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Protocol Administrative Letter October 1, 2020 "A Phase II Study of Neratinib Alone and in Combination with Fulvestrant *in Metastatic*
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her2 non-amplified but her2 mutant

The purpose of this letter is to clarify an error regarding the duration of follow-up after the last dose of protocol therapy. In Protocol Amendment 13.0, 16MAR2020, Section 6.7 Duration of Follow-up currently reads,
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6.7 duration of follow-up

Patients will be followed every 3 months for 2 years or until death, whichever occurs first. Patients removed from study for unacceptable adverse events will be followed until resolution or stabilization of the adverse event. Patients will be followed through either review of medical records, phone calls, or office visits. This section is incorrect, as it should read,  
Patients will be followed until 30 days after last dose of study therapy. Patients removed from study for unacceptable adverse events will be followed until resolution or stabilization of the adverse event. Patients will be followed through either review of medical records, phone calls, or office visits. If you have any questions, please contact me or the regulatory coordinator (Natalie Benford at natalienconner@wustl.edu, 314-747-8365) Regards, Dr. Cynthia Ma, MD, PhD. Professor of Medicine Washington University School of Medicine Internal Medicine - Medical Oncology Phone: 314-362-8903 Email: cynthiaxma@wustl.edu A Phase II Study of Neratinib **Alone and in Combination with Fulvestrant**  
  
in Metastatic HER2 Non-amplified but HER2 Mutant Breast Cancer Protocol\#: 201209135  
  
  
Version date: 03/16/2020  
| Coordinating Center:            | Washington University School of Medicine 660 S. Euclid Ave, Campus Box 8056 St. Louis, MO 63110                          |                  |
|---------------------------------|--------------------------------------------------------------------------------------------------------------------------|------------------|
| Principal Investigator:         | Cynthia X. Ma, M.D., Ph.D. Washington University School of Medicine  Telephone: 314-362-9383 Email: cynthiaxma@wustl.edu |                  |
| Sub-Investigators:              | Institution:                                                                                                             | Division:        |
| Matthew J.C. Ellis, M.B., Ph.D. | Baylor College of Medicine                                                                                               | Medical Oncology |
| Ron Bose, M.D., Ph.D.           | Washington University                                                                                                    | Medical Oncology |
| Carey Anders, M.D.              | Lineberger Cancer Center                                                                                                 | Medical Oncology |
| Rachel Freedman, M.D.           | Dana Farber Cancer Institute                                                                                             | Medical Oncology |
| Philippe Bedard, M.D.           | Princess Margaret Cancer Centre                                                                                          | Medical Oncology |
| Kimberly Blackwell, M.D.        | Duke Cancer Institute                                                                                                    | Medical Oncology |
| Melody Cobleigh, M.D.           | Rush University Medical Center                                                                                           | Medical Oncology |
| Andres Forero, M.D.             | University of Alabama                                                                                                    | Medical Oncology |
| Daniel Hayes, M.D.              | University of Michigan                                                                                                   | Medical Oncology |
| Janice Lu, M.D.                 | University of Southern California                                                                                        | Medical Oncology |
| Mark Pegram, M.D.               | Stanford Medicine Cancer Institute                                                                                       | Medical Oncology |
| Timothy J. Pluard, M.D.         | St. Luke's Cancer Institute                                                                                              | Medical Oncology |
| Matthew Goetz, M.D.             | Mayo Clinic                                                                                                              | Medical Oncology |
| Julie Nangia, M.D.              | Baylor College of Medicine                                                                                               | Medical Oncology |
| Jason Jones, M.D.               | Avera Cancer Institute                                                                                                   | Medical Oncology |
| Marc E. Lippman, M.D.           | University of Miami                                                                                                      | Medical Oncology |
| Massimo Cristofanilli, M.D.     | Northwestern University                                                                                                  | Medical Oncology |
| Foluso Ademuyiwa, M.D.          | Washington University                                                                                                    | Medical Oncology |
| Michael J. Naughton, M.D.       | Washington University                                                                                                    | Medical Oncology |
| Rama Suresh, M.D.               | Washington University                                                                                                    | Medical Oncology |
| Leonel Hernandez-Aya, M.D.      | Washington University                                                                                                    | Medical Oncology |
| Katherine Weilbaecher, M.D.     | Washington University                                                                                                    | Medical Oncology |
| Ashley Frith, M.D.              | Washington University                                                                                                    | Medical Oncology |
| Haeseong Park, M.D.             | Washington University                                                                                                    | Medical Oncology |
| Matthew Cherian, M.D.           | Washington University                                                                                                    | Medical Oncology |
| Lindsay Peterson, M.D.          | Washington University                                                                                                    | Medical Oncology |
| Feng Gao, M.D., Ph.D.           | Washington University                                                                                                    | Biostatistics    |
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washington university research coordinator

Caroline Bumb Telephone: 314-362-7249 Email: cbumb@wustl.edu A Phase II Study of Neratinib **Alone and in Combination with Fulvestrant**
in Metastatic HER2 Non-amplified but HER2 Mutant Breast Cancer Study Drug(s): Neratinib (PB-272)
IND\#: 116297 **// Clinical Trials.gov\#:** NCT01670877 Protocol Revision History  
Initial Approval Version **09/27/12** Amendment #1 **01/02/13** Amendment #2 03/22/13 Amendment #3 04/22/13 Amendment #4 **09/04/13** Amendment #5 **11/08/13** Amendment #6 12/26/13 Amendment #7 02/05/15 Amendment #8 **12/11/15** Amendment #9 09/14/16 Amendment #10 **02/07/17** Amendment #11 **10/05/17** Amendment #12 **06/15/18** Amendment #13 03/16/2020
a phase ii study of
principal investigator signature page

| Principal Investigator:                                                                                                                                                                                                                                                                                                                                                                                                         | Cynthia X. Ma, M.D., Ph.D. Signature of Investigator   | Date   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------|--------|
| Printed Name of Investigator By my signature, I agree to personally supervise the  conduct of this study and to ensure its conduct in  compliance with the protocol, informed consent,  IRB/HRPO procedures, the Declaration of Helsinki, ICH  Good Clinical Practices guidelines, and the applicable  parts of the United States Code of Federal Regulations or  local regulations governing the conduct of clinical  studies. |                                                        |        |
a phase ii study of
contact information

Ron Bose, M.D., Ph.D. Washington University School of Medicine Telephone: 314-747-9308 Email: rbose@wustl.edu Matthew J. Ellis, M.B., Ph.D. Baylor College of Medicine Email: matthew.ellis@bcm.edu Rachel Freedman, M.D.  
Dana-Farber Cancer Institute Harvard University Email: rachel_freedman@dcfi.harvard.edu Kimberly Blackwell, M.D.  
Duke Cancer Institute Duke University Medical Center Email: kimberly.blackwell@duke.edu Janice Lu, M.D. Keck School of Medicine University of Southern California Email: Janice.Lu@med.usc.edu Philippe Bedard, M.D. Princess Margaret Cancer Centre Email: philippe.bedard@uhn.ca Mark Pegram, M.D. Stanford Medicine Cancer Institute Email: mpegram@stanford.edu Matthew Goetz, M.D. Mayo Clinic Email: goetz.matthew@mayo.edu Amy Krie, M.D.  
Avera Cancer Institute Email: Amy.Krie@avera.org Carey **Anders, M.D.** Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Email: canders@med.unc.edu Timothy Pluard, M.D. St. Luke's Cancer Institute Email: tpluard@saint-lukes.org Melody Cobleigh, M.D.  
Rush University Medical Center Email: melody_cobleigh@rush.edu Andres Forero, M.D. University of Alabama Cancer Center Email: aforero@uabmc.edu Daniel Hayes, M.D. University of Michigan Email: hayesdf@umich.edu Marc Lippman, M.D. University of Miami Email: mlippman@med.miami.edu Julie Nangia, M.D. Baylor College of Medicine Email: nangia@bcm.edu Massimo Cristofanilli, M.D.  
Northwestern University Email: Massimo.Cristofanilli@nm.org  
  
  
Trastuzumb 6mg/kg IV cycle 1 day 1, followed by 4mg/kg IV cycle 1 day 15, then 4 mg/kg IV on days 1-15 of subsequent cycles Continue therapy until disease progression, unacceptable adverse events, or patient refusal
a phase ii study of
glossary of abbreviations

| Glossary of Abbreviations         |                                                                  |
|-----------------------------------|------------------------------------------------------------------|
| AE                                | Adverse event                                                    |
| ALT (SGPT)                        | Alanine transaminase (serum glutamate pyruvic transaminase)      |
| ANC                               | Absolute neutrophil count                                        |
| AST (SGOT)                        | Aspartate transaminase (serum glutamic oxaloacetic transaminase) |
| BUN                               | Blood urea nitrogen                                              |
| CBC                               | Complete blood count                                             |
| CLIA                              | Clinical Laboratory Improvement Amendments                       |
| CNS                               | Central nervous system                                           |
| CR                                | Complete remission                                               |
| CRF                               | Case report form                                                 |
| CT                                | Computed tomography                                              |
| CTCAE                             | Common Terminology Criteria for Adverse Events                   |
| DLTs                              | Dose Limiting Toxicities                                         |
| DNA                               | deoxyribonucleic acid                                            |
| DSM                               | Data and Safety Monitoring                                       |
| DSMC                              | Data Safety Monitoring Committee                                 |
| EC                                | Ethics Committee                                                 |
| ECG (or EKG)                      | Electrocardiogram                                                |
| ECOG                              | Eastern Cooperative Oncology Group                               |
| EDTA                              | ethylenediaminetetraacetic acid                                  |
| EGFR                              | Epidermal growth factor receptor                                 |
| ER                                | Estrogen Receptor                                                |
| FDA                               | Food and Drug Administration                                     |
| FISH                              | fluorescent in situ hybridization                                |
| GCP                               | Good Clinical Practice                                           |
| HER2                              | Human epidermal growth factor receptor 2                         |
| HHS                               | Department of Health and Human Services'                         |
| HIV                               | Human Immunodeficiency Virus                                     |
| HRPO                              | Human Research Protection Office (IRB)                           |
| IHC                               | Immunohistochemistry                                             |
| IND                               | Investigational New Drug                                         |
| IRB                               | Institutional Review Board                                       |
| ITT                               | Intent-to-treat                                                  |
| IULN                              | Institutional Upper Limit of Normal                              |
| ILLN                              | Institutional Lower Limit of Normal                              |
| IV                                | Intravenous (i.v.)                                               |
| LDH                               | Lactate dehydrogenase                                            |
| LPS                               | lipopolysaccharide                                               |
| MedDRA                            | Medical Dictionary for Regulatory Activity                       |
| MRI                               | Magnetic resonance imaging                                       |
| MTD                               | Maximum tolerated dose                                           |
| Version 03/16/2020 - Amendment 13 | Page 7 of 87                                                     |  
| NCCN   | National Cancer Center Network                           |
|--------|----------------------------------------------------------|
| NCI    | National Cancer Institute                                |
| NIH    | National Institutes of Health                            |
| OHRP   | Office of Human Research Protections                     |
| ORR    | Overall response rate                                    |
| OS     | Overall survival                                         |
| PB     | Peripheral blood                                         |
| PBMC   | Peripheral blood mononuclear cell                        |
| PD     | Progressive disease                                      |
| PI     | Principal investigator                                   |
| PR     | Partial response (Partial remission)                     |
| QASMC  | Quality Assurance and Safety Monitoring Committee        |
| RBC    | Red blood cell (count)                                   |
| RECIST | Response Evaluation Criteria in Solid Tumors (Committee) |
| RFS    | Relapse free survival                                    |
| RR     | Response rate                                            |
| SAE    | Serious adverse event                                    |
| SCC    | Siteman Cancer Center                                    |
| SD     | Stable disease                                           |
| TF     | Treatment failure                                        |
| TTP    | Time to progression                                      |
| WBC    | White blood cell (count)                                 |
| WHO    | World Health Organization                                |  
| Table of Contents                                                                                                              |                                                                                                                                                              |
|--------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|
| SCHEMA - Part I (closed)                                                                                                       |                                                                                                                                                              |
| SCHEMA - Part II .                                                                                                             |                                                                                                                                                              |
| 1.0                                                                                                                            | BACKGROUND AND RATIONALE                                                                                                                                     |
| 1.1                                                                                                                            | Metastatic HER2 Negative Breast Cancer                                                                                                                       |
| 1.2                                                                                                                            | Neratinib                                                                                                                                                    |
| Fulvestrant                                                                                                                    |                                                                                                                                                              |
| 1.3                                                                                                                            |                                                                                                                                                              |
| 1.4                                                                                                                            | Trastuzumab (Herceptin)                                                                                                                                      |
| 1.5                                                                                                                            | HER2 (ERBB2)                                                                                                                                                 |
| 1.6                                                                                                                            | M Mutation in HER2 (ERBB2)                                                                                                                                   |
| 1.7                                                                                                                            | Preliminary Results from Part I with Single Agent Neratinib ................................................................20                               |
| 1.8                                                                                                                            | Correlative Studies Background .                                                                                                                             |
| 2.0                                                                                                                            | OBJECTIVES .                                                                                                                                                 |
| Primary Objectives .                                                                                                           |                                                                                                                                                              |
| 2.1                                                                                                                            |                                                                                                                                                              |
| 2.2                                                                                                                            | Secondary Objectives                                                                                                                                         |
| 2.3                                                                                                                            | Exploratory Objectives .                                                                                                                                     |
| 3.0                                                                                                                            | PATIENT SELECTION                                                                                                                                            |
| 3.1                                                                                                                            | Pre-registration (for patients with unknown HER2 mutation status to have tumor tissue                                                                        |
| screened centrally by Wash U GPS laboratory)                                                                                   |                                                                                                                                                              |
| 3.2                                                                                                                            | Registration (for patients positive for HER2 mutation to receive study treatments) ......................23                                                  |
| 3.3                                                                                                                            | Inclusion of Women and Minorities                                                                                                                            |
| 4.0                                                                                                                            | REGISTRATION PROCEDURES                                                                                                                                      |
| Pre-Registration (for patients with unknown HER2 mutation status to have tumor tissue                                          |                                                                                                                                                              |
| 4.1                                                                                                                            |                                                                                                                                                              |
| screened centrally by Wash U GPS laboratory)                                                                                   |                                                                                                                                                              |
| 4.2                                                                                                                            | Registration for Study Therapy .                                                                                                                             |
| 4.3                                                                                                                            | Recruitment through Army of Women                                                                                                                            |
| 4.4                                                                                                                            | Recruitment from Outside Practice.                                                                                                                           |
| 5.0                                                                                                                            | TUMOR HER2 SEQUENCING                                                                                                                                        |
| Tumor HER2 sequencing at Washington University CLIA lab following pre-registration for                                         |                                                                                                                                                              |
| 5.1                                                                                                                            |                                                                                                                                                              |
| eligibility                                                                                                                    |                                                                                                                                                              |
| Tumor or circulating tumor DNA HER2 Mutation/Variants Identified at Laboratories other                                         |                                                                                                                                                              |
| 5.2                                                                                                                            |                                                                                                                                                              |
| than GPS@WU                                                                                                                    |                                                                                                                                                              |
| 5.3                                                                                                                            | Eligible HER2 mutations                                                                                                                                      |
| 5.4                                                                                                                            | Germline DNA Sequencing for HER2 to determine eligibility .....................................................................30                            |
| 5.5                                                                                                                            | HER2 Mutation Testing by ctDNA                                                                                                                               |
| 6.0                                                                                                                            | TREATMENT PLAN .                                                                                                                                             |
| 6.1                                                                                                                            | Neratinib Administration                                                                                                                                     |
| Concurrent Prophylactic Administration of Loperamide .......................................................................31 |                                                                                                                                                              |
| 6.2                                                                                                                            | Fulvestrant Administration .                                                                                                                                 |
| 6.3                                                                                                                            | General Concomitant Medication and Supportive Care Guidelines ....................................................31                                         |
| 6.4                                                                                                                            |                                                                                                                                                              |
| 6.5                                                                                                                            | W Women of Childbearing Potential ........................................................................................................................37 |
| 6.6                                                                                                                            | D DURATION OF THE RAPY .                                                                                                                                     |
| 6.7                                                                                                                            | D DURATION OF FOLLOW-UP                                                                                                                                      |
| 7.0                                                                                                                            | DOSE DELAYS/DOSE MODIFICATIONS AND AE MANAGEMENT ........................................................38                                                  |
| 7.1                                                                                                                            | Neratinib Dose Administration Table                                                                                                                          |
| 7.2                                                                                                                            | General Dose Adjustments for Neratinib-Related Toxicities ......................................................................38                           |
| 7.3                                                                                                                            | Guidelines for the Management of Diarrhea .....................................................................................................39            |
| 7.4                                                                                                                            | Guidelines for the Management of Changes in Liver Function Tests ................................................42                                          |
| 7.5                                                                                                                            | Guidelines for the Management of LVEF Declines                                                                                                               |
| 7.6                                                                                                                            | SUBJECT COMPLIANCE .                                                                                                                                         |
| 7.7                                                                                                                            | Fulvestrant Dose Modifications                                                                                                                               |
| 7.8                                                                                                                            | Trastuzumab (or FDA Approved Biosimilar) Dose Modifications .............................................................4                                   |
| 8.0                                                                                                                            | REGULATORY AND REPORTING REQUIREMENTS ..............................................................................................44                       |
| Reporting to the Human Research Protection Office (HRPO) at Washington University.............45                               |                                                                                                                                                              |
| 8.1                                                                                                                            | Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at Washington                                                                     |
| 8.2                                                                                                                            |                                                                                                                                                              |
| UNIVERSITY .                                                                                                                   |                                                                                                                                                              |
| 8.3                                                                                                                            | Secondary Sites Reporting Requirements ....................................................................................................45                |
| Version 03/16/2020 - Amendment 13                                                                                              | Page 9 of 87                                                                                                                                                 |  
| 8.4                                                                                                                                              | Reporting to Secondary Sites ...............................................................................................................................45            |
|--------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 8.5                                                                                                                                              | Reporting to the FDA...................................................................................................................................................46 |
| 8.6                                                                                                                                              | Reporting to Puma Biotechnology, Inc. ...........................................................................................................47                       |
| 8.7                                                                                                                                              | Timeframe for Reporting Required Events....................................................................................................47                             |
| 9.0                                                                                                                                              | PHARMACEUTICAL INFORMATION                                                                                                                                                |
| 9.1                                                                                                                                              | Neratinib                                                                                                                                                                 |
| 9.2                                                                                                                                              | Fulvestrant                                                                                                                                                               |
| 10.0                                                                                                                                             | CORRELATIVE STUDIES                                                                                                                                                       |
| 10.1                                                                                                                                             | Required archival tumor sample for research: ............................................................................................................50               |
| Required research blood collection .                                                                                                             |                                                                                                                                                                           |
| 10.2                                                                                                                                             | Optional research tumor biopsy (kits will be provided): .                                                                                                                 |
| 10.3                                                                                                                                             |                                                                                                                                                                           |
| 10.4                                                                                                                                             | Reporting of a CLIA multi-gene NGS on tumor biopsy collected at disease progression on study                                                                              |
| drug:                                                                                                                                            | 50                                                                                                                                                                        |
| 10.5                                                                                                                                             | Correlative Science Study Calendar .                                                                                                                                      |
| 11.0                                                                                                                                             | STUDY CALENDAR                                                                                                                                                            |
| 12.0                                                                                                                                             | DATA SUBMISSION SCHEDULE .                                                                                                                                                |
| 13.0                                                                                                                                             | MEASUREMENT OF EFFECT                                                                                                                                                     |
| Antitumor Effect - Solid Tumors                                                                                                                  |                                                                                                                                                                           |
| 13.1                                                                                                                                             |                                                                                                                                                                           |
| 13.2                                                                                                                                             | Disease Parameters .                                                                                                                                                      |
| 13.3                                                                                                                                             | Methods for Evaluation of Measurable Disease                                                                                                                              |
| 13.4                                                                                                                                             | Response Criteria                                                                                                                                                         |
| 14.0                                                                                                                                             | DATA AND SAFETY MONITORING .................................................................................................................................61            |
| 14.1                                                                                                                                             | Independent Research Monitor ..........................................................................................................................................62 |
| 15.0                                                                                                                                             | A AUDITING .                                                                                                                                                              |
| STATISTICAL CONSIDERATIONS                                                                                                                       |                                                                                                                                                                           |
| 16.0                                                                                                                                             |                                                                                                                                                                           |
| 16.1                                                                                                                                             | STUDY Objectives .                                                                                                                                                        |
| 16.2                                                                                                                                             | Study Design and Sample Size Justification .....................................................................................................64                        |
| 16.3                                                                                                                                             | DATA ANALYSIS                                                                                                                                                             |
| 17.0   MULTICENTER REGULATORY REQUIREMENTS                                                                                                       |                                                                                                                                                                           |
| 18.0                                                                                                                                             | REFERENCES .                                                                                                                                                              |
| APPENDIX A: ECOG PERFORMANCE STATUS SCALE ....................................................................................................74 |                                                                                                                                                                           |
| APPENDIX B PATIENT'S MEDICATION DIARY                                                                                                            |                                                                                                                                                                           |
| APPENDIX C: PATIENT INSTRUCTIONS FOR THE MANAGEMENT OF DIARRHEA ..............................76                                                 |                                                                                                                                                                           |
| APPENDIX D: RECIST 1.1 Tumor Evaluation Form                                                                                                     |                                                                                                                                                                           |
| APPENDIX E: Definitions                                                                                                                          |                                                                                                                                                                           |
| APPENDIX F: Reporting Timelines.                                                                                                                 |                                                                                                                                                                           |
| APPENDIX G: Serious Adverse Event Reporting Cover Sheet                                                                                          |                                                                                                                                                                           |
a phase ii study of
1.0 background and rationale 1.1

