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Enhancing Efficacy of Chemotherapy in Triple Negative/Basal-Like Breast Cancer by Targeting Macrophages: A Multicenter Phase Ib/II study of PLX 3397 and Eribulin in Patients with Metastatic Breast Cancer UCSF Protocol No.: 12751 Version Number: 10.0 Version Date: 03/03/2016  
  
  
Lisa Coussens, M.D. Shelley Hwang, M.D.  
Page 1 of 85   
Collaborating Project Investigators  
  
| Version 10.0   | 03-03-2016   |
|----------------|--------------|
| Version 9.0    | 09-18-2015   |
| Version 8.0    | 03-10-2015   |
| Version 7.0    | 01-06-2014   |
| Version 6.0    | 02-07-2013   |
| Version 5.0    | 09-20-2012   |
| Version 4.0    | 06-25-2012   |
| Version 3.0    | 05-29-2012   |
| Version 2.0    | 03-26-2012   |
| Version 1.0    | 12-14-2011   |
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table of contents

| Table of Contents                                                                                                                                       |                                                                                                                                                    |            |
|---------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| 1.0                                                                                                                                                     | Background and Rationale                                                                                                                           |            |
| 1.1                                                                                                                                                     | Background .                                                                                                                                       |            |
| 1.2                                                                                                                                                     | Clinical Studies ............................................................................................................................18    |            |
| 1.3                                                                                                                                                     | RATIONALE                                                                                                                                          |            |
| 2.0                                                                                                                                                     | Objectives .                                                                                                                                       |            |
| 2.1                                                                                                                                                     | Primary Objective                                                                                                                                  |            |
| 2.1.2                                                                                                                                                   | Phase II .                                                                                                                                         |            |
| 2.2                                                                                                                                                     | Secondary Objectives                                                                                                                               |            |
| 2.2.1                                                                                                                                                   | Phase Ib                                                                                                                                           |            |
| 2.2.2                                                                                                                                                   | Phase II .                                                                                                                                         |            |
| 3.0                                                                                                                                                     | Study Design and Eligibility Criteria .....................................................................................................24      |            |
| 3.1                                                                                                                                                     | Study Design .                                                                                                                                     |            |
| 3.1.1                                                                                                                                                   | Phase Ib                                                                                                                                           |            |
| 3.1.2                                                                                                                                                   | Phase II .                                                                                                                                         |            |
| 3.2                                                                                                                                                     | Inclusion Criteria                                                                                                                                 |            |
| 3.3                                                                                                                                                     | Exclusion Criteria .                                                                                                                               |            |
| 4.0                                                                                                                                                     | Patient Registration                                                                                                                               |            |
| 4.1                                                                                                                                                     | Stratification                                                                                                                                     |            |
| 4.2                                                                                                                                                     | Randomization and Blinding ................................................................................................................27      |            |
| 4.3                                                                                                                                                     | Registration .                                                                                                                                     |            |
| 4.3.1                                                                                                                                                   | UCSF Helen Diller Family Comprehensive Cancer Center .........................................27                                                   |            |
| 4.3.2                                                                                                                                                   | Participating sites                                                                                                                                |            |
| Investigational Treatment Plan ......................................................................................................................28 |                                                                                                                                                    |            |
| 5.0                                                                                                                                                     |                                                                                                                                                    |            |
| 5.1                                                                                                                                                     | Dose and Schedule                                                                                                                                  |            |
| 6.0                                                                                                                                                     | General Concomitant Medication and Supportive Care Guidelines                                                                                      |            |
| 6.1                                                                                                                                                     | Use of Myeloid Growth Factors                                                                                                                      |            |
| 7.0                                                                                                                                                     | Toxicity Management & Dose Modifications ............................................................................30                            |            |
| 7.1                                                                                                                                                     | PLX3397                                                                                                                                            |            |
| 7.2                                                                                                                                                     | Eribulin                                                                                                                                           |            |
| 8.0                                                                                                                                                     | Schedule of Assessments .......................................................................................................................35  |            |
| 8.1                                                                                                                                                     | Screening Assessments (same for both the Phase Ib and Phase II study)......................38                                                      |            |
| 8.2                                                                                                                                                     | Assessments During Treatment ...............................................................................................38                     |            |
| Follow-Up Assessments                                                                                                                                   |                                                                                                                                                    |            |
| 8.3                                                                                                                                                     |                                                                                                                                                    |            |
| 9.0                                                                                                                                                     | Criteria for Evaluation ................................................................................................................40         |            |
| 9.1                                                                                                                                                     | Response Definitions .................................................................................................................40           |            |
| 9.2                                                                                                                                                     | Toxicity Definitions ....................................................................................................................45        |            |
| 10.0                                                                                                                                                    | C Conserved to the Conserved Conse                                                                                                                 |            |
| 10.1                                                                                                                                                    | Conditions for Terminating the Study .................................................................................45                           |            |
| 10.2                                                                                                                                                    | Conditions for Individual Patient Termination ................................................................45                                   |            |
| 11.0                                                                                                                                                    | Drug Information ...............................................................................................................................45 |            |
| 11.1                                                                                                                                                    | Eribulin mesylate (E7389)                                                                                                                          |            |
| 11.1.3                                                                                                                                                  | Mode of Action                                                                                                                                     |            |
| 11.1.4                                                                                                                                                  | Storage and Stability..........................................................................................................47                  |            |
| 11.1.5                                                                                                                                                  | Metabolism ..............................................................................................................................47        |            |
| 11.1.6                                                                                                                                                  | Preparation & Administration                                                                                                                       |            |
| Protocol Version #10                                                                                                                                    | Page 3 of 85                                                                                                                                       | 03/03/2016 |
| Protocol #: 12751                                                                                                                                       |                                                                                                                                                    |            |  
| 11.1.7                                                                                                                            | Side Effects ...............................................................................................................................47               |            |
|-----------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| 11.1.8                                                                                                                            | Nursing Implications ......................................................................................................48                                |            |
| 11.2                                                                                                                              | PLX3397 ...............................................................................................................................48                    |            |
| 11.2.1                                                                                                                            | PLX3397 Administration..............................................................................................48                                       |            |
| 11.2.2                                                                                                                            | PLX3397 Packaging and Labeling ...............................................................................48                                             |            |
| 11.2.3                                                                                                                            | PLX3397 Storage and Stability .......................................................................................49                                      |            |
| 11.2.4                                                                                                                            | PLX3397 Accountability, Reconciliation, and Return ................................................49                                                        |            |
| 11.2.5                                                                                                                            | PLX3397 Compliance ....................................................................................................49                                    |            |
| 11.2.6                                                                                                                            | PLX3397 Toxicity ............................................................................................................49                              |            |
| 12.0                                                                                                                              | Statistical Considerations                                                                                                                                   |            |
| 12.1                                                                                                                              | Endpoint Definitions .                                                                                                                                       |            |
| 12.2                                                                                                                              | Analysis Plan                                                                                                                                                |            |
| 12.3                                                                                                                              | Accrual Objectives ..................................................................................................................................54      |            |
| 12.4                                                                                                                              | Estimated Duration of Study .............................................................................................................54                  |            |
| 12.5                                                                                                                              | DATA SAFETY Monitoring ...................................................................................................................................54 |            |
| 12.6                                                                                                                              | Replacement Policy .                                                                                                                                         |            |
| 12.7                                                                                                                              | Pharmacokinetic studies ......................................................................................................................54             |            |
| 12.7                                                                                                                              | Correlative studies                                                                                                                                          |            |
| 13.0                                                                                                                              | Data Collection and Management                                                                                                                               |            |
| 14.0                                                                                                                              | Correlative studies                                                                                                                                          |            |
| 14.1                                                                                                                              | T TUMOR biopsies                                                                                                                                             |            |
| 14.2                                                                                                                              | CSF1 levels                                                                                                                                                  |            |
| 14.3                                                                                                                              | Blood for leukocyte subtyping .................................................................................................................56            |            |
| 14.4                                                                                                                              | Pharmacokinetic sampling ..................................................................................................................56                |            |
| 14.5.                                                                                                                             | Archival tumor samples                                                                                                                                       |            |
| Evaluation of dynamic tissue and blood biomarkers most likely to show early                                                       |                                                                                                                                                              |            |
| 14.6.2                                                                                                                            |                                                                                                                                                              |            |
| biologic changes that predict clinical response ...............................................................................58 |                                                                                                                                                              |            |
| 14.6.3 Determine magnitude of change in serum CSF1 .................................................................58            |                                                                                                                                                              |            |
| Reporting Adverse Drug Reactions                                                                                                  |                                                                                                                                                              |            |
| 15.0                                                                                                                              |                                                                                                                                                              |            |
| 15.1                                                                                                                              | Adverse Event Definitions ....................................................................................................................58             |            |
| 15.2                                                                                                                              | Recording of an Adverse Event                                                                                                                                |            |
| 15.3                                                                                                                              | Follow-up of Adverse Events                                                                                                                                  |            |
| 15.4                                                                                                                              | Adverse Events Monitoring..................................................................................................60                                |            |
| 15.5                                                                                                                              | Expedited Reporting ..........................................................................................................................61             |            |
| 15.6                                                                                                                              | Reporting DLTs...........................................................................................................................63                  |            |
| 16.0                                                                                                                              | Data Safety Monitoring Plan ..........................................................................................................63                     |            |
| 16.1                                                                                                                              | Oversight and Monitoring Plan ..............................................................................................63                               |            |
| 16.2                                                                                                                              | Monitoring and Reporting Guidelines ....................................................................................63                                   |            |
| 16.3                                                                                                                              | Review and Oversight Requirements ..................................................................................64                                       |            |
| 17.0                                                                                                                              | S Study Management .                                                                                                                                         |            |
| 17.1                                                                                                                              | Pre-study Documentation ..........................................................................................................65                         |            |
| 17.2                                                                                                                              | Independent Ethics Committees/Institutional Review Board .......................................65                                                           |            |
| 17.3                                                                                                                              | Informed Consent ............................................................................................................................65              |            |
| 17.4                                                                                                                              | Oversight and Monitoring Plan ................................................................................................65                             |            |
| 17.5                                                                                                                              | Coordinating Center Processing and Documenting FDA Correspondence ................66                                                                         |            |
| 17.6                                                                                                                              | Record Keeping and Record Retention ...................................................................................66                                    |            |
| 17.7                                                                                                                              | Regulatory Documentation                                                                                                                                     |            |
| Protocol Version #10                                                                                                              | Page 4 of 85                                                                                                                                                 | 03/03/2016 |
| Protocol #: 12751                                                                                                                 |                                                                                                                                                              |            |  
| 17.8                                                                                                                                     | Patient Enrollment 67                                |
|------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------|
| 17.9                                                                                                                                     | Study Drug Supply and Accountability 68              |
| 17.10                                                                                                                                    | Coordinating Center Documentation of Distribution 68 |
| 17.10.1                                                                                                                                  | Approval of Protocol and Amendments 68               |
| 18.0                                                                                                                                     | References 69                                        |
| Appendix 1: Strong CYP3A4 Inhibitors and Inducers 74 Appendix 2: Drugs With A Risk Of Torsades De Pointes 75 Appendix 3: Drug Diaries 77 |                                                      |
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list of abbreviations

| AE   | Adverse event                            |
|------|------------------------------------------|
| ALT  | Alanine transaminase                     |
| ANC  | Absolute neutrophil count                |
| AST  | Aspartate transaminase                   |
| BUN  | Blood urea nitrogen                      |
| CCC  | Comprehensive Cancer Center (UCSF)       |
| CEC  | Circulating endothelial cell             |
| CHR  | Committee on Human Research (UCSF IRB)   |
| CR   | Complete response                        |
| CSF  | Colony-stimulating factor                |
| CT   | Computerized tomography                  |
| CTX  | Chemotherapy                             |
| CTC  | Circulating tumor cell                   |
| CTMS | Clinical Trials Management System        |
| DLT  | Dose limiting toxicity                   |
| DSMB | Data safety monitoring board             |
| DSMC | Data safety monitoring committee (UCSF)  |
| ECOG | Eastern Cooperative Oncology Group       |
| EF   | Ejection Fraction                        |
| FDA  | Food and Drug Administration             |
| FISH | Fluorescent in-situ hybridization        |
| HER2 | Human Epidermal Growth Factor Receptor 2 |
| IHC  | Immunohistochemistry                     |
| MCB  | Metastatic breast cancer                 |
| MRI  | Magnetic Resonance Imaging               |
| MTD  | Maximum tolerated dose                   |
| NCI  | National Cancer Institute                |
| OS   | Overall survival                         |
| PET  | Positron emission tomography             |
| PK   | Pharmacokinetics                         |
| PFS  | Progression-free survival                |
| PR   | Partial response                         |
| RFS  | Relapse-free survival                    |
| RR   | Response rate                            |
| SAE  | Serious adverse event                    |
| TAM  | Tumor associated macrophage              |
| TN   | Triple negative                          |
| TNBC | Triple negative breast cancer            |
| TTP  | Time to progression                      |
| UCSF | University of California San Francisco   |
| ULN  | Upper limit of normal                    |
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1.0 background and rationale 1.1

