eric M. Snyder, Ryan Sprissler, and Thomas P. Olson
         Introduction,  117
        Significance,  117
        The Beta-Adrenergic Receptors and β-Blockade in Hypertension,  118
        Genetic Variation of the β-AR and β-AR and β-Blocker Responsiveness in Hypertension,  120
        Present Relevance and Future Directions,  122
         Summary,  122
         Clinics care points,  122
         Author contributions,  123
         Disclosure,  123

        Informatics

         Blood Group Genotyping,  127

        By Jensyn K. Cone Sullivan, Nicholas Gleadall, and William J. Lane
         Introduction,  128
        Red Blood Cell Antigen Phenotypes and Blood Groups,  128
        Blood Group Genetic Changes,  128
         Significance,  132
        Next-Generation Sequencing,  132
        DNA Microarrays,  134
         Present relevance and future avenues to consider or to investigate,  136
        Clinical Indications for Genotyping,  136
         Summary,  139
         Definitions,  140
        Call Out 1: Blood Group Terms,  140
        Call Out 2: Allele Terminology,  140
        Call Out 3: Blood Group Governance,  140
        Call Out 4: Next Generation Sequencing Terminology,  140
        Call Out 5: Array Terminology,  140
         Clinic care points,  141
         Disclosure,  141

        Artificial Intelligence in Anatomic Pathology,  145

        By Joshua J. Levy and Louis J. Vaickus
         Introduction,  145
         Slides at scale,  146
         Introduction to machine learning,  147
         Deep learning,  148
         Training and validating model,  151
         Emerging technologies, applications, and challenges,  153
        Patch-Based Approaches,  153
        Cell-Based Approaches,  153
        Unsupervised and Self-Supervised Approaches,  154
        Weakly Supervised Approaches,  155
        Graph-Based Learning,  156
        Multimodal Approaches,  157
        Spatial Omics Information,  159
        Image Registration, Generative Adversarial Networks (GANs), and Virtual Staining,  161
        Text/EHR-Based Analyses,  163
        Explainable Artificial Intelligence,  163
        Hierarchical Bayesian Methods,  164
        Data Stewardship and Federated Learning,  166
         Summary,  167
         Acknowledgements,  167
         Clinics care points,  168

        Solid Tumors

         Operationalizing Genomic Medicine: Laboratory Practice Considerations Beyond the Assay,  173

        By Nikoletta Sidiropoulos
         Introduction,  173
        Significance,  174
         Summary,  183
         Disclosure,  184

        Cell-free Nucleic Acids in Cancer: Current Approaches, Challenges, and Future Directions,  187

        By Liron Barnea Slonim, Kathy A. Mangold, Mir B. Alikhan, Nora Joseph, Kalpana S. Reddy, Linda M. Sabatini, and Karen L. Kaul
         Preanalytical requirements,  188
         Technical approaches,  188
        Limited Target Analysis,  188
        Broader Genomic Analysis,  191
        Other Markers Found in Plasma,  192
         Clinical applications,  192
        Screening and Early Detection of Cancer,  192
        Guiding Treatment,  193
        Challenges in the Implementation of Cell-free Nucleic Acid Testing,  194
         Summary,  195
         Clinics care points,  196
         Disclosure,  196

        Tumor Mutational Burden Calculation and Microsatellite Instability Detection in Clinical Next-Generation Sequencing Assays,  199

        By Ashkan Bigdeli, Amanda Oran, and Robyn Sussman
         Checkpoint blockade therapy, tumor mutational burden, and microsatellite instability,  199
         Tumor mutational burden algorithms,  200
         Microsatellite instability detection,  201
         Validation of microsatellite instability determination and tumor mutational burden calculation,  201
         Clinics care points,  203
         Disclosure,  203

        COVID-19

         A PENNdemic Year in Review,  205

        By Sarah E. Herlihy and Caren Gentile
         Introduction,  205
         The first phase: navigating the emergency,  205
         The second phase: building a testing strategy,  206
         The third phase: expansion,  210
         The fourth phase: looking forward,  213
         Summary,  214
         Disclosure,  215

        Review of SARS-CoV-2 Antigen and Antibody Testing in Diagnosis and Community Surveillance,  217

        By Robert D. Nerenz, Jacqueline A. Hubbard, and Mark A. Cervinski
         Introduction,  217
         Antibody testing,  218
        Assay Format,  218
        Serology Correlation with Neutralizing Antibody Titer,  219
         Antibody response,  222
         COVID-19 vaccines,  222
        Pfizer-BioNTech Vaccine,  223
        Moderna Vaccine,  223
        Janssen Biotech, Inc, Vaccine,  223
         SARS-CoV-2 antigen testing,  224
        BinaxNOW,  224
        Sofia,  225
        Becton Dickinson Veritor,  225
        Effectiveness of Antigen Testing in Controlling Viral Spread,  226
        Antigen Conclusions,  226
         Summary,  227
         Clinics care points,  227
         Disclosure,  227

        The Genomic Landscape of Severe Acute Respiratory Syndrome Coronavirus 2: Surveillance of Variants of Concern,  231

        By M. Shaheen S. Malick and Helen Fernandes
         Variants of concern in resource-limited settings,  232
         Seroprevalence and efficacy of vaccines,  233
         The role of genomic surveillance for monitoring a pandemic,  234