Over 80% of breast cancers are HER2 negative (HER2-), defined by the lack of *HER2* gene amplification, for which targeted therapies are lacking. The prognosis for the estrogen receptor negative (ER-)/HER2- subset is particularly poor[1]. Although patients with ER+/HER2- disease fair better, resistance to endocrine manipulation develops almost invariably[2]. The median survival is only 18-24 months when chemotherapy is necessary in the metastatic setting[3]. There is a significant unmet clinical need to uncover new drug targets to improve the outcome of these patients.  
HER2/neu (ERBB2) is a well established proto-oncogene that encodes a receptor tyrosine kinase of the Epidermal Growth Factor Receptor (EGFR) family[4]. Receptor activation via heterodimerization between HER2 and EGFR and other family members leads to strong mitogenic signals and cellular proliferation[5]. HER2-targeted agents are among the most successful cancer therapeutics in the clinic, revolutionizing the care for patients with HER2+ breast cancer [6-11]. HER2+ breast cancer is defined clinically by either HER2 protein overexpression on immunohistochemistry (IHC) or HER2 gene amplification by fluorescent in situ hybridization (FISH) testing and occurs in approximately 15-20% of all breast cancer cases. HER2- breast cancers as a whole have not been shown to derive benefit from these highly effective agents[12]. Results from The Cancer Genome Atlas (TCGA) breast cancer project demonstrated that a small subpopulation of HER2- breast cancer carry mutations in HER2, with majority being in the kinase domain and compilation of data on 1,391 breast cancers documented 22 cases of somatic HER2 mutation, a mutation rate of 1.6% [13-15]. In addition, laboratory investigations indicated that many of these mutations are activating mutations, with the capacity to induce cellular transformation and importantly an enhanced susceptibility to the inhibitory effects of anti-HER2 agents (see Preliminary Investigation section). These discoveries raised the possibility that mutations in HER2 could be effectively targeted by the well established anti-HER2 agents in the clinic. The objective of this application is to establish HER2 mutation as a therapeutic target in HER2- breast cancer. We therefore propose a phase II study of neratinib, a pan HER inhibitor, in metastatic HER2-/HER2 mutated breast cancers for tumor response. As of January 17, 2015, the study enrolled 11 patients with HER2 mutations, among which 10 patients had ER+ breast cancer. Considering the well-established signaling crosstalk between ER
and HER2 in ER+ HER2+ breast cancer16,17, we propose to add the ER down regulator fulvestrant to neratinib in the ER+ subpopulation in the second part of this trial. Although the prevalence of HER2 mutation is low, because breast cancer is so common this would account for approximately 4,000 new cases each year in the US alone, an annual incidence that is very similar to chronic myeloid leukemia (CML), a disease transformed by the introduction of tyrosine kinase inhibitors.
a phase ii study of
1.2 neratinib 1.2.1 mechanism of

Neratinib (PB-272) is a potent irreversible pan HERB inhibitor. Neratinib is an orally available small molecule that inhibits HER-1, HER-2, and HER-4 at the intracellular tyrosine kinase domains, a mechanism of action that is different from trastuzumab[16-18]. Neratinib reduces HER-1 and HER-2 autophosphorylation, downstream signaling, and the growth of HER-1 and HER-2 dependent cell lines. Preclinical data suggest that neratinib will have antitumor activity in HER-1 - and/or HER-2-expressing carcinoma cell lines, with cellular IC50 <100 nM.
a phase ii study of
1.2.2 preclinical data

Neratinib is highly active against cell lines overexpressing either HER-2 or HER-1. Neratinib blocks HER receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation. Neratinib most likely inhibits kinase activity through irreversible binding at a targeted cysteine residue in the adenosine triphosphate binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, neratinib treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. In vivo, neratinib is active in HER-2- and HER-1-dependent tumor xenograft models, when administered orally on a once-a-day schedule. Overall, neratinib is less potent against HER1 dependent tumors than HER-2-dependent tumors in vivo, even though it has equivalent activity against the 2 kinases in vitro.
a phase ii study of
1.2.3 neratinib phase 1 and

Preliminary pharmacokinetic analyses demonstrated that neratinib absorption was relatively slow, and the maximum concentration (Cmax) was generally attained within 3 to 6 hours. After oral administration, the neratinib Cmax and area under the concentration versus time curve (AUC) increased in a dose-dependent manner in general. Mean steadystate Cmax and AUC values were 70.1 ng/mL and 975 ng·h/mL for the 180-mg dose group, respectively, 73.5 ng/mL and 939 ng·h/mL for the 240-mg dose group, respectively, 90.4 ng/mL and 1333 ng·h/mL for the 320-mg dose group, respectively, and 105 ng/mL and 1704 ng·h/mL for the highest dose of 400 mg, respectively. The neratinib exposure (AUC) increased 1.2- to 2.7-fold (mean accumulation ratio) when comparing the steady-state exposure on day 21 after repeated daily administration with the exposure on day 1 after administration of 80 to 400 mg of neratinib. The mean accumulation ratio was 1.2 after a 240-mg dose, indicating no significant accumulation of neratinib after repeated daily dose administration at the dose to be used in this proposed trial. The data indicated a slow distribution of neratinib with a large apparent volume of distribution (Vz/F on day 1: about 3188 to 6181 L) after oral absorption. After oral administration on day 1, neratinib was eliminated with a mean apparent terminal half-life (t½) of approximately 13 to 17 hours.  
There was moderate to large variability in neratinib t½, Cmax, and AUC; coefficients of variation generally ranged from 8% to 90%. In the single agent, first in human, phase 1 study 3144A1-102-US, neratinib single agent administered orally was given in increasing doses from 40 mg to 400 mg. Diarrhea was the main dose limiting toxicity (DLT) observed. At the 400-mg dose level, 4 of 6 subjects had DLT of grade 3 diarrhea. Thus, the maximum tolerated dose (MTD) was determined to be 320 mg per day. However, due to the frequency and severity of neratinib-related diarrhea observed in subsequent subjects treated at the MTD (34% of subjects reported grade 3 diarrhea), the MTD was reevaluated to 240 mg per day. Neratinib monotherapy phase 2 data showed that oral daily doses of 240 mg of neratinib were generally well tolerated with significant antitumor activity in subjects with HER-2-positive advanced breast cancer [19]. In this trial, patients will receive 240 mg continuous daily oral doses of neratinib. Since diarrhea has been tolerable with prophylactic use of loperamide during the first cycle therapy in the first 3 patients treated in this trial, we elected to allow dose escalation to 320mg in subsequent cycles, with initial prophylactic use of loperamide, in patients who tolerated the 240mg daily dosing.
a phase ii study of
1.2.4 single-agent activity of neratinib

Neratinib as a single agent has been studied in a phase 2 trial in subjects with metastatic HER-2 positive breast cancer [19]. Sixty-six (66) subjects with prior trastuzumab based therapy were enrolled into arm A; 70 subjects without any prior trastuzumab exposure were enrolled into arm B. Objective response rate and median progression free survival were used as estimates of antitumor activity. For patients with prior trastuzumab treatment, the objective response rate was 24% (95% CI, 14% to 36%); for patients with no prior trastuzumab treatment, the objective response rate was 56% (95% CI, 43% to 69%). Six and eight patients with and without prior trastuzumab treatment, respectively, had stable disease for at least 24 weeks that yielded clinical benefit rates of 33% and 69%. The median duration of objective tumor response was 39.3 weeks (95% CI, 32.3 weeks to 93.7 weeks) for patients with prior trastuzumab treatment and 52.4 weeks (95% CI, 33.1 weeks to not estimable) for patients with no prior trastuzumab treatment. The median onset of first complete response or partial response was rapid at 7.1 weeks for patients both with and without prior trastuzumab treatment (prior trastuzumab range, 3.0 to 24.0 weeks; no prior trastuzumab range, 3.1 to 15.6 weeks; 75th percentile, 7.1 weeks for both). Similar efficacy results were obtained by investigator assessment.
a phase ii study of
1.2.5 adverse event profile of

In the neratinib monotherapy phase II breast cancer study, diarrhea was the only grades 3 to 4 adverse event that occurred in more than 10% of patients (28 patients with grade 3; one patient with grade 4) and was the predominant adverse event associated with dose reductions, which were required in 29% of patients with prior trastuzumab treatment and in 4% of patients with no prior trastuzumab treatment. However, only one patient (with prior trastuzumab treatment) discontinued treatment because of grade 2 diarrhea. The onset of diarrhea occurred early in the course of therapy (median time to onset, 2 to 3 days) and lasted a median of 5 to 7 days per event. Diarrhea severity abated during multiple weeks of treatment. During study week 1, diarrhea occurred in 70% to 90% of patients and decreased to 10% to 15% of patients after month 2. Only one patient had grade 3 diarrhea after month 2. With use of antidiarrheals and dose modification, neratinib treatment was continued in 99% of patients, despite this common lower gastrointestinal adverse effect. Mild, often transient, skin rashes were observed in approximately one quarter of the patients. Other common AEs are nausea, vomiting, fatigue, anorexia, abdominal pain, asthenia, dehydration, rash, elevated alanine aminotransferase (ALT), and elevated aspartate
Page 13 aminotransferase (AST). The above AEs are considered to be adverse drug reactions for neratinib. As opposed to treatment with other HER-2 targeted agents, such as trastuzumab, cardiotoxicity has not been observed with neratinib, even in subjects previously treated with anthracyclines and/or trastuzumab.  
Interstitial lung disease, which can sometimes be fatal, has been reported with other oral tyrosine kinase inhibitors that target EGFR (HER-1) +/- HER2 (HER-2), including lapatinib, gefitinib, and erlotinib. Pneumonitis which was considered to be drug related, according to the investigator's assessment, has been reported in clinical studies of neratinib. Subjects receiving neratinib should be monitored for acute onset or worsening of pulmonary symptoms such as dyspnea, cough, and fever and treated appropriately. There has been one event of bone marrow suppression reported on December 9, 2009, in a subject who was taking neratinib monotherapy for 4 months without any other clear cause. This resulted in neutropenia, and thrombocytopenia. Refer to the most recent version of the investigator's brochure for a summary of findings from nonclinical studies that potentially have clinical significance and from clinical studies that are relevant to the study. Also, refer to the most recent version of the investigator's brochure for a summary of the known and potential risks and benefits to human subjects.
a phase ii study of
1.3 fulvestrant 1.3.1 fulvestrant in