T**RIPLE NEGATIVE BREAST CANCER** The marked difference in breast cancer (BC) outcome between black women and white women in the United States has long been recognized as an important health care concern[1]. Although BC incidence is lower overall among black women, the mortality rate is consistently higher[2-4]. Recent data has demonstrated a cross-over in age adjusted incidence of BC, with a higher rate in black women under age 40 than white women (number of BCs per 100,000 woman-years 15.5 versus 13.1), than with subsequent cross over with higher rates in white women than black women aged 40 or older (281.3 versus 239.5)[5]. Many of the determinants resulting in racial differences in BC outcome have been attributed to disparities in screening, diagnosis and treatment. However, studies of racial differences in BC treatment now indicate that factors apart from health care access contribute to disparities in BC outcome. A significant finding to emerge has been the discovery that African American women have biologically more aggressive disease, independent of social factors[1]. In a SEER-based cancer registry of over 100,000 women, 57% of African American women had high-grade disease, compared to 41% in white women[6]. In both SEER and multi-center studies, estrogen receptor (ER)-negative, progesterone receptor (PR)-negative disease has been found to be more prevalent among African Americans. Carey et al evaluated 496 BCs in the Carolina Breast Cancer study, and found a significantly higher incidence of basal-like BCs in premenopausal compared to postmenopausal African American women (39 versus 14%) and non-African American women of any age (16%)[7]. In contrast, luminal A or low-grade, hormone receptor-positive BC, were found less commonly in younger black women. In the California Cancer Registry, women with ER, PR and HER2 negative BC (triple negative, TNBC) were more likely to be under age 40 and non-Hispanic black or Hispanic[8]. Non-Hispanic black women had the poorest survival, with a 5-year survival for late-stage tumors of only 14%. There is now evidence that biological features of BC affect response to specific therapies. Analysis of gene expression patterns has led to classification of BCs into a number of subsets correlated with biologic behavior, outcome, and therapeutic response[9]. Perou et al used specific gene expression patterns to classify BC into four groups: luminal type, which are often ER positive, basal-like type, which are often ER negative, a type which over-expresses HER-2, and the *normal* breast type[10]. These groupings have been extensively validated, including one series of over 1000 BCs[11]. Further sub-classifications have been defined. TNBC refers to a heterogeneous group of tumors defined on the basis of negative ER, PR, and lack of HER-2 gene amplification associated with a poor prognosis, some of which exhibit abnormalities in the *BRCA* genes. Many of these tumors are highly proliferative with short duration of response to therapy with either primary or rapid development of resistance to standard therapy[12]. The basal-like classification is based on gene expression data, and most, but not all TNBC are categorized as basal-like[13, 14]. Basal-like or TNBC account for ~15% of BC diagnoses. Compared to other BCs, TNBC is commonly of higher grade, occurs more frequently in younger women and those of African American descent, and is associated with Page 7 of 85 increased visceral and central nervous system metastasis as well as inferior survival[15, 16]. Despite initial high responses to combination neoadjuvant chemotherapy (CTX), relapses are seen early following diagnosis among women with TNBC[17]. C*HEMOTHERAPY FOR ADVANCED STAGE BREAST CANCER* Approximately 30% of women diagnosed with early stage BC will develop systemic recurrence and 5% will present with de novo metastatic disease, with only a few patients achieving longterm survival with standard CTX. A disproportionate percent of patients with metastatic BC
(MBC) will have TNBC. CTX is a critical component of treatment for metastatic BC (MBC), particularly for hormone resistant disease, resulting in improved cancer related symptoms and prolonged survival. Many combination CTX regimens have been studied in an effort to improve outcome. Although these have demonstrated improved response rates compared to single agents, this has been at the expense of increased toxicity without improved survival[18]. International guidelines recommend use of sequential single agent CTX except in the case of visceral crisis or rapidly progressing disease[19]. Microtubule inhibitors are one of the most effective classes of agents available for treating early and late stage BC, and are considered a standard of care for MBC treatment. Microtubule function is vital to cell survival and plays an important role in proliferation and motility, maintenance of cell shape and protein trafficking. Several agents affecting microtubule dynamics are active anti-tumor agents and induce polymerization, or cause non-functional tubulin aggregates blocking cell division by interfering with mitotic spindle function, consequently resulting in cell cycle arrest and cell death[20]. Paclitaxel (PTX) is one of the most widely used agents in this class, although its Cremaphor solvent causes bone marrow and peripheral nerve toxicity, and requires steroid premedication to prevent anaphylaxis. In advanced disease, weekly dosing is superior to an every 3 wk schedule[21]. Nab-paclitaxel (nab-PTX) is albumin-bound PTX delivered without premedication, and is associated with less toxicity than Cremaphor-based PTX. Weekly therapy is effective in PTX- and docetaxel-resistant disease[22]. Treatment with the novel halichondrin analogue, eribulin, improved survival (OS) compared to treatment of physician's choice (TPC) in patients with heavily pre-treated and taxane plus anthracycline-resistant MBC (eribulin:13.1 mo vs. TPC: 10.7 mos, HR 0.81, p=0.041)[23], and has been recently approved by the FDA for treatment of advanced breast cancer (see specific data below). Response rates were higher in the eribulin arm (12.2 vs. 4.7%, p=0.002), and although PFS was longer by investigator assessment, by central review PFS was not significantly different (3.7 versus 2.2 months, HR 0.87, p=0.14). Subset analysis suggested benefit from eribulin across risk groups. In the overall study, grade 3-4 toxicities included higher rates of neutropenia (21% versus 14%) and febrile neutropenia (3% versus 0.8%); anemia, asthenia/fatigue, nausea, mucositis and hand-foot syndrome were more prevalent in the TPC arm. Within patients with TNBC, current data indicates no difference in response to therapy based on race, although this remains controversial[24, 25]. Nonetheless, median time to progression (TTP) remains short for patients with this aggressive subtype of BC[12]; in the recent phase III eribulin trial, 144 patients were identified with TNBC, and in this group the PFS for eribulin was 2.23 mos, compared to 1.9 mos for TPC. Clearly, additional treatment approaches for patients with chemotherapy-resistant TNBC are critical. MYELOID CELLS, *MAMMARY GLAND DEVELOPMENT AND BREAST CANCER* Leukocytes are normal cellular components of all tissues, are critical for regulating normal tissue homeostasis, and are significant paracrine regulators of all physiologic and pathologic tissue Page 8 of 85 repair processes. In mammary glands, every stage of development is accompanied by changes in the surrounding stroma that is populated by immune cells particularly those of the innate lineage. Mechanisms whereby innate immune cells are recruited to developing mammary epithelial structures have not been fully elucidated, although the process is clearly triggered by estrogen[26]. Studies of mice in which CSF-1 is absent owing to a *Csf1* homozygous null mutation (Csf1*op/op*) have shown that many tissues, including mammary glands, are severely depleted of macrophages (Ms), indicating an essential requirement for CSF-1for Mmaturation and function[27, 28]. Csf1*op/op* mice have inhibited mammary development characterized by fewer numbers of terminal end buds, reduced branching and diminished ductal length compared to wild type mice[26]. Thus, in M-deficient glands, although a ductal tree eventually develops that fills the fat pad, the resulting gland is atrophic[28]. A similar defect was reported in CSF-1 receptor (CSF1R)-null mutant mice[29]. While BC has not historically been linked to underlying inflammation or infection, it exhibits tumor-associated inflammation characterized by infiltration of leukocytes into developing tumors where increases in T cells and myeloid cells in neoplastic stroma parallels progression[30-32]. In BCs, Ms are the most abundant innate immune cell type present where they regulate angiogenic processes via production of pro-angiogenic factors including vascular endothelial growth factor (VEGF) and proteases[33]. In a transgenic mouse model of mammary adenocarcinoma development, e.g., MMTV-PyMT mice[34], increased M infiltration in premalignant tissue occurs immediately before activation of angiogenesis and onset of malignancy[35, 36]. CSF-1 is broadly expressed by mammary tumor cells, and its expression correlates with extent of M infiltration[37]. MMTV-PyMT mice carrying the Csf1*op/op* mutation exhibit 95% decreased infiltration of Ms in tumors, inhibited angiogenesis, significantly delayed tumor progression and diminished pulmonary metastasis[35]. Another myeloid population implicated in BC development are the so-called immature myeloid suppressor cells (IMCs)[38, 39] that express low to undetectable levels of major histocompatibility complex (MHC)-II and costimulatory molecules, thus they cannot induce antitumor responses similar to Ms activated by type 1 cytokines (M1-Ms). Rather, IMCs promote tumor development by exerting inhibitory activity on both tumor-specific and nonspecific T cells and by providing factors for tumor growth and neovascularization[40, 41].  
In BC, like in mammary gland development, Ms are reregulated in part by CSF1, mediated by the CSF1R[42]. A second CSF1R ligand, interleukin (IL)-34, possesses similar binding affinities and also regulates M recruitment to tissues, but exhibits distinct tissue distribution[43-45]. Paracrine interactions between Ms and MECs form positive feed-forward loops involving M-
expressed EGF, and CSF1 expressed by neoplastic cells, that together regulate carcinoma cell chemotaxis along collagen fibers towards blood vessels directed by perivascular Ms[46, 47].  
Based on these findings, it seems reasonable to postulate that blockade of cellular and/or molecular programs enhancing M recruitment in BC may represent tractable targets for anticancer therapy. Indeed, blockade of CSF1 or CSF1R results in decreased M presence in tissues and in experimental tumors, correlates with diminished angiogenesis, reduced tumor growth and metastasis in some models[35, 48-52]. T CELLS REGULATE PROTUMOR MACROPHAGE/*MONOCYTE ACTIVITY* Experimental studies have revealed that B and T cells exert pro-tumor activity indirectly by regulating myeloid cell bioactivity, including Ms, IMCs and mast cells, resulting in resistance to endocrine therapies and enhanced development of metastasis[32, 53-55]. We reported that interleukin (IL)-4-expressing TH2 CD4+ T cells promote invasion and metastasis of mammary adenocarcinomas by regulating protumor M and IMC activity, i.e., increased expression of EGF, transforming growth factor (TGF), reactive oxygen species (ROS), inducible nitrogen oxygen synthase (iNOS)[32, 56], and repression of cytotoxic CD8+ T lymphocytes (CTL)[32].  
Using the MMTV-PyMT mouse model of mammary carcinogenesis[34], we revealed that PyMT/CD4+ T cell-deficient mice (CD4-/-) exhibited a significantly attenuated metastatic phenotype[32] similar to that of CSF1*op/op*/PyMT mice[35]. IMCs and Ms in tumors of CD4deficient/PyMT mice expressed elevated levels of type 1 cytokines, e.g., tumor necrosis factor
(TNF), IL-6, IL-12p40, IL-1, and *Nos2* mRNA, indicative of a prevalent TH1 immune microenvironment and M1-M phenotype[32], whereas IMCs and Ms from CD4proficient/PyMT mice were instead indicative of alternatively activated M2-Ms that expressed higher levels of *arginase-1* (Arg-1) and Tgf, thus characterizing a protumor TH2 microenvironment[32]. PyMT/IL4R-deficient and PyMT mice treated with neutralizing antibodies to IL-4, phenocopied PyMT/CD4-/- mice with diminished metastasis and presence of M1-IMCs and M1-Ms in carcinomas[32]. Together this data indicates that TH2-CD4+ T cells promote metastasis by enhancing pro-tumor bioactivities of Ms and IMCs, and that depletion of Ms and/or IMCs, or blockade of their IL-4-regulated pathways, may provide a survival advantage by limiting progression and metastasis. R*ATIONALE FOR MACROPHAGE TARGETING IN TNBC* Patients with metastatic TNBC represent a significant challenge as many have CTX-resistant disease at relapse, and others develop resistance quickly after initial response. Despite promising advances with agents inhibiting DNA repair, (PARP inhibitors), all patients with advanced TN disease will eventually die from their disease[57, 58]. Recently, Martin and colleagues reported that BCs in African Americans displayed different expression profiles correlating with the TN phenotype[59]. Importantly however, differences in the tumor microenvironment were also identified. Specifically, tumor-associated Ms were independently increased in tumors of African American women. Supporting this finding, a response signature for colony stimulating factor (CSF)1, a primary regulator of tissue M maturation and infiltration, was identified in 1725% of BCs associated with decreased expression of ER and PR[60]. Campbell and colleagues studied tissue M infiltration in two independent cohorts and found a significant correlation between intratumoral Ms and specific tumor features, including high grade, hormone receptornegativity, basal-like subtype, and the number of Ms was associated with increased risk of death from cancer[61]. Studies in transgenic mouse models of mammary carcinogenesis have revealed that Ms promote carcinogenesis and enhance metastasis by high-level expression of epidermal growth factor (EGF) and activation of EGF-regulated signaling cascades in mammary epithelial cells (MECs)[62]. These data, along with others, indicate that M infiltration may be a prognostic indicator, and could serve as a potential target for novel therapies. Since high-grade, TN tumors characterized by increased M infiltration are disproportionately represented among women of African American ancestry, identifying an alternate treatment approach to this biologic group of tumors has potential to close the outcome disparity observed among African Page 10 of 85
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preliminary studies leukocyte profiling in

Based on our previous studies demonstrating that CD4+ T cells and M2-Ms enhance development of pulmonary metastasis in MMTV-PyMT mice[32, 56], we postulated that the density of CD4, CD8 and CD68-positive immune cells in human BC would provide prognostic information. To evaluate this, we established a classification and regression tree algorithm to define a "signature" in a screening cohort of BC tissues (n=179, cohort I). High and low thresholds for each marker were established through a decision tree analysis with 10-fold crossvalidation of each tree model. All BC samples were categorized as having either a CD68high/CD4high/CD8low or a CD68low/CD4low/CD8high *immune signature,* and the same thresholds were then applied to a validation BC cohort (cohort II, n=498). Kaplan Meyer analysis in the two cohorts (677 pts) demonstrated significantly reduced relapse-free survival (RFS) for pts bearing the CD68high/CD4high/CD8low signature (**Fig 1A-B**). Multivariate Cox regression analysis revealed that the CD68high/CD4high/CD8low signature was an independent predictor of decreased OS and RFS after controlling for grade, nodal status, tumor size, ER, PR and HER2 status in both cohorts (p<0.05 and p=0.001, respectively). Kaplan-Meier analysis of Cohort II
also demonstrated significantly reduced RFS in node-positive pts (**Fig 1C**). Analysis of the prognostic value of the immune signature within individual tumor subtypes revealed that the CD68high/CD4high/CD8low phenotype was associated with reduced OS and RFS in TNBCs (**Fig**
1D). This correlation in TN disease is particularly important given the aggressive nature of TNBCs that often becomes refractory to CTX. These results indicate that infiltration by Ms and T cells may influence BC recurrence in lymph node-positive pts.
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neoadjuvant ctx correlates with myeloid

To identify biomarkers or parameters of immune cell presence in BCs in a prospective manner, and to identify pts most likely to benefit from M-targeted therapy, we have started to evaluate freshly isolated, unfixed human BC tissue, resected from women undergoing surgery at UCSF, to reveal leukocyte complexity by fluorescent-activated cell sorting (FACS). Thus far, we have evaluated 4 invasive ductal carcinomas (IDC) and one ductal carcinoma in situ (DCIS), with adjacent normal breast tissue also available from 3 of the IDCs (**Fig 1D**). With regards to variances in immune complexity in these tumors, the most obvious variance identified thus far is that the samples from women who had received neoadjuvant CTX contained ~6-fold higher % of Ms (CD14+CD11b+), as compared to BC tissue from women who had not received neoadjuvant CTX (**Fig 1E-F**). Since immunosuppressive monocytes (IMCs) regulate cancer development in murine models, and since these would not be efficiently depleted by therapeutic strategies targeting CSF1 or CSF1R, we evaluated samples for lineage markers that would identify accumulation of this population and thus far, have not found evidence of their accumulation within BC or adjacent normal tissue (**Fig 1E,G**).
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cytotoxics induce expression of m/monocyte

To reveal the molecular mediators involved in CTX-associated M recruitment, we evaluated MECs from murine mammary and human breast tumors *in vitro* for mRNA expression of monocyte/M cytokines/chemokines following exposure to PTX, cisplatin or radiation therapy, and found increased expression of important M/IMC chemoattractant mRNAs, including Page 11 of 85 CSF1, CCL8/MCP2 and *IL34* (**Fig 2A** shows analysis of PyMT-derived carcinoma cells treated with PTX *in vitro*). Analysis of *CSF1* mRNA expression *in vivo* revealed that mammary tumors of MMTV-PyMT mice had a 2-fold higher expression of *CSF1* mRNA following PTX exposure
(**Fig 2B**) that correlated with increased density of Ms in tumor stroma (**Fig 2C**).  
PLX3397: A  
  
POTENT ANTAGONIST OF
THE *CSF1R*
TYROSINE KINASE
PLX3397 is a competitive ATP inhibitor with potent (nM) specificity for CSF1 and cKIT receptor tyrosine kinases, with 10100 fold selectivity for these target kinases as opposed to other related kinases (e.g. KDR)[64]. Specifically PLX3397 is a selective inhibitor of Fms (CSF1R, the receptor for colony stimulating factor [CSF-1, also known as macrophagecolony stimulating factor, M-CSF], as well as the ligand interleukin 34 [IL34]), Kit (the receptor for stem cell factor, SCF), and oncogenic Flt3 (the receptor for Flt3 ligand) activity intended for oral administration. When screened against a broad panel of kinases, IC50 values were > 1.0 μM for all, with the majority > 10 μM. Ligand-dependent proliferation of M-NFS-60 and BAC1.2F5 cells is inhibited by PLX3397 with IC50s of 0.33, 0.23 and 0.31 μM, respectively. In THP-1 cells, CSF1R autophos-phorylation induced by M-CSF is inhibited by 7.0 nM PLX3397. Human osteoclast precursor cells were induced to differentiate into mature osteoclasts by RANK-L and M-CSF, and inhibited by PLX3397 with an IC50 of 33 nM. By contrast, in RS4:11 cells, Flt3 auto-phosphorylation was induced by Flt3 ligand with autophosphorylation requiring high levels of PLX3397 (1500 nM) for inhibition. PLX3397 is currently in a phase 1 trial (ClinicalTrials.gov Identifier: NCT01004861) under Protocol PLX108-01 entitled, "A Phase 1 Study to Assess Safety, Pharmacokinetics, and Pharmacodynamics of PLX3397 in Pts with Advanced, Incurable, Solid Tumors in which the Target Kinases Are Linked to Disease Pathophysiology." The PLX3397 clinical formulation has demonstrated appreciable bioavailability and dose-dependent exposures to date, and is supplied as gel caps of 100 mg each. The plasma half-life of PLX3397 appears to be greater than 10 hours, and the trial has achieved exposures that are within the efficacious range predicted by preclinical studies. Nineteen pts have been enrolled to date, and the current dose level is 600 mg by mouth given once daily on an empty stomach. A maximum tolerated dose (MTD) has not yet been defined, and dose escalation is ongoing without consistent clinically significant toxicity. One younger pts experienced a reversible change in hair color to white, attributed to inhibition of KIT. The majority of the biomarker studies are ongoing; flow studies have demonstrated a reduction in monocytes that appear sensitive to CSF1 stimulation in pts treated with PLX3397. MACROPHAGE-*DEPLETION IMPROVES CHEMOSENSITIVITY*
The combined implication of our preclinical data is that high levels of Ms such as observed in high grade TNBC, may limit response to CTX. Thus, we treated late-stage MMTV-PyMT mice with PTX, and agents blocking M and/or IMC infiltration. MMTV-PyMT mice were treated with PLX3397 starting at d80 followed with one cycle of PTX (10 mg/kg, Q5Dx3, i.v.) and monitored to endpoint (2.0 cm primary tumors or day 100). While PTX increased the Ms
(CD45+CD11b+Ly6C-Ly6G-F4/80+) in tumor stroma, combined treatment with PLX3397 significantly reduced PTX-induced M infiltration (**Fig 3A**) accompanied by reduced primary tumor growth (**Fig 3B**) and pulmonary metastasis (**Fig 3C**). Using a histopathological staging criteria[35, 65], PLX3397/PTX-treated mice developed fewer late-stage carcinomas containing large areas of necrosis and extensive cell death (p<0.05, cleaved caspase 3+ cells), decreased density of CD31+ vessels (p<0.05), with no accompanying change in epithelial proliferation (data not shown). PLX33897 treatment had no effect on infiltration of tumors by IMCs
(CD45+CD11b+Ly6CHi) or DCs (CD45+CD11blo/-Ly6C-CD22-Ly6G-CD11cHiMHCIIHi) (data not shown). Since PLX3397 inhibits both CSF1R and cKIT kinases, we treated mice bearing orthotopic mammary tumors (derived from carcinoma cells of MMTV-PyMT mice) with neutralizing monoclonal antibody (mAB) to CSF1 as monotherapy, and in combination with PTX.  
PLX3397/PTX and CSF1 mAB/PTX similarly depleted ~ 80% of M∅s with no diminution in the presence of IMCs (data not shown), and significantly reduced primary tumor growth (Fig 4A), thus indicating that the cKIT inhibitory effects of PLX3397 were insignificant. Neither PLX3397 nor CSF1 mAB depletes IMCs within PyMT tumors; thus, we evaluated CD11b mAB/PTX to eradicate IMCs. CD11b is an integrin cell adhesion molecule expressed on granulocytes, M∅s, IMCs, DCs, and NK cells that in part, regulates transendothelial migration of cells into tissue and tumor parenchyma. CD11b mAB treatment efficiently depletes both the M∅ and IMC population in tumors[66]. PyMT tumor-bearing mice treated with either CD11b mAB/PTX exhibited significant reduction in primary tumor growth similar to CSF1mAB/PTX
or PLX3397/PTX (**Fig 4B**). Similar results were observed when tumor-bearing mice were treated with PLX3397/carboplatin and PLX3397/cisplatin (data not shown).
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anti-tumor response in plx3397/ptx-treated mice

