Molecular Pathology of Breast Cancer (eBook)

Bio Data: Prof. Sunil Badve. MBBS, MD. FRCPath. Sunil Badve is Professor in departments of Pathology and Laboratory Medicine, and Internal Medicine and the Director of Translational Genomics Core at the Indiana University Simon Cancer Center. He received his medical degree and pathology residency at Grant Medical College and Tata Memorial Hospital for cancer. He did further training at the St George’s Medical School and at Royal Marsden Hospital, England. In USA, he trained at the Albert Einstein School of Medicine (NY) and at Yale. He served on the faculty of Northwestern University prior to joining Indiana University.  Dr. Badve’s main research and clinical expertise is in breast cancer. He is the breast Pathologist for ECOG group and serves on the Breast Cancer Steering Committee. He has been a PI and co-investigator on several NIH (R01; R21), DOD and foundation grants. He is recognized as a Komen Scholar by the Susan G Komen for the Cure. He has over publication of over 170 peer-reviewed scientific articles in addition to invited reviews and book chapters including WHO Classifications of Breast and Thymic tumors. He has several patents and has developed and commercialized a gene signature for the prognostication of thymic cancers. He serves as the associate editor of Clinical Breast Cancer and on the editorial board of six additional journals (Modern Pathology, Analytical and Cellular Pathology, Pathology, BMC Cancer, Journal of Cancer, and Journal of Molecular Biomarkers and Diagnosis). Dr. Badve is a regular speaker at national and international pathology meetings and has conducted short courses on breast pathology for the CAP and USCAP. He has served ASCO-CAP Guidelines Committee for hormone receptor assessment in breast cancer and has been a faculty presenter at the ASCO annual meeting.Bio Data: Assistant Prof. Yesim Gökmen-Polar, Ph.D Dr. Yesim Gökmen-Polar is an Assistant Research Professor in the Department of Pathology and Laboratory Medicine at Indiana University School of Medicine. She received her Ph.D. in Molecular Biology at Bogazici University, Department of Molecular Biology and Genetics, Istanbul, Turkey. She further continued her training in cancer biology at the Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas. She joined the Division of Hematology & Oncology at Indiana University as a research associate, and specialized in targeted therapeutics for breast cancers and thymic cancers. She serves in several working groups such as TCGA Thymoma working group, ITMIG Tumor Bank/Biology Working group and Thymoma Tissue Bank Oversight Committee at IU School of Medicine. She also serves as a reviewer for several journals including BMC Cancer, Neoplasia, Clinical Breast Cancer, Pathology-Research and Practice, and Oncotarget. She is also a speaker for Affymetrix and a faculty presenter in meetings including USCAP and AACR. Her research interests are centered on the identification and characterization of novel prognostic and therapeutic targets in breast cancer and thymic cancer. She has approximately 50 publications mainly in the field of cancer biology. She has patents in breast cancer prognostication and has developed and commercialized a gene signature for the prognostication of thymic cancers. Her specific expertise is in development of novel therapeutic targets (i.e. targeting EphA2 receptor tyrosine kinase, protein kinase C beta, anti-tubulins and TORC1/2) in colon, breast cancer and thymic malignancies. Her lab developed and characterized a novel human thymoma model, designated as IU-TAB-1, which was derived from a patient with the stage II thymoma (WHO type A/B). Her current research uses next generation sequencing (NGS), microarrays, and computational tools to understand the transcriptional regulation by non-coding RNAs and alternative splicing that contribute to breast cancer recurrence/metastasis. Her lab uses these approaches to develop novel therapeutics to target breast cancer and thymic cancer.