Fulvestrant (Faslodex) is an estrogen receptor antagonist that binds to ER and leads to its degradation, a distinct mechanism of action from other endocrine therapy agents that target ER. Fulvestrant blocks ER action without agonist effect. It is FDA approved for patients who have disease progression on other antiestrogen agents in the advanced breast cancer setting. In two phase III trials, fulvestrant 250 mg monthly was found to be at least as effective as anastrozole with respect to disease progression and overall survival [20, 21]. In the EFECT trial, fulvestrant 250 mg monthly was found to be at least as effective as exemestane in patients with metastatic ER+ breast cancer resistant to non-steroidal AIs[22]. In the CONFIRM trial, high-dose fulvestrant at 500 mg monthly was found to be more effective compared to the previous dosing of 250 mg monthly, which prompted FDA's subsequent approval of fulvestrant 500 mg every 28 days, following a loading dose of 500 mg on days 1 and 15 during the first month of therapy for refractory metastatic breast cancer[23]. Overall, fulvestrant demonstrated an overall response rate of approximately 10% in aromatase inhibitor resistant, fulvestrant naive ER+ HER2- breast cancers [24, 25]. There is no data at this time regarding the activity of fulvestrant in ER+ HER2- breast cancer with HER2 mutations. However its efficacy in ER+ HER2+ breast cancer has been described in a retrospective study [26]. In this study, fulvestrant resulted in an overall response rate of 9% and clinical benefit rate of 42% (43/101 patients), with anti-tumor activity observed in up to the fourth line of endocrine therapy and seventh line of overall therapy [26]. Since the response rate approximated 10% in both HER2+ and HER2- population based on the studies described above, we assume a similar response rate of 10% in fulvestrant naïve HER2 mutant patient population in this trial.
a phase ii study of
1.3.2 fulvestrant in combination with

Although there is no data in the literature for the combination of fulvestrant and neratinib in ER+ breast cancer, fulvestrant was combined with the HER1/HER2 inhibitor lapatinib in a randomized phase III trial of fulvestrant with or without lapatinib in patients with AI
resistant metastatic ER+ HER2 1+, 2+, 3+ [25]. The study demonstrated an overall response rate of 9% in the fulvestrant plus placebo arm and 20% in the fulvestrant plus lapatinib arm, with anticipated side effects observed with single agents alone [25]. Since fulvestrant is well tolerated, there is no obvious theoretical pharmacokinetic interaction between neratinib and fulvestrant, and the mechanism of action of neratinib is similar to lapatinib, we do not anticipate the side effects profile be different from single agent therapy with neratinib and fulvestrant.
a phase ii study of
1.3.3 adverse event profile of

The most common adverse reactions reported in the fulvestrant study groups, regardless of causality, were GI symptoms including nausea and vomiting (26%/13%), constipation (12.5%), diarrhea (12.3%) and abdominal pain (11.8%); as well as headache (18.9%); back pain (15.4%); hot flashes (17.7%); and pharyngitis (16.1%). Vaginal bleeding was reported in < 1% of patients and occurred most commonly during the first 6 weeks after changing from existing hormonal treatment to fulvestrant. If vaginal bleeding persists, further evaluation is required. Injection site reaction with mild transient pain (9.1-11.6%) and inflammation were seen with fulvestrant. Seven percent of patients (1% of treatments) given a single 5 ml IM injection and 27% of patients (4.6% of treatments) given 2 x 2.5 ml IM injections experienced reactions. Additional adverse reactions occurring in > 5% of patients treated with fulvestrant during clinical trials include asthenia (weakness) (68.3%), bone pain (15.8%), dyspnea (14.9%), increased cough (10.4%), pelvic pain (10%), anorexia (9%), peripheral edema (9%), rash (unspecified) (7.3%), chest pain (unspecified) (7.1%), dizziness (6.9%), insomnia (6.9%), fever (6.4%), paresthesias (6.4%), urinary tract infection (6.1%), depression (5.7%), anxiety (5%), diaphoresis (5%), and anemia (4.5%). Other adverse events reported as fulvestrant-related and occurring infrequently (< 1%) include thromboembolism, myalgia, vertigo, and leukopenia. In addition, hypersensitivity reactions such as angioedema and urticaria have been infrequently reported.
a phase ii study of
1.4 trastuzumab (herceptin) 1.4.1 mechanisms

Trastuzumab is an IgG1 kappa that selectively binds to the extracellular domain of the HER2 receptor. Trastuzumab inhibits HER2+ breast cancer cell proliferation in preclinical models[27, 28]. In addition, trastuzumab is a mediator of antibody-dependent cellular cytotoxicity (ADCC)[29, 30].
a phase ii study of
1.4.2 clinical studies

Trastuzumab is effective either as a single agent or in combination with a variety of chemotherapy agents for HER2 positive breast cancer [31]. In a study of single agent
Page 15 trastuzumab, administered 4mg/kg loading dose followed by 2mg/kg weekly, or 8mg/kg loading dose and 4mg/kg weekly, as first-line therapy for HER2-overexpressing breast cancer, the objective response rate was 35% in HER2 3+ and 34% in HER2 amplified cases[32]. In another phase II trial of single agent trastuzumab in HER2+ breast cancer, trastuzumab was administered 8 mg/kg then 6 mg/kg Q3 weeks and the clinical benefit rate was 33%[33]. In patients who had received extensive prior therapies, the objective response rate was found to be around 15% [34]. Trastuzumab also demonstrated its ability to reduce cancer recurrence and death in the adjuvant setting as demonstrated by the large multicenter randomized controlled trials [8, 35]. Trastuzumab is a recombinant DNA-derived humanized monoclonal antibody against HER2 and is approved by FDA for treatment of metastatic and early stage breast cancer that overexpresses HER2.
a phase ii study of
1.4.3 dual targeting in her2+

Approximately 15% of HER2+ breast cancer patients relapse after initial therapy with trastuzumab. [36] Preclinical evidence has shown synergistic interaction between trastuzumab and other HER-2 targeted agents, and several clinical studies have shown that dual HER2 targeting may prolong time to resistance and improve survival outcomes.[37] A double-blind phase III study (CLEOPATRA) compared first-line trastuzumab plus pertuzumab plus docetaxel with trastuzumab plus placebo plus docetaxel in 808 patients with metastatic HER2+ breast cancer. The pertuzumab arm had longer median progression free survival (PFS) (19 months vs. 12 months; HR=0.62; 95% CI, 0.51-0.75; P <0.001) and overall survival (OS) (not yet reached in pertuzumab arm vs. 37.6 months; HR 0.66; 95% CI, 0.52-0.84). [38] A randomized phase III, study of single agent lapatinib vs. combination of trastuzumab plus lapatinib in patients with HER2+ metastatic breast cancer who had disease progression on prior trastuzumab-based therapy showed superior PFS (HR=0.73; 95% CI, 0.57-0.93; P =0.008) and clinical benefit rate (CBR) (24.7% in the combination arm vs. 12.4% in the monotherapy arm; P =0.01) for the combination arm. There was also a median OS advantage seen in the combination arm (14 months vs 9.5 months; HR 0.74; 95% CI 0.75-0.97; P=0.026). The side effect profile is favorable with the combination arm showing a higher incidence of diarrhea (P=0.03). [39] A meta-analysis examining the cardiac toxicity in breast cancer patients treated with dual HER2 blockade showed no significant increase in cardiotoxicity, although the trials generally only included patients with good baseline cardiac function and patients who have tolerated prior treatment with trastuzumab,[40] Long term outcomes on cardiac toxicity remain to be seen.
a phase ii study of
1.4.4 neratinib in combination with

A phase I/II, nonrandomized, open label study of neratinib in combination with trastuzumab was performed in women with advanced HER2+ breast cancer.[41] The phase I, doseescalation phase enrolled 8 patients. No DLT was reported and the recommended dose of neratinib was 240mg daily in combination with trastuzumab 4mg/kg loading dose followed by 2mg/kg weekly thereafter. In phase II, 37 patients received neratinib and trastuzumab at the recommended dose and 28 patients were evaluable for efficacy. In the evaluable population, the 16-week PFS rate was 44.8% (95% CI, 25.9 - 62.1%), and the median PFS was 15.9 weeks (95% CI 15.1 - 31.3), with data censored for 5 patients. Two patients had the best overall response of complete response (CR), 6 patients had partial response (PR) and 14 patients had stable disease (SD). The overall response rate was 28.6% (95% CI, 13.2 - 48.7%) and the clinical benefit rate (CBR) was 35.7% (95% CI, 18.6 - 55.9%).[41] In this trial, trastuzumab (or FDA approved biosimilar) will be added to the study drug neratinib or neratinib and fulvestrant (ER+ cohort, Part II of the trial) at disease progression if insurance coverage is obtained to explore the activity of combined HER2 targeting in HER2 mutant breast cancer.
a phase ii study of
1.5 her2 (erbb2)

HER2/neu (ERBB2), located on chromosomal 17q21.1, is a well established proto-oncogene that encodes HER2, a transmembrane tyrosine kinase receptor of the EGF receptor (EGFR) family composed of four members (EGFR/HER1, HER2, HER3 and HER4) that contain an extracellular ligand-binding domain, a lipophilic transmembrane domain, and an intracellular tyrosine kinase domain[4]. Receptor activation via heterodimerization between HER2 and EGFR and other members leads to strong mitogenic signals and cellular proliferation[5].  
The identification of *HER2* gene amplification and the subsequent invent of HER2-targeted agents have revolutionized the care of patients with breast cancer [11]. Approximately 20% of invasive breast cancer cases carry *HER2* gene amplification, leading to increased HER2 protein expression, an aggressive clinical behavior and a decreased disease free and overall survival [42-44]. Trastuzumab, a monoclonal antibody against HER2, was the first anti-HER2 agent which was found to markedly improve the survival of patients with HER2 positive breast cancer in both metastatic and early stage setting [6-10], establishing one of the first molecular cancer therapies. Multiple agents are currently available in clinical practice either approved by FDA or through clinical trials for patients with HER2 positive breast cancer defined by either HER2 overexpression or gene amplification [11].
a phase ii study of
1.6 mutation in her2 (erbb2)

The vast majority of HER2 mutations were identified in patients with HER2 negative disease who would not have been eligible to receive HER2 targeted drugs under the current standard of care. In addition to the TCGA-breast cancer project patients, we compiled data from 6 other breast cancer sequencing projects[15, 45-49] and identified 15 HER2 somatic mutations in 22 out of the 1391 patients, an overall incidence of 1.6% (Fig. 1). Mild enrichment for HER2 mutations may occur in lobular breast cancer as three HER2 somatic mutations were observed in 39 lobular breast cases in the TCGA study[50] and three were observed among 113 lobular cases in Shah et al., suggesting a prevalence of 4% in this histological subtype[46]. Mutations clustered in two locations, the extracellular domain (ECD) at aa 309-310 (5 of 25) and the kinase domain between aa 755-781 (17 of 25).  
Blue circles represent individual cases.
a phase ii study of
1.6.2 preclinical functional characterization of

Effects on kinase activities: We successfully expressed HER2 kinase domain constructs for WT, and V777L, D769H, V842I mutants and purified them to >80% purity. *In vitro* kinase assays demonstrated that both the D769H-HER2 and the V777L-HER2 had greater tyrosine kinase activity than WT-HER2 (Figure 2, left) and were sensitive to lapatinib in vitro (Figure 2, right). In contrast, V842I-HER2 showed modestly increased kinase activity and a small shift in lapatinib *in vitro* IC50 (22 vs 10 nM).  
  
Effects **on Signaling and Growth:** HER2 mutations were introduced into nontransformed, MCF10A breast epithelial cells using a retroviral vector. The V777L, D769H, D769Y, P780ins, V842I, and R896C mutants are activating mutations that increased HER2 autophosphorylation and substrate protein (phospholipase γ C1, PLCγ) phosphorylation relative to WT HER2 (Fig. 3). Modest increases in HER2, PLCγ, and EGFR phosphorylation were seen with G309A, L755S, and R678Q. HER2 del.755-759 showed a marked decrease in HER2 and PLCγ phosphorylation, but increased EGFR phosphorylation, which suggests that del.755-759 has an increased ability to activate other HER/ErbB family members. The ability of various TKI drugs to block signaling from HER2 mutations was tested (not shown) and we observed that L755S mutation is a lapatinib-resistant mutation that is sensitive to neratinib and canertinib. While all three drugs are HER2/EGFR selective, lapatinib is a reversible inhibitor[52]. In contrast, neratinib and canertinib are irreversible inhibitors that form a covalent bond with a Cys residue in HER2's active site[53].  
Fig. 3. Activation of downstream signaling by HER2 mutations. KD indicates kinase domain mutations; JM: juxtamembrane; ECD: extra-cellular domain.  
The effect on cell growth and  
  
 morphology in three dimensional cultures was measured. As previously reported[54, 55],
MCF10A-HER2 WT formed spherical structures (Fig. 4, upper left). MCF10A cells bearing HER2 V777L, D769H, D769Y, P780ins, V842I, R896C, or G309A mutations formed irregular structures with spiculated, invading protrusions (Fig. 4, top row). Addition of the HER2 antibody, trastuzumab, restored the spherical morphology to these mutations (Fig. 4, middle row). HER2 L755S, del.755-759, and R678Q formed spherical structures, similar to WT HER2. MCF10A cells are an EGF-dependent cell line and require supplementary EGF in their media. Addition of lapatinib, a dual HER2/EGFR inhibitor, blocked the growth of MCF10A-HER2 WT cells and most of the HER2 mutant cells (Fig. 4, lower row). Interestingly, L755S and del.755-759 were resistant to this dose of lapatinib and continued to form large spherical structures, but they were sensitive to the irreversible inhibitor, neratinib (Fig. 5). The del.755-759 mutation was sensitive to gefitinib (Fig. 5), consistent with the increased activation of EGFR observed in HER2 del.755-759 mutant cells (Fig. 3). The ability of the HER2 mutations to  
 increase tumor formation in xenografts was also tested (Fig. 6). NIH3T3-HER2 WT cells served as the control cell line. NIH3T3- HER2 V777L, D769H, and G309A mutant cell lines had more rapid tumor growth than the HER2 WT control (p-values all <0.01, as indicated in the figure by **). The L755S, V842I, R678Q and mutations showed tumor growth indistinguishable from the HER2 WT control (Fig. 6). These mutations did have more rapid tumor growth than empty vector cells, suggesting that these mutations do not impair HER2 functioning. Xenograft experiments using MCF7 cell lines are in progress (not shown).  
Summary of HER2 preclinical **data:** To date, we have tested thirteen HER2 somatic mutations, of which seven are found to be activating mutations that are sensitive to HER2 targeted drugs: G309A, D769H, D769Y, V777L, P780ins, V842I and R896C[51]. In addition, a recent HER2 functional study by Greulich et al. also identified G309E and S310F as activating mutations[56]. Notably, the L755S mutation showed a lapatinibresistant phenotype, but was sensitive to neratinib[51].  
We hypothesize that tumors with mutations in HER2 can be effectively treated with neratinib. As a pilot study, we will conduct a 2-stage phase II single arm trial of neratinib in patients with HER2-/HER2 mutation+ metastatic breast cancer. Neratinib is chosen as it is an irreversible inhibitor of HER1, HER2 and HER4 rather than lapatinib which is a reversible inhibitor of HER1 and HER2. Importantly, neratinib has shown activity in lapatinib resistant HER2 mutations in our preclinical studies (Figure 5). HER2 mutation is present in 1.6% of primary breast cancer overall, but certain subpopulation of breast cancer may have higher incidence. We therefore plan to analyze the clinical and pathological features of tumors with or without HER2 mutation during this trial and preferentially screen those that are most likely to carry the HER2 mutations.
a phase ii study of
1.7 preliminary results from part

