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UAB 1514- A Phase I/II Study of Preoperative (Neoadjuvant) Combination of Letrozole (Femara®), Everolimus (Afinitor®), and TRC105 in Postmenopausal Women with Newly Diagnosed Local or Locally Advanced Potentially Resectable Hormone-Receptor Positive and Her2 Negative Breast Cancer PI: ERICA STRINGER-REASOR, MD
NCT: 02520063 February 7, 2023
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University Of Alabama At Birmingham Local Protocol #: Uab 1514  
A Phase I/II Study of Preoperative (Neoadjuvant) Combination of Letrozole (Femara®), Everolimus (Afinitor®), and TRC105 in Postmenopausal Women with Newly Diagnosed Local or Locally Advanced Potentially Resectable Hormone-Receptor positive and Her2 negative Breast Cancer Brief **Title:** Letrozole, everolimus, and TRC105 in postmenopausal HR positive Breast Cancer  
| Principal Investigators:   | Erica M. Stringer Reasor, M.D. Division of Hematology/Oncology Department of Medicine University of Alabama at Birmingham UAB Comprehensive Cancer Center 1802 Sixth Avenue South North Pavilion 2501 Birmingham, AL, 35294-3300 email: esreasor@uabmc.edu                                                                                                                                                                                                                                                                          |
|----------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Co-Investigator:           | Christos Vaklavas, M.D.  cvaklava@yahoo.com                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| Sub-investigators:         | Lisle M Nabell, M.D. Division of Hematology/Oncology Department of Medicine University of Alabama at Birmingham UAB Comprehensive Cancer Center 1802 Sixth Avenue South North Pavilion 2540 Birmingham, AL, 35294-3300 Phone: (205) 934-3061 Fax: (205) 975-9002 email: lisle@uab.edu Carla I Falkson, M.D. Division of Hematology/Oncology Department of Medicine University of Alabama at Birmingham UAB Comprehensive Cancer Center 1802 Sixth Avenue South North Pavilion 2540 Birmingham, AL, 35294-3300 Phone: (205) 975-2691 |  
email: cfalkson@uab.edu Statistician: **Study Coordinator:**  
  
Kimberly V. Lamaster, RN, BSN
Yufeng Li, Ph.D. Division of Preventive Medicine Department of Medicine University of Alabama at Birmingham 1720 2nd Avenue South Medical Tower 644 Birmingham, AL, 35294- 4410 Phone: (205) 996-2866 and (205) 934-9520 Fax: (205) 934-4262 email: yufengli@uab.edu Data Manager: To Be Named NCI Supplied Agent: None Protocol Type / Version \# / Version Date Amendment 1, version 2\ 6-29-2016 Amendment 2, version 3\ 7-25-19
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protocol synopsis

Title of Study: A Phase I/II Study of Preoperative (Neoadjuvant) Combination of Letrozole  
  