Tumors from PLX3397/PTX-treated MMTV-PyMT mice revealed increased % of CD4+, CD8+
T cells (**Fig 5A**) and DCs (data not shown) correlating with significantly (p<0.05) increased expression of cytotoxic effector molecules (interferon (IFN), granzyme A (GRZA), granzyme B
(GRZB), perforin-1 (PRF1), and "Type-1" DC effectors molecules IL12p35 and interferon
(IFN))[67], and decreased expression of immunosuppressive *ARG1* (**Fig 5B**), thus indicating an overall reprogramming of the immune microenvironment where anti-tumor immune pathways had been bolstered. To evaluate the significance of this, we depleted MMTVPyMT/PLX3397/PTX-treated mice of CD8+ T cells with a neutralizing mAB to CD8a, and found that the improved outcome following PLX3397/PTX therapy was lost (**Fig 5C**) indicating enhanced chemosensitivity accompanying M-depletion also involved bolstering productive CD8+
T cell-dependent CTL responses.
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cd68 and cd8 mrna expression

To determine if stratification of BCs by *CD68* and CD8 mRNA expression predicted response to CTX, we analyzed *CD68* and *CD8a* mRNA expression in a cohort of 311 pts constructed from 2 independent data sets[68, 69]. All pts provided fine needle aspirates (FNA) taken prior to neoadjuvant CTX and pathological response was assessed at the time of surgery. Using median expression as a threshold, examination of CD8 and *CD68* mRNA in FNA samples demonstrated 3 specific groups CD68>CD8, CD68<CD8 and CD68=CD8 (denoted CD68high/CD8low, CD68/CD8equal and CD68low/CD8high respectively). Analysis of the rate of pathological complete response (pCR) in these groups revealed that the CD68high/CD8low group had a significantly lower rate of pCR (7%) as compared to the other two groups, with the CD68low/CD8high exhibiting the highest rate of pCR at 27%, thus indicating that the balance between M and CD8 infiltration can be used as a biomarker to predict response to neoadjuvant CTX.
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nonclinical pharmacology

PLX3397 is a selective inhibitor of Fms (CSF1R, the receptor for colony stimulating factor [CSF-1, also known as macrophage-colony stimulating factor, M-CSF], as well as the ligand interleukin 34 [IL-34]), Kit (the receptor for stem cell factor, SCF), and oncogenic Flt3 (the receptor for Flt3 ligand) activity intended for oral administration. The effects of PLX3397 on multiple aspects of tumorigenesis have been characterized in cellular and in vivo assays. The proliferation of cell lines that depend on CSF-1, SCF, or endogenous Flt3-ITD (internal tandem duplications) is inhibited at inhibitory concentration of 50% (IC50) values below 1 µM.  
Furthermore, CSF-1-induced autophosphorylation of Fms and SCF-induced autophosphorylation of Kit are potently inhibited by PLX3397. Finally, the RANK-L- and CSF-1-dependent differentiation of osteoclast precursors is also potently inhibited by PLX3397. These in vitro results translate to PLX3397 effects in a variety of in vivo models for Fms-dependent proliferation, Fms-dependent osteoclast differentiation, Flt3-ITD dependent tumor growth, and Kit-dependent mast cell proliferation. While pharmacologic effects due to the inhibition of Fms and Kit are expected, the relative selectivity of PLX3397 against other kinases suggests that offtarget effects against other kinases should be reduced. The safety pharmacology of PLX3397 has been evaluated in 5 Good Laboratory Practice (GLP) studies (2 in vitro and 3 in vivo). Three of these studies addressed the potential adverse cardiovascular or cardiac electrophysiological effects of PLX3397.  
An inhibitory concentration of 50% (IC50) of 0.7 μM was obtained in the human ether-a-go-go  
  
(hERG) channel assay. However, no PLX3397-related prolongation of action duration was observed in an isolated rabbit Purkinje fiber study. In an in vivo dog telemetry study, electrocardiographic parameters were unchanged by PLX3397 treatment, but left ventricular contractility (LV dP/dtmax) and arterial pulse pressure were significantly lower compared to controls in dogs that received 300 and 1000 mg/kg. Because PLX3397 binds to the hERG channel yet no QT prolongation has been identified nonclinically, an investigative safety pharmacology study was conducted in order to examine potential binding to hCav1.2 whose inhibition has Fig 4: Combined treatment with CSF1 mAB or CD11b mAB and PTX
reduces primary orthotopic PyMT-tumor growth (A,B). Higher percentage of IMCs in 4T1 tumors compared to PyMT tumors(C). PLX3397 plus PTX reduces 4T1 tumor growth (D). (*) p<0.05, (**) p <0.01. FVB/n: mammary tissue from nontransgenic mice.  
Page 15 of 85  
  
  

been negatively correlated with risk for QT prolongation. IC50 for the inhibitory effect of PLX3397 on hCav1.2 calcium current was determined to be 0.2 μM. Central nervous system
(CNS) and respiratory function were not affected in rats.
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nonclinical pharmacokinetics

PLX3397 binds extensively to albumin in 4 species (mouse, rat, dog and human). After intravenous (IV) administration in nonclinical studies, elimination terminal half-life (t1/2) values were 3.5 hours in mice, 5.1 hours in rats, 1.9 hours in dogs, and 3.7 hours in monkeys. PLX3397 is metabolically stable and not susceptible to rapid metabolic degradation. CYP3A4 is the main CYP enzyme that metabolizes PLX3397. The results also suggest that CYP1A2 and 2C9 might also play a minor role in its metabolism. PLX3397 had no inhibitory effect on CYP1A2 and 3A4 with an IC50 > 30uM. It had modest effects on the other major CYPs, (2C9, 2C19, and 2D6)
with an IC50 ranging from 11.1 to 22.2 μMPLX3397, at 10 μM, did not significantly inhibit BCRP, BSEP, SMVT, OCT1, OCT2, OAT1,OAT3-mediated transport of probe substrate. Compared to vehicle control, PLX3397 inhibited the transport of probe substrates of uptake transporters OATP1B1, OATP1B3 and OATP2B1 with respective inhibition of 27.7%, 21.4% and 37.1%. PLX3397 also inhibited P-gp mediated transport of probe substrates with a small but statistically significant inhibition of 9.35% at 10 μM.
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plx3397 appears to penetrate into

Two high-dose GLP 4-week general toxicology studies were conducted with daily oral gavage administration of PLX3397 (once daily [QD] in rats and twice daily [BID] in dogs) at doses of 20, 60, and 200 mg/kg/day in rats and 50, 100, and 300 mg/kg/day in dogs, with 16-day (rat) or 14-day (dog) recoveries. Neither a noeffect-level (NOEL) nor a no-adverse-effect-level (NOAEL) of PLX3397 could be determined in either species due to toxicity. Significant adverse test article-related observations appear to be related to PLX3397-mediated inhibition of Fms and Kit kinases. PLX3397-related histopathologic observations included testicular spermatagonia reduction, bone marrow hypocellularity, thymic lymphoid reduction, bone hyperostosis and hypertrophy, ovarian follicular degeneration, and liver hepatocellular hypertrophy. All findings were partially or fully reversible. Two additional GLP toxicology studies at lower dose levels involved daily oral gavage administration of PLX3397 HCl for 4 weeks (QD in rats and dogs) at doses of 0.5, 2, and 10 mg/kg/day in rats and 1, 6, and 30 mg/kg/day in dogs, with 8-week recoveries. The NOAELs of PLX3397 were determined to be 10 mg/kg/day in rats and 6 mg/kg/day in dogs in these additional studies. All adverse findings were fully reversible, including testicular spermatagonia reduction in dogs. Two 13-week GLP toxicology studies involved daily oral gavage administration of PLX3397 HCl for 13 weeks (QD in rats and dogs), with 8-week recoveries at doses of 0.5, 4 and 20 mg/kg/day in rats and 1, 6, and 30 mg/kg/day in dogs. NOAELs were determined to be 4 mg/kg/day in rats and 6 mg/kg/day in dogs. No new target organ toxicities were seen in either study In rats, anemia, and bone marrow depletion, and hepatocellular vacuolation associated with increased liver enzymes were seen. In dogs, findings of reproductive toxicity (spermatogonial reduction) and increased incidence of emesis were seen at the t 30 mg/kg dose level, which were reversible. Potential effects of PLX3397 on embryofetal development in rats were assessed at 4, 10, and 40 mg/kg/day. Based on changes in hematology parameters at 40 mg/kg/day in the dams, and fetal external and visceral malformations and skeletal developmental variations (findings primarily related to decreases in ossification) in the fetuses at 40 mg/kg/day, a dose level of 10 mg/kg/day was considered to be the NOAEL. PLX3397 was not mutagenic or clastogenic in the Ames,chromosomal aberration and micronucleus) tests, andshowed no potential to cause phototoxicity in vitro in the NIH 3T3 fibroblast assay.
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conclusions from preclinical data

Results from these studies support a fundamental and functional interplay between M
presence/bioactivity and CTX response and demonstrate that mouse modeling can guide development of clinical studies, aid in clinical trial design, and identify pts most likely to benefit from M-targeted therapy. PLX3397 is a novel oral highly selective kinase inhibitor that inhibits M, osteoclasts and mast cells, and mice treated with PLX3397 phenocopy mice similarly treated with neutralizing mABs against CSF1 or CD11b and CTX. Interestingly, a number of studies suggest that TNBC may be relatively resistant to taxanes, particularly following prior exposure in the early stage setting. These preclinical data imply that leveraging either inhibition of the bioactivity of Ms or eradicating their presence in tumor tissue will provide a survival advantage to women receiving CTX in a neoadjuvant setting, and may benefit women in an adjuvant setting when treated in combination with CTX. The combination of PLX3397 with CTX, therefore, is an entirely novel treatment strategy that has the potential to improve outcome not just for pts with advanced TNBC, which is the main goal for this clinical trial, but also as initial, potentially curable therapy in high risk TNBC, which is our long-term goal.
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1.2 clinical studies clinical pharmacokinetics

PLX3397 human exposure has been evaluated in 8 clinical studies. PLX3397 HCl was administered as single agent in 5 studies and in combination with paclitaxel, temozolomide and vemurafenib in the other 3 studies. The PK of PLX3397 at dose levels between 200 mg/day and 1200 mg/day has been evaluated in the dose-escalation and extension study, PLX108-01, using QD or BID dosing in the fasting state. The Tmax is approximately 2 hours, and the mean accumulation ratio compared to Day 1 values is approximately 2-fold. In general, there is doseproportional exposure. In the PLX108-05 acute myeloid leukemia (AML) doseescalation study at dose levels between 800 mg/day and 5000 mg/day, saturation of exposure was observed at 2000 mg/day. The steady state exposure of 900 mg QD cohort in PLX108-03 Hodgkin's lymphoma study and of 1000 mg BID cohortsin all the other single agent studies (PLX108-04 glioblastoma , PLX108-05 AML, PLX108-06 prostate cancer) were similar to, exposure of the respective dose groups in PLX108-01 solid tumors study. The mean PLX3397 plasma concentrations for the 800 mg BID cohorts at 2, 4 and 6 hours in the combination studies with paclitaxel (PLX108-07), temozolomide (PLX108-08), and vemurafanib (PLX108-09) were comparable to those seen in PLX3397 single agent studies. Subjects administered 600 mg of PLX3397 HCl with a high-fat, high-calorie meal displayed an approximately 2-fold increase in Cmax and AUC compared to subjects administered PLX3397 HCl in the fasted state (PLX108-11). When PLX3397 HCl was administered in the presence of esomeprazole, overall exposure was reduced approximately 30%. PLX3397 is currently in a Phase 1 trial (ClinicalTrials.gov Identifier: NCT01004861) under Protocol PLX108-01 entitled, "A Phase 1 Study to Assess Safety, Pharmacokinetics, and Pharmacodynamics of PLX3397 in Patients with Advanced, Incurable, Solid Tumors in which the Target Kinases Are Linked to Disease Pathophysiology." The PLX3397 clinical formulation has demonstrated appreciable bioavailability and dose-dependent exposures to date, and is supplied as gel caps of 100 mg each. The plasma half-life of PLX3397 appears to be greater than 10 hours, and the trial has achieved exposures that are within the efficacious range predicted by preclinical studies. Nineteen patients have been enrolled to date, and the current dose level is 600 mg by mouth given once daily on an empty stomach. A maximum tolerated dose has not yet been defined, and dose escalation is ongoing without consistent clinically significant toxicity. One younger patient experienced a reversible change in hair color to white, attributed to inhibition of KIT. The majority of the biomarker studies are ongoing; flow studies have demonstrated a reduction in monocytes that appear sensitive to CSF1 stimulation in patients treated with PLX3397. No clinical studies on drug-drug interactions have been performed. However, preclinical biochemical and cellular studies predict that the drug-drug interaction potential is low. The plasma PK of paclitaxel (measured at multiple time points after 10 mg/kg injection) was determined to have a half-life of 1 hour in mice receiving vehicle chow, and was unchanged by 7 previous days consumption of the PLX3397 drug chow (measured 15 and 45 minutes after paclitaxel injection. It is assumed that eribulin will have a similar profile, however pharmacokinetics will be performed in the phase I portion of this trial.  
Fig 6: A) Increased CD4 and CD8 T cells in tumors of PLX3397/PTX treated MMTV-PyMT
mice. B) Mean fold change in cytokine mRNA
expression of PyMT tumors from mice treated with PTX alone or in combination with PLX3397.  
C) 85-day-old MMTV-PyMT mice were treated with PTX and/or PLX3397 and anti-CD8 IgG. Total tumor burden/animal was assessed every 5 days. (*) p<0.05, (**) p <0.01.  
The ongoing Phase 1 dose escalation study PLX108-01 in patients with solid tumors is designed to evaluate the safety and PK of PLX3397 administered orally in order to establish a maximum tolerated dose (MTD). As of June 2011, a total of 41 patients have been treated with PLX3397 PO. The dose levels have been 200 mg/day (n=3), 300 mg/day (n=6), 400 mg/day (n=6), 600 mg/day (n=6), 900 mg/day (n=7), 1000 mg/day (n=7) and 1200 mg/day (n=6). In summary, the Tmax is approximately 2 hr, the mean elimination half-life is approximately 20 hr, and the mean accumulation ratio compared to Day 1 values is approximately 1.6. In general, there is increasing exposure with increasing dose. On Day 15, the AUC0-24hr was 62 uM•hr for the 200 Page 18 of 85 mg/day dose group, 122 uM•hr for the 300 mg/day dose group, 98 uM•hr for the 400 mg/day dose group, 258 uM•hr for the 600 mg/day dose group, 254 uM•hr for the 900 mg/day dose group and 329 uM•hr for the 1200 mg/day dose group (pharmacokinetic data for the 1000 mg/day cohort is not currently available). Dose-limiting toxicities (DLTs) of Grade 3 AST
elevation and Grade 4 neutropenia occurred at 1200 mg/day. A 1000 mg/day cohort was opened and 1 out of 7 patients experienced a DLT (Grade 3 AST), **establishing 1000 mg/day as the** MTD.  
There have been no safety signals in vital signs, physical examinations, or ECGs (including careful evaluation of potential QT prolongation). A reduction in hemoglobin (usually G1) has been observed in several patients, but this has not resulted in treatment discontinuation in any patients. There have been a total of 6 patients with DLTs, as follows: G3 increased INR (300 mg/day) in a patient on warfarin; G3 lymphopenia (600 mg/day), subsequently exempted as a DLT; G3 lymphopenia and G4 hyponatremia (600 mg/day); G3 AST (1000 mg/day); G3 AST (1200 mg/day) which recovered after study drug discontinuation; and G4 neutropenia (1200 mg/day) which recovered after holding study drug and management with G-CSF. As this is a population of patients with metastatic solid tumors that have been heavily pretreated with cytotoxic therapies, adverse events are anticipated due to either the disease or previous treatments. The most common AEs have been nausea and fatigue. Please see the Investigator's Brochure for more details on adverse events and tolerability. Response biomarkers are being assayed in order to profile the inhibitory activity of PLX3397 on Fms and Kit activity as a function of dose and exposure. These biomarkers include circulating tumor cells (CTCs) and CD14+/CD16+ proinflammatory monocyte cell numbers, IL-6, IL-1β, MMP3, and markers of osteoclast activity. Most of the soluble markers are not elevated at Baseline in the patients treated to date, so no decrease with treatment can be anticipated. However, in 4 patients with elevated CTCs at Baseline, 3 patients have shown a clinically relevant reduction during treatment with PLX3397. Marked reductions in the CD14+/CD16+ cell populations have also been observed in the majority of patients, with no change in the remainder. CLINICAL PHARMACOKINETICS *PLX3397* As of June 30, 2014, data are available on 345 patients who have been enrolled in the PLX3397 clinical program across 8 clinical studies PLX108-01, PLX108-03, PLX108-04, PLX108-05, PLX108-06, PLX108-07, PLX108-08, and PLX108-09. Of the 345 patients, 181 patients with solid tumors received as studies are a single agent (which included advanced, incurable, solid tumors; relapsed or refractory Hodgkin's lymphoma; recurrent glioblastoma multiforme; or progressive castration-resistant prostate cancer with high circulating tumor cell counts). Ninety patients received PLX3397 as a single agent for relapsed or refractory AML. Seventy-four patients received PLX3397 in combination with other therapies. Most of these studies are ongoing. The most frequent TEAEs (>20%) among all treated patients included fatigue, nausea, , decreased appetite, diarrhea, anemia, vomiting, and increases in aspartate aminotransferase (AST). Hair color changes (depigmentation) and constipation also commonly occurred in solid tumor patients receiving single agent PLX3397. With the exception of febrile neutropenia (which occurred at a high rate in the AML study), less than 10% of these common AEs were considered as Grade 3 or higher Treatment related SAEs) reported more than once included, neutropenia Page 19 of 85
(including febrile neutropenia), anemia, pneumonia, increased AST or ALT, increased INR, dehydration, hyponatremia, and maculo-papular rash.
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eribulin