As of Jan 17, 2015, we have identified 16 patients with metastatic breast cancer carrying HER2  
 mutations (Table 1). Fifteen of these patients (94%) had ER+ disease. Among the first 10 patients with somatic Her2 mutations (9 with activating mutations and 1 with unknown significance), 1 patient with known activating mutation HER2 L755S had a partial response to neratinib. The study passed the efficacy criteria to continue enrollment to the 2nd stage (refer to statistical section).  
Because of the high incidence of ER positivity in HER2 mutated breast cancer and the well established crosstalk between ER and HER2 pathways, we propose to examine the combination of neratinib and fulvestrant in patients with ER+, HER2-/HER2 mutated breast cancers in Part II of the trial.  
Figure 6. HER2 induces growth of tumors in nude mice. 5 x 10 5NIH3T3 cells expressing indicated construct were injected subcutaneously.  
Histology N %  
Ductal 9 56%  
Lobular 6 38%  
Unknown 1 6%  
| Biomarkers   |
|--------------|  
ER +, PR + 7 44%  
ER+, PR - 8 50%  
  
ER-, PR - 1 6%
a phase ii study of
1.8 correlative studies background

Metastatic HER2- breast cancer is a heterogeneous group of diseases. Understanding the molecular and genomic structure of these tumors is the first step toward individualized therapy. In this protocol, additional tumor specimens at metastatic sites will be collected at baseline if possible when the biopsy is performed for sequencing analysis of HER2. All patients are required to consent to research on archival primary tumor specimens. Patients will be consented for research related molecular studies including genomic studies, such as whole or targeted genomic sequencing analysis, gene expression studies, for novel therapeutic target discovery. In addition, blood samples will be collected at baseline and post therapy for studies of circulating markers that correlate with breast cancer prognosis and treatment response.
a phase ii study of
2.0 objectives 2.1 primary objectives

1. Part I: To determine the clinical benefit (CR+PR+SD>6 months) of neratinib alone in patients with metastatic HER2- breast cancer that carry HER2 mutations.  
2. Part II fulvestrant-naïve ER+ cohort: To examine the clinical benefit (CR+PR+SD>6 months)
of neratinib in combination with fulvestrant in patients with fulvestrant-naïve metastatic HER2- , ER+ breast cancer carrying activating HER2 mutations.  
3. Part II fulvestrant-treated ER+ cohort: To examine the clinical benefit (CR+PR+SD≥6 months)
of neratinib in combination with fulvestrant in patients with metastatic HER2-, ER+ breast cancer carrying activating HER2 mutations previously treated with fulvestrant.  
4. Part II ER- cohort: To determine the clinical benefit rate of neratinib alone in patients with metastatic HER2-, ER- breast cancer carrying activating HER2 mutations.
a phase ii study of
2.2 secondary objectives

1. To determine the PFS of patients treated with neratinib alone in patients with metastatic HER2but HER2 mutated breast cancer by ER status and by HER2 mutations (activating vs. unknown significance).  
2. To assess the PFS and response rate of neratinib in combination with fulvestrant in patients with fulvestrant-naïve metastatic ER+ HER2- breast cancer carrying activating HER2 mutations.  
3. To assess the PFS and response rate of neratinib in combination with fulvestrant in patients with metastatic ER+ HER2- breast cancer carrying activating HER2 mutations previously treated with fulvestrant.  
4. To assess the safety profile and tolerability of neratinib in combination with fulvestrant in patients with metastatic ER+ HER2- breast cancer carrying activating HER2 mutations.  
5. To correlate the presence of HER2 mutation with histology subtype (invasive lobular vs.  
invasive ductal cancer), tumor grade (grade 1-2 vs 3), tumor staging at initial diagnosis (I vs. II or III vs. IV), disease free survival in HER2- breast cancer.
a phase ii study of
2.3 exploratory objectives

1. To compare the occurrence of HER2 mutation in paired primary and metastatic sites 2. To collect peripheral blood plasma samples for circulating HER2 mutation analysis 3. To investigate other potential therapeutic targets in HER2- breast cancer 4. To explore potential mechanisms of treatment resistance 5. To explore anti-tumor response after adding trastuzumab (or FDA approved biosimilar) to neratinib or neratinib plus fulvestrant when tumor progresses on single agent neratinib or neratinib in combination with fulvestrant.
a phase ii study of
3.0 patient selection

3.1 Pre-registration (for patients with unknown HER2 mutation status to have tumor tissue screened centrally by Wash U GPS laboratory)
a phase ii study of
3.1.1 inclusion criteria for pre-registration

1. Histologically or cytologically confirmed HER2-negative (0 or 1+ by IHC or nonamplified by FISH) breast cancer that is stage IV.  
2. Agree to provide archival tumor material for research 3. There is no limitation on the number of prior lines of systemic therapy. 4. Presence of measurable or non-measurable disease by RECIST 1.1 is acceptable, except to be eligible for the Part II fulvestrant-naïve ER+ cohort, at least one measurable disease by RECIST 1.1 is required.  
5. At least 18 years of age. 6. ECOG performance status < 2 (see Appendix A). 7. Adequate organ function as defined below within 8 weeks of pre-registration:
- Serum creatinine: ≤1.5 x ULN - Child-Pugh class A if with liver disease 8. Able to understand and willing to sign an IRB approved written informed consent document.  
Note: HER2 mutation testing may be performed while the patient is receiving active systemic therapy for metastatic breast cancer so that the result can be used to determine eligibility for study drug therapy in the future.
a phase ii study of
3.1.2 exclusion criteria for pre-registration

1. Testing for LVEF is not required for pre-registration, but patient must not have a recent LVEF < LLN or have symptoms of congestive heart failure.  
2. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements.  
3. Acute or currently active hepatic or biliary disease requiring antiviral therapy (with the exception of Gilbert's syndrome, asymptomatic gallstones, liver metastases, or stable chronic liver disease per investigator assessment).  
4. History of significant cardiac disease, cardiac risk factors, or uncontrolled arrhythmias. 5. Symptomatic intrinsic lung disease or extensive tumor involvement of the lungs resulting in dyspnea at rest.
a phase ii study of
3.2 registration (for patients positive

1. Tumor tissue tested positive for HER2 mutation. See the list of mutations eligible for Part II enrollment in Table 2 (Section 5.1.6). Mutations outside the list will be assessed on a case-by-case basis by the study team to determine eligibility. Note: HER2 mutations listed in Table 2 detected by Guardant360 are also eligible 2. Agree to provide archival tumor material for research 3. ECOG performance status < 2 (see Appendix A). 4. Adequate organ function as defined below within 2 weeks of registration:
- Absolute neutrophil count: ≥1.5 × 109/L (1500/mm3) - Platelet count: ≥100 × 109/L (100,000/mm3)
- Serum creatinine: ≤1.5 x ULN
- Child-Pugh class A if with liver disease 5. The patient must have completed radiation therapy and be at least 1 week from the last systemic therapy administration, with adequate recovery of bone marrow and organ functions, before starting neratinib.  
6. Presence of disease progression on the most recent disease evaluation. 7. Patients with known treated brain metastasis are eligible, but must have received radiation and be off steroids and stable (without evidence of disease progression by imaging or exam) for 3 months.  
8. QTc interval ≤ 450 msec for men or ≤ 470 msec for women within 2 weeks of registration.  
9. LVEF > institutional LLN within 4 weeks of registration. 10. Women of childbearing potential and men must agree to use adequate contraception
(hormonal or barrier method of birth control, abstinence) prior to study entry and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while participating in this study, she must inform her treating physician immediately. Men must agree and commit to use a barrier method of contraception while on treatment and for 3 months after the last dose of the investigational product (see Section 6.3).  
11. Able to understand and willing to sign an IRB approved written informed consent document.  
12. There is no limitation on the number of prior lines of systemic therapy. 13. To be eligible for the Part II fulvestrant-naïve ER+ cohort, prior treatment with fulvestrant is not allowed. In addition, ER and/or PR positivity by institutional standard is required on pathology from the most recent tumor specimen if biopsy was done unless the tissue source (for example, pleural effusion or ascites or bone biopsy) may yield false negative ER and/or PR result, in which case the pathology from an earlier time point could be used and a discussion with the study chair is required.  
14. To be eligible for the Part II fulvestrant-treated ER+ cohort, prior disease progression on fulvestrant is required. In addition, ER and/or PR positivity by institutional standard is required on pathology from the most recent tumor specimen unless the tissue source (for example, pleural effusion or ascites or bone biopsy) may yield false negative ER and/or PR result, in which case the pathology from an earlier time point could be used and a discussion with the study chair is required.
a phase ii study of
3.2.2 inclusion criteria for registration

1. Histologically or cytologically confirmed HER2-negative (0 or 1+ by IHC or nonamplified by FISH) breast cancer that is stage IV.  
2. Tumor tissue or circulating tumor DNA tested positive for HER2 mutation. See the list of mutations eligible for Part II enrollment in Table 2 (Section 5.1.6). Mutations outside the list will be assessed on a case-by-case basis by the study team to determine eligibility.  
3. Presence of measurable or non-measurable disease by RECIST 1.1 is acceptable, except to be eligible for the Part II fulvestrant-naïve ER+ cohort, at least one measurable disease by RECIST 1.1 is required.  
4. At least 18 years of age. 5. ECOG performance status < 2 (see Appendix A). 6. Adequate organ function as defined below within 2 weeks of registration:
- Absolute neutrophil count: ≥1.5 × 109/L (1500/mm3)
- Platelet count: ≥100 × 109/L (100,000/mm3)
- Serum creatinine: ≤1.5 x ULN - Child-Pugh class A if with liver disease 7. The patient must have completed radiation therapy and be at least 1 week from the last systemic therapy administration, with adequate recovery of bone marrow and organ functions, before starting neratinib.  
8. Presence of disease progression on the most recent disease evaluation. 9. Patients with known treated brain metastasis are eligible, but must have received radiation and be off steroids and stable (without evidence of disease progression by imaging or exam) for 3 months.  
10. QTc interval ≤ 450 msec for men or ≤ 470 msec for women within 2 weeks of registration.  
11. LVEF > institutional LLN within 4 weeks of registration. 12. Women of childbearing potential and men must agree to use adequate contraception
(hormonal or barrier method of birth control, abstinence) prior to study entry and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while participating in this study, she must inform her treating physician immediately. Men must agree and commit to use a barrier method of contraception while on treatment and for 3 months after the last dose of the investigational product (see Section 6.3).  
13. Able to understand and willing to sign an IRB approved written informed consent document.  
14. There is no limitation on the number of prior lines of systemic therapy. 15. To be eligible for the Part II fulvestrant-naïve ER+ cohort, prior treatment with fulvestrant is not allowed. In addition, ER and/or PR positivity by institutional standard is required on pathology from the most recent tumor specimen if biopsy was done unless the tissue source (for example, pleural effusion or ascites or bone biopsy) may yield false negative ER and/or PR result, in which case the pathology from an earlier time point could be used and a discussion with the study chair is required.  
16. To be eligible for the Part II fulvestrant-treated ER+ cohort, prior disease progression on fulvestrant is required. In addition, ER and/or PR positivity by institutional standard is required on pathology from the most recent tumor specimen unless the tissue source (for example, pleural effusion or ascites or bone biopsy) may yield false negative ER and/or PR result, in which case the pathology from an earlier time point could be used and a discussion with the study chair is required.
a phase ii study of
3.2.3 exclusion criteria for registration

1. Currently receiving any other investigational agents or systemic cancer therapy. 2. Currently taking medications and herbal or dietary supplements that are strong cytochrome P450 (CYP) 3A4 inducers or inhibitors (refer to Section 6.4.4). A washout period of at least 5 days is required and must have been completed prior to the start of neratinib if the patient was taking any of these agents. If unavoidable, patients taking CYP3A4 inhibitors should be monitored closely.  
3. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements.  
4. Acute or currently active hepatic or biliary disease requiring antiviral therapy (with the exception of patients with Gilbert's syndrome, asymptomatic gallstones, liver metastases, or stable chronic liver disease per investigator assessment).  
5. Pregnant and/or breastfeeding. 6. History of significant cardiac disease, cardiac risk factors, or uncontrolled arrhythmias. 7. Symptomatic intrinsic lung disease or extensive tumor involvement of the lungs resulting in dyspnea at rest.  
8. Experiencing grade 2 or greater diarrhea. 9. Prior treatment with neratinib. 10. Child-Pugh class B or C liver dysfunction.
a phase ii study of
3.3 inclusion of women and

Both men and women and members of all races and ethnic groups are eligible for this trial.
a phase ii study of
4.0 registration procedures 4.1 pre-registration

Patients must not start any protocol intervention prior to pre-registration through the Siteman Cancer Center. All eligible and consenting patients will be pre-registered to this protocol for the purposes of HER2 sequencing; if HER2 mutation is identified, the patient will then be registered if eligible for study treatment. The following steps must be taken:
1. Confirmation of patient pre-registration eligibility by Washington University 2. Pre-Registration of patient in the Siteman Cancer Center database 3. Assignment of unique patient number (UPN)
Once the patient has been entered in the Siteman Cancer Center database, the WUSM coordinator will forward verification of enrollment and the UPN via email.
a phase ii study of
4.1.1 confirmation of patient eligibility

Confirm patient eligibility by faxing the information listed below to the research coordinator listed in the *Siteman Cancer Center Clinical Trials Core Protocol Procedures* for Secondary Sites packet and email Caroline Bumb (cbumb@wustl.edu) at least one business day prior to registering patient:
1. Your name and contact information (telephone number, fax number, and email address)
2. Your site PI's name, the registering MD's name, and your institution name 3. Patient's race, sex, and DOB 4. Three letters (or two letters and a dash) for the patient's initials 5. Currently approved protocol version date 6. Copy of signed consent form (patient name may be blacked out) 7. Planned date of sample shipment 8. Completed pre-registration eligibility checklist (Appendix B), signed and dated by a member of the study team 9. Copy of appropriate source documentation confirming patient eligibility
a phase ii study of
4.1.2 patient pre-registration in the

Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent late afternoon or early morning enrollments should be planned in advance and coordinated with the Washington University research coordinator. Registration will be confirmed by the research coordinator or his/her delegate by email or fax within one business day. Verification of eligibility and pre-registration should be kept in the patient chart. Patients at all sites must be pre-registered through the Siteman Cancer Center OnCore database at Washington University.
a phase ii study of
4.1.3 assignment of upn