(Femara®), Everolimus (Afinitor®), and TRC105 in Postmenopausal Women with Newly Diagnosed Local or Locally Advanced Potentially Resectable Hormone-Receptor positive and Her2 negative Breast Cancer Brief Title: Letrozole, everolimus, and TRC105 in postmenopausal hormone receptor positive Breast Cancer Investigators: Erica M Stringer-Reasor, M.D., Christos Vaklavas, M.D.; (principal co-investigators) Lisle M Nabell, M.D., Carla I Falkson, M.D. (sub-investigators), Yufeng Li, Ph.D. (statistician) Phase: Pilot **Type of Investigation:** drug Study Center(s): UAB, Comprehensive Cancer Center Rationale: Our group has demonstrated in 2 different and independent studies that the addition of an anti-angiogenic agent to hormonal therapy in the neoadjuvant setting increased not only the proportion of patients with a pathologic complete remission (pCR) and near pCR (invasive residual disease less than 5 mm in the primary tumor) but also the proportion of patients that were down-staged (from stage 2 and 3 to 0 and 1)1,2 (a prognostic factor in neoadjuvant hormonal therapy). In addition, preclinical and clinical studies have shown that the addition of an mTOR inhibitor to hormonal therapy is able to reverse resistance to hormonal agents; these studies led to the approval of everolimus in combination with exemestane by the FDA for hormone receptor positive metastatic breast cancer 3-6. However, preclinical studies have shown that mTOR
inhibition results in VEGF inhibition which leads to overexpression of CD105 in the endothelial cells as a compensatory mechanism7,8. Thus, the addition of an anti-CD105 agent may increase the efficacy observed in the randomized trial of letrozole and everolimus in patients with stage 2 and 3 breast cancer published by Baselga recently 4.  
Background: The achievement of a pCR with neoadjuvant therapy in early-stage breast cancer has a favorable impact on long-term outcomes and is an excellent prognostic surrogate marker913. Responses to neoadjuvant therapy however, are intrinsically dependent on the molecular subtype14 with the immunophenotypically luminal (hormone-receptor positive and HER2 negative) breast cancers rarely achieving a pCR12. Nonetheless, in the latter subgroup, downstaging to at least pathologic stage 1 or 0 is equally important as it has been associated with a significant low risk of recurrence15. With chemotherapy yielding similar benefits with hormonal therapy in the neoadjuvant setting16,17, the latter is generally preferred as it is associated with fewer adverse events and is overall better tolerated among postmenopausal women who frequently have other comorbidities18. In the neoadjuvant setting, aromatase inhibitors have been consistently shown to be superior over tamoxifen19-23, making the former the agents of choice in postmenopausal women with hormone-receptor positive [HR(+)] breast cancer. Despite the high rates of clinical and radiographic response achieved with hormonal therapy19-23, the rates of downstaging are still low15. Collectively, there is a **need to improve**
the efficacy of aromatase inhibitors in the neoadjuvant setting with minimal additional toxicities as it directly impacts clinical outcomes. Targeting angiogenesis in HR(+) breast cancer: Angiogenesis is an integral step in tumor progression24. In mouse xenograft models of HR(+) breast cancer, enhanced angiogenesis accelerated tumor growth25, and overexpression of the Vascular Endothelial Growth Factor  
 (VEGF) either by the tumor26 or stromal cells27 was associated with hormone independent growth and resistance to endocrine therapies. The concept of targeting angiogenesis to restore or enhance the efficacy of neoadjuvant hormonal therapy in breast cancer has been successfully translated in the clinical arena. The Translational Breast Cancer Research Consortium completed a study comparing preoperative letrozole versus letrozole plus bevacizumab in postmenopausal women with operable HR(+)
breast cancer. The study (TBCRC002) showed that bevacizumab "deepened" the responses obtained with letrozole alone. Downstaging from stage 2/3 to 0/1 was achieved in 45% of patients in the letrozole-bevacizumab arm as compared to 25% in the letrozole-alone group.  
Remarkably, the combination induced a pCR in 11.1% of patients 1(and 8% near pCR), comparable to the 12% pCR rate reported in a prior pilot single-institution study 2; no complete or near-complete pathologic remissions were observed in the letrozole-alone group. Taken together, enhanced angiogenesis is associated with hormone resistance and there is a successful clinical precedence of rationally combining an antiangiogenic agent with hormonal therapy that even led to an unprecedented pCR rate in an intrinsic subgroup where pCRs are rarely observed. Endoglin (CD105) as a rational target of malignant angiogenesis: Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo28. It diverts TGF-β downstream signaling toward proliferation and migration as well as transcription of proangiogenic genes including endoglin itself29-31. Preclinical evidence suggests that CD105 is highly and exclusively or mainly expressed in *de novo* tumor vessels32,33, as opposed to the normal quiescent endothelium which has a very low turnover. In preclinical murine xenograft models of HR(+) breast cancer, anti-endoglin monoclonal antibodies selectively inhibited tumor angiogenesis and delayed the growth of established tumors, while sparing normal vasculature34.  
Endoglin expression has been investigated in multiple malignancies and has been consistently associated with adverse clinical outcomes31. This observation is in line with the premise that enhanced tumor vasculature correlates with adverse prognosis and the fact that endoglin is a marker of activated angiogenic endothelium31. Collectively, its exclusive expression in the activated endothelium and its pathophysiologic role in promoting angiogenesis make endoglin a rational target of tumor angiogenesis. Pharmacologic targeting of endoglin (CD105) with TRC105: TRC105 is a chimeric IgG1 monoclonal antibody that binds with high avidity to CD105 (endoglin). It inhibits TGF-β mediated pro-angiogenic signaling and induces potent antibody-dependent cell-mediated cytotoxicity. In a phase I study, the maximum tolerated dose was determined at 10 mg/kg weekly; 15 mg/kg every two weeks was equally well tolerated and also resulted in continuous serum concentrations known to saturate CD105 binding sites on proliferating endothelium35.  
The most common adverse events associated with the investigational agent were infusion reactions that were successfully mitigated with pre-medications and prolongation of the infusion time, hemorrhages (mostly grade 1 and 2), headaches, and hypoproliferative anemia35.  
Importantly, the median age in this phase I clinical trial was 6335, making this study highly informative in terms of adverse events for the patient population intended to be used in our proposed study. Considering the non-overlapping toxicity profile with aromatase inhibitors and the preclinical evidence highlighting malignant angiogenesis being in the core of  
 hormone independence, there is an unprecedented potential of synergism between TRC105 and letrozole that is worth clinical investigation. Rationale for incorporating everolimus in the neoadjuvant setting: The phosphatidyl inositol-3 kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway is an intracellular signal transduction pathway downstream of receptor tyrosine kinases36. Class IA
PI3K molecules are heterodimers composed of a regulatory subunit (p85) and a catalytic subunit
(p110), the α isoform of which (encoded by *PIK3CA*) is widely mutated or amplified in human cancers37,38, including breast cancer39,40. Comprehensive genomic analyses of breast cancer samples in the context of The Cancer Genome Atlas Project also highlighted the high frequency of mutations along the PI3K/AKT/mTOR pathway41. In particular, activating mutations in the PIK3CA, inactivating mutations or loss of *PTEN,* and loss of *INPP4B* (a negative regulator of the PI3K/AKT pathway) were detected in 49%, 13%, and 9% (luminal A) and 32%, 24%, and 16% (luminal B), respectively. At the preclinical level, breast cancer cells with upregulated PI3K/AKT/mTOR pathway were shown to be resistant to hormonal therapy and this resistance was restored by everolimus and other mTOR inhibitors 42,43. Taken together, the PI3K/AKT/mTOR pathway can be activated by various genetic events in HR (+) breast cancer and its inhibition with the FDA-approved agent everolimus has been proven at the clinical level to restore3 or enhance4**sensitivity to endocrine therapies.**
Synergism between TRC105 and everolimus. The molecular basis for combining antiangiogenic therapy with everolimus is provided by preclinical studies 44-46. In animal models, blocking mTOR was shown to reduce the VEGF levels and to inhibit significantly the response of endothelial cells to stimulation by VEGF 45. mTOR signaling was also shown to play a key role in hypoxia-triggered smooth muscle and endothelial cell proliferation and angiogenesis in vitro; this response (triggered by growth factors other than VEGF, such as FGF and PDGF) was abrogated with rapamycin, the prototypic mTOR inhibitor46. Moreover, VEGF inhibition leads to reactive upregulation of endoglin7and large established CD105(+) vessels lying in the core of the tumors have been shown to be insensitive to VEGF
inhibitors8. In those studies, CD105 expression was increased following inhibition of the VEGF
pathway. CD105 expression increased more than two-fold in human pancreatic cancers grown in mice treated with an antibody that binds VEGF7. Similarly, treatment of human bladder cancers grown in mice with an antibody that blocks activation of the VEGF receptor increased CD105 expression within the core tumor vasculature8. Finally, a study of spontaneously occurring neuroendocrine tumors in RIP-Tag2 mice treated with antibody to VEGF indicated that TGF- β was the most highly upregulated factor, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)47. These studies indicate that the CD105 pathway is a significant mechanism of escape from VEGF inhibitor therapy. Rationale for ovarian suppression with gonadotropin-releasing hormone analogs in premenopausal women. The IMPACT trial (Immediate Preoperative Anastrozole, Tamoxifen or Combined with Tamoxifen) established the superiority of aromatase inhibition over tamoxifen or tamoxifen + aromatase inhibition in the preoperative setting 48 . In addition, suppression of the proliferation marker Ki67 (which in itself has been shown to be predictive of favorable outcome) was statistically greater with anastrozole as compared with tamoxifen or the combination 49. Aromarase inhibitors in premenopausal women are contraindicated as they may  
  