Preface 5
Contents 6
1 Translation of Biomarkers into Clinical Practice 8
1.1 From Biomarker to Biomarker Test 9
1.2 Clinical Uses for Biomarker Tests 10
1.3 Principles in Determination of Fitness of a Biomarker Test for an Intended Clinical Use 12
1.3.1 Analytical validity 13
1.3.2 Clinical validity 13
1.3.3 Clinical Utility 14
1.4 Evaluation of Clinical Utility 14
1.4.1 Prognostic Biomarker Utility 14
1.4.2 Predictive Biomarker Utility 17
1.4.2.1 Considerations for Treatment-Selection or Enrichment-Predictive Biomarker Utility 17
1.4.2.2 Considerations for Response-Predictive Biomarker Utility 20
1.4.3 Monitoring Biomarker Utility 21
1.5 Regulatory Considerations 22
1.6 Discussion 23
References 23
2 Preanalytic Variables, Tissue Quality and Clinical Samples from Breast Cancer Patients: Implications for Treatment Planning, Drug Discovery and Translational Research 26
Abstract 26
2.1 Introduction 26
2.2 Pre-analytical Variables 28
2.2.1 The Rapid Tissue Acquisition Program 29
2.2.2 How Important Is Standardizing Tissue Handling? 29
2.3 Pre-analytical Variable and Breast Cancer Diagnosis 30
2.3.1 Conclusions 31
Acknowledgments 32
References 32
3 Impact of Analytical Variables in Breast Cancer Biomarker Analysis 34
Abstract 34
3.1 Introduction 34
3.2 Preanalytical Stage 37
3.2.1 Ischaemic Phase 37
3.2.2 Tissue Fixation 37
3.2.3 Processing and Embedding 38
3.2.4 Achieving Optimal Conditions 38
3.3 Analytical Stage 39
3.3.1 Sectioning and Storage 39
3.3.2 Staining 39
3.3.2.1 Morphological Analysis 39
3.3.2.2 Immunohistochemistry and In Situ Hybridisation 40
3.3.3 Internal Quality Control 41
3.4 Postanalytical Stage 42
3.4.1 Assessment 42
3.4.2 Reporting 43
3.4.3 Quality Assurance 45
3.5 Conclusions and Future Directions 47
References 48
4 Hormone Receptors in Breast Cancer 51
Abstract 51
4.1 Introduction 51
4.2 Hormone Receptor Assessment-A Historical View 52
4.3 General Principles of Hormone Receptor Testing and Standardization of Methodology 53
4.4 The Estrogen Receptor 54
4.4.1 Variants 54
4.4.2 Definition of Positivity 55
4.4.3 Detection Methods 56
4.4.4 ER Mutations and Promoter Methylation 57
4.5 The Progesterone Receptor 57
4.5.1 Variants 57
4.5.2 Added Value to ER? 58
4.5.3 Definition of Positivity 58
4.5.4 Detection Methods 58
4.6 The Androgen Receptor 58
4.7 Intrinsic Subtype and Hormone Receptor Expression 59
4.8 Image Analysis 59
4.9 Testing of Breast Cancer Recurrences 60
4.10 Hormone Receptors in Ductal Carcinoma In Situ 60
4.11 Hormone Receptors in Male Breast Cancer 60
4.12 Conclusion 60
References 61
5 Human Epidermal Growth Factor Receptor 2 (HER2): Translating the Lab to the Clinic 65
Abstract 65
5.1 Introduction 65
5.2 HER2 Testing 65
5.3 Approach to Metastatic HER2-Positive Breast Cancer 67
5.4 Approach to Early HER2-Positive Breast Cancer 68
5.5 Mechanisms of Resistance to HER2-Directed Therapy 71
5.6 Conclusions 73
References 74
6 Triple-Negative Breast Cancer 77
Abstract 77
6.1 Introduction 78
6.2 Insights from Gene Expression Profiling 78
6.3 Insights from Cancer Genetics 80
6.4 Role of Host Antitumor Immunity in TNBC 82
6.5 Conclusions 84
References 84
7 Proliferation Markers in Breast Cancer 87
Abstract 87
7.1 Introduction 87
7.2 Mitotic Index/SBR Grade 88
7.3 S-Phase Fraction and the Related Tools 89
7.4 Nuclear Antigens 89