Each patient will be identified with a unique patient number (UPN) for this study. Patients will also be identified by first, middle, and last initials. If the patient has no middle initial, a dash will be used on the case report forms (CRFs). All data will be recorded with this identification number on the appropriate CRFs.
a phase ii study of
4.2 registration for study therapy

Patient who initially pre-registered and found to have HER2 mutation in the tumor will be registered to receive study therapy if the patient remains eligible. The same UPN will be used. Patients with HER2 mutation identified by another CLIA certified laboratory and meet all eligibility will be registered prior to study drug therapy. An UPN will be assigned as described in Section 4.1.3.
a phase ii study of
4.2.1 confirmation of patient eligibility

Confirm patient eligibility by faxing the information listed below to the research coordinator listed in the Siteman Cancer Center Clinical Trials Core Protocol Procedures for Secondary Sites packet and email Caroline Bumb (cbumb@wustl.edu) at least two business days prior to registering patient:  
1. Your name and contact information (telephone number, fax number, and email address)
2. Your site PI's name, the registering MD's name, and your institution name 3. Patient's race, sex, and DOB 4. Three letters (or two letters and a dash) for the patient's initials 5. Currently approved protocol version date 6. Copy of signed consent form (patient name may be blacked out) 7. Planned date of treatment to start 8. Completed registration eligibility checklist (Appendix C), signed and dated by a member of the study team 9. Copy of appropriate source documentation confirming patient eligibility
a phase ii study of
4.2.2 patient registration in the

Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent late afternoon or early morning enrollments should be planned in advance and coordinated with the Washington University research coordinator. Registration will be confirmed by the research coordinator or his/her delegate by email or fax within one business day if all required documentation to determine patient eligibility is provided. Verification of eligibility and registration should be kept in the patient chart. Patients at all sites must be registered through the Siteman Cancer Center OnCore database at Washington University.
a phase ii study of
4.3 recruitment through army of

The following procedures describe how women recruited via Army of Women (AOW) will be screened and registered for this trial:
1. HRPO-approved Eblast will be sent by AOW coordinator to women signed up through AOW to receive recruitment messages.  
2. Women responding to the Eblast will be referred by AOW coordinator to WUSM study coordinator at HER2MutationTrial@dom.wustl.edu.  
3. WUSM study coordinator will send a HRPO-approved introductory email to each AOW
recruit requesting a time to discuss the trial and attaching the version of the screening consent form drafted specifically for outside recruits.  
4. WUSM study coordinator will consent the AOW recruit over the phone. If the recruit consents to be screened for this trial, the WUSM study coordinator will mail the patient info sheet and a SASE, requesting that the patient complete the info sheet and return it along with the screening consent form.  
5. Once the screening consent form and patient info sheet have been received by the WUSM
study coordinator, the medical records for the AOW recruit will be requested by the WUSM study coordinator.  
6. Once the medical records have been received, the pre-registration process described in Section 4.1 will be followed and the pre-registration eligibility criteria described in Section 3.1 will be checked. The patient will be informed of eligibility for HER2 mutation testing via email.  
7. If the patient isshown to be eligible for screening, the WUSM study coordinator will request a tumor specimen for HER2 mutation testing. The WUSM study coordinator will send the report to the patient after reviewing with the PI. If negative, the patient is not eligible to continue in the study. If positive, the PI will discuss with the patient and the patient's oncologist if the patient desires it and will refer the patient to a participating institution for consent to the main study.
a phase ii study of
4.4 recruitment from outside practice

An informational card with the title of the study and contact information of the WUSM study coordinator will be made and distributed to medical oncologists from outside practices at scientific meetings and lectures. These physicians will be encouraged to refer potentially eligible patients to the WUSM study coordinator, who will begin the pre-registration screening process as described in \#3 in Section 4.3. Screening and testing will continue as described in Section 4.3.
a phase ii study of
5.0 tumor her2 sequencing 5.1

Patients meeting the pre-registration eligibility criteria with unknown tumor *HER2* mutation status could undergo tumor *HER2* sequencing centrally at the CAP/CLIA certified Genomic and Pathology Services at Washington University (GPS@WU) after preregistration to the study. Sanger sequencing of Exons 18, 19, 20, 21, 22, 23 and 24 was initially used for the study. Since October 2014, this was switched to a next generation
Page 28 sequencing (NGS) HER2 assay that covers all coding exons of the *HER2* gene.  
Archival tumor specimens from the primary breast cancer or from a metastatic site is required for the sequencing analysis. Samples will be need to be shipped to the *"Clinical* Support Services Office at the Washington University Department of Pathology &
Immunology". Please refer to the Correlative Science Procedures Manual Section 1.0 for tumor sample requirement and shipping instructions. Note that sample shipment and sequencing analysis can be performed while the patient is receiving other systemic therapies so the results could be used to determine whether the patient is eligible to receive the study therapy when disease progresses on the current treatment. The study coordinator from the participating institution listed on the sample shipment form will be notified of the results per email within 3-4 weeks. The study chair and the Washington University study coordinator have access (requiring password) to a secure folder on the WUSM pathology department network drive where the clinical reports are stored. For remotely screened participants, the reports will be faxed or emailed to the participant or the treating physician as applicable.
a phase ii study of
5.2 tumor or circulating tumor

Patients with tumor or circulating tumor DNA *HER2* mutations meeting the criteria defined in Section 5.3 identified at other CAP/CLIA-certified laboratories are eligible for study entry if they meet all other eligibility criteria. To determine the eligibility of the reported mutation from laboratories other than Wash U GPS, the full report will need to be sent to Caroline Bumb (cbumb@wustl.edu) and Cynthia Ma (cynthiaxma@wustl.edu). Pre-registration process is not required for patients with known mutations as above but the archival tumor sample must be submitted to Wash U Alliance/ACOSOG-CSB/TPC, not to the *Clinical Support Service Office*, for future confirmation of the *HER2* mutation and correlative studies (also refer to the Section 10 for Correlative Study). Please refer to the Correlative Science Procedure Manual regarding the sample requirement and shipping instructions for Correlative Studies.
a phase ii study of
5.3 eligible her2 mutations

HER2 mutation is defined as a single nucleotide variant (SNV) or an indel variation that changes the encoded amino acid in the coding region of *HER2* or an intronic mutation known to affect the splicing of *HER2*. Identified SNVs will be compared to known single nucleotide polymorphisms (SNPs) identified in the SNP data base as well as to the variants identified in cancer mutation databases including the Catalogue Of Somatic Mutations In Cancer (COSMIC) database. *HER2* SNPs would be not considered as an *HER2* mutation. Germ-line DNA will be sequenced to rule out single nucleotide polymorphisms if needed. All somatic *HER2* mutations were eligible for Part I of the trial. However, due to the lack of response in patients with *HER2* mutations of unknown significance observed in Part I of the trial [68], only known activating mutations listed in Table 2 are considered eligible for patients enrolling to Part II of the trial unless approved by the study chair. Mutations outside the list will be assessed on a case-by-case basis by the study team to determine eligibility.  
| Table 2 Known Activating HER2 Mutations                                               |              |
|---------------------------------------------------------------------------------------|--------------|
| HER2 mutation                                                                         | Reference    |
| G309A/E                                                                               | [56, 57]     |
| S310F/Y                                                                               | [56]         |
| S653C                                                                                 | [58]         |
| L755S                                                                                 | [57, 59, 60] |
| L755P                                                                                 | [60]         |
| Del. 755-759                                                                          | [57]         |
| D769Y/H                                                                               | [57]         |
| V777L                                                                                 | [57, 60]     |
| V842I                                                                                 | [57]         |
| T862A                                                                                 | [60]         |
| L869R                                                                                 | [62]         |
| H878Y                                                                                 | [60, 63, 64] |
| G776V/C                                                                               | [65]         |
| All exon 20 insertions, including: A771_Y772insYVMA A775_G776insYVMA  P780_Y781insGSP | [66, 67]     |
a phase ii study of
5.4 germline dna sequencing for

In cases where it is unclear whether the identified HER2 variant is somatic (tumor specific) versus germline in origin, the study team at WUSM will notify the site investigator to submit a blood sample of the patient for germline DNA sequencing at Wash U GPS laboratory. Please refer to Section 2.0 of the Correlative Science Procedure Manual for the submission and shipment.
a phase ii study of
5.5 her2 mutation testing by

Plasma in cancer patients often carries small amounts of fragmented cell-free DNA of 160-180 base pairs, which are originated from the necrosis or apoptotic process of cancer cells. Advances in the next generation sequencing (NGS) technology and digital genomic techniques support the clinical validity of cell-free circulating DNA (ctDNA) sequencing analysis to non-invasively identify actionable genomic alterations, monitor treatment response, and investigate resistance mechanisms [69]. ctDNA sequencing is particularly helpful in cases that tumor DNA sequencing is not possible due to insufficient quality or quantity of the tumor tissue. In collaboration with Guardant Health, Inc., we analyzed the plasma ctDNA collected from patients enrolled in this trial using the CLIA certified digital sequencing panel (Guardant 360), which utilizes hybrid capture followed by NGS of all exons in 30 genes (including HER2) and critical exons (those reported as having a somatic mutation in COSMIC) of 40 additional genes to detect and report single nucleotide variants (SNVs) and small indels in 70 genes, copy number amplifications in 18 genes, and select fusions [70]. The plasma ctDNA sequencing successfully identified HER2 mutation in 11/14 (79%; 90% CI: 53-94%) patients tested positive for HER2 mutation by tumor DNA NGS of HER2. Importantly, none of the 32 patients who were negative for HER2 mutation by tumor DNA sequencing was positive by ctDNA sequencing. Therefore, the specificity of the Guardant 360 ctDNA sequencing for HER2 mutation was 100% (90% CI: 91100%). These data are being prepared for publication. Based on these results and the fact that tumor DNA sequencing for HER2 was unsuccessful in over 25% of patients pre-registered to this
Page 30 trial, we will allow patients with HER2 mutation detected in plasma by a CLIA lab to enroll on the study to receive neratinib.
a phase ii study of
6.0 treatment plan

To be eligible to receive neratinib, mutation in HER2 either in the primary or metastatic tumor or circulating tumor DNA is required. However, data that have been collected during pre-registration from patients who have HER2 non-mutant tumors will also be used for studies to correlate HER2 mutation with histology subtype (invasive lobular vs. invasive ductal cancer), tumor grade (grade 1-2 vs. 3), tumor staging at initial diagnosis (I vs. II or III vs. IV), and disease-free survival in HER2-negative breast cancer. The left over tumor specimens from HER2 sequencing analysis from all patients will be assayed in future studies to understand the biology of HER2-negative breast cancer. The future studies could include tumor DNA, RNA or protein analysis.
a phase ii study of
6.1 neratinib administration

Neratinib is administered in the outpatient setting. It will be taken by mouth with food, preferably in the morning daily, starting at 240mg per day. Neratinib will be given on a 28-day cycle (+/- 3 days). The dose modification guidelines are provided in Section 7.0. In Cycle 2 or subsequent cycles, patients may begin taking 320mg per day of neratinib with prophylactic loperamide provided the patients did not experience any intolerable grade 2 or higher treatment-related AEs during previous cycles (including diarrhea while receiving prophylactic loperamide).
a phase ii study of
6.2 concurrent prophylactic administration of

Investigators must ensure that subjects have loperamide on hand when starting to take the investigational product. Loperamide is the recommended standard therapy to treat diarrhea in this study. If alternative antidiarrheal medication is used, the reason must be documented in the source documents. Acceptable reasons are non-tolerance of loperamide or lack of efficacy. Loperamide will be dispensed directly by the site on day 1 (preferred option), or a prescription for loperamide will be provided for the subject with the instruction to have loperamide on hand prior to taking the first dose of investigational product. If none of the above is feasible, the subject must obtain loperamide "over-the-counter" prior to taking the first dose of investigational product on Day 1.
a phase ii study of
6.3 fulvestrant administration

Fulvestrant will be given on a 28-day cycle (+/- 3 days). It is administered at a dose of 500 mg (2 x 250 mg injections) IM on Days 1 and 15 of Cycle 1, and 500 mg IM on Day 1 of each subsequent cycle. Administer intramuscularly slowly in the buttock. (NOTE: 500 mg dose will require one 250 mg injection in each buttock). Immediately activate needle protection device upon withdrawal from patient by pushing lever arm completely forward until needle tip is fully covered. Visually confirm that the lever arm has fully advanced and the needle tip is covered. If unable to activate, discard immediately into an approved sharps container.
a phase ii study of
6.4 general concomitant medication and

Patients should receive full supportive care, including transfusions of blood and blood products, antibiotics, antinausea/diarrhea, etc., when appropriate. Subjects on digoxin, a P-glycoprotein substrate with a narrow therapeutic window, should be monitored closely and their digoxin dose adjusted as needed, since neratinib is an inhibitor of Pglycoprotein. Co-administration of neratinib and digoxin could result in increased digoxin levels and associated digoxin toxicity.  
Elective surgery should be avoided while receiving investigational product. If surgery is required the recommendation is to hold investigational product for 3 to 4 days prior to surgery, and then resume product when the subject is tolerating a full diet post-operatively (no later than 3 weeks after holding it). The start date and stop date and indication for concomitant treatments and/or therapies and medications given because of an AE occurring from the signing of the consent form until the end of treatment will be recorded on the Concomitant Medications Form in REDCap.
a phase ii study of
6.4.1 prohibited concomitant treatment

The following treatments are prohibited during neratinib therapy:
- Any chemotherapy, radiation therapy, immunotherapy, biotherapy, or surgery for breast cancer.  
- Any other investigational agent.
a phase ii study of
6.4.2 permitted concomitant treatment during

The following treatments are permitted during the study:
- Standard therapies for preexisting medical conditions, and for medical and/or surgical complications. All medication should be recorded.  
- Bisphosphonates, regardless of the indication.
a phase ii study of
6.4.3 addition of trastuzumab (or