increase gonadotropin secretion because of the reduced feedback of estrogen to the hypothalamus and pituitary50. To that end, in order for premenopausal women to benefit from aromatase inhibition, they should become postmenopausal through ovarian suppression with gonadotropin-releasing hormone analogs. For this clinical trial goserelin subcutaneously q 4 weeks will be used. Collectively, considering the non-overlapping toxicity profiles and the preclinical evidence highlighting malignant angiogenesis and the PI3K/AKT/mTOR pathway being in the core of resistance to and failure of endocrine therapies, there is an unprecedented potential of synergism among letrozole, TRC105, and everolimus that is worth clinical investigation.  
Primary Objective: Determine the tolerability and feasibility of combining everolimus and TRC105 with letrozole in postmenopausal women with newly diagnosed stage 2 and 3 (T2-T4ac, N0-2, M0; excluding patients with T4d or inflammatory breast cancer) estrogen and/or progesterone receptor positive, HER2/neu negative breast cancer, treated for 24 weeks in the preoperative (neoadjuvant) setting. Secondary Objective: 1. Determine the efficacy of the combination of letrozole, everolimus, and TRC105 2. Determine the pharmacokinetic parameters of everolimus and TRC105 when used in combination with letrozole.  
3. Determine the pharmacodynamic parameters of everolimus and TRC105 when used in combination with letrozole.  
Exploratory objective: Evaluate specific biomarkers of potential prognostic value and biomarkers potentially predictive of response or resistance to the combination of everolimus and TRC105 with letrozole.  
Primary Endpoint: Determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D) and rates of adverse events associated with the combination of letrozole with everolimus and TRC105. Secondary Endpoints: 1. Determine the rates of complete pathologic remission and downstaging achieved with the investigational treatment at the time of surgery.  
2. Determine the Cmax, Tmax, AUC, T½, clearance of everolimus and TRC105 when used in combination with letrozole.  
3. Determine the changes in tumor cell proliferation by means of changes in Ki67 expression and changes in serum concentration of markers of angiogenesis (VEGF-A, soluble VEGFR2, activin A and activin B, angiopoietin-1 and -2, TGF-β, soluble endoglin) before and after the investigational treatment and correlate with clinical, radiographic, and pathologic response.  
Exploratory Endpoints: 1. Correlate the levels of PTEN immunostaining with response 2. Correlate levels of phosphorylated AKT (pAKT) immunostaining with response 3. Correlate expression of endothelial (CD105 and CD31/CD34) and pericyte (NG2, and MCAM [CD146]) markers in the initial diagnostic and final surgical specimens with response.  
4. Determine translational (mRNA to protein) profiles by means of next-generation whole  
  