7.5 Ki67 91
7.5.1 Analytical Validity 92
7.5.2 Clinical Validity—Prognostic or Predictive? 94
7.6 Molecular Signatures 96
7.7 Conclusion 97
References 100
8 Novel Immunohistochemical Based Biomarkers in Breast Cancer 105
Abstract 105
8.1 Introduction 105
8.2 Diagnostic Use of Immunohistochemistry 106
8.2.1 Myoepithelial Markers 107
8.2.2 Epithelial Markers 110
8.2.3 Distinction of Lobular and Ductal Carcinoma 113
8.2.4 Metastases to the Breast 114
8.3 Predictive and Prognostic IHC Based Markers 115
8.4 Novel Biomarkers in Breast Cancer Research 117
8.5 IHC-Based Prognostic Gene Signatures 118
8.5.1 Mammostrat 119
8.5.2 IHC4 Score 121
8.5.3 Nottingham Prognostic Index plus (NPI+) 121
8.6 Conclusion 121
References 121
9 Tumor Heterogeneity in Breast Cancer 126
Abstract 126
9.1 Introduction 126
9.2 Extent of Tumor Heterogeneity? 127
9.3 Origins of Tumor 128
9.3.1 Origins of Intratumor Heterogeneity 129
9.3.2 Cancer Stem Cells 129
9.3.3 Clonal Evolution 130
9.3.4 Codependency of Clones 131
9.4 Heterogeneity in Primary and Metastatic Tumors 131
9.5 Implications of Tumor Heterogeneity 132
9.6 Summary and Future Directions 133
Disclosure 133
References 133
10 Breast Cancer Stem Cells 138
Abstract 138
10.1 Cancer Stem Cells 138
10.2 Discovery of Breast Cancer Stem Cells 139
10.3 The Epithelial-to-Mesenchymal Transition (EMT) and Breast Cancer Stemness 140
10.4 CSCs and the Development of Resistance to Conventional Therapies 140
10.5 Signaling Pathways Involved in Breast Cancer Stem Cells 141
10.5.1 JAK2-STAT3 Signaling Pathway 141
10.5.2 Wnt/Beta-Catenin Pathway 142
10.5.3 Autophagy 143
10.5.4 Induced Nitric Oxide Synthase (INOS) Pathway 144
10.6 CSCs and the Development of Mechanistically-Based Therapies 145
10.7 Heterogeneity of Cancer or CSCs and Clinical Significance 146
10.8 Methods Used to Determine Cancer Stem Cells 146
10.8.1 Sphere-Forming Assay 146
10.8.2 In Vivo Limiting Dilution Assay 147
10.8.3 Lineage Tracing 147
10.8.4 Patient-Derived Xenografts (PDXs) as Preclinical Small Animal Models 148
10.9 Conclusions 149
References 149
11 The Tumor Microenvironment as a Metastasis Biomarker in Breast Cancer 157
Abstract 157
11.1 Introduction 158
11.2 The Hallmarks of Cancer and the Tumor Microenvironment 159
11.3 Epithelial-to-Mesenchymal Transition (EMT), Mena, and Metastasis 159
11.4 Role of Mena and Cofilin in Promoting Tumor Cell Motility and Metastasis 160
11.5 Interaction Between Migratory Tumor Cells and the Microenvironment 161
11.6 Motility and Transendothelial Migration (TREM) Are Required for Development of Metastasis 162
11.7 TREM and Intravasation Occur at TMEM Sites in Primary Tumors 163
11.8 Invasive Mena Isoforms, TMEM Structures, and Recurrence in Human Breast Cancer 165
11.9 Conclusions 167
References 167
12 Tumor Infiltrating Lymphocytes as a Prognostic and Predictive Biomarker in Breast Cancer 170
Abstract 170
12.1 Introduction 171
12.2 Characterization of TILs in Breast Cancer 171
12.3 TILs Are an Immunologic Response to Tumor Neoantigens 174
12.4 Composition of TILs and Subpopulations 175
12.5 The Tumor Microenvironment and TIL Function 175
12.6 Clinical Validity of TILs as a Prognostic Biomarker in Patients with Breast Cancer Treated with Adjuvant Chemotherapy 176