Eribulin mesylate (E7389) is a nontaxane, microtubule dynamics inhibitor with a novel mechanism of action. Eribulin suppresses polymerization, has no effect on microtubule depoly merization, and sequesters tubulin into nonfunctional aggregates[70, 71]. Preclinical studies have demonstrated antitumor activity in cell lines that are taxane resistant as a result of β-tubulin mutations[72]. In addition, eribulin appears to have less neurotoxicity than other microtubule antagonists. A murine model compared the effect on nerve conduction of paclitaxel and ixabepilone to eribulin. This data, presented by Wozniak and colleagues at the annual European Society for Medical Oncology found that treatment with paclitaxel and ixabepilone resulted in a reduction in caudal nerve conduction velocity as well as caudal and digital amplitude, but eribulin had no deleterious effect on these endpoints. Eribulin also caused less severe morphological changes in dorsal root ganglion and sciatic nerves on pathological assessment. Nine Phase 1 clinical studies have been completed. The National Institutes of Health (NIH) sponsored two Phase 1 studies of E7389 (National Cancer Institute [NCI] Study 5730 and Study 7444), and Eisai has sponsored seven additional studies: E7389-A001-101 and E7389-A001-102 in the United States (US); E7389-E044-103, E7389-E044-108, E7389-E044-109, and E7389 E044-110 in Europe; and E7389-J081-105 in Japan. In general, the PK of E7389 is characterized by a rapid distribution phase, with a prolonged elimination phase after intravenous infusion. The disposition of E7389 follows linear kinetics over the dose range studied, as shown by consistent dose-independent pharmacokinetic parameters (terminal half-life [t½], clearance [CL], steadystate volume of distribution [Vss]) and similar dose-normalized parameters (Cmax/Dose, AUC0t/Dose and AUC0-∞/Dose) between E7389 doses ranging from 0.25 to 1.4 mg/m2 (E7389-A001101) and from 0.25 to 4.0 mg/m2 (E7389-A001-102). NCI Phase 1 Study 5730 included subjects with advanced solid tumors and was designed to evaluate the toxicity and PK of E7389 and to determine the maximum tolerated dose (MTD) using a bolus injection on Days 1, 8 and 15 of a 28-day cycle. Dose limiting toxicities (DLTs) were observed in approximately one third of subjects that received 1.4 mg/m2 and in four out of five subjects at 2 mg/m2. The principal investigators considered 1.4 mg/m2 as the MTD.  
This schedule was selected as the initial schedule for the Phase 2 program. Two Phase 1 dosefinding studies were conducted by Eisai, administering E7389 as a 1-hour infusion. Study E7389-A001-101 determined the safety, toxicity, PK, and MTD of E7389 administered on Days 1, 8, and 15 of a 28-day cycle. The MTD was determined to be 1.0 mg/m2. Dose limiting toxicities were primarily Grade 3 and 4 neutropenia, but also included Grade 3 fatigue and Grade 3 anorexia. Study E7389-A001-102 determined the safety, PK, MTD and tumor response of E7389 administered on Day 1 of a 21-day cycle in subjects with advanced solid tumors. The MTD was determined to be 2.0 mg/m2 and Grade 4 febrile neutropenia was observed as the DLT. The DLT was observed in all three subjects who received 4.0 mg/m2 (Cycle 1, Days 7, 8 or 11); in two out of three subjects who received 2.8 mg/m2 (Cycle 1, Day 9 or 10); and in two out of seven subjects who received 2.0 mg/m2 (Cycle 1, Day 7 or 8). Although the study was not designed or powered to establish efficacy, of 21 subjects, one had an unconfirmed partial response (PR) at 12 weeks, and 12 subjects had stable disease (SD). Study E7389-J081-105 was conducted at National Cancer Center Hospital East in Japan to evaluate the safety, toxicity, PK, and MTD of E7389 administered as a 2 to 5 minute bolus infusion on Days 1 and 8 of a 21-day cycle. DLTs were observed in five subjects at 1.4 mg/m2 and 2.0 mg/m2, and the MTD was determined to be 1.4 mg/m2. Studies E7389-E044-103, E7389-E044-108, E7389-E044-109 and E7389-E044-110 were also performed with E7389 administered as a 2 to 5 minute bolus infusion on Days 1 and 8 of a 21-day cycle. These studies investigated the excretion balance and metabolic pathway of E7389, the influence of hepatic impairment on exposure to E7389, the PK and tolerance of E7389 when co-administered with oral multiple doses of ketoconazole (a potent CYP3A4 inhibitor) and the impact of E7389 on ECG. NCI Phase 1 Study 7444, included subjects with refractory or advanced solid tumors and was designed to evaluate safety, tolerability, toxicity, and anti-tumor activity of E7389 and gemcitabine administered in combination as a 2 to 5 minute intravenous bolus on Days 1, 8, and 15 of a 28-day cycle or Days 1 and 8 of a 21-day cycle. The 21-day cycle was better tolerated. DLTs with E7389 at 1.4 mg/m2 and gemcitabine at 1000 mg/m2 included Grade 3 diarrhea, Grade 3 dizziness and fatigue. The recommended Phase 2 dose was E7389 at 1.0 mg/m2 and gemcitabine at 1000 mg/m2. In the clinical setting, eribulin has demonstrated efficacy in patients with heavily pre-treated metastatic breast cancer, and is given in a 21 day cycle of an intravenous infusion weekly for two weeks followed by one week off. The first phase II trial treated 103 patients with a median of 4 prior chemotherapy regimens for advanced disease[73]. 70 subjects received 1.4 mg/m2 eribulin administered as an IV bolus on days 1, 8, and 15 of a 28-day cycle; another 33 subjects received eribulin administered as an IV bolus on days 1 and 8 of a 21-day cycle). The 21-day cycle cohort was added because 63% of subjects in the 28-day cycle cohort experienced dose delays, reductions, or omissions due to neutropenia; in most cases the day 15 dose was being omitted. The response rate (RR) was 11.5% (all PR), the clinical benefit rate (CBR) was 17.2%, and progression free survival (PFS) was 2.6 months. The second trial enrolled 299 patients with prior therapy or resistance to anthracyclines, taxanes and capecitabine and the same median prior number of treatment regimens[74]; all patients received treatment on the day 1 and 8 schedule every 21 days. Results were similar by independent review, with an RR of 9.3%, a CBR of 17.1% and PFS of 2.6 months. Between the two trials, overall survival (OS) ranged from 9 to 10.4 months. The most common drug-related grades 3 to 4 toxicities were neutropenia (5464%), fatigue (5-10%), peripheral neuropathy (5-6.9%), and febrile neutropenia (4-5.5%). The results of a phase III trial comparing eribulin to treatment of physician's choice (TPC) in patients with anthracyline and taxane pre-treated MBC and at least two prior chemotherapy regimens for advanced disease has recently led to FDA approval of this novel chemotherapy agent [75]. The Embrace trial randomized 508 women to eribulin, and 254 women to TPC; the primary endpoint was OS, which was prolonged with eribulin to 13.12 months versus 10.65 months in the TPC arm (HR 0.81, p=0.041). The RR was also longer in the eribulin arm (12.2 versus 4.7%, p=0.002), and although PFS was significantly longer by investigator assessment, by central review PFS was not significantly different, likely due to the inclusion of patients with evaluable rather than solely measurable disease (3.7 versus 2.2 months, HR 0.87, p=0.14). Subset analysis suggested benefit from eribulin across identified risk groups. 144 patients were identified with ER/PR and HER2 negative disease, determined locally, and in this group the PFS for eribulin was 2.23 months, compared to 1.9 months for TPC. In the overall study, grade 3-4 toxicities included higher rates of neutropenia (21% versus 14%) and leukopenia (11.7% versus Page 21 of 85 5%) as well as febrile neutropenia (3% versus 0.8%). However, anemia, asthenia/fatigue, nausea, mucositis and hand-foot syndrome were more prevalent in the TPC arm. 1.3 Rationale TNBC is a highly proliferative BC subtype associated with poor outcome, and acquisition of CTX resistance. This subtype of BC is found more frequently in young African American women, and the presence of Ms correlates with more aggressive tumor biology and worse survival. Concordant with this data, the M-associated TH1 signature (CD4lo/CD68lo/CD8hi)
predicted worse RFS in patients with TNBC (**Fig 1**). Preclinical data indicate that inhibition of CSF1R with PLX3397 enhances responsiveness to standard CTX, including PTX (**Fig 3-5**).  
However, in humans, therapy with PTX requires premedication with immune-suppressing steroids that may negate effects of macrophage-depletion. In addition, the majority of patients with TNBC presenting in the pre-treated metastatic setting will have already been exposed to taxanes as well as other CTX/targeted agent combinations. Eribulin is a well-tolerated, novel halichondrin analogue recently approved by the FDA for treatment of MBC not requiring steroid premedication and is therefore an ideal CTX choice to partner with PLX3397 in this population of patients. The phase Ib portion of the trial will include all BC subtypes in order to efficiently complete accrual and proceed to a phase II trial specifically designed for patients with TNBC. Both trials will be conducted at three Comprehensive Cancer Centers, with breast cancer investigators who have expertise in all phases of clinical trials, as well as acquisition of tumor biopsies. Breast cancer advocates from each center will be actively involved in the study design and clinical trial execution, as well as all patient education and recruitment.
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phase i trial results (preliminary)

Twenty-eight patients were enrolled in the Phase Ib trial, with a final MTD of 1000 mg of PLX3397 in two divided doses given each day combined with eribulin at 1.4 mg/m2 day 1 and 8 every 21 days. The primary toxicities included rash, bone marrow suppression and transaminitis which were overall manageable with dose reduction, dose delays, growth factor support, and antihistamines and steroids for rash.
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2.0 objectives 2.1 primary objective

2.1.1 Phase Ib:
To determine the maximum tolerated dose of PLX3397 given in combination with standard dose eribulin in patients with metastatic breast cancer.
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2.1.2 phase ii

To determine the percentage of patients with chemotherapy pre-treated triple negative metastatic breast cancer treated with PLX3397 in combination with eribulin who are progression free at 12 weeks.
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2.2 secondary objectives 2.2.1 phase

To determine the safety and tolerability of PLX3397 in combination with eribulin in patients with metastatic breast cancer.  
Correlative studies:
To determine the pharmacokinetics of the combination of PLX3397 and eribulin. To correlate change in CSF1 levels during treatment with specific dose levels of PLX3397.  
Make a preliminary assessment of the relationship between immune profile, tumor subtype, and tumor associated macrophages with response to therapy.  
Correlate immune profile and tumor associated macrophages from primary tumor blocks with metastatic tumor tissue.
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2.2.2 phase ii

To determine the response rate of PLX3397 in combination with eribulin in patients with chemotherapy pre-treated triple negative metastatic breast cancer.  
To determine the duration of response from the above therapy in this patient population.  
To determine the safety and tolerability of PLX3397 in combination with eribulin in this patient population.  
Correlative studies:
Correlation of CSF1 levels and response/duration of response to treatment.  
Correlation of immune profiling in blood with PLX3397 treatment. Correlate tumor immune profile before and after therapy with tumor subtype and response to therapy.
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3.0 study design and eligibility

This is a nonrandomized, open label phase Ib/II study evaluating the safety and efficacy of eribulin in combination with PLX3397, a novel CSF1 inhibitor, in patients with metastatic breast cancer. The phase II portion of this trial will be limited to patients with triple negative disease.
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3.1.1 phase ib

The phase I portion of this trial is a dose escalation of PLX3397 to determine the maximum tolerated dose (MTD) of PLX3397 when given in combination with standard dose eribulin. Patients will be enrolled in cohorts of three, using the dose levels and plan outlined in the statistical section, with 6 patients enrolled at the MTD. All patients with accessible tumor will be required to have a tumor biopsy at study start before starting therapy. Pharmacokinetics of PLX3397 and eribulin, and blood levels of CSF1 will be obtained as outlined in section 14. To allow rapid accrual to phase Ib, and an earlier start to the phase II trial, patients will be enrolled in phase I with both hormone receptor positive and negative disease, and at any line of therapy assuming eligibility criteria are otherwise met. Dose limiting toxicity (DLT) will be defined as any treatment-related toxicity meeting the criteria below and occurring within the first 21 days of combination therapy. Patients must receive at least 14 days of PLX3397 and 2 doses of eribulin during the first cycle in order to be considered evaluable for DLT (unless the missed doses are due to a DLT). Patients discontinuing study therapy in Cycle 1 for reasons other than treatment-related or a DLT may be replaced.
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hematologic dlts