exome sequencing and sequencing of ribosome-protected mRNA associated with resistance to the investigational combination.
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study design the study consists

The first part of the study will follow the conventional 3+3 dose escalation design. Up to 18 patients will be enrolled. During the first part (phase I, **dose escalation**), the Maximum Tolerated Dose (MTD) and the Recommended Phase 2 Dose (RP2D) of the combination will be determined. The dose of letrozole will be kept the same for all cohorts (2.5 mg PO daily) (Table 1). The dose of everolimus will be escalated from 5 mg PO daily (cohort 1) to 10 mg PO daily (cohort 2, *continuous arrow*). The dose of everolimus will be de-escalated to 5 mg PO daily (cohort 1) if the dose of 10 mg PO daily is not well tolerated (*dashed arrow*). The dose of TRC105 will be 15 mg/kg q 2 weeks (cohorts 1 and 2, *continuous arrow*). The dose of TRC105 will be de-escalated to 10 mg/kg q 2 weeks (cohort -1) if 15 mg/kg q 2 weeks in combination with everolimus 5 mg PO daily are not well tolerated (*dashed arrow*). Different doses of the 3 agents evaluated in this trial have shown to be active against breast cancer in clinical trials. Premenopausal women at the time of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression.  
| Cohort   | TRC105 (mg/kg IV q 2 weeks)   | Everolimus (mg PO daily)   |
|----------|-------------------------------|----------------------------|
| 1        | 15                            | 5                          |
| 2        | 15                            | 10                         |
| -1       | 10                            | 5                          |  
The second part of the study (phase II, **expansion phase**), will ensue once the MTD and RP2D have been determined. In order to minimize exposure of patients to ineffective therapy, a Gehan's two-stage design will be used51. The study will enroll 10 patients first, and if no patient has pCR or downstaging, the study will close on the basis that it is unlikely (p=0.1074) that 0/10 responses would occur if the true response rate were >20%. If at least one patient has pCR or downstaging, an additional 10 patients will be treated (total of 20). Accrual will be halted as a result of any grade ≥3 hemorrhage, cerebrovascular and vasovagal adverse events; the FDA will be contacted to discuss further accrual or potential need for protocol amendment.  
  