12.7 Clinical Validity of TILs as a Prognostic and Predictive Biomarker in Patients with Breast Cancer Treated with Neoadjuvant Chemotherapy 180
12.8 Conclusion 186
References 187
13 Novel Imaging Based Biomarkers in Breast Cancer 190
13.1 Introduction 190
13.2 Glucose Metabolism Imaging 191
13.3 Steroid Receptor Imaging 194
13.3.1 Estrogen Receptor Imaging 194
13.3.2 Progesterone Receptor Imaging 197
13.3.3 Androgen Receptor Imaging 197
13.4 HER2 Receptor Imaging 198
13.5 Cell Proliferation Imaging 199
13.5.1 Thymidine Analogs, Including Fluorothymidine (FLT) 199
13.5.2 Alternative Approaches to Proliferation Imaging: Sigma-2 Receptor Imaging 201
13.6 Other Novel Imaging Agents 202
13.6.1 Membrane Lipid Synthesis 202
13.6.2 Amino Acid Transport 202
13.6.3 PARP-1 202
13.6.4 Angiogenesis 203
13.7 Conclusion 203
Acknowledgments 203
References 203
14 Circulating Tumor Markers for Breast Cancer Management 210
Abstract 210
14.1 Introduction 210
14.2 Traditional Tumor Markers 211
14.2.1 Carcinoembryonic Antigen (CEA) 211
14.2.2 Mucin 1 or MUC1 (CA15.3/CA27.29) 212
14.2.3 HER2/Neu Oncogene 213
14.3 Tumor Markers in Development: Protein Markers 214
14.3.1 Tissue Polypeptide Antigens (TPA) 214
14.3.2 Serum Autoantibodies Against Tumor Associated Antigens (TAA) 215
14.4 Challenges in Utility of Circulating Tumor Markers 217
14.4.1 Cost Effectiveness 217
14.4.2 Poor Specificity 217
14.4.3 Lack of Reproducibility 218
14.5 Conclusion 218
REFERENCES: 218
15 Circulating Tumor Cells 222
Abstract 222
15.1 Introduction 222
15.2 Isolation and Characterization of CTCs 223
15.2.1 CTC Enrichment 223
15.2.2 CTC Detection 225
15.2.2.1 Antibody-Based Methods 225
15.2.2.2 Molecular Methods 225
15.2.3 CTC Characterization 227
15.2.3.1 Epithelial-Mesenchymal Transition 227
15.2.3.2 Stem Cell Theory 227
15.2.3.3 Genotype of CTCs 227
15.3 Clinical Role of CTCs 227
15.3.1 Prognostication 227
15.3.1.1 Early Breast Cancer 227
15.3.1.2 Metastatic Breast Cancer 229
15.3.2 Therapy Monitoring 230
15.3.2.1 Early Breast Cancer 231
15.3.2.2 Metastatic Breast Cancer 231
15.3.3 Treatment Selection Based on CTCs—Liquid Biopsy 232
15.3.3.1 Early Breast Cancer 232
15.3.3.2 Metastatic Breast Cancer 232
15.4 Conclusion/Future 233
Acknowledgments 233
References 233
16 Circulating Nucleic Acids (RNA/DNA) in Breast Cancer 238
Abstract 238
16.1 Introduction 239
16.2 Circulating Tumoral DNA 239
16.2.1 Circulating Tumor DNA: Preanalytical Considerations and Technologies for ctDNA Detection 239
16.2.2 Clinical Applications of CtDNA in Breast Cancer 240
16.2.2.1 Assessment of Tumor Burden 240
16.2.2.2 Tumor Genomic Profiling 243
16.2.2.3 Epigenetic Profiling 245
16.3 Circulating microRNA 245
16.3.1 Origin of microRNA and Methods for microRNA Analysis 245
16.3.2 Clinical Application of cmiRNA in Breast Cancer 249
16.3.3 Targeting Circulating miRNAs in Breast Cancer 252
16.4 Conclusion 253
References 253
17 Prognostic Factors for Ductal Carcinoma in Situ of the Breast 260
Abstract 260
17.1 Introduction 260
17.2 Randomized Trials of Radiation Treatment and Tamoxifen 261
17.3 Risk Stratification Using Clinical and Pathologic Factors 261
17.4 Risk Stratification Using Molecular Profiling 262
17.5 Development and Validation of the Oncotype DX DCIS Score 264
17.6 Conclusions and Future Directions 268
References 268
18 Prognostic and Predictive Gene Expression Signatures in Breast Cancer 271