If a patient experiences disease progression following neratinib or neratinib in combination with fulvestrant, trastuzumab may be added to the treatment regimen provided it is covered by the patient's insurance company. Trastuzumab has available biosimilar products FDA approved for use. Trastuzumab or any of the FDA approved commercially available biosimilar products for trastuzumab may be administered to patients based on their insurance requirements or institutional practice. If a biosimilar product is required, dosing, schedule of administration, and premedications are identical to the guidance described throughout the protocol for trastuzumab. Preparation of the drug product will be dependent on the biosimilar selected for use. Patients should start trastuzumab (or FDA approved biosimilar) with their next scheduled fulvestrant injection. Neratinib does not need to be held between progression and Day 1 of trastuzumab (or FDA approved biosimilar) treatment. Palliative radiation for pain is allowed prior to starting trastuzuamb with prior approval from the Washington University PI. Neratinib should be held during radiation. AE monitoring, dose modifications for neratinib, and tumor response on combination therapy will be followed per protocol. For these patients, treatment cycle will be restarted at cycle 1 at the start of trastuzumab. Research blood collection will follow the study calendar (beginning at C1D1). Trastuzumab (or FDA approved biosimilar) is administered intravenously with a loading dose of 6 mg/kg then 4 mg/kg every 2 weeks +/- 3 days. A cycle will be defined as 28 days. For the first (loading) dose of trastuzumab (or FDA approved biosimilar), premedication with diphenhydramine 50 mg PO or other antihistamine equivalent and acetaminophen 650 mg PO will be given.
a phase ii study of
6.4.4 effect of other drugs

| Gastric Acid Reducing Agents Clinical Impact - Concomitant use of neratinib with a proton pump  inhibitor (PPI, lansoprazole) resulted in a decrease  of neratinib Cmax by 71% and AUC by 65% - Concomitant use with other pH lowering agents was  not studied but a decrease in neratinib AUC is also  considered likely - Decreased neratinib AUC may reduce neratinib  activity Prevention or  Management - PPIs Avoid concomitant use - H2-receptor antagonists Avoid concomitant use - Antacids Separate neratinib dosing  by 3 hours after antacids Strong and Moderate CYP3A4 Inhibitors Clinical Impact - Concomitant use of neratinib with a strong CYP3A4  inhibitor (ketoconazole) increased neratinib Cmax by  321% and AUC by 481% - Concomitant use of neratinib with other strong or  moderate CYP3A4 inhibitors may increase  neratinib concentrations - Increased neratinib concentrations may increase the  risk of toxicity Preventation or  Avoid concomitant use of neratinib with strong or moderate  Management CYP3A4 inhibitors. Examples1 Strong CYP3A4 inhibitors: boceprevir, clarithromycin,  cobicistat, conivaptan, danoprevir and ritonavir, diltiazem,  elvitegravir and ritonavir, grapefruit juice, idelalisib,  indinavir and ritonavir, itraconazole, ketoconazole,  lopinavir and ritonavir, nefazodone, nelfinavir, paritaprevir  and ritonavir and (ombitasvir and/or dasabuvir),  posaconazole, ritonavir, saquinavir and ritonavir, tipranavir  and ritonavir, troleandomycin, voriconazole Moderate CYP3A4 inhibitors: aprepitant, cimetidine,  ciprofloxacin, clotrimazole, crizotinib, cyclosporine,  dronedarone, erythromycin, fluconazole, fluvoxamine,  imatinib, tofisopam, verapamil Strong or Moderate CYP3A4 Inducers Clinical Impact - Concomitant use of neratinib with a strong CYP3A4  inducer (rifampin) reduced neratinib Cmax by 76%  and AUC by 87%   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| -                                                                                                                                                   | Concomitant use of neratinib with other strong or  moderate CYP3A4 inducers may decrease neratinib  concentrations   |                              |
|-----------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|------------------------------|
| -                                                                                                                                                   | Decreased neratinib AUC may reduce neratinib  activity                                                               |                              |
| Prevention or                                                                                                                                       | Avoid concomitant use of neratinib with strong or moderate                                                           |                              |
| Management                                                                                                                                          | CYP3A4 inducers                                                                                                      |                              |
| Examples1                                                                                                                                           | Strong CYP3A4 inducers:                                                                                              | carbamazepine, enzalutamide, |
| mitotane, phenytoin, rifampin, St. John's wort Moderate CYP3A4 inducers: bosentan,                                                                  | efavirenz,                                                                                                           |                              |
| etravirine, modafinil                                                                                                                               |                                                                                                                      |                              |
| 1 These examples are a guide and not considered a comprehensive list of all possible drugs that may fit this  category. Please contact study chair. |                                                                                                                      |                              |  
If unavoidable, patients taking CYP3A4 inhibitors should be monitored closely. Subjects on coumarin-derivative anticoagulants (eg, warfarin) should be monitored closely and their anticoagulant dose adjusted as needed. Subjects on chronic laxatives should be followed closely and consideration should be given to decreasing or stopping laxatives prior to starting investigational agent, given the potential for neratinib-related diarrhea to be worsened by concomitant laxative use. Drugs known to cause QTc prolongation which are being given concomitantly require close monitoring of the subject with serial ECGs.
a phase ii study of
medications that may cause qtc

The following table presents a list of drugs that prolong, may prolong, or are unlikely to prolong the QTc. Please note that this list is frequently updated. For the most current list of medications, users should be directed to the following website:
http://www.azcert.org/medical-pros/drug-lists/drug-lists.cfm.  
Patients using drugs known to cause QT/QTc prolongation should be monitored closely with serial electrocardiograms (ECG) at the Investigator's discretion.  
| Possible  Washout           |                                               |                         |
|-----------------------------|-----------------------------------------------|-------------------------|
| Period - Days               |                                               |                         |
| Alfuzocin                   | ~10 hours                                     | 7                       |
| Amantadine                  | 17 +/- 4 hours (10-25)                        | 4                       |
| Amiodarone (cordarone)      | 58 days (15-142)                              | 180                     |
| 36 days (active metabolite) | Possible                                      |                         |
| Compound                    | Compound Half Life                            | Washout Period  - Hours |
| Amitriptyline*              | > 24 hours, wide interpatient variability     |                         |
| Arsenic trioxide            | Not characterized                             |                         |
| Azithromycin                | 40 hours                                      |                         |
| Bepridil                    | 42 hr (26-64)                                 | 10                      |
| Chloral hydrate             | Readily converted to Trichloroethanol (active | 48                      |
| metabolite T1/2=7-10 hour)  |                                               |                         |
| Chloroquine                 | Prolonged (days to weeks)                     |                         |
| Chlorpromazine              | 30 +/- 7 hours                                | 7                       |
| Cisapride                   | 6 - 12 hour, up to 20 hour                    | 60                      |
| Clarithromycin              | Non linear PK3-4 hr (250mg Q12)               | 36                      |
| 5-7 hr (500mg Q12)          |                                               |                         |
| Cloroquine                  | 6 to 60 days; mean 20 days                    |                         |
| Desipramine*                | > 24 hours, wide interpatient variability     |                         |
| Disopyramide                | 6.7 hr (4-10)                                 | 36                      |
| Dofetilide                  | 10 hr                                         | 48                      |
| Dolesetron                  | 8.1 hr                                        |                         |  
| Domperidone                             | 7-8 hr                                                                                                                                | 48     |
|-----------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|--------|
| Doxepin*                                | > 24 hours, wide interpatient variability                                                                                             |        |
| Droperidol                              | 2.2 hours                                                                                                                             | 10     |
| Erythromycin                            | * Each salt form has different Half life*                                                                                             |        |
| Felbamate                               | 20-23 hr                                                                                                                              | 5      |
| Flecainide                              | 20 hr (12-27)                                                                                                                         | 5      |
| Foscarnet                               | 87.5+/-41.8 hours                                                                                                                     | 20     |
| *distribution and release from bone*    |                                                                                                                                       |        |
| Fosphenytoin                            | 12-29 hr                                                                                                                              | 6      |
| Gatifloxacin                            | 7-14 hr                                                                                                                               | 48     |
| Gemifloxacin                            | 7 hours                                                                                                                               | 48     |
| Grepafloxacin                           | 16 hr                                                                                                                                 | 3      |
| Halofantrine                            | 6-10 days ( variable among individual)                                                                                                | 45     |
| Haloperidol                             | 18 +/-5 hr                                                                                                                            | 5      |
| Ibutilide                               | 6 hours (2-12) * variable among subject*                                                                                              | 36     |
| Imipramine*                             | > 24 hours, wide interpatient variability                                                                                             |        |
| Indapamide                              | 14 hours (biphasic elimination)                                                                                                       | 3      |
| Isradipine                              | 8 hours ( multiple metabolites)                                                                                                       | 48     |
| Levofloxacin                            | 6-8 hours                                                                                                                             | 48     |
| Levomethadyl                            | Multiple compartment PK with active metabolite 2.6 day for LAAM, 2 day for nor-LAAM, 4 day for  dinor-LAAM                            | 20     |
| Lithium                                 | 24 hour (10-50)                                                                                                                       | 7      |
| Mesoridazine                            | 24-48 hours ( animal study)                                                                                                           | 10     |
| Methadone                               | 15-30 hours                                                                                                                           | 7      |
| Moexipril/HCTZ                          | 2-9 hour (include active metabolite) for moexipril;                                                                                   | 48     |
| 5.6-14.8 hours for HCTZ                 |                                                                                                                                       |        |
| Moxifloxacin                            | 12 +/-1.3 hours                                                                                                                       | 72     |
| Naratriptan                             | 6 hours                                                                                                                               | 36     |
| Nicardipine                             | ~ 2 hour post IV infusion                                                                                                             | 12     |
| Nortriptyline*                          | > 24 hours, wide interpatient variability                                                                                             |        |
| Octreotide                              | 1.7 hours                                                                                                                             | 12     |
| Ofloxacin                               | 5 to 7.5 hours                                                                                                                        | 2      |
| Ondansetron                             | 4 hours (IV/IM); 3 hours (PO)                                                                                                         | 1 to 3 |
| Pentamidine                             | 6.4+/-1.3 hours                                                                                                                       | 36     |
| Pimozide                                | 55 hours                                                                                                                              | 10     |
| Procainamide                            | 3-4 hour for PA and NAPA (active metabolite)                                                                                          | 24     |
| Protiptyline*                           | > 24 hours, wide interpatient variability                                                                                             |        |
| Quetiapine                              | 6 hours                                                                                                                               | 36     |
| Quinidine                               | 6-8 hours in adult; 3-4 hours in children                                                                                             | 36     |
| Quinine                                 | 4-5 hours                                                                                                                             |        |
| Risperidone                             | 3-20 hours (extensive to poor metabolizer) 9-hydroxyrisperidone (active metabolite) T ½ =21-30  hours (extensive to poor metabolizer) | 4      |
| Salmeterol                              | 5.5 hours ( only one datum)                                                                                                           | 36     |
| Sotalol                                 | 12 hours                                                                                                                              | 72     |
| Sparfloxacin                            | 20 hours (16-30)                                                                                                                      | 4      |
| Sumatriptan                             | 2.5 hours                                                                                                                             | 12     |
| Tacrolimus                              | ~34 hours in healthy; ~19 hours in Kidney transplant                                                                                  | 7      |
| Tamoxifen                               | 5-7 days (biphasic)                                                                                                                   | 30     |
| Telithromycin                           | 2-3 hr                                                                                                                                | 24     |
| Thioridazine                            | 20-40 hours (Phenothiazines)                                                                                                          | 7      |
| Tizanidine                              | 2.5 hours                                                                                                                             | 12     |
| Vardenifil                              | 4 to 5 hours                                                                                                                          |        |
| Venlaflaxine                            | 5 +/-2 hours for parent comp.                                                                                                         | 60     |
| 11+-2 hours for OVD (active metabolite) |                                                                                                                                       |        |
| Voriconizole                            | 6 hours; dose dependent                                                                                                               |        |
| Ziprasidone                             | 7 hr                                                                                                                                  | 36     |
| Version 03/16/2020 - Amendment 13       | Page 36 of 87                                                                                                                         |        |  
Zolmitriptan 2.8-3.7 hours (higher in female) 18
*Weakly associated with Torsades de Pointes and/or QT prolongation but that are unlikely to be a risk for Torsades de Pointes when used in usual recommended dosages and in patients without other risk factors (*e.g.*, concomitant QT prolonging drugs, bradycardia, electrolyte disturbances, congenital long QT syndrome, concomitant drugs that inhibit metabolism).
a phase ii study of
references

1. **Physician's Desk Reference 2002**
2. **Facts and Comparisons ( update to June 2005)**
3. **The Pharmacological Basis of Therapeutics 9th Edition, 1996**
a phase ii study of
6.5 women of childbearing potential

Women of childbearing potential (women with regular menses, women with amenorrhea, women with irregular cycles, women using a contraceptive method that precludes withdrawal bleeding, and women who have had a tubal ligation) are required to have a negative serum or urine pregnancy test within 7 days prior to the first dose of neratinib. Female and male patients (along with their female partners) are required to use two forms of acceptable contraception, including one barrier method, during participation in the study and for one month following the last dose of the neratinib. Men must agree and commit to use a barrier method of contraception while on treatment and for 3 months after the last dose of the investigational product. If a patient is suspected to be pregnant, neratinib should be immediately discontinued. In addition a positive urine test must be confirmed by a serum pregnancy test. If it is confirmed that the patient is not pregnant, the patient may resume dosing. If a female patient or female partner of a male patient becomes pregnant during therapy or within one month after the last dose of neratinib, the investigator must be notified in order to facilitate outcome follow-up.
a phase ii study of
6.6 duration of therapy

If at any time the constraints of this protocol are considered to be detrimental to the patient's health and/or the patient no longer wishes to continue protocol therapy, the protocol therapy should be discontinued and the reason(s) for discontinuation documented in the case report forms. In the absence of treatment delays due to adverse events, treatment may continue until one of the following criteria applies:  
- Documented and confirmed disease progression - Death
- Adverse event(s) that, in the judgment of the investigator, may cause severe or permanent harm or which rule out continuation of study drug
- General or specific changes in the patient's condition render the patient unacceptable for further treatment in the judgment of the investigator
- Suspected pregnancy - Serious non-compliance with the study protocol
- Lost to follow-up
- Patient withdraws consent - Investigator removes the patient from study
- The Siteman Cancer Center decides to close the study
Patients who prematurely discontinue treatment for any reason will be followed as indicated in the study calendar.
a phase ii study of
6.7 duration of follow-up

Patients will be followed every 3 months for 2 years or until death, whichever occurs first. Patients removed from study for unacceptable adverse events will be followed until resolution or stabilization of the adverse event. Patients will be followed through either review of medical records, phone calls, or office visits.
a phase ii study of
7.0 dose delays/dose modifications and

The allowed dose levels for dose reduction(s) due to toxicity which is deemed possibly, probably, or definitely related to neratinib are listed in the below dose administration table.  
| Neratinib (mg)    | Number of 40 mg   |    |
|-------------------|-------------------|----|
| Dose Level        | tablets           |    |
| 2                 | 320               | 8  |
| Starting dose (1) | 240               | 6  |
| -1                | 200               | 5  |
| -2                | 160               | 4  |
| -3                | 120               | 3  |
a phase ii study of
7.2 general dose adjustments for

Dose escalation of neratinib to 320 mg per day **with prophylactic loperamide** is allowed to start at Cycle 2 or subsequent cycles in patients who experienced no intolerable grade 2 or higher treatment-related AEs during the previous cycle(s) of therapy (including diarrhea while receiving prophylactic loperamide). The guideline for diarrhea management outlined in Section 7.3 is to be followed, except that patients will be contacted over the phone within the first week of escalation to 320 mg, and then on a weekly basis thereafter during the first month at the higher dose of neratinib. Subjects should be withdrawn from the study if the 120-mg dose level of neratinib is not tolerable, or if the subject fails to recover to NCI grade 0 to 1 (or to within baseline of starting values for preexisting laboratory abnormalities) from treatment-related toxicity, leading to treatment delay of >3 weeks. Missed dose(s) of the investigational product will not be made up. Dose adjustments are described below. Additional clinical situations may result in dose adjustments as clinically indicated (e.g., intolerable, persistent grade 2 drug-related AE). For general guidelines for the management of diarrhea, refer to Section 7.3. The management of LFT changes is specifically addressed in Section 7.4. For the management of LVEF declines, follow the guidelines in Section 7.5.  
| Event                                                                                                                                                                                                |                                                                                                                                                                                                     |             |       |                 |         |       |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-------|-----------------|---------|-------|
| (Based on NCI CTC4.0)                                                                                                                                                                                | Action                                                                                                                                                                                              |             |       |                 |         |       |
| Pneumonitis/Interstitial Lung Disease:                                                                                                                                                               | -                                                                                                                                                                                                   | Symptomatic | Grade | 2:              | Hold    | the   |
| Symptomatic Grade 2 or Grade ≥3                                                                                                                                                                      | investigational product until resolution to ≤  grade 1 or baseline within 3 weeks of  stopping the investigational product. Then  resume the investigational product at the  next lower dose level. |             |       |                 |         |       |
| -                                                                                                                                                                                                    | Grade ≥3: Discontinue the investigational  product permanently.                                                                                                                                     |             |       |                 |         |       |
| Grade 4 (Hematologic or  Nonhematologic)                                                                                                                                                             | -                                                                                                                                                                                                   | Hold        | the   | investigational | product | until |
| recovery to ≤grade 1 or baseline within 3  weeks of stopping the investigational  product, then resume the investigational  product with 2 dose levels reduction.                                    |                                                                                                                                                                                                     |             |       |                 |         |       |
| (Including nausea and/or vomiting despite  optimal medical therapy) Other Grade 3 Nonhematologic  (Including nausea and/or vomiting  despite optimal medical therapy, and  asthenia lasting >3 days) | -                                                                                                                                                                                                   | Hold        | the   | investigational | product | until |
| recovery to ≤grade 1 or baseline within 3  weeks of stopping the investigational  product. Then resume the investigational  product at the next lower dose level.                                    |                                                                                                                                                                                                     |             |       |                 |         |       |
a phase ii study of
7.3 guidelines for the management