Study Schema
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everolimus

Premenopausal women at the time of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression Drug Dose (doses, frequency, duration):
Letrozole 2.5 mg PO daily, continuously until the day prior to surgery. TRC105 15 or 10 mg/kg IV every two weeks until 4 weeks prior to surgery. The first dose of TRC105 will be split into two doses; see Table 7-1 ( Study Calendar ) for additional details. Everolimus 5 or 10 mg PO dily, continuously until 4 weeks prior to surgery. Premenopausal women at the time of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression.  
Main Criteria for Inclusion/Exclusion:
Key Inclusion Criteria - Recent diagnosis of hormone receptor positive and HER2 negative breast cancer.  
- Stage 2 and 3 hormone receptor positive and HER2 negative breast cancer (stage T2-4 but not inflammatory, N0-2, M0).  
- Histological grade I, II or III according to the modified Bloom Richardson scale52.  
  
- No prior treatment specific for breast cancer. - Menopausal status as defined by the National Comprehensive Cancer Network. Premenopausal women are eligible if ovarian suppression is achieved with goserelin - ECOG performance status < 2 (Karnofsky > 60%). - Must have signed study-specific informed consent. - Liver Function Tests < 2.5 times the upper normal limit (UNL). - Staging workup: medical/surgical/radiation therapy evaluation, CT scans and bone scan. - Lipid profile (cholesterol and triglycerides).  
- ANC ≥ 1,500/mm3, platelets ≥ 100,000/mm3, Hemoglobin ≥ 10g%.  
- Renal function: serum creatinine < 1.5 institutional UNL or creatinine clearance > 40 cc/min.
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key exclusion criteria

- Inflammatory breast cancer. - Pregnancy. - Metastatic disease. - HER2 positive breast cancer by immunohistochemistry or FISH. - Triple negative breast cancer (hormone receptor and Her2 negative). - Disease that cannot be followed by imaging studies. Safety Assessments:
Vitals signs Weight ECOG performance status Hematology Chemistry Lipid profile Hepatitis panel Adverse events Concomitant medications
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other assessments

a. Pharmacokinetic (PK) studies. To investigate the pharmacokinetic interaction between TRC105 and everolimus, samples will be collected and concentrations of the agents measured on cycle 1 day 1 and cycle 2 day 1 at the following time points: pre-dose, 1, 2, 4, 6, and 24 hours and also cycle 1 day 4 predose. Pharmacokinetic parameters to be estimated include maximum serum concentration (Cmax), time of maximum serum concentration (Tmax), area under the serum concentration versus time curve (AUC), terminal half-life (T½), and clearance. Serum for assessment of TRC105 anti-product antibody formation will be collected before dosing on cycle 1 day 1, every 4 weeks thereafter, 4 and 12 weeks following the last dose of TRC105 and concentrations will be determined by a validated ELISA53.  
b. Pharmacodynamic (PD) studies. Markers of angiogenesis (VEGF-A, soluble VEGFR2, activin A and activin B, angiopoietin-1 and -2, TGF-β, soluble endoglin) will be measured on samples collected pre-dose, cycle 1 day 15, cycle 2 day 1, and prior to surgery. c. Research biopsy. A research biopsy will be obtained at baseline, on cycle 2 day 1, and the time of the surgery. At least 2 core biopsies will be taken, one will be embedded in paraffin and the other one will be immediately frozen. Immunohistochemistry for pAKT and PTEN (markers  
Efficacy **Assessments:**  
Physical examination  
Imaging studies (mammography,  
breast ultrasound, breast magnetic resonance imaging)  
Surgical Pathology  
of the PI3K/AKT/mTOR pathway), CD105 and CD31/CD34 (pan-endothelial markers), and  
 NG2 and MCAM/CD146 (pericyte markers) will be conducted on the research biopsies, and the final surgical specimen. Next Generation Sequencing. Determine translational (mRNA to protein) profiles by means of next-generation whole exome sequencing and sequencing of ribosome-protected mRNA associated with resistance to the investigational combination. Statistical Methods: this is a phase I study and primarily descriptive statistics will be used. (a) **Sample size justification:** The dose escalation part of the study will follow a conventional 3+3 dose escalation design according to the schema above. Up to 18 patients will be enrolled in this part anticipating that dose level 1 or 2 will be the RP2D. The number of patients to be enrolled ranges from 3 (≥2/3 DLTs in cohort 1) to 18 (enrollment of 6 patients in all cohorts).  
The sample size of 20 in Phase II part study is NOT determined by the statistical power but the feasibility and relative precision of estimate, e.g. standards error of the estimation is ≤10%. We will estimate the pCR of the combination to generate hypotheses for further clinical investigations. A Gehan's two-stage design will be used51 which allows for the rapid rejection of an ineffective treatment at the end of the first stage, and provides an estimation of the success rate with a given precision, at the end of the second stage. The study will enroll 10 patients first. If no patient has pCR or downstaging, it is unlikely (p=0.1074) that 0/10 responses would occur if the true response rate were >20% and the phase II portion will be closed. If at least one patient has pCR or downstaging, an additional 10 patients will be treated (total of 20). With an accrual of 20 patients to phase II portion of the study, estimated two-sided 95% confidence intervals (CI) would be 1.2%-31.7%, 5.7% -43.6% and 11.8%-54.3% if pCR is 10%, 20% or 30% respectively. The confidence intervals are calculated using the exact method Clopper-Pearson intervals54. Final analysis will include all patients enrolled in the trial.  
(b) **Analytic plan for primary objective:** The rate of adverse events will be estimated at the end of the study along with two-sided 95% exact CIs (Clopper-Pearson intervals).
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(c) analytic plan for secondary