Abstract 271
18.1 Introduction 271
18.2 Classical Prognostic Factors 272
18.3 Molecular Prognostic Tests in ER+ Breast Cancer 273
18.3.1 Intrinsic Classification/Prosigna (Nanostring, Seattle, Washington) 273
18.3.2 Mammaprint (Agendia, Amsterdam) 273
18.3.3 Oncotype DX (Genomic Health, Inc., Redwood City, California) 275
18.3.4 Breast Cancer Index (BioTheranostics, San Diego, California) 276
18.3.5 MapQuant DX (Ipsogen, Marseilles, France) 276
18.3.6 EndoPredict (Sividon Diagnostics GmbH, Koln, Germany) 276
18.3.7 IHC4 277
18.4 Comparisons of Tests for ER+ Breast Cancer 277
18.4.1 Lack of Overlap of Gene Panel 277
18.4.2 Discrete Classifiers Versus Continuous Predictor 278
18.4.3 Concordance Between Assays 278
18.5 Late Relapse 279
18.6 Future Directions 279
18.7 Conclusion 280
References 280
19 Genomic Markers in ER-Negative Breast Cancer 284
Abstract 284
19.1 Breast Cancer Subtypes 285
19.2 Hormone Receptor Subtypes Within ER-Negative BC 285
19.3 Gene Expression Based Genomic Markers in Different Breast Cancer Subtypes 287
19.4 Gene Expression Signatures Developed in ER-Negative Breast Cancer 288
19.5 The Role of Immune Cell Infiltration as a Marker in ER-Negative Breast Cancer 288
19.6 Complexity of Immune Cell Markers in ER-Negative Breast Cancer 289
19.7 Gene Mutations as Markers in ER-Negative Breast Cancer 289
19.8 Somatically Mutated Genes in ER-Negative Breast Cancer 290
19.9 Global Genome Alterations in ER-Negative Breast Cancer 292
19.10 Current Clinical Utility of Genomic Tests for ER-Negative Breast Cancer 293
19.11 Conclusions 294
References 294
20 Next-Generation Sequencing Based Testing for Breast Cancer 300
Abstract 300
20.1 Introduction 300
20.2 Next-Generation Sequencing 301
20.3 NGS Panels in Risk Assessment 302
20.3.1 Clinical Validity and Utility of Risk Assessment 302
20.3.2 Genes Commonly Found in NGS Gene Panels 303
20.3.3 Clinical Use of Multi-gene Panel Sequencing for Risk Assessment 307
20.4 Multigene Panels for Analysis of Somatic Tumor Mutations and Therapy Selection 312
20.4.1 Advantages and Limitations of Mutation Panels: What is actionable mutation? 312
20.4.2 Genomic Landscape of Breast Cancer Subclasses 313
20.4.2.1 Luminal Breast Cancers 313
20.4.2.2 HER2 Amplified Breast Cancer 315
20.4.2.3 Triple Negative Breast Cancer 316
20.4.3 Clinical Utility of Mutation Panels for Therapeutic Purposes 317
20.5 Summary 318
References 319
21 Epigenetic Mechanism in Breast Cancer 330
Abstract 330
21.1 Introduction 331
21.2 Basic Epigenetic Mechanisms: Four Major Components 331
21.2.1 Methylation 332
21.2.1.1 DNA Methyl Transferases (DNMTs) 334
21.2.1.2 Methyl-CpG-Binding Proteins 334
21.2.1.3 Ten-Eleven Translocation Cytosine Dioxygenases (TETs) 335
21.2.2 Histone Modifications 335
21.2.3 Noncoding RNA Profiling 336
21.2.4 Chromatin Remodeling 336
21.3 Epigenetic Biomarkers in Breast Cancer Diagnosis and Prognosis 337
21.3.1 DNA Methylation Biomarkers in Breast Cancer 337
21.3.2 Histone Modification Biomarkers in Breast Cancer 337
21.3.3 Alterations in Chromosomal Conformation and Nucleosome Repositioning in Breast Cancer 338
21.4 Clinical Utility of Epigenetic Mechanisms 338
21.4.1 Epigenetic Inhibitors in Breast Cancer Treatment 338
21.4.1.1 DNA Methyltransferase Inhibitors 338
21.4.1.2 Histone Deacetylase Inhibitors 339