Diarrhea is the major DLT of neratinib with onset typically occurring early in the course of treatment (during the first few weeks of treatment). Primary prophylactic use of antidiarrheal medication is **mandatory** for all enrolled subjects receiving neratinib at either 240mg or 320mg daily. Loperamide is the recommended standard therapy to treat diarrhea in this study. If alternative antidiarrheal medication is proposed, this should be discussed with the Principal Investigator and the reason documented in the source documents. Second-line antidiarrheal treatments and adjunctive therapies (i.e., octreotide [SANDOSTATIN®]) are also recommended for use when appropriate. The investigator must review with the subject the patient instructions for the management of diarrhea and the Medication Diary (Appendix B) for the subject's daily recording of investigational product dose, any adverse reactions, number of stools and use of loperamide and/or other antidiarrheals. Both the subject and the investigator must sign the IRB-approved patient instructions (template is Appendix C) for the management of diarrhea. Copies of both documents are handed to the subject before leaving the site with investigational product on Day 1 with clear instructions to contact the investigator in the event of de novo onset or persistent ≥ grade 2 diarrhea to discuss the appropriate course of treatment. In addition, the Investigator or designee will contact the subject within 24, 48, and 72 hours after the first dose of study drug is administered to inquire about any diarrhea. Documentation of any occurrences of stools or diarrhea must be as precise as possible and captured in the Medication Diary (Appendix B). For adverse event recording, documentation of "Intermittent" events of diarrhea is limited to grade 1. If events of grade 1 diarrhea are separated by 3 days without any diarrhea, then each event must be documented as separate adverse events with corresponding start and stop dates. The entries on the Medication Diary should be reviewed together with the subject at every visit and the data provided on the diary serves as source data for adverse event documentation on the CRF.  
If the subject has experienced diarrhea since the last visit, details of the daily number of unformed stools provided on the diary help to grade the diarrhea as precisely as possible (per NCI CTC 4.0). Also, daily dose of loperamide (or other antidiarrheals, if applicable) noted on the diary will be reviewed and recorded on the CRF. Loperamide will be dispensed directly by the site (either as a script or from over the counter) on day 1 with neratinib. It is very important to initiate treatment with loperamide concomitantly with the first dose of neratinib to minimize occurrence and severity of diarrhea.
a phase ii study of
for new onset uncomplicated grade

- Stop all lactose-containing products
- Drink 8 to 10 large glasses of clear liquids per day
- Eat frequent small meals - Recommend low fat regimen enriched with bananas, rice, applesauce and toast (BRAT diet)
until resolution of diarrhea.
a phase ii study of
pharmacological treatment

- For days 57 through 365, administer 4mg (2 tablets/capsules) as needed (not to exceed 8 tablets capsules or 16 mg per day)
- For patients with persistent grade 1 diarrhea on loperamide, Lomotil (diphenoxylate hydrochloride and atropine sulfate) 1 tablet (2.5 mg) every 6 hours to 8 hours may be added.  
- For grade 2 diarrhea (4 to 6 stools per day above baseline, despite intensive anti-diarrheal therapy), consider adding octreotide (short-acting) 150 micrograms SC TID; or after initial dose of short-acting octreotide, if well tolerated, a single dose of octreotide LAR 20 mg IM.
a phase ii study of
prophylactic dosing instructions (cycles 1

- Inform patients that they will experience diarrhea while taking neratinib
- Administer loperamide: initial dose of 4 mg (2 tablets/capsules) with the first dose of neratinib
- For days 1 through 14, administer 4 mg (2 tablets/capsules) three times daily - For days 15 through 56, administer 4 mg (2 tablets/capsules) twice daily - If diarrhea occurs despite prophylaxis, treat with additional antidiarrheals, fluids and electrolytes as clinically indicated. NERLYNX dose interruptions and dose reductions may also be required to manage diarrhea
- For patients with persistent grade 1 diarrhea (<4 stools per day above baseline) on loperamide, Lomotil (diphenoxylate hydrochloride and atropine sulfate) 1 tablet (2.5 mg) every 6-8 hours may be added.  
- Prophylactic loperamide administration may continue for subsequent cycles at the physician's discretion.  
- For grade 2 diarrhea during cycle 1 (4 to 6 stools per day above baseline, despite intensive anti-diarrheal therapy as described above), consider adding octreotide (short-acting) 150 micrograms SC TID; or after initial dose of short-acting octreotide, if well tolerated, a single dose of octreotide LAR 20 mg IM.  
Page 40
a phase ii study of
for grade 3 or grade

- Administer loperamide: initial dose of 4 mg (2 tablets) with the first bout of diarrhea followed by 2 mg (1 tablet) every 4 hours or after every unformed stool (maximum 16 mg a day) and continue loperamide at this frequency until diarrhea free for 12 hours. Then titrate the amount of loperamide used to keep diarrhea controlled (< 4 stools/day).  
- Administer octreotide (SANDOSTAINE®) [100-150 µg SC BID or IV (25-50 µg/h) if dehydration is severe, with dose escalation up to 500 µg SC TID]
- Use intravenous fluids as appropriate
- Consider prophylactic antibiotics as needed (e.g., fluoroquinolones) especially if diarrhea is persistent beyond 24 hours or there is fever or grade 3-4 neutropenia.  
Stool cultures should be done to exclude infectious causes of grade 3 or 4 diarrhea or diarrhea of any grade with complicating features (dehydration, fever, and/or grade 3 or 4 neutropenia) per the investigator's discretion. Results from occult blood, fecal leukocyte stain, Clostridium difficile, Campylobacter, Salmonella, and Shigella testing, when performed, should be reported. Subjects with significant diarrhea who are unresponsive to medical treatment may require treatment interruption or dose reduction.  
| NCI CTCAE V4.0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Action                                                                                                                   |                                                                                                                     |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Adjust anti-diarrheal treatment, as per the  guidelines for management of diarrhea at the  first onset of diarrhea.      |                                                                                                                     |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Continue neratinib at full dose.                                                                                         |                                                                                                                     |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Instruct patient to follow dietetic  recommendations in the guidelines for  management of diarrhea.                      |                                                                                                                     |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Fluid intake of ~2L should be maintained to  avoid dehydration.                                                          |                                                                                                                     |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Once the event resolved to ≤ grade 1 or  baseline, start loperamide 4 mg with each  subsequent neratinib administration. |                                                                                                                     |
| Grade 1 Diarrhea [Increase of < 4 stools per  day over baseline; mild increase in ostomy  output compared to baseline.] OR Grade 2 Diarrhea [Increase of 4-6 stools per  day over baseline; moderate increase in ostomy  output compared to baseline;] lasting ≤ 5 days OR Grade 3 Diarrhea [Increase of ≥7 stools per day  over baseline; incontinence; hospitalization  indicated; severe increase in ostomy output  compared to baseline limiting self-care activities  of daily living {ADL};] lasting ≤ 2 days Persisting and intolerable Grade 2 Diarrhea lasting >5 days despite being treated with  optimal medical therapy, or associated with  fever, dehydration, or grade 3-4 neutropenia | -                                                                                                                        | Adjust anti-diarrheal treatment, as per the  guidelines for management of diarrhea at the  first onset of diarrhea. |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Hold neratinib until recovery to ≤ grade 1 or  baseline.                                                                 |                                                                                                                     |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Instruct patient to follow dietetic  recommendations of the guidelines for  management of diarrhea.                      |                                                                                                                     |
| OR Grade 3 Diarrhea lasting > 2 days despite being  treated with optimal medical therapy, or                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                          |                                                                                                                     |
| Version 03/16/2020 - Amendment 13                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Page 41 of 87                                                                                                            |                                                                                                                     |  
Gastrointestinal Toxicities Requiring Dose Adjustment of Neratinib  
| NCI CTCAE V4.0                                                                          | Action                                                                                                                                                                                             |                                                                     |
|-----------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------|
| associated with fever, dehydration, or grade 3-4  neutropenia                           | -                                                                                                                                                                                                  | Fluid intake of ~2L should be maintained,  intravenously if needed. |
| -                                                                                       | If recovery occurs:  o ≤1 week after withholding treatment,  resume same dose of neratinib.  o Within 1-3 weeks after withholding  treatment, reduce neratinib dose to the  next lower dose level. |                                                                     |
| -                                                                                       | If event recurs and the neratinib dose has  not already been decreased, reduce neratinib dose to the next lower dose level.                                                                        |                                                                     |
| -                                                                                       | If subsequent events occur, reduce neratinib dose to the next lower dose level.                                                                                                                    |                                                                     |
| -                                                                                       | Once the event resolved to ≤ grade 1 or  baseline, start loperamide 4 mg with each  subsequent neratinib administration.                                                                           |                                                                     |
| -                                                                                       | If event recurs and the neratinib dose has  already been reduced at the lowest level,  discontinue neratinib.                                                                                      |                                                                     |
| OR Any Grade 4 diarrhea [Life-threatening  consequences; urgent intervention indicated] |                                                                                                                                                                                                    |                                                                     |
a phase ii study of
7.4 guidelines for the management

Changes in LFTs have been reported in subjects taking neratinib. Subjects experiencing grade 3 or 4 diarrhea requiring IV hydration and associated with any signs or symptoms of hepatotoxicity such as worsening of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash or eosinophilia should be evaluated for LFT changes. Abnormal values in aspartate transaminase (AST) and/or alanine transaminase (ALT) concurrent with abnormal elevations in total bilirubin that meet the criteria in the table below in the absence of other causes of liver injury are considered potential cases of drug-induced liver injury (potential Hy's Law cases) and should always be considered important medical events. The possibility of hepatic neoplasia (primary or secondary) should also be considered. During evaluation of potential hepatotoxicity, bilirubin should be fractionated and prothrombin time should be measured. Also, liver imaging should be obtained for subjects with any signs or symptoms of hepatotoxicity and/or grade 3 or greater LFT elevations, or as clinically indicated.  
| Event                                                                                                                                                                                  |                              |                                                                                                                                    |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|------------------------------------------------------------------------------------------------------------------------------------|
| (Based on NCI CTC4.0)                                                                                                                                                                  | Action                       |                                                                                                                                    |
| Grade 3 ALT (>5-20x ULN), or Grade 4 ALT (>20x ULN), or Grade 3 Bili (>3-10x ULN) and direct bili ≥ 35% of total bili, or Grade 4 Bili (>10x ULN) and direct bili ≥ 35% of total bili. | -                            | Permanently discontinue  investigational product (IP)                                                                              |
| -                                                                                                                                                                                      | Evaluate alternative causes. |                                                                                                                                    |
| ALT > 3x ULN and Total bilirubin > 2x ULN and Alkphos < 2x ULN.                                                                                                                        | -                            | Hold IP and immediately contact the  PI to discuss next steps, including  evaluation of alternative causes, and  management of IP. |
| -                                                                                                                                                                                      | Report as SAE.               |                                                                                                                                    |
| Signs or symptoms related to liver injury (abdominal  pain, fever, jaundice, rash, eosinophilia, or drop in  performance status) with either:                                          | -                            | Permanently discontinue IP.                                                                                                        |
| -                                                                                                                                                                                      | Evaluate alternative causes. |                                                                                                                                    |
| Grade 2 ALT (>3-5x ULN), or Grade 2 ALT (>3-5x ULN) and Grade 2 Bili (>1.5-3x ULN) and direct bili ≥ 35% of total bili.                                                                |                              |                                                                                                                                    |
a phase ii study of
7.5 guidelines for the management

LVEF assessments will be performed at the completion of every four cycles during the treatment period. It is strongly recommended to use the same method of cardiac evaluation (ECHO or MUGA) at each time point for each subject. In case of symptomatic heart failure, the investigational product must be discontinued permanently.  
Asymptomatic decline in LVEF that warrants intervention is defined as:
- LVEF < LLN AND absolute decrease in LVEF of ≥ 10% compared with pre-treatment LVEF, OR
- Absolute decrease in LVEF of ≥ 15% compared with pre-treatment LVEF
If asymptomatic decline in LVEF that warrants intervention is experienced, then:
- Temporarily HOLD neratinib and - Repeat LVEF in 3-4 weeks
If asymptomatic decline in LVEF as defined above persists, permanently discontinue neratinib, repeat LVEF in 3-4 weeks and consider cardiology consult. If LVEF recovers to ≥ LLN and absolute decrease in LVEF of resolves to < 15%, resume neratinib at the same dose, and resume LVEF monitoring per this protocol. If a subject has a second episode of asymptomatic decline in LVEF that meets either of the above criteria, permanently discontinue neratinib, repeat LVEF in 3-4 weeks and consider cardiology consult. Note that, for adverse events other than asymptomatic LVEF decline, if neratinib is held for > 3 weeks, subject should be withdrawn from the treatment phase of the study. In case of asymptomatic LVEF decline, subjects may resume neratinib within one week after LVEF recovery is documented as above, even if the timeframe exceeds 3 weeks. If a site does not provide normal ranges for ECHO or MUGA, the LLN should be defaulted to 50%.
a phase ii study of
7.6 subject compliance

Compliance is monitored by study personnel at the site by using subject-completed diaries and verbal information from the subject that is recorded on source documents, the drug inventory record, and CRFs. A subject must have taken the prescribed investigational product dosage 75% of the days in the treatment period to be compliant. Dose adjustments must follow the recommendations in this section. Site personnel will review subject-completed diaries at every visit and will document on drug accountability forms provided to the sites. Dose administration will be recorded on sponsor drug accountability forms.
a phase ii study of
7.7 fulvestrant dose modifications

There are no planned dose modifications for fulvestrant. Dosing may be modified or discontinued at the discretion of the treating physician.
a phase ii study of
7.8 trastuzumab (or fda approved

There are no planned dose modifications for trastuzumab (or FDA approved biosimilar). Dosing may be modified or discontinued at the discretion of the treating physician.
a phase ii study of
8.0 regulatory and reporting requirements

The entities providing oversight of safety and compliance with the protocol require reporting as outline below. Please refer to Appendix E for definitions and Appendix F for a grid of reporting timelines. Adverse events will be tracked from start of treatment up to 28 days following the last day of study treatment. All adverse events must be recorded on the toxicity tracking case report form (CRF) with the exception of: - Baseline adverse events, which shall be recorded on the medical history CRF Refer to the data submission schedule in Section 12 for instructions on the collection of AEs in the EDC.The Washington University Human Research Protection Office (HRPO) requires that all events meeting the definition of unanticipated problem or serious noncompliance be reported as outlined in Section 8.1.5 and 8.1.6. The FDA requires that all serious and unexpected adverse events considered related to the study drug be reported as outlined in Section 8.6. In addition, any fatal or life-threatening adverse experiences where there is a reasonable possibility of relationship to study intervention must be reported. Puma Biotechnology requires that all adverse events be reported as outlined in Section 8.7.
a phase ii study of
8.1 reporting to the human

Reporting will be conducted in accordance with Washington University IRB Policies.
a phase ii study of
pre-approval of all protocol exceptions