1. pCR or downstaging rate will be estimated along with two-sided 95% CIs with the exact method of Clopper-Pearson intervals.  
2. We will use descriptive statistics (mean, median, geometric mean, coefficient of variation, and 95% confidence intervals) and graphical displays to evaluate the pharmacokinetic parameters of the TRC105 and everolimus.  
3. We will use descriptive statistics (mean, median, geometric mean, coefficient of variation, and 95% confidence intervals) and graphical displays to analyze the changes in markers of proliferation and angiogenesis with the investigational treatment.  
4. Exploratory analysis will be conducted on the correlations between clinical outcomes and pharmacodynamic biomarkers or translation profiles.
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table of abbreviations

| 4-EBP-1 ACE AdEERS ADR AE ANC AUC AKT ALT APA AST BAL BCG BIRADS BUN Cmax CBC CC CD CD105 CI CoA CPK CR CRF CT CTCAE CTEP CYP3A4 DEXA DLCO DLT DM DS&E ECOG EKG ELISA ER FDA FGF FISH FSH GCP GERD GI   | 4E Binding Protein 1 Angiotensin-Converting Enzyme Adverse Event Expedited Reporting System Adverse Drug Reaction Adverse events Absolute Neutrophil Count Area Under the Curve Protein Kinase B Alanine aminotransferase/glutamic pyruvic transaminase/GPT Anti-Product (TRC105) Antibody Aspartate aminotransferase/glutamic oxaloacetic transaminase/GOT Bronchoalveolar lavage Bacillus Calmette-Guérin Breast Imaging Reporting and Data System Blood Urea Nitrogen Maximum Serum Concentration Complete Blood Count Craniocaudal Cluster of Differentiation Endoglin  Confidence Intervals Coenzyme A Creatine Phosphokinase Complete Response Case Report Form Computed Tomography Common Terminology Criteria for Adverse Events Cancer Therapy Evaluation Program Cytochrome P450 3A4 Dual-Energy X-ray Absorptiometry Diffusing capacity of the Lung for Carbon Monoxide Dose Limiting Toxicity Diabetes Mellitus Drug Safety and Epidemiology Department Eastern Cooperative Oncology Group Electrocardiogram Enzyme-linked Immunosorbent Assay Estrogen Receptor Food and Drug Administration Fibroblast Growth Factor Fluorescence in situ Hybridization Follicle Stimulating Hormone Good Clinical Practice Gastroesophageal Reflux Disease Gastrointestinal- 11 - | P a g e   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
  