21.4.1.3 Chromatin Remodeling Based Therapies 340
21.5 Concluding Remarks 340
References 341
22 Noncoding RNAs in Breast Cancer 346
Abstract 346
22.1 Introduction 347
22.2 Classes of Noncoding RNAs 348
22.2.1 Small Noncoding RNAs 348
22.2.1.1 miRNAs, siRNAs and piRNAs 348
22.2.1.2 Small Nuclear RNAs (snRNAs) 349
22.2.1.3 Ribosomal RNAs (rRNAs) 349
22.2.1.4 Transfer RNAs (tRNAs) 349
22.2.2 Functional Relevance of Small Noncoding RNAs in Breast Cancer: miRNAs as Prognostic and Predictive Markers 350
22.2.3 Long Noncoding RNAs 351
22.2.4 Functional Relevance of Long Noncoding RNAs in Breast Cancer: lncRNAs as Prognostic and Predictive Markers 352
22.2.4.1 Chromatin Modifying LncRNAs 352
22.2.4.2 LncRNAs Organizing Nuclear Structure 354
22.2.4.3 Protein or miRNA Decoys 354
22.2.4.4 LncRNAs as SnoRNA Hosts 355
22.2.4.5 Ribosome Associated LncRNAs 355
22.2.4.6 LncRNAs Playing a Role in Estrogen Receptor Signaling and Endocrine Resistance 356
22.3 Challenges and Future Directions: The Clinical Relevance of Noncoding RNAs? 356
22.4 Conclusions and Future Directions 357
References 357
23 Alternative Splicing in Breast Cancer 366
Abstract 366
23.1 Introduction 366
23.2 Alternative Splicing in Breast Cancer 369
23.2.1 Mutations in RNA Splicing Factors 369
23.2.2 Altered Gene Expression Levels in RNA Splicing Factors 370
23.3 Alternative Splicing Events in Breast Cancer 370
23.3.1 Cassette Exons 370
23.3.1.1 Exon Skipping 370
23.3.1.2 Exon Inclusion and Complex Splicing Patterns 371
23.3.2 Mutually Exclusive Exons 372
23.3.3 Intron Retention 372
23.3.4 Alternative 5? Splice Sites 372
23.3.5 Alternative 3? Splice Sites 373
23.4 Future Directions Promises and Limitations
References 373
24 Pharmacogenomics of Breast Cancer 379
Abstract 379
24.1 Introduction 379
24.2 Tamoxifen Metabolism—The CYP2D6 Story 380
24.3 SULT1A1 385
24.4 CYP19A1 and Estrogen Metabolism 386
24.5 Estrogen Receptors 1 and 2 389
24.6 Pharmacogenetics and Taxane-Related Toxicities 391
24.7 Pharmacogenomics of Bevacizumab Treatment Breast Cancer 394
24.8 GWAS and Aromatase Inhibitor-Associated Adverse Effects 395
24.9 Conclusion 396
References 396
25 Applied Proteomics in Breast Cancer 402
Abstract 402
25.1 Introduction 403
25.2 Types of Platforms for Multiplex Protein Profiling 403
25.2.1 Proteomic Technologies 403
25.2.2 Array-Based Technologies 405
25.2.3 Mass Spectrometry (MS) Based Methods 405
25.2.3.1 Label-Based and Label-Free MS Methods 406
25.2.3.2 Selected and Multiple Reaction Monitoring (SRM and MRM) 406
25.3 Protein–Protein Interaction (PPI) Profiling 406
25.3.1 Issues Related to Sample Preparation 407
25.4 Applications 407
25.4.1 Biomarker for Breast Cancer Risk 407
25.4.2 Biomarker for Classification 408
25.4.3 Biomarker for Prognostics 409
25.4.4 Biomarker for Treatment Response Prediction 409
25.5 Challenges to Proteomics 409
25.6 Conclusions 410
References 410
26 Biomarkers in the Clinic 414
Abstract 414
26.1 Introduction 414
26.2 Why Biomarkers? 414
26.3 The Established Targets: ER, PR, and HER2 415
26.4 Endocrine Resistance 416
26.5 HER2 417
26.6 HER2 Resistance: When the Biomarker Fails Us 418
26.7 Triple Negative Breast Cancer 419
26.8 The Multigene Assays 420
26.9 Proliferation as a Clinical Biomarker: Ki67 421
26.10 Biomarkers of the Future: The “Omics” 421
References 423