CTCAEv4 Grade 4 neutropenia lasting for ≥5 days in duration CTCAEv4 Grade 4 neutropenia with fever >38.5oC and/or infection requiring antibiotic or anti-fungal treatment CTCAEv4 Grade 4 thrombocytopenia ( platelets ≤25.0/µL)
CTCAEv4 Grade 3 thrombocytopenia associated with significant bleeding Non-hematologic DLTs Any CTCAEv4 Grade ≥3 non-hematologic toxicity (except alopecia), **unless the event is**
clearly **unrelated** to treatment with PLX3397 in combination with eribulin Grade ≥3 nausea, vomiting, or diarrhea that resolves to Grade ≤2 within 48 hours, with or without medical intervention or prophylaxis, will not be considered a DLT
Grade 3 fatigue that resolves within 14 days, with or without medical intervention or prophylaxis, will not be considered a DLT
Grade ≥3 hyperglycemia will not be considered a DLT
A treatment delay of greater than 7 days for PLX3397 or inability to get two doses of eribulin, even if a dose reduction is required on day 8 in the first cycle due to toxicity that is not related to cancer worsening or intercurrent illness will be considered a DLT. A dose reduction required on Day 8 is not considered a DLT. Patients in each cohort will be followed for at least 3 weeks (one full cycle) before opening accrual to the next dose level. If one patient in any cohort develops a DLT, an additional 3 patients will be enrolled at that level. A minimum of 12 patients will be enrolled in the phase I study. The phase II trial will not open until the last patient in the phase I study has been followed for at least 3 weeks.
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3.1.2 phase ii

The phase II portion of this trial will evaluate PFS in patients with TNBC treated with PLX3397 and eribulin, using the dose of PLX3397 determined in the phase Ib study in a two-step design. Please see the statistical section for details regarding enrollment and statistical design. Treatment is preceded by a 6 to 7 day lead-in phase, in which patients will take PLX3397 800mg (400 mg in the morning and 400 mg at night) for 5 days, followed by 1 to 2 days off before starting eribulin combined with PLX3397. PLX3397 will continue to be given on a 5 days on, 2 days off schedule, repeated weekly, throughout the course of treatment. Patients with accessible tumor will undergo a core biopsy of tumor before the start of PLX3397 treatment, and then a fine needle aspiration or core biopsy will be performed on the day of or the day before the start of eribulin (day -2 to day 0).
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3.2 inclusion criteria

Pathologically confirmed diagnosis of breast cancer with documented progressive disease.  
Patients with stable brain metastases are eligible for this trial. Stable brain metastases defined as stable disease for one month and not on active treatment including steroids.  
Concomitant therapy with bisphosphonates is allowed. Stable dose coumadin anticoagulation is allowed, providing that anticoagulation can be safely held to an INR within normal range for the purpose of tumor biopsy. LMWH is the preferred method of anticoagulation.  
PT/INR and PTT ≤ Grade 1 within two weeks before initial biopsy of visceral organs.  
Measurable disease, as defined by RECIST v1.1 guidelines or evaluable disease. Bone metastases must be evaluable.  
Disease amenable to core biopsy. Patients with pulmonary metastases as their only site of disease may enroll on this trial and will not undergo biopsy.  
For Phase Ib: patients with HER2 overexpressing disease must have been previously treated with trastuzumab (patients with HER2 overexpressing disease are not eligible for the Phase II trial).  
Age eighteen years or older. ECOG performance status <2.  
Life expectancy of > 12 weeks.  
Patients with ≤ Grade 1 peripheral neuropathy are eligible for this trial using the CTCAE
v4.0, regardless of use of therapy for neuropathy including gabapentin.  
Adequate bone marrow reserve: ANC > 1000, platelets > 100,000.  
Adequate renal function: serum creatinine < 1.5x upper limit of normal OR calculated creatinine clearance ≥ 50 ml/min.  
Sodium and potassium levels ≤ Grade 1. Adequate hepatic function: AST and ALT <2.5 x ULN, and total bilirubin < 1.5x upper limit of normal. In patients with liver dysfunction due to hepatic metastases, AST and ALT are permitted to be < 5 times the ULN.No clinical evidence of heart failure or history of untreated ejection fraction below the lower limit of normal per institutional standards, or significant QT prolongation (> Grade 1, 480 msec) no history of congenital long QT syndrome, and no use of drugs known to increase the risk of Torsades de Point - patients may be eligible for study if the drug can be changed to another agent with less risk.  
Able to take oral medications and maintain hydration.  
Ability to give written informed consent and willingness to comply with the requirements of the protocol.  
Women of child-bearing potential must agree to use an effective method of birth control during treatment and for six months after receiving their last dose of study drug.
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specific inclusion criteria for phase

Patients enrolling on the phase II portion of this trial must have ER, PR and HER2 negative disease defined as less than 10% staining for ER and PR, and HER2 0-1+ by IHC, or 2+ by IHC and no evidence of amplification by FISH using local laboratory testing.
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3.3 exclusion criteria

Treatment with another chemotherapy or hormonal therapy within the past 2 weeks.  
Treatment with trastuzumab, bevacizumab or other targeted therapies within the past 2 weeks.  
Concurrent radiation therapy is not allowed with the exception of brain metastases developing on study treatment (see section 5.2 for details).  
Ongoing treatment with any other investigational therapy.  
Prior treatment with eribulin. Severe, concurrent illness including congestive heart failure, significant cardiac disease and uncontrolled hypertension, that would likely prevent the patient from being able to comply with the study protocol.  
Inadequate bone marrow, renal, or hepatic function as defined above, or an active coagulopathy that precludes tissue biopsy.  
Pregnant or lactating women and women of child-bearing potential who are not using an effective method of birth control. Women of childbearing potential must undergo a pregnancy test within seven days of starting the study drug.
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4.3 registration 4.3.1 ucsf helen

Patients who have consented and are eligible for the study will be registered in the UCSF Comprehensive Cancer Center Clinical Trials Management System (CTMS). The CTMS is password protected and complies with HIPAA standards. The assigned CRC at UCSF will issue the patient a unique study number.  
Page 26 of 85 A log of patients who are screened but ineligible for the study will also be kept at each site.
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4.3.2 participating sites

To register patients on study, the site CRC will complete an eligibility form and fax it to the designated UCSF CRC (contact information to be provided by the start of study). Written confirmation of enrollment will be faxed to the enrolling site within 24 hours, along with assigned study number. Study numbers for each patient will be assigned serially. Pertinent information will be entered from the patient medical record.
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5.0 investigational treatment plan 5.1

Phase Ib: In the phase Ib study, patients will start both PLX3397 and eribulin on day 1. Patients will take PLX3397 in two divided doses (twice daily), the first dose immediately followed by the IV administration of eribulin. A tumor biopsy will be obtained before treatment start. Pharmacokinetics will be obtained as outlined in section 14.4. The study will use a single-arm, open-label, phase I trial design with expanded cohort for response assessment. Eligible patients must have confirmed metastatic carcinoma of the breast and have received at least one prior cytotoxic chemotherapy regimen for advanced disease. The study will follow a standard dose-escalation schema with 3 to 6 patients per cohort (3+3 design). The starting dose level will consist of PLX3397 at 600 mg by mouth daily. Enrollment to successive cohorts up to dose level 2 will be performed according to the table shown below, to establish the MTD. If 2 or more dose-limiting toxicities (DLTs) are observed at level 0, one dose reduction (to level -1) is built in to the study design. All patients at a given dose level will be followed on treatment for at least 3 weeks before accrual to the next cohort can commence. There will be no intra-patient dose escalation allowed. For any dose cohort, if a patient is removed from study for reasons that are clearly not treatment-related, then an additional patient will be accrued to that dose level. For the purposes of Phase Ib dose escalation, DLTs will be defined as any treatment-related toxicity occurring within the first 21 days of combination therapy as noted in section 3.1.1.  
| Dose Escalation Schedule   | Dose                                    |                       |
|----------------------------|-----------------------------------------|-----------------------|
| Dose Level                 | PLX3397                                 | Eribulin              |
| Level -2                   | 400 mg                                  | 0.7 mg/m2, 2-5 min IV |
| (200 mg BID)               | Day 1, 8 q21 days                       |                       |
| Level -1                   | 400 mg                                  | 1.1 mg/m2, 2-5 min IV |
| (200 mg BID)               | Day 1, 8 q21 days                       |                       |
| 600 mg                     |                                         |                       |
| (400 mg AM;  200 mg PM)    |                                         |                       |
| Level 0                    |                                         |                       |
| (starting dose)            | 1.4 mg/m2, 2-5 min IV Day 1, 8 q21 days |                       |
| Level 1                    | 800 mg                                  | 1.4 mg/m2, 2-5 min IV |
| (400 mg BID)               | Day 1, 8 q21 days                       |                       |
| Protocol Version #10       | Page 27 of 85                           | 03/03/2016            |  
  
| 1000 mg                 |                                         |
|-------------------------|-----------------------------------------|
| (600 mg AM;  400 mg PM) |                                         |
| Level 2 (MTD)           | 1.4 mg/m2, 2-5 min IV Day 1, 8 q21 days |  
  
Due to the bone marrow suppression known to occur with eribulin, patients may receive myeloid growth factors at any time, at physician discretion (for guidelines regarding use of neupogen, see section 6.1). An eribulin premedication regimen of intravenous dexamethasone (6 mg, or physician-selected dose), or other equivalent steroid, <60 minutes prior to eribulin. This may be tapered with subsequent doses per investigator discretion. Note: Patients enrolled on to the trial under protocol version 6.0 which allowed for Eribulin dosing schedule of Day 1 and Day 15 every 28 days will remain on that treatment and assessment schedule. Phase II: The phase II portion will begin after the MTD for PLX3397 is determined and we will enroll a total of 56 patients with triple (ER/PR/HER2) negative metastatic breast cancer to evaluate the safety and efficacy of the proposed treatment. In the phase II study, treatment is preceded by a 6 to7 day lead-in phase, in which patients will take PLX3397 800mg (400 mg in the morning and 400 mg at night) alone for 5 days, followed by 1 to 2 days off before starting eribulin combined with PLX3397. PLX3397 will continue to be given on a 5 days on, 2 days off schedule, repeated weekly, throughout the course of treatment. Concomitant antihistamines are recommended to reduce the incidence of rash. A tumor core biopsy will be obtained before treatment start, and then again before the start of eribulin. Patients will be treated until clinical or radiographic disease progression or cumulative toxicity and will be evaluated regularly in clinic. Evaluation of disease response or progression will be accomplished with CT scans, and bone scans as indicated, every 6 weeks. Visits will take place before each dose of eribulin (Day 1 and Day 8) during cycle 1, and then on Day 1 before cycle 2 and beyond. 5.2 Duration of Therapy Treatment with the combination of PLX3397 and eribulin will continue until patients experience disease progression based on RECIST v1.1 criteria, toxicity that prevents continued treatment, or in the case of patient decision. Patients who have evidence of systemic response or disease control after at least one full cycle of eribulin and PLX3397 and who develop brain metastases may stay on study with a break in study therapy of up to one month in order to complete brain directed therapy. Brain directed therapy may include whole brain radiation or stereotactic radiosurgery. Eribulin should be held during whole brain radiation but need not be adjusted for stereotactic radiation. Stereotactic radiation should be scheduled for the week off of eribulin if at all possible. Patients may resume treatment, if deemed safe per the PI.
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6.0 general concomitant medication and

Concomitant treatment is permitted if the medication is not expected to interfere with the evaluation of safety or efficacy of the study drug. During the study, if the use of any concomitant treatment becomes necessary (e.g., for treatment of an adverse event), the treatment must be Page 28 of 85 recorded on the CRF, including the reason for treatment, generic name of the drug, dosage, route, and start and stop dates of administration. Antihistamines (i.e. loratadine or cetirizine) are recommended as pre-meds for all pts to prevent rash, and growth factor use is strongly recommended as prophylaxis for patients who have a history of bone marrow suppression on past chemotherapy and is allowed at the treating physicians discretion. Although PLX3397 does not appear to inhibit CYP drug-metabolizing enzymes to an important extent, caution is warranted when administering PLX3397 to subjects taking drugs that are highly dependent on CYP3A4 for metabolism and have a narrow therapeutic index. Of the five major CYP isoforms, 3A4 (BFC) may be involved in phase I metabolism (first pass)
of PLX3397, with possibly CYP1A2 playing a minor role. Until information regarding exposuretoxicity and exposure-response relationships are available with PLX3397, concomitant CYP3A4 inhibitors and inducers should be administered with caution, in the event they alter the systemic exposure to PLX3397 (see Appendix I for a list of common CYP3A4 inhibitors and inducers). In general, strong inhibitors or inducers of CYP3A4 should be avoided unless absolutely clinically necessary without effective alternatives. These include anticonvulsants, mycin antimicrobials, and antiretrovirals. Some common examples include inhibitors such as erythromycin, fluoxetine, gemfibrozil, and inducers such as rifampicin, carbamazepine, phenytoin, efavirenz, and nevirapine.
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6.1 use of myeloid growth

The primary toxicity of eribulin is bone marrow suppression, specifically neutropenia. Patients enrolling on this study will have had prior exposure to chemotherapy so will be at risk for bone marrow suppression. Myeloid growth factors will be allowed at physician discretion at any time, including during the DLT period. It is recommended that NCCN guidelines are followed. Myeloid growth factors will be allowed at any time following NCCN guidelines. In particular, prophylactic neupogen should be considered for patients requiring growth factor support with prior systemic chemotherapy.
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7.0 toxicity management & dose

The following dose modification rules will be used with respect to potential toxicity. Toxicity will be assessed continuously according to the NCI Common Terminology Criteria for Adverse Events Version 4.0 (CTCAE v4.0) The AE may be due to PLX3397 or eribulin alone in which case the respective agent should be reduced. If overlapping toxicity is suspected both drugs may be reduced with the potential to reescalate the non-offending agent. Given the different nature of PLX3397 and eribulin toxicities, separate tables are shown for individual agents.
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7.1 plx3397 phase i

PLX3397 dose reductions and interruptions will be permitted during the first 21 days of Cycle 1 only if a patient experiences a protocol-defined DLT. If a patient experiences a DLT during Cycle 1, PLX3397 treatment continuation at a lower dose may be permitted at the discretion of the Investigator including following Cycle Day 1 chemotherapy. In particular, prophylactic neupogen should be considered for patients requiring growth factor support with prior systemic chemotherapy. After Cycle 1 Day 21, dose reductions or interruptions for adverse events may take place at any time.
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phase ii