| GnRH HAMA HACA HbA1c HBcAb HbsAb HbsAg HBV HCV HHT-1 HMG HR HUVEC IB ICH IgG1 IHC IRB IUD IUS IV kg LD LH log10 MedDRA mg MLO MMP-14 MRI mRNA MTD mTOR MUGA NCI pAKT pCR PCR PD PD PDGF PET P-gp PHI PJP PNET PI3K   | Gonadotropin-releasing hormone Human Anti-Murine Antibody  Human Anti-Chimeric Antibody Hemoglobin A1c Hepatitis B core antibody Hepatitis B surface antibody Hepatitis B surface antigen Hepatitis B virus Hepatitis C virus Hereditary Hemorrhagic Telangiectasia T1 (Rendu/Osler/Webber  syndrome) 3-hydroxy-3-methyl-glutaryl Hormone Receptor Human Umbilical Vein Endothelial Cells Investigator's Brochure International Conference on Harmonization Immunoglobulin G1 Immunohistochemistry Institutional Review Board Intrauterine Device Intrauterine System Intravenous kilogram Longest Diameter Luteinizing Hormone Decadic logarithm (common logarithm) Medical Dictionary for Regulatory Activities milligram Mediolateral Oblique  Matrix Metalloproteinsae - 14 Magnetic Resonance Imaging messenger RNA Maximum Tolerated Dose mammalian Target Of Rapamycin Multi Gated Acquisition Scan National Cancer Institute phosphorylated Protein Kinase B Pathologic Complete Remission  Polymerase Chain Reaction Pharmacodynamic  Progressive Disease Platelet Derived Growth Factor Positron-Emission Tomography P-glycoprotein Protected Health Information Pneumocystis jirovecii Pneumonia Pancreatic Neuroendocrine Tumor- 12 - | P a g e   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| PK PO PR PTEN RAD001 RECIST RP2D RT-PCR SAE SD SEGA SUSARs T½  Tmax TAC TBCRC TGF-β TGF-βR TSC UA ULN US VEGF VEGF-A VEGFR2 WHO   | Phosphatidyl Inositol-3 Kinase Pharmacokinetic Oral Partial Response Phosphatase and Tensin Homolog everolimus Response Evaluation Criteria for Solid Tumors Recommended Phase 2 Dose Reverse Transcriptase-Polymerase Chain Reaction Serious Adverse Event Stable Disease Subependymal Giant cell Astrocytoma Suspected Unexpected Serious Adverse Reactions Half-life time of maximum serum concentration docetaxel 75 mg/m 2 , doxorubicin 50 mg/m2 , cyclophosphamide 500  mg/m2 Translational Breast Cancer Research Consortium Transforming Growth Factor-beta Transforming Growth Factor-beta Receptor Tuberous Sclerosis Complex Urinalysis Upper Level of Normal Ultrasound Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor - A  Vascular Endothelial Growth Factor Receptor 2 World Health Organization   |
|-----------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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glossary of terms

| Assessment         | A procedure used to generate data required by the study                                                                                                                                                                                                                                                                                                                                                                                        |
|--------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Baseline           | For efficacy evaluations, the baseline assessment will be the last available  assessment before or on the date of randomization. For safety evaluations (i.e. laboratory assessments and vital signs), the  baseline assessment will be the last available assessment before or on the  start date of study treatment. The value obtained at baseline assessments, referred to as "baseline value"  will be used as reference for the patient. |
| Control drug       | A study treatment used as a comparator to reduce assessment bias,  preserve blinding of investigational drug, assess internal study validity,  and/or evaluate comparative effects of the investigational drug                                                                                                                                                                                                                                 |
| Dose level         | The dose of drug given to the patient (total daily or weekly etc.)                                                                                                                                                                                                                                                                                                                                                                             |
| Enrollment         | Point/time of patient entry into the study; the point at which informed  consent must be obtained (i.e. prior to starting any of the procedures  described in the protocol)                                                                                                                                                                                                                                                                    |
| Investigational    | The study treatment whose properties are being tested in the study; this                                                                                                                                                                                                                                                                                                                                                                       |
| drug               | definition is consistent with US CFR 21 Section 312.3 and is synonymous  with "investigational new drug."                                                                                                                                                                                                                                                                                                                                      |
| Investigational    | Drug whose properties are being tested in the study as well as their                                                                                                                                                                                                                                                                                                                                                                           |
| treatment          | associated placebo and active treatment controls (when applicable). This  also includes approved drugs used outside of their indication/approved  dosage, or that are tested in a fixed combination. Investigational treatment  generally does not include other study treatments administered as  concomitant background therapy required or allowed by the protocol  when used in within approved indication/dosage                          |
| Medication         | A unique identifier on the label of each study treatment package which is                                                                                                                                                                                                                                                                                                                                                                      |
| number             | linked to one of the treatment groups of a study                                                                                                                                                                                                                                                                                                                                                                                               |
| Other study        | Any drug administered to the patient as part of the required study                                                                                                                                                                                                                                                                                                                                                                             |
| treatment          | procedures that was not included in the investigational treatment                                                                                                                                                                                                                                                                                                                                                                              |
| Patient            | A unique identifying number assigned to each patient/subject/healthy  volunteer who enrolls in the study                                                                                                                                                                                                                                                                                                                                       |
| Period             | A subdivision of the study timeline; divides stages into smaller functional  segments such as screening, baseline, titration, washout, etc.                                                                                                                                                                                                                                                                                                    |
| Premature          | Point/time when the patient exits from the study prior to the planned  completion of all study treatment administration and/or assessments; at  this time all study treatment administration is discontinued and no further  assessments are planned, unless the patient will be followed for  progression and/or survival                                                                                                                     |
| patient withdrawal | - 14 - | P a g e                                                                                                                                                                                                                                                                                                                                                                                                                               |  
  