The Sponsor-Investigator (or designee) is required to notify the QASMC of any unanticipated problem involving risks to participants or others occurring at WU or any BJH or SLCH institution that has been reported to and acknowledged by HRPO. Unanticipated problems reported to HRPO and withdrawn during the review process need not be reported to QASMC. QASMC must be notified within **10 days** of receipt of IRB acknowledgment via email to qasmc@wustl.edu. Submission to QASMC must include the MyIRB form and any supporting documentation sent with the form. For events that occur at secondary sites, the Washington University Sponsor Investigator (or designee) is required to notify the QASMC within 10 days of Washington University notification via email to qasmc@wustl.edu. Submission to QASMC must include either the myIRB form and supporting documentation or (if not submitted to myIRB) the date of occurrence, description of the event, whether the event is described in the currently IRB approved materials, the event outcome, determination of relatedness, whether currently enrolled participants will be notified, and whether the informed consent document and/or any study procedures will be modified as a result of this event.
a phase ii study of
8.3 secondary sites reporting requirements

The research team at each secondary site is required to promptly notify the Washington University Sponsor-Investigator and designee of all serious adverse events (refer to Appendix E) **within 1** working day of the occurrence of the event or notification of the secondary site's PI of the event. This notification may take place via email if there is not yet enough information for a formal written report (using FDA Form 3500a (MedWatch) and Washington University's cover sheet (Appendix G). A formal written report must be sent to the Washington University Sponsor-Investigator and designee **within 4 calendar days** (for fatal or life-threatening suspected adverse reactions) or 11 calendar days (for serious unexpected suspected adverse reactions) of the occurrence of the event or notification of the secondary site's PI of the event. The research team at a secondary site is responsible for following its site's guidelines for reporting applicable events to its site's IRB according to its own institutional guidelines. The research team at Washington University is responsible for reporting all applicable events to the FDA, IBC, and Puma Biotechnology, Inc. as needed. Washington University pre-approval of all protocol exceptions must be obtained prior to implementing the change. Local IRB approval must be obtained as per local guidelines. Washington University IRB approval is not required for protocol exceptions occurring at secondary sites. 8.4 Reporting to Secondary Sites The Washington University Sponsor-Investigator (or designee) will notify the research team at each secondary site of all reportable events that have occurred at other sites within 10 working days of the occurrence of the event or notification of the Sponsor-Investigator of the event. This includes events that take place both at Washington University and at other secondary sites, if applicable. Refer to Section 16.0 (Multicenter Management) for more information.
a phase ii study of
8.5 reporting to the fda

The conduct of the study will comply with all FDA safety reporting requirements. **PLEASE NOTE** THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER FROM REPORTING REQUIREMENTS FOR HRPO/QASMC. It is the responsibility of the Washington University principal investigator to report to the FDA as follows:
- Report any unexpected fatal or life-threatening suspected adverse experiences (Refer to Appendix E for definitions) no later than **7 calendar days** after initial receipt of the information.  
- Report a suspected adverse reaction that is both serious and unexpected (SUSAR,
refer to Appendix E) no later than **15 calendar days** after it is determined that the information qualifies for reporting. Report an adverse event (refer to Appendix E) as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the drug and the adverse event, such as: o A single occurrence of an event that is uncommon and known to be strongly associated with drug exposure o One or more occurrences of an event that is not commonly associated with drug exposure but is otherwise uncommon in the population exposed to the drug o An aggregate analysis of specific events observed in a clinical trial that indicates those events occur more frequently in the drug treatment group than in a concurrent or historical control group
- Report any findings from epidemiological studies, pooled analysis of multiple studies, or clinical studies that suggest a significant risk in humans exposed to the drug no later than **15 calendar days** after it is determined that the information qualifies for reporting.  
- Report any findings from animal or in vitro testing that suggest significant risk in humans exposed to the drug no later than **15 calendar days** after it is determined that the information qualifies for reporting.  
- Report any clinically important increase in the rate of a serious suspected adverse reaction of that listed in the protocol or IB within **15 calendar days** after it is determined that the information qualifies for reporting.  
Submit each report as an IND safety report in a narrative format or on FDA Form 3500A or in an electronic format that FDA can process, review, and archive. Study teams must notify the Siteman Cancer Center Protocol Development team of each potentially reportable event within 1 business day after initial receipt of the information, and must bring the signed 1571 and FDA Form 3500A to the Siteman Cancer Center Protocol Development team no later than 1 business day prior to the due date for reporting to the FDA. Each notification to FDA must bear prominent identification of its contents ("IND Safety Report") and must be transmitted to the review division in the Center for Drug Evaluation
Page 46 and Research (CDER) or in the Center for Biologics Evaluation and Research (CBER) that has responsibility for review of the IND. Relevant follow-up information to an IND safety report must be submitted as soon as the information is available and must be identified as such ("Follow-up IND Safety Report").
a phase ii study of
8.6 reporting to puma biotechnology

Any serious adverse events which occur during the clinical study or within 28 days of receiving the last dose of study medication, whether or not related to the study drug, must be reported by the investigator. In addition, any SAEs which occur as a result of protocol specific diagnostic procedures or interventions must also be reported. All serious adverse events, in addition to being reported to the FDA by the investigator, must be reported by email to **Puma Biotechnology, Inc.** as follows: For expedited reports, (Site) will send the MEDWATCH report to the Company no later than seven (7) days for initial life-threatening and death reports, and fifteen (15) days for all other initial or follow-up serious and unexpected suspected adverse reaction (SUSAR) as assessed by the investigator for causality and by the Investigator-Sponsor for causality and expectedness based on the adverse reaction table in the most current Investigator Brochure, from the time of receipt of the SAE by (Site). For non-expedited reports (i.e., unrelated to study drugs or listed/expected event), (Site) will send the MEDWATCH report to the Company no later than thirty (30) days from the time of receipt of the SAE by (Site).
a phase ii study of
by e-mail to pumasae@parexel.com

SAEs brought to the attention of the investigator at any time after cessation of neratinib and considered by the investigator to be related or possibly related to neratinib must be reported to Puma Biotechnology, Inc. if and when they occur. Additionally, in order to fulfill international reporting obligations, SAEs that are related to study participation (e.g., procedures, invasive tests, changes from existing therapy) or are related to a concurrent medication will be collected and recorded from the time the subject consents to participate in the study until he/she is discharged from the study.
a phase ii study of
8.7 timeframe for reporting required

Adverse events will be tracked for 28 days following the last day of study treatment.
a phase ii study of
9.0 pharmaceutical information 9.1 neratinib

Neratinib (PB-272) is a potent irreversible pan erbB inhibitor. Neratinib is an orally available small molecule that inhibits erbB-1, erbB-2, and erbB-4 at the intracellular tyrosine kinase domains, a mechanism of action that is different from trastuzumab.
a phase ii study of
9.1.3 supplier(s)

Neratinib will be provided free of charge by **Puma Biotechnology, Inc**.
a phase ii study of
9.1.4 dosage form and preparation

Neratinib 40-mg tablets will be supplied by the sponsor. There are 210 pills per bottle. Repackaging of product is not allowed.
a phase ii study of
9.1.5 storage and stability

Investigational products will be stored by sites in a secure location at the storage conditions listed on the label; store at 25°C (77°F) or below with desiccant (bottle contains the dessicant); do not freeze. Correspondingly, subjects must be instructed to store investigational products in a safe place at these same storage conditions. Storage in a refrigerator or cold room is acceptable as long as the temperature remains above 0 °C. The investigational product must be stored as indicated. Deviations from the storage requirements, including any actions taken, must be documented and reported to the sponsor. Once a deviation is identified, the investigational product must be quarantined and not used until the sponsor provides documentation of permission to use the investigational product. The investigational product is only to be administered to subjects who have provided informed consent. Once the investigational product has been assigned to a subject it must not be reassigned to another subject.
a phase ii study of
9.1.6 administration

The number of tablets of neratinib taken will depend on the prescribed dose per the dose administration table. The investigational product will be taken by mouth with food, preferably in the morning.
a phase ii study of
9.2 fulvestrant 9.2.1 description

Fulvestrant (Faslodex) is an estrogen receptor antagonist which down-regulates the ER protein in breast cancer cells.
a phase ii study of
9.2.2 availability

Fulvestrant injection is commercially available as a sterile single-patient pre-filled syringe containing 250 mg at a concentration of 50 mg/ml. The solution is a clear, colorless to yellow, viscous liquid. In addition to the fulvestrant, each injection contains as inactive ingredients alcohol, USP, benzyl alcohol, NF, and benzyl benzoate, USP as co-solvents and castor oil, USP as a co-solvent and release rate modifier. For patients who cannot obtain fulvestrant commercially, such as those who are without medical insurance or with a limited income, AstraZeneca Foundation Patient Assistance Program can be reached at 1-800-424-3727 or by downloading a PDF application from the AstraZeneca US web site at www.astrazeneca-us.com. Completed applications should be mailed to:
AstraZeneca Foundation Patient Assistance Program PO Box 66551 St. Louis, MO 63166-66551
a phase ii study of
9.2.3 storage and stability

The syringes of fulvestrant for all cycles of treatment should be stored in the original container and refrigerated at 2°- 8°C (36°- 46°F).
a phase ii study of
9.2.4 preparation

Remove glass syringe barrel from tray and check that it is not damaged. Peel open the safety needle (SafetyGlide™) outer packaging. Break the seal of the white plastic cover on the syringe luer connector to remove the cover with the attached rubber tip cap. Twist to lock the needle to the luer connector. Remove needle sheath. Remove excess air from the syringe (a small gas bubble may remain).
a phase ii study of
9.2.5 administration

Fulvestrant will be administered at a dose of 500 mg (2 x 250 mg injections) IM on Cycle 1 Day 1 and Day 15, then on Day 1 of each subsequent cycle. Administer intramuscularly slowly in the buttock. (NOTE: 500 mg dose will require one 250 mg injection in each buttock.) Immediately activate needle protection device upon withdrawal from patient by pushing lever arm completely forward until needle tip is fully covered. Visually confirm that the lever arm has fully advanced and the needle tip is covered. If unable to activate, discard immediately into an approved sharps container.
a phase ii study of
9.2.6 drug interactions

In vitro studies using human hepatocytes, fulvestrant was metabolized predominantly by conjugation. The metabolites thus formed are thought to possess no estrogenic activity and minimal anti-estrogenic activity. In studies using human liver microsomes, fulvestrant inhibited the activity of CYP1A2, 2C9 and 3A4 minimally. CYP3A4 did metabolize fulvestrant in these studies, but the human hepatocyte studies noted above indicate conjugation is a more important metabolic pathway. In addition, studies in healthy volunteers indicate that fulvestrant metabolism is not significantly affected by inducers or inhibitors of CYP3A4, nor does fulvestrant affect the metabolism of 3A4 substrates. Thus, fulvestrant is not expected to be involved in significant drug interactions mediated by CYP3A4.
a phase ii study of
10.0 correlative studies 10.1 required

- Archival tumor specimens that are left over from the HER2 mutation analysis at Washington University are not returned and will be used for investigations of potential therapeutic targets in HER2-negative breast cancer.  
- Additional archival tumor specimens may be requested for studies that compare mutations in primary versus metastatic disease sites, and predictors of treatment response to neratinib.  
- For patients who had *HER2* mutation identified in another laboratory and registered to the study, please submit archival tumor specimens to Wash U Alliance/ACOSOG-CSB/TPC.  
- Please refer to the Correlative Science Procedure Manual Section 3.1 for sample requirement and shipping instructions.
a phase ii study of
10.2 required research blood collection

Peripheral blood samples will be collected in patients who have *HER2* mutation and are registered to receive study drug therapy. Blood will be collected at baseline, during therapy as well as at disease progression (Also refer to the Correlative Science Calendar **Section 10.5**), for **ctDNA** sequencing and other circulating markers that may affect disease outcome. **Please ship samples** to **Wash U Alliance/ACOSOG-CSB/TPC.** Please refer to the Correlative Science Procedure Manual Section 3.2 for sample requirement and shipping instructions.
a phase ii study of
10.3 optional research tumor biopsy

If a patient is registered to this trial to receive neratinib and has tumors that are safe for biopsy, we encourage consenting for tumor collection prior to study drug administration and/or at the time of disease progression on neratinib for investigations that assess predictors of response to therapy and mechanisms of resistance. Note that the tumor biopsy collected at disease progression on study treatment will be subjected to the CLIA NGS panel of 122 genes (the Comprehensive Cancer Gene Set, verion 3.2 (CCGSv3.2)), at the Wash U GPS and a clinical report will be issued to the site investigator to provide to the treating physician and the patient within 3-4 weeks of receiving the specimen. The sequencing report will provide interpretations of genomic findings, but it is at the discretion of the treating physician and the patient in regards to any need for referral to clinical genetic counseling service. The report may be helpful in assessing patient eligibility for clinical trials that require mutation screening. Please refer to the Correlative Science Procedure Manual Section 3.3 in regards to sample requirements and shipping instructions.
a phase ii study of
10.4 reporting of a clia

This will follow a full clinical workflow including evaluation of the FFPE biopsy specimen by a qualified surgical pathologist (WU Anatomic and Molecular Pathology Core lab), extraction of DNA, NGS library preparation and sequencing, NGS data analysis and variant interpretation for the CCGSv3.2 Solid Tumor gene set (122 genes). Cases with negative results (no level 1 or 2 variants identified) will proceed to additional reflex testing for the hematopoietic disorders gene set (54 genes).  
| Specimen Type                                                                                          | Collection/Submission Time Point     | Shipping                                         | Ship       |         |     |
|--------------------------------------------------------------------------------------------------------|--------------------------------------|--------------------------------------------------|------------|---------|-----|
| Condition                                                                                              |                                      |                                                  |            |         |     |
| Pre-                                                                                                   | To                                   |                                                  |            |         |     |
| Pre                                                                                                        |                                      |                                                  |            |         |     |
| treatment /                                                                                            |                                      |                                                  |            |         |     |
| registration                                                                                           | C1D1 (prior  to starting  treatment) | C1D15, C2D1,  C3D1 and day  1 of each odd  cycle | EOT / Prog |         |     |
| Archival Tissue for HER2  mutation testing (see CSPM 1.0)                                              | X                                    | Ambient                                          | GPS        |         |     |
| X1                                                                                                     | Ambient                              | GPS                                              |            |         |     |
| 10 ml Whole Blood in EDTA  for germline DNA sequencing  (see CSPM 2.0) Archival Tissue for correlative | X2                                   | Ambient                                          | TPC        |         |     |
| studies (See CSPM 3.0) 10 ml Whole Blood in EDTA                                                       | X                                    | Ambient                                          | TPC        |         |     |
| tube 3 ml plasma (from 10 ml                                                                           | X                                    | X                                                | X          | Frozen3 | TPC |
| EDTA tube)                                                                                             | X                                    | X                                                | X          | Frozen3 | TPC |
| 3 ml serum (from 10 ml SST  tube) (2) 8 ml Streck tubes                                                | X                                    | X                                                | X          | Ambient | TPC |
| Optional Fresh Biopsies (see                                                                           | X                                    | X                                                | Frozen and | TPC     |     |
| CSPM 3.3)                                                                                              | Ambient                              |                                                  |            |         |     |
| 1 Only required if notified by WUSM study team                                                         |                                      |                                                  |            |         |     |  
1 Only required if notified by WUSM study team 2 FFPE tumor rich block/slides are required - due within 60 days of registration for patients who receive treatment on trial. 3Store frozen samples at -70oC to -80oC. Ship frozen samples quarterly on dry ice.  
CSPM: Correlative Science Procedure Manual