Guidelines for dosage modification for PLX3397–related toxicities as well as guidelines for their management are noted in Table 7-1. Please notify the Principal Investigator or UCSF study coordinator about all dose modifications. Dose reductions should occur in increments of 200 mg. These parameters are only suggestions and are not intended to supersede the clinical judgment of the treating physician. All adjustments should be made in consultation with the site specific Principal Investigator.  
| Table 7-1. Dose Modifications Guidelines for PLX3397 Toxicity Grade PLX3397 dose changes during                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Dose adjustments for     |                         |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|-------------------------|
| (CTCAE v4)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | current treatment period | resumption of treatment |
| Non-Hematologic Toxicity Drug Related Grade 3 (start symptomatic treatment when possible) 1 st Appearance Continue treatment and provide  Reduce by one dose level if symptoms persist  supportive care for ≥5 days despite supportive management. 2 nd Appearance Interrupt until resolved (grade 0 -1  Reduce by an additional dose level.  or baseline) 3 rd Appearance Discontinue permanently N/A Drug Related Grade 4 (start symptomatic treatment when possible) 1 st Appearance Interrupt until resolved (grade 0 -1  Reduce by one dose level.  or baseline) 2 nd Appearance Discontinue permanently N/A Hematologic Toxicity Grade 4 neutropenia 1 st Appearance Interrupt until ANC recovers;  Once recovered to ANC ≥500X106 /L, resume  provide growth factor support at same dose. If ANC does not recover to ≥ 1X109 /L after 7 days, reduce dose by one dose  level 2 nd Appearance Interrupt until ANC recovers;  Once resolved to ANC ≥500X106 /L, reduce  provide growth factor support dose by one dose level. If ANC does not  recover to ≥1X109 /L after 7 days, reduce dose  by an additional dose level .  3 rd Appearance Interrupt until ANC recovers;  If ANC does not recover to ≥500X106 /L after  provide growth factor support 14 days, discontinue permanently. Grade 2 and > 2x Baseline elevation of liver enzymes (ALT, AST, AlkPhos) 1 st Appearance Interrupt until recovers to less than No ChangeProtocol Version #10 Page 30 of 85 03/03/2016 Protocol #: 12751 |                          |                         |  
| Table 7-1. Dose Modifications Guidelines for PLX3397 Toxicity Grade PLX3397 dose changes during                                | Dose adjustments for                                                         |                                                                                       |
|--------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|---------------------------------------------------------------------------------------|
| (CTCAE v4)                                                                                                                     | current treatment period                                                     | resumption of treatment                                                               |
| Grade 2                                                                                                                        |                                                                              |                                                                                       |
| 2 nd Appearance                                                                                                                | Interrupt until recovers to less than                                        | Reduce byone dose level                                                               |
| Grade 2                                                                                                                        |                                                                              |                                                                                       |
| 3 rd Appearance                                                                                                                | Interrupt until recovers to less than                                        | Reduce by an additional dose level                                                    |
| Grade 2                                                                                                                        |                                                                              |                                                                                       |
| 4 th Appearance                                                                                                                | Discontinue permanently                                                      |                                                                                       |
| Grade 3 or Grade 4 febrile neutropenia 1 st Appearance Interrupt until ANC and fever  recovery; provide growth factor  support | Once resolved to ANC ≥500X106 /L and T ≤ 38˚C, reduce dose by one dose level |                                                                                       |
| 2 nd Appearance                                                                                                                | Interrupt until ANC and fever  recover; provide growth factor  support       | Once resolved to ANC ≥500X106 /L and T ≤38˚C, reduce dose by an additional dose level |
| 3 rd Appearance                                                                                                                | Discontinue permanently                                                      | N/A                                                                                   |
| Grade 4 thrombocytopenia 1 st Appearance                                                                                       | Interrupt until PLT ≥ 25X109 /L                                              | Reintroduce at same dose                                                              |
| 2 nd Appearance                                                                                                                | Interrupt until PLT ≥ 25X109 /L                                              | Reduce dose by one dose level                                                         |
| 3 rd Appearance                                                                                                                | Interrupt until PLT ≥ 25X109 /L                                              | Reduce dose by one dose level                                                         |
| th Appearance                                                                                                                  | Discontinue permanently                                                      | N/A                                                                                   |
| 4                                                                                                                              |                                                                              |                                                                                       |  
Dose interruptions for Grade 2 non-hematologic toxicity for up to 1 week can be implemented at the discretion of the treating physician to manage intolerable or clinically significant toxicity. No dose reduction is required when resuming treatment. A dose delay of greater than 21 days from the intended date of eribulin administration or a 21 day hold in administration of PLX3397 requires removal from study therapy. Every effort should be made to adhere to the intended schedule whenever possible.
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7.2 eribulin phase i

Eribulin dose reductions will be permitted during the first 21 days of Cycle 1 only if a patient experiences a protocol-defined DLT. Patients starting at 1.4 mg/m2, eribulin dose reductions will be permitted at physician discretion on Cycle 1, Day 8 - this will not be considered a DLT. If a patient experiences a protocol-defined DLT during Cycle 1, eribulin dose reduction will be permitted at physician discretion. After Cycle 1 Day 21, dose reductions or interruptions for adverse events may take place at any time.
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phase ii

Guidelines for dosage modification for eribulin–related toxicities as well as guidelines for their management are noted in Table 7-2.  
| Table 7-2. Guidelines for dosage modification for eribulin–related toxicities NCI-CTC grade,  unless otherwise  Management of  specified eribulin Dose upon resumption Neutropenia  1 st episode Hold until afebrile and  No change and add filgrastim (ANC <1000) +/- fever (T>38) ANC >1000 Toxicity 2 nd episode Hold until afebrile and  No change ANC >1000 If ANC does not recover to ≥ 1X109 /L after 7 days despite  growth factor use, reduce by  one dose level  3 rd episode Hold until afebrile and    Reduce by one dose level ANC >1000 4 th episode Remove from study Remove from study Thrombocytopenia Grade 2 or greater  Hold until >75K No change (<75K) Recurrent grade 2  Hold until >75K  Reduce by one dose level or greater 3 rd occurrence  Hold until >75K No change grade 2 or greater 4 th occurrence  Remove from study Remove from study grade 2 or greater Rash Grade 1 or 2 Continue treatment;  No change** supportive care Grade 3 Hold until grade1 or  No change** less; supportive care Second episode  Hold until grade1 or   Reduce by one dose level grade 3 less; supportive care 3 rd occurrence  Remove from study;  Remove from study grade 3 supportive care GI toxicity: diarrhea, nausea,  Grade 1, 2 Continue treatment No change; maximize  vomiting supportive treatment Grade 3 Hold until grade 1 or  No change; maximize  less  supportive treatment Recurrent grade 3 Hold until grade 1 or  No change less  3 rd occurrence Hold until grade 1 or   Reduce by one dose level less  4 th occurrence  Remove from study Remove from study grade 3 or 4 Liver function abnormalities  Grade 1, 2 Continue treatment No change (ALT, AST, Alk Phos only) Grade 3 and >2x  Hold until less than  No change baseline grade 3 recurrent 3 and  Hold until less than   Reduce by one dose level >2x baseline grade 3 Neuropathy (motor and Grade 1, 2 Continue treatment No changeProtocol Version #10 Page 32 of 85 03/03/2016 Protocol #: 12751   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| NCI-CTC grade,  unless otherwise   | Management of     |                      |                            |
|------------------------------------|-------------------|----------------------|----------------------------|
| specified                          | eribulin          | Dose upon resumption |                            |
| sensory)                           | Grade 3           | Hold until less than | No change                  |
| grade 3                            |                   |                      |                            |
| Toxicity                           | Recurrent grade 3 | Hold until less than |  Reduce by one dose level |
| grade 3                            |                   |                      |                            |
| 3 rd occurrence                    | Remove from study | Remove from study    |                            |
| grade 3                            |                   |                      |                            |  
Dose interruptions for Grade 2 non-hematologic toxicity for up to 1 week can be implemented at the discretion of the treating physician to manage intolerable or clinically significant toxicity. No dose reduction is required when resuming treatment. A dose delay of greater than 21 days from the intended date of eribulin administration or a 21 day hold in administration of PLX3397 requires removal from study therapy. Every effort should be made to adhere to the intended schedule whenever possible.  
| Dose Reduction Levels for PLX3397 and Eribulin  Dose                                 |                                                                                     |                                         |
|--------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|-----------------------------------------|
| Dose Level                                                                           | PLX3397                                                                             | Eribulin                                |
| 800 mg x 5 days/week (400 mg BID x 5days,  followed by 2 days off,  repeated weekly) | 1.4 mg/m2, 2-5 min IV Day 1, 8 q21 days                                             |                                         |
| Starting Dose                                                                        | 600 mg x 5 days/week                                                                |                                         |
| (400mg AM and 200mg PM x  5days, followed by 2 days  off, repeated weekly)           | 1.1 mg/m2, 2-5 min IV Day 1, 8 q21 days                                             |                                         |
| 1 st dose reduction                                                                  | 400 mg x 5 days/week (200mg BID x 5days,  followed by 2 days off,  repeated weekly) | 0.7 mg/m2, 2-5 min IV Day 1, 8 q21 days |
| 2 nd dose reduction                                                                  |                                                                                     |                                         |
8.0 schedule of assessments
phase ii

| Table 8-1 Study Calendar: Phase Ib                     |                                            |                                                      |         |         |           |      |      |     |     |      |     |     |      |
|--------------------------------------------------------|--------------------------------------------|------------------------------------------------------|---------|---------|-----------|------|------|-----|-----|------|-----|-----|------|
| Cycle                                                  | Comment                                    | Screening                                            | Cycle 1 | Cycle 2 | Cycle 3 & |      |      |     |     |      |     |     |      |
| (1 cycle =                                             | Beyond                                     | EOT                                                  |         |         |           |      |      |     |     |      |     |     |      |
| 21 days) Real life                                     | ≤4                                         | ≤2                                                   | ≤ 30    |         |           |      |      |     |     |      |     |     |      |
| time clock                                             | wks                                        | wks                                                  | D 11    | D 2     | D 8       | D 15 | D 11 | D 8 | D15 | D 11 | D 8 | D15 | days |
| Informed  Consent                                      | X                                          |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| Registration                                           | X                                          |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| History & Exam                                         | X                                          | X                                                    | X       | X       | X         | X    |      |     |     |      |     |     |      |
| ECOG                                                   | X                                          | X                                                    | X       | X       | X         |      |      |     |     |      |     |     |      |
| ConMed  Review                                         | X                                          | X                                                    | X       | X       | X         | X    | X    | X   |     |      |     |     |      |
| Peripheral Neuropathy                                  | X                                          | X                                                    | X       | X       |           |      |      |     |     |      |     |     |      |
| CBC w/ 5 part  diff.                                   | X                                          | X                                                    | X       | X       | X         | X    | X    | X   |     |      |     |     |      |
| BUN, CO2, Cl, K,  Na, Creatinine,  Glucose (nonfasting), &  Magnesium                                                        |                                            |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| Chemistry Panel                                        | X                                          | X                                                    | X       | X       | X         |      |      |     |     |      |     |     |      |
| Liver Function                                         | ALT, AST, AlkPhos,                         | X10                                                  | X10     | X10     |           |      |      |     |     |      |     |     |      |
| Total Bilirubin, &                                     |                                            |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| Tests                                                  | Albumin                                    |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| PT/PTT                                                 | Completed prior to  tumor biopsy           | X                                                    |         |         |           |      |      |     |     |      |     |     |      |
| Urine Pregnancy  Within 7 days of  beginning treatment | X                                          | X                                                    |         |         |           |      |      |     |     |      |     |     |      |
| Test 2 EKG 3                                           | X                                          | X                                                    |         |         |           |      |      |     |     |      |     |     |      |
| Eribulin                                               | Day 1 & 8,  q 21 days                      | X                                                    | X5      | X       | X         | X    | X    |     |     |      |     |     |      |
| Therapy, IV4 PLX3397  Therapy                          | Oral                                       | Beginning Cycle 1, Day 1: Daily  (self-administered) |         |         |           |      |      |     |     |      |     |     |      |
| Bone Scan &                                            | Baseline, then as  indicated               | X                                                    |         |         |           |      |      |     |     |      |     |     |      |
| other imaging  CT (C/A/P)                              | Every 6 weeks                              | X                                                    | X       | X       |           |      |      |     |     |      |     |     |      |
| PK Draws                                               | X6                                         | X7                                                   | X8      |         |           |      |      |     |     |      |     |     |      |
| Blood for  CSF19                                       | X                                          | X                                                    | X       |         |           |      |      |     |     |      |     |     |      |
| Blood for  Leukocyte                                   | X                                          |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| Subtyping9 Tumor biopsy                                | Required for patients  w/ accessible tumor | X                                                    |         |         |           |      |      |     |     |      |     |     |      |
| Archived Tumor  Requested of all  participants         | X                                          |                                                      |         |         |           |      |      |     |     |      |     |     |      |
| Tissue                                                 |                                            |                                                      |         |         |           |      |      |     |     |      |     |     |      |  
1. Allowed a window of ± 3 days for Day 1 procedures 2. In women of child bearing potential 3. EKGs performed at the start of every other cycle (even numbered), at 6 week intervals 4. The following eribulin treatment delays are allowed for *patient coordination:*
C1 D8 (up to 3 days), C2+ D1 (up to 14 days), and C2+ D8 (up to 3 days)
5. A delay in C1 D8 of up to 7 days is allowed for resolution of toxicity 6. Plasma samples for PK on C1 D1 will occur pre-PLX3397 administration and at 1, 2, and 5 hours post treatments (± 15 minutes)
7. Plasma samples for PK on C1D2 will occur 24 hours post eribulin treatment, but pre-PLX3397 administration (± 30 minutes)
8. Plasma samples for PK on C2D1 will occur pre-PLX3397 and at 2 hours post treatment (± 15 minutes) 9. All blood should be drawn before administration of IV eribulin 10. Liver function tests will be completed on D15 of the firsts three cycles only (C1D15, C2D15, and C3D15).
8.0 schedule of assessments
table 8-2 study calendar phase

| Cycle                          | Cycle 3                                                                               |                                                                         |         |      |     |     |      |     |     |     |    |
|--------------------------------|---------------------------------------------------------------------------------------|-------------------------------------------------------------------------|---------|------|-----|-----|------|-----|-----|-----|----|
| Comment                        | Screenin                                                                              | Lead-In                                                                 |         |      |     |     |      |     |     |     |    |
| (1 cycle =                     | Phase                                                                                 | Cycle 1                                                                 | Cycle 2 | &    |     |     |      |     |     |     |    |
| g                              |                                                                                       |                                                                         |         |      |     |     |      |     |     |     |    |
| 21 days)                       | Beyond                                                                                | EO T  ≤ 30  days 7                                                      |         |      |     |     |      |     |     |     |    |
| Informed  Consent              | X                                                                                     |                                                                         |         |      |     |     |      |     |     |     |    |
| Registration                   | X                                                                                     |                                                                         |         |      |     |     |      |     |     |     |    |
| History & Exam                 | X                                                                                     | X                                                                       | X       | X    | X   | X   |      |     |     |     |    |
| ECOG                           | X                                                                                     | X                                                                       | X       | X    | X   |     |      |     |     |     |    |
| ConMed Review                  | X                                                                                     | X                                                                       | X       | X    | X   | X   | X    | X   |     |     |    |
| Peripheral Neuropathy          | X                                                                                     | X                                                                       | X       | X    |     |     |      |     |     |     |    |
| CBC w/ 5 part  diff.           | X                                                                                     | X                                                                       | X       | X    | X   | X   | X    | X   |     |     |    |
| Day                            |                                                                                       |                                                                         |         |      |     |     |      |     |     |     |    |
| -7 to -6                       | D 11                                                                                  | D 8                                                                     | D15     | D 11 | D 8 | D15 | D 11 | D 8 | D15 |     |    |
| Real life                      | ≤4                                                                                    | ≤2                                                                      |         |      |     |     |      |     |     |     |    |
| time clock                     | wks                                                                                   | wks                                                                     |         |      |     |     |      |     |     |     |    |
| BUN, CO2, Cl, K,  Na, Creatinine,  Glucose (nonfasting), &  Magnesium                                |                                                                                       |                                                                         |         |      |     |     |      |     |     |     |    |
| Chemistry Panel                | X                                                                                     | X                                                                       | X       | X    | X   |     |      |     |     |     |    |
| ALT, AST,  AlkPhos, Total      | X                                                                                     | X                                                                       | X       | X10  | X   | X   | X10  | X   | X   | X10 | X  |
| Bilirubin, &  Albumin          |                                                                                       |                                                                         |         |      |     |     |      |     |     |     |    |
| Liver Function  Tests PT/PTT11 | Completed prior to  tumor biopsy                                                      | X                                                                       |         |      |     |     |      |     |     |     |    |
| Pregnancy Test 2               | Within 7 days of  beginning treatment                                                 | X                                                                       | X       |      |     |     |      |     |     |     |    |
| EKG 3                          | X                                                                                     | X                                                                       |         |      |     |     |      |     |     |     |    |
| Eribulin                       | Day 1 & 8,  q 21 days                                                                 | X                                                                       | X5      | X    | X   | X   | X    |     |     |     |    |
| Therapy, IV4 PLX3397  Therapy  | Oral                                                                                  | Beginning With Lead-In Phase (Day -7 to -6):  Daily (self-administered) |         |      |     |     |      |     |     |     |    |
| Bone Scan &                    | Baseline, then as  indicated                                                          | X                                                                       |         |      |     |     |      |     |     |     |    |
| other imaging  CT (C/A/P)      | Every 6 weeks9                                                                        | X                                                                       | X       | X    |     |     |      |     |     |     |    |
| Blood for                      | X                                                                                     | X                                                                       | X8      |      |     |     |      |     |     |     |    |
| CSF16                          | X                                                                                     |                                                                         |         |      |     |     |      |     |     |     |    |
| Blood for  Leukocyte           | X                                                                                     | X                                                                       | X       | X8   |     |     |      |     |     |     |    |
| Subtyping6                     | Required for                                                                          | X                                                                       |         |      |     |     |      |     |     |     |    |
| Tumor biopsy                   | patients w/                                                                           | X                                                                       | (Day    |      |     |     |      |     |     |     |    |
| accessible tumor               | 0/ -1)                                                                                |                                                                         |         |      |     |     |      |     |     |     |    |
| Archived Tumor                 | Requested of all  participants                                                        | X                                                                       |         |      |     |     |      |     |     |     |    |
| Tissue                         | 1.                                                                                    | Allowed a window of ± 3 days for Day 1 procedures                       |         |      |     |     |      |     |     |     |    |
| 2.                             | In women of child bearing potential. Urine or Serum test allowed.                     |                                                                         |         |      |     |     |      |     |     |     |    |
| 3.                             | EKGs performed at the start of every other cycle (even numbered), at 6 week intervals |                                                                         |         |      |     |     |      |     |     |     |    |  
6. Blood should be drawn before the first dose of PLX3397 during the lead-in phase, and before each administration of eribulin.  
7. EOT procedures should occur within 30 days of last dose, after which the patient will be Off-Study. 8. Cycle 4 Day 1 only 9. Patients with either CR or PR and who have received at least 6 cycles of therapy may reduce the frequency of scans to every 9 weeks.  
10. Liver function tests will be completed on D15 of the first three cycles only (C1D15, C2D15, and C3D15). 11. PT/PTT is only required for patients getting visceral organ biopsies
8.0 schedule of assessments
8.1 screening assessments (same for