| Stop study                            | Point/time at which the patient came in for a final evaluation visit or when                                                                                                                                                                                                                                                                                                                                                                        |    |         |                 |     |                |     |
|---------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|---------|-----------------|-----|----------------|-----|
| participation                         | study treatment was discontinued whichever is later                                                                                                                                                                                                                                                                                                                                                                                                 |    |         |                 |     |                |     |
| Study treatment                       | Includes any drug or combination of drugs in any study arm administered  to the patient (subject) as part of the required study procedures, including  placebo and active drug run-ins. In specific examples, it is important to judge investigational treatment  component relationship relative to a study treatment combination; study  treatment in this case refers to the investigational and non-investigational  treatments in combination. |    |         |                 |     |                |     |
| Study                                 | Point/time when patient permanently stops taking study treatment for any  reason; may or may not also be the point/time of premature patient  withdrawal                                                                                                                                                                                                                                                                                            |    |         |                 |     |                |     |
| treatment  discontinuation Supportive | Refers to any treatment required by the exposure to a study treatment, e.g.                                                                                                                                                                                                                                                                                                                                                                         |    |         |                 |     |                |     |
| treatment                             | premedication                                                                                                                                                                                                                                                                                                                                                                                                                                       | of | vitamin | supplementation | and | corticosteroid | for |
| pemetrexed disodium.                  |                                                                                                                                                                                                                                                                                                                                                                                                                                                     |    |         |                 |     |                |     |
| Variable                              | Identifier used in the data analysis; derived directly or indirectly from data  collected using specified assessments at specified time points                                                                                                                                                                                                                                                                                                      |    |         |                 |     |                |     |
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1. objectives and endpoints 1.1

2.1.1 Rationale for improving outcomes with neoadjuvant treatment 2.1.2 Rationale for incorporating everolimus in the neoadjuvant setting 2.1.3 Background: Clinical studies with everolimus in HR+ breast cancer 2.1.4 Rationale for incorporating antiangiogenic therapy in the neoadjuvant setting 2.1.5 Background: Clinical studies with antiangiogenic agents in HR+ breast cancer 2.1.6 Rationale for targeting endoglin in cancer therapeutics 2.1.7 Clinical studies with TRC105 2.1.8 Synergism between TRC105 and everolimus 2.1.9 Duration of neoadjuvant treatment 2.1.10 Rationale for ovarian suppression with gonadotropin-releasing hormone analogs in premenopausal women 2.2 Correlative Studies Background 2.2.1 PI3K/AKT/mTOR pathway in breast cancer 2.2.2 Regulation of gene expression at the translational level
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5. treatment plan 3. patient

3.1 Study Population 3.2 Eligibility Criteria 3.3 Exclusion Criteria 3.4 Inclusion of Women and Minorities 3.5 Subjects Who Fail to Meet the Eligibility Criteria.
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4. study design

4.1 Phase I, dose escalation 4.2 Phase II, expansion phase 4.3 Study accrual and treatment considerations 4.4 Rationale for the Study Doses and Design 4.5 Rationale for the Preoperative Setting 4.6 Rationale for the Drug Combination 4.7 Rationale for Tumor Assessments by Both Physical Examination and Imaging 4.8 Rationale for the duration of preoperative treatment  
5.1 Subject Selection and Registration  
 5.2 Study Medications 5.2.1 Letrozole 5.2.2 Everolimus 5