The following screening procedures must be performed within 2 weeks (or 4 weeks where noted) of entering the clinical trial:
Patients must have reviewed and signed the consent form (within 4 weeks)
Complete medical history and physical to determine if patient meets the study entry criteria Review of concomitant medications Baseline laboratories, including: complete blood count with 5 part differential, chemistry panel, liver functions tests, PT/INR, and PTT
Pregnancy test if patient has childbearing potential (within 7 days) Tumor imaging, including a CT scan of the chest, abdomen, and pelvis and bone scan, or a whole body PET CT for patients with measurable disease. PET CT scans must include a diagnostic CT to be study eligible (cannot use CT scans for attenuation only purposes).  
12-lead EKG
Request archived tumor tissue Tumor biopsy for patients with accessible tumor Blood for correlative studies
8.0 schedule of assessments
8.2 assessments during treatment

Once the patient is confirmed to be eligible based on screening assessments, the following study assessments will be performed. See Table 8-1 and 8-2 for summary. One cycle is defined as 21 days. Day 1 visit procedures for any cycle of therapy are allowed a window of ± 3 days unless otherwise noted. Following the first cycle of therapy, patients may delay the start of a new cycle of therapy up to 2 weeks as necessary for personal activities, such as travel or vacation although every attempt should be made to keep patients on schedule. Treatment or visit delays due to public holidays, weather conditions, or natural disasters, do not constitute protocol violations. The following treatment delays are allowable for patient coordination:
Cycle 1, Day 8 - delay eribulin administration up to 3 days Cycle 2+, Day 1 - delay eribulin administration up to 14 days Cycle 2+, Day 8 - delay eribulin administration up to 3 days
8.0 schedule of assessments
8.2.1 lead in phase -

Blood for CSF1 and leukocyte phenotyping o For CSF1: 5 cc of blood will be drawn into a serum separator tube (gold top) o For leukocyte phenotyping: 10 cc of blood will be drawn into an EDTA tube
(purple top)
Page 37 of 85
8.0 schedule of assessments
tumor biopsy day 0/-1 8.2.2

The following assessments will be performed prior to receiving treatment on Day 1:
Medical history and physical examination Review of concomitant medications Laboratories, including complete blood count, chemistry panel and liver function tests. Blood for CSF1 and leukocyte phenotyping Blood for pharmacokinetics (Phase I only)
The following assessments will be performed after receiving treatment on Day 1 (Phase Ib only):
Blood for pharmacokinetics will be drawn at 1 hour, 2 hours and 5 hours post treatment The following assessment will take place on Day 2 (Phase Ib only):
Blood for pharmacokinetics and CSF1 will be drawn 24 hours post Day 1 treatment The following assessment will take place on Day 8:
Medical history and symptom-driven physical examination Laboratories, including complete blood count with differential Review of concomitant medications The following assessment will take place on Day 15:
Liver function tests
8.0 schedule of assessments
8.2.3 during subsequent cycles (cycle

The following assessments will be performed prior to receiving treatment on Day 1:
Medical history and physical examination Review of concomitant medications Laboratories, including complete blood count, chemistry panel and liver function tests. Urine Pregnancy test will only be done for women of childbearing potential (Cycle 3+)
Blood for CSF1 and leukocyte phenotyping (Cycle 2 and 4 Day 1only)
Blood for pharmacokinetics (Cycle 2, Phase Ib only)
The following assessments will be performed after receiving treatment on Day 1:
Blood for pharmacokinetics will be drawn at 2 hours post treatment (C2, Phase Ib only)
The following assessments will take place on Day 8:
Laboratories, including complete blood count with differential Review of concomitant medications The following assessment will take place on Day 15:
Liver function tests (Cycles 2 and 3 only)
Scans including all measurable and/or evaluable disease will be obtained every 6 weeks and at End of Treatment. Patients with either CR or PR and who have received at least 6 cycles of therapy may reduce the frequency of scans to every 9 weeks.
8.0 schedule of assessments
8.3 follow-up assessments

Patients will be permitted to stay on treatment until they have disease progression. Patients who develop progressive disease or Grade 3/4 toxicity that does not resolve after discontinuation of all study medications for 14 days will be removed from the study. All patients who leave the study must have a follow up visit within 30 days after their last dose of study medication. At this time, patients will have the following:
Full medical history and physical exam for assessment of residual toxicities Blood draw for complete blood count with differential, chemistry panel, and liver function tests If patients continue to have evidence of persistent toxicities at the first follow up visit, they will have continued monitoring at the discretion of the principal investigator until these symptoms are resolved or considered stable.
8.0 schedule of assessments
9.0 criteria for evaluation 9.1

Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their first treatment with PLX3397 Evaluable for objective response: Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.)
Evaluable Non-Target Disease Response: Patients who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for nontarget disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions.
8.0 schedule of assessments
disease parameters

Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as ≥20 mm (≥2 cm) by chest x-ray or as ≥10 mm (≥1 cm) with CT scan, MRI, or calipers by clinical exam. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).  
Malignant lymph nodes: To be considered pathologically enlarged and measurable, a lymph Page 39 of 85 node must be ≥15 mm (≥1.5 cm) in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm [0.5 cm]). At baseline and in follow-up, only the short axis will be measured and followed.  
Non-measurable disease: All other lesions (or sites of disease), including small lesions (longest diameter <10 mm [<1 cm] or pathological lymph nodes with ≥10 to <15 mm [≥1 to <1.5 cm]
short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions.  
Target lesions: All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as *target lesions* and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.  
Non-target lesions: All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.
8.0 schedule of assessments
methods for evaluation of measurable

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.  
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(*e.g.*, skin nodules and palpable lymph nodes) and ≥10 mm (≥1 cm) diameter as assessed using calipers (*e.g.*, skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.  
Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.  
Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm (0.5 cm) or less. If CT scans have slice thickness greater than 5 mm (0.5 cm), the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (*e.g.* for body scans).  
Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible.  
PET-CT: At present, the low dose or attenuation correction CT portion of a combined PET-CT
is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed.  
Tumor markers: Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Specific guidelines for both CA-125 response (in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been published [*JNCI* 96:487-488, 2004; *J Clin Oncol* 17, 3461-3467, 1999; *J Clin Oncol* 26:1148-1159, 2008]. In addition, the Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be integrated with objective tumor assessment for use in first-line trials in ovarian cancer [*JNCI*
92:1534-1535, 2000].  
Cytology, Histology: These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (*e.g.*, residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens Page 41 of 85 during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.
8.0 schedule of assessments
response criteria evaluation of target

Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes
(whether target or non-target) must have reduction in short axis to <10 mm (<1 cm).  
Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.  
Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm). (Note: the appearance of one or more new lesions is also considered progressions).  
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
8.0 schedule of assessments
evaluation of non-target lesions

Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm [<1 cm] short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.  
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.  
Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal* progression of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
8.0 schedule of assessments
evaluation of best overall response

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
Page 42 of 85  
| Target                                                                                                                   | Non-Target                                                                         | New       | Overall   | Best Overall Response when                        |
|--------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------|-----------|-----------|---------------------------------------------------|
| Lesions                                                                                                                  | Lesions                                                                            | Lesions   | Response  | Confirmation is Required*                         |
| CR                                                                                                                       | CR                                                                                 | No        | CR        | >4 wks. Confirmation**                            |
| CR                                                                                                                       | Non-CR/NonPD                                                                                    | No        | PR        |                                                   |
| CR                                                                                                                       | Not evaluated                                                                      | No        | PR        |                                                   |
| PR                                                                                                                       | Non-CR/NonPD/not  evaluated                                                                                    | No        | PR        | >4 wks. Confirmation**                            |
| SD                                                                                                                       | Non-CR/NonPD/not  evaluated                                                                                    | No        | SD        | Documented at least once >4  wks. from baseline** |
| PD                                                                                                                       | Any                                                                                | Yes or No | PD        |                                                   |
| Any                                                                                                                      | PD***                                                                              | Yes or No | PD        | no prior SD, PR or CR                             |
| Any                                                                                                                      | Any                                                                                | Yes       | PD        |                                                   |
|                                                                                                                         | See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. |           |           |                                                   |
| **                                                                                                                       | Only for non-randomized trials with response as primary endpoint.                  |           |           |                                                   |
| *** In exceptional circumstances, unequivocal progression in non-target lesions may be  accepted as disease progression. |                                                                                    |           |           |                                                   |
8.0 schedule of assessments
for patients with measurable disease

Note: Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as "*symptomatic deterioration."* Every effort should be made to document the objective progression even after discontinuation of treatment.
8.0 schedule of assessments
for patients with non-measurable disease

| Non-Target Lesions   | New Lesions                                                                                                                                                                                                                                  | Overall Response   |
|----------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| CR                   | No                                                                                                                                                                                                                                           | CR                 |
| Non-CR/non-PD        | No                                                                                                                                                                                                                                           | Non-CR/non-PD*     |
| Not all evaluated    | No                                                                                                                                                                                                                                           | not evaluated      |
| Unequivocal PD       | Yes or No                                                                                                                                                                                                                                    | PD                 |
| Any                  | Yes                                                                                                                                                                                                                                          | PD                 |
|                     | 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since SD is  increasingly used as an endpoint for assessment of efficacy in some trials so to assign this  category when no lesions can be measured is not advised |                    |
8.0 schedule of assessments
duration of response

Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented.  
Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.
8.0 schedule of assessments
9.2 toxicity definitions

All study toxicities will be graded by the NCI Common Terminology Criteria for Adverse Events Version 4.0.
8.0 schedule of assessments
10.0 criteria for termination 10.1

The Principal Investigator may terminate the study for any of the following reasons:
Significant toxicities: Patients with any grade 3/4 toxicity that persists and does not improve 21 days after discontinuation of all study medications, despite pharmacologic management, will be removed from the study. Any patient who experiences any grade 4 toxicity can be removed from the study at the discretion of the study investigator.  
If it becomes clear that the study treatment is less effective than standard treatment. The phase II study has a two-stage design (see section 3 for more details). If fewer than 8 out of the first 20 pts are progression-free at 12 weeks, this would be considered an unacceptably low PFS rate (<39%), the study will not continue and accrual will cease. Study accrual will continue while efficacy is being determined. Conversely, if at least 7 patients in the first stage of the phase II study are free of progressive disease at 12 weeks, then enrollment will continue to the second stage of the study. .
Once all data collection has been completed.
8.0 schedule of assessments
10.2 conditions for individual patient

The Principal Investigator may terminate the participation of an individual patient for any of the following reasons:
Disease progression Need for exclusionary concurrent treatment Withdrawn consent Protocol non-compliance Lost to follow-up
8.0 schedule of assessments
11.0 drug information

Eribulin is supplied in single-use vials containing 1 mg of the drug at a concentration of 0.5 mg/mL. The single-vial cartons should be stored at 25 °C.  
Page 44 of 85 The recommended dosage of eribulin is 1.4 mg/m2 administered intravenously over two to five minutes on days 1 and 8 of a 21-day cycle. Eribulin may be administered undiluted or diluted in 100 mL of 0.9% Sodium Chloride Injection, USP. The drug should not be administered through an IV line that is delivering a dextrose-containing solution. Syringes containing undiluted eribulin and containers of diluted eribulin may be stored up to 4 hours at room temperature or up to 24 hours refrigerated at 25 °C. The labeling includes recommendations for dosage reductions in patients with liver or kidney impairment. Also included in the labeling are recommendations for reducing and delaying scheduled doses in response to blood-count abnormalities that develop during treatment. The labeling states that febrile or afebrile neutropenia occurred in 82% of eribulin recipients in the company's pivotal Phase III clinical trial, compared with 53% of patients in the control group. Other adverse events that affected at least 25% of eribulin recipients were anemia, weakness or tiredness, alopecia, peripheral neuropathy, nausea, and constipation. According to the labeling, blood-cell counts should be monitored during eribulin therapy and dosages reduced or delayed in patients with low red- or white-blood-cell counts. Patients should also be periodically assessed for peripheral neuropathy. QT-interval assessments are recommended during eribulin therapy for patients who have heart failure, bradyarrhythmia, or electrolyte abnormalities or are receiving concomitant treatment with drugs that prolong the QT interval. PLX3397 is supplied in 200 mg gelcaps.
8.0 schedule of assessments
11.1 eribulin mesylate (e7389) 11.1.2

Eribulin mesylate (E7389) is a synthetic analog of Halichondrin B (HalB), a natural product isolated from the marine sponge Halichondria okadai.1 HalB is a large polyether macrolide that exerts potent anticancer effects in cell-based and animal models of cancer. The structurally simplified synthetic analog eribulin mesylate (E7389) encompasses the biologically active macrocyclic portion of HalB, and shows similar or identical anticancer properties in preclinical models.
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11.1.3 mode of action

E7389 exerts its antiproliferative effects via a tubulin-based antimitotic mechanism. Currentlyavailable data indicate that its mechanism of action is similar or identical to that of HalB.4,5 Like HalB, E7389 was shown to be a potent inhibitor of tubulin polymerization into microtubules as well as microtubule dynamics in vitro and in whole cells.4,6 Nonclinical data show that sub- to low-nmol/L levels of E7389 inhibit cancer cell proliferation via induction of irreversible cell cycle blocks at G2/M, disruption ofmitotic spindles, and initiation of apoptosis. Eribulin inhibits the growth phase of microtubules without affecting the shortening phase and sequesters tubulin into nonproductive aggregates. Eribulin exerts its effects via a tubulin-based Page 45 of 85 antimitotic mechanism leading to G2/M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage.
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11.1.4 storage and stability

The long-term storage condition for E7389 drug product is to store at 2°C to 8°C (do not freeze). Clinical supplies labeled for storage at temperatures up to 25°C have been reassigned to refrigerated storage (2°C to 8°C) to provide an extended provisional shelf life. The drug product may be administered without dilution or diluted with 0.9% Sodium Chloride Injection to concentrations between 0.005 mg/mL and 0.2 mg/mL, and administered per Eisai's directions. Photostability studies have demonstrated that protection from light is not necessary for the E7389 drug product.
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11.1.5 metabolism

Cytochrome 3A4 (CYP3A4) appears to be the major enzyme responsible for the human hepatic metabolism of E7389, and forms mainly isomeric monohydroxylates in vitro.
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11.1.6 preparation & administration

Aseptically withdraw the required amount of HALAVEN from the single-use vial and administer undiluted or diluted in 100 mL of 0.9% Sodium Chloride Injection, USP. **Do not dilute in or** administer through an intravenous line containing solutions with **dextrose.** Do not administer in the same intravenous line concurrent with the other medicinal products. Store undiluted HALAVEN in the syringe for up to 4 hours at room temperature or for up to 24 hours under refrigeration (40°F or/ 4°C). Store diluted solutions of HALAVEN for up to 4 hours at room temperature or up to 24 hours under refrigeration. Discard unused portions of the vial.
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11.1.7 side effects

The most common adverse reactions (≥25%) reported in patients receiving eribulin were neutropenia, anemia, asthenia/fatigue, alopecia, peripheral neuropathy, nausea, and constipation. The most common serious adverse reactions reported in patients receiving eribulin were febrile neutropenia (4%) and neutropenia (2%). The most common adverse reaction resulting in discontinuation of eribulin was peripheral neuropathy (5%). Because clinical trials are conducted under widely varying conditions, the adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in other clinical trials and may not reflect the rates observed in clinical practice. In clinical trials, eribulin has been administered to 1,222 patients with multiple tumor types, including 240 patients exposed to eribulin for 6 months or longer. The majority of the 1,222 patients were women (82%) with a median age of 58 years (range: 26 to 91 years). The racial and ethnic distribution was Caucasian (83%), Black (5%), Asian (2%), and other (5%). The adverse reactions were identified in 750 patients treated in Study 1 [see Clinical Studies (14)]. In Study 1, patients were randomized (2:1) to receive either eribulin (1.4 mg/m2 on Days 1 and 8 of a 21-day cycle) or single agent treatment chosen by their physician(control group). A total of 503 patients received eribulin, and 247 patients in the control group received therapy consisting of chemotherapy [total 97% (anthracyclines 10%, capecitabine 18%, gemcitabine 19%, taxanes 15%, vinorelbine 25%, and other chemotherapies 10%)] or hormonal therapy (3%). The median duration of exposure was 118 days for patients receiving eribulin and 63 days for patients receiving control therapy. Table 2 reports the most common adverse reactions occurring in at least 10% of patients in either group.
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11.1.8 nursing implications

There are no premedications required for eribulin infusion and eribulin is not a significant vesicant.
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11.2 plx3397 11.2.1 plx3397 administration

PLX3397 will be administered orally using a capsule formulation (200 mg per capsule). PLX3397 will be administered only to patients who have signed and dated an informed consent form. PLX3397 will be administered on a 5 day on, 2 day off schedule, repeated weekly, ,
beginning C1D1 (baseline) for Phase Ib, or Day -7/-6 for Phase II. PLX3397 should be taken orally with 240 mL (8 oz) of room-temperature water in the fasting state. The patient should fast at least 1 hour before administration and 1 hour after administration of PLX3397. Patients will be permitted to eat a low-fat, bland snack (e.g., crackers, toast, tea) during the fasting period if needed. PLX3397 should be taken by the patient at approximately the same time of day. Instructions for BID Dosing: The time between BID doses should be approximately 12 hrs. Dosing may occur once daily (QD) under certain circumstances (e.g. dose reductions) and at the discretion of the Investigator in consultation with the Principal Investigator. On C1D1, C1D2 and C2D1, the patients should take the morning dose of PLX3397 at the clinical site after the pre-dose PK blood sample is obtained– patients should be instructed not to take the study drug at home prior to these clinic visits. The time of dosing will be recorded in the clinic. The evening dose of PLX3397 should be taken by the patient at home. On all other visit days, patients may administer the study drug at home and record dosing information in the PLX3397 administration diary.  
Missed doses of greater than 2 hrs before or after the appropriate dosing time should be skipped and not taken as a double dose at the next dosing timepoint. For example, a patient who takes their PLX3397 at 8 a.m. and 8 p.m. each day should not take their dose if they are outside of the ±2 hr window period. Patients who vomit their dose should be instructed to NOT make up that dose.
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11.2.2 plx3397 packaging and labeling

PLX3397-HCl capsules (200 mg strength of the active free base of PLX3397) are manufactured, packaged, and labeled according to GMP and GCP at the following address:
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11.2.3 plx3397 storage and stability

PLX3397-HCl capsules will be stored at the clinical site, as indicated on the study drug label, i.e., room temperature (between 15-30°C, or 59-86°F). Patients will be requested to store PLX3397 at the recommended storage conditions noted on the label, out of the reach of children or other cohabitants.
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11.2.4 plx3397 accountability reconciliation and

The investigator is accountable for all test article supplied by the Sponsor. Copies of the completed source dispensing and inventory record(s) will be returned to the Sponsor (or designee) after the Sponsor (or designee) has performed accountability procedures. PLX3397 accountability will also be captured in the EDC throughout the course of the trial.  
All PLX3397 capsules must be returned to the contract distribution center with the appropriate form if there is evidence that the product has been tampered within transit. Returned and unused PLX3397 capsules may also be destroyed and documented at the investigative site in accordance with GCP.
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11.2.5 plx3397 compliance

At each clinic visit, patients will be questioned about their PLX3397 compliance. PLX3397 compliance will also be tracked through a patient diary.
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11.2.6 plx3397 toxicity

PLX3397 treatment-emergent Adverse Events occurring in ≥ 10% of patients with solid tumors are listed in the table on the following page (combined data from 5 studies, including Phase I,
Phase I/II, and Phase II studies).  
| Overall AEs          | Related AEs   |            |
|----------------------|---------------|------------|
| (any Grade)          | (ny Grade)    |            |
| Preferred Term       | (N=92)        | (N=92)     |
| ANY EVENT            | 87 (94.6%)    | 69 (75.0%) |
| FATIGUE              | 36 (39.1%)    | 23 (25.0%) |
| DECREASED APPETITE   | 35 (38.0%)    | 21 (22.8%) |
| NAUSEA               | 30 (32.6%)    | 19 (20.7%) |
| VOMITING             | 2 (23.9%)     | 9 (9.8%)   |
| ANA                  | 21 (22.8%)    | 1 (12.0%)  |
| HAIR COLOR CHANGES   | 17 (18.5%)    | 17 (18.5%) |
| DIARRHOEA            | 16 (17.4%)    | 7 (7.6%)   |
| AST INCREASED        | 13 (14.1%)    | 12 (13.0%) |
| PYREXIA              | 13 (14.1%)    | 3 (3.3%)   |
| CONSTIPATION         | 1 (12.0%)     | 4 (4.3%)   |
| DIZZINESS            | 1 (12.0%)     | 4 (4.3%)   |
| DYSPNOEA             | 10 (10.9%)    | 1 (1.1%)   |
| rash                 | 10 (10.9%)    | 6 (6.5%)   |
| Protocol Version #10 | Page 48 of 85 |            |
| Protocol #: 12751    |               |            |  
8 serious adverse events (SAEs) possibly or probably related to study drug have been reported in 5 patients in the pooled solid tumor studies (listed in table below). No SAE has been reported more than once. Please refer to the PLX3397 Investigator's Brochure (version 6 dated October 27, 2014) for more detailed information. One patient experienced constrictive pericarditis. The finding was seen on one echocardiogram and resolved on the first follow-up echocardiogram 2 weeks later. This patient had cancer cells in the fluid around the heart, which may have contributed to the inflammation. The study doctor could not rule out the possibility of this event being related to study drug.
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12.0 statistical considerations 12.1 endpoint

The study will follow a standard dose-escalation schema (Phase Ib portion) with 3 to 6 patients per cohort (3+3 design). The starting dose level will consist of PLX3397 600 mg. Enrollment to successive cohorts up to dose Level 2 will be performed according to the table shown below, to establish the MTD. If 2 or more dose-limiting toxicities (DLTs) are observed at level 0, one dose reduction (to Level -1) is built in to the study design. All patients at a given dose level will be followed on treatment for at least 3 weeks before accrual to the next cohort can commence. There will be no intra-patient dose escalation allowed. For any dose cohort, if a patient is removed from study for reasons that are clearly not treatment-related, then an additional patient will be accrued to that dose level.  
| Dose Escalation Schedule Dose   |         |          |
|---------------------------------|---------|----------|
| Dose Level                      | PLX3397 | Eribulin |  
  
| Dose Escalation Schedule   |                                         |                                         |
|----------------------------|-----------------------------------------|-----------------------------------------|
| Level -2                   | 400 mg                                  | 0.7 mg/m2, 2-5 min IV                   |
| (200 mg BID)               | Day 1, 8 q21 days                       |                                         |
| Level -1                   | 400 mg                                  | 1.1 mg/m2, 2-5 min IV                   |
| (200 mg BID)               | Day 1, 8 q21 days                       |                                         |
| 600 mg                     |                                         |                                         |
| (400 mg AM;  200 mg PM)    |                                         |                                         |
| Level 0                    |                                         |                                         |
| (starting dose)            | 1.4 mg/m2, 2-5 min IV Day 1, 8 q21 days |                                         |
| Level 1                    | 800 mg                                  | 1.4 mg/m2, 2-5 min IV                   |
| (400 mg BID)               | Day 1, 8 q21 days                       |                                         |
| 1000 mg                    |                                         |                                         |
| Level 2                    | (600 mg AM;  400 mg PM)                 | 1.4 mg/m2, 2-5 min IV Day 1, 8 q21 days |
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phase ii

The Phase II portion will begin after MTD is determined and we will enroll a total of 56 patients with TNBC to evaluate the safety and efficacy of the proposed treatment. Patients will be treated until clinical or radiographic disease progression or cumulative toxicity. Patients will be evaluated regularly in clinic with history, physical examination, and CT scans every 6 weeks. Visits will take place weekly during the first cycle (one cycle = 21 days) of study treatment, and then on day 1 and 8 of every 3 week cycle during the second cycle and beyond. For Phase II, sample size is based under the Simon's 2-stage optimal design for progression-free survival at 3 months as a single proportion. The study is designed to determine the following hypothesis with >80% power and 2-sided type I error of 5% using binomial approximation: H0: PFS% <39% versus Ha: PFS% > 57% (with PLX3397), where PFS% is the proportion of progression-free (PFS) at 3 months. The PFS% of >57% would represent a clinically relevant improvement with the treatment in triple-negative patients with locally recurrent or metastatic breast cancer. Based on these considerations, a sample size of 51 evaluable patients is required for the study. We plan for a targeted enrollment of 56 patients with 10% dropout rate.
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interim assessment and stopping rules

Safety and SAE data will be routinely monitored. Because of the possibility of unacceptable or excessive defined serious adverse events during treatment or the study enrollment period, stopping rules for safety/tolerability are also developed. We have set the maximum rate of treatment-related SAE at 15% in patient enrollment. Thus, we may consider terminating the study if the number of patients with SAE is greater than the number expected under the binomial distribution. The following table shows the early stopping guideline based on the number of SAE observed under the rate of unacceptable SAE in the group sequential enrolment for the study. For example, assuming the unacceptable rate of SAE is greater than 15%, if the study observes 3 or more SAE in the first 17 treated patients, then we may consider if the study should be stopped early. Safety Stopping Rules: Number of enrolled patients with excessive SAE to consider early study termination.  
| Number patients in study   | 1-17   | 18-29   | 30-41   |
|----------------------------|--------|---------|---------|
| Unacceptable SAE - 15%     | 3      | 4       | 6       |  
Efficacy Stopping Rules Failure to obtain early responses at the scheduled tumor assessments will be taken as evidence that the regimen in question is ineffective. If fewer than 8 out of the first 20 pts are progressionfree at 12 weeks, this would be considered an unacceptably low PFS rate (<39%), the study will not continue and accrual will cease. Accrual will contiue while efficacy data is being determined. If 25 or fewer pts are progression free at 12 weeks at full accrual, then the treatment would be deemed ineffective. If the true PFS rate at 12 weeks is 39% for TNBC pts, the probability of terminating the study during the interim evaluation is 0.63; if the true PFS rate is 57% for TNBC pts, the probability that the trial will be stopped in the first stage is 0.10.
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12.2 analysis plan primary efficacy

Demographic data (age, sex, race, ethnicity, body weight) and baseline disease characteristics will be tabulated and summarized and presented in data listings.
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progression free survival

Progression-free survival at 3 months (PFS) is defined as the proportion of patients alive and progression-free 90 days after Study Day 1. Duration of PFS is defined as the time from Study Day 1 to the earlier of disease progression or death due to any cause. Progression free survival will be calculated from the first administration of PLX3397 with eribulin. The analyses of PFS will be performed using the Per Protocol population. These analyses are designed to include only objective progression events per the RECIST v1.1 criteria. Patients who do not have any post-baseline tumor assessments will be right censored on the date of Study Day 1. Patients who receive subsequent anti-cancer therapy before experiencing an event will be right censored at the date of the last tumor assessment prior to the date of initiation of subsequent therapy. Patients who have not experienced an event (and are not otherwise censored) at the time of data cutoff will be right censored on the date of their last tumor assessment PFS will be estimated as a simple proportion based upon the results of the 12-week tumor assessment. Patients for whom this assessment is not performed will be included as failures, even if known to be alive at this timepoint. Confidence intervals will be provided. PFS and duration of PFS will also be estimated using Kaplan-Meier methods with associated confidence intervals.
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secondary and supportive efficacy endpoints

The objective response rate (ORR) is defined as the proportion of patients for whom the best overall response at the time of data cutoff is confirmed CR or confirmed PR as assessed per RECIST v 1.1criteria. A table defining the derivation of best overall response to be applied will be provided in the SAP. The analysis of ORR will employ the Per Protocol population. Patients who do not have any post-baseline tumor assessments will be counted as non-responders.  
Point estimates of ORR and confidence intervals will be provided. As a basic measure of doseresponse, ORR in the escalation phase will also be presented by dose cohort. Objective response will be assessed using the RECIST v 1.1 response criteria. The response will be collected on CRFs. The number and percent of subjects with each type of response will be summarized and presented in data listings. A 95% confidence interval will also be calculated using exact bionomial method. The 95% confidence interval for the difference in objective response rates will be calculated using exact binomial method. Time to onset of the response and duration of response will be also summarized using descriptive statistics.
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duration of response

Duration of response is defined as the time from first documentation of objective response that is subsequently confirmed to PD by the criteria or death due to any cause. Responders who have not been documented to have progressed or died at time of data cutoff will be right censored at the last available adequate tumor assessment. Median duration of response for each treatment arm and its associated confidence interval will be estimated using the Kaplan-Meier method.
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time to progression

Time to progression will be estimated using the Kaplan-Meier method. Efficacy responses, disease progression and relapse classified based on RECIST v1.1 criteria will be used to determine progression. Time to progression will be calculated from the first administration of PLX3397 with eribulin. Patients who do not have disease progression will be censored at the date of the last evaluation for study disease or at the time of initiation of the new therapy, whichever is earlier. Patients lacking any response assessment after randomization will be censored at Day 1.
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12.3 accrual objectives phase i

A minimum of 12 patients will enroll in the Phase I trial, following a 3+3 design for 3 cohorts. We estimate that 10 patients will enroll at UCSF, and 7 patients each at Duke and Vanderbilt. Phase I completed at UCSF with 28 patients enrolled as 03/10/2015
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phase ii

This study will enroll 56 patients (n=22 in the first stage and n=34 in the second stage) in the Phase II portion with the assumed 10% dropouts. We estimate that 26 patients will enroll at UCSF and 15 each at Vanderbilt and Duke.
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12.4 estimated duration of study

The Phase I study is expected to complete accrual within 1.25 years, and the Phase II study is expected to complete accrual over a time course of 2.5 years.
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p h ase i

F or a n y d ose c o h ort, if a patie nt is re m o ve d fr o m st u d y f or reas o ns t hat are clearl y n ot treat me ntrelate d, t he n a n a d diti o nal patie nt will be accr ue d t o t hat d ose le vel.
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p h ase ii

Patie nts w h o ha ve n o e val uati o n meas ures after starti n g t hera p y, a n d st o p t hera p y f or reas o ns ot her t ha n pr o gressi o n or t o xicit y will be re place d. Patie nts w h o recei ve o nl y o ne d ose of eri b uli n or less t ha n 1 4 da ys of P L X 3 3 9 7 f or reas o ns ot her t ha n pr o gressi o n or t o xicit y will be re place d.
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1 2. 7 p h

P har mac o ki netic st u dies will be perf or me d as o utli ne d i n secti o n 1 4. 4. Ser u m c o nce ntrati o ns of P L X 3 3 9 7 wit h eri b uli n will be assesse d b y E LI S A at Ple x xi k o n I nc. a n d will be use d t o calc ulate t he val ues of P K para meters, i ncl u di n g ma xi m u m ser u m dr u g c o nce ntrati o n ( C ma x), area u n der t he c o nce ntrati o n-ti me c ur ve fr o m ti me zer o t o i nfi nit y ( A U C 0-i nf), s yste mic cleara nce ( C L),  
v ol u me of distri b uti o n ( V), a n d eli mi nati o n half-life (t 1/ 2). Descri pti ve statistics (i ncl u di n g n u m ber, mea n a n d/ or me dia n, sta n dar d de viati o n, a n d c oefficie nt of variati o n) will be use d t o descri be t he c o nce ntrati o n ti me data. W here a p pr o priate, t hese data ma y be c o m bi ne d wit h data fr o m ot her st u dies as part of a metaa nal ysis. T he i nfl ue nce of e x p os ure o n cli nical safet y para meters (e. g., selecte d A Es) will be e x pl ore d. Res ults of t he P K a nal ysis will be e val uate d i n c o nj u ncti o n wit h a vaila ble safet y a n d p har mac o d y na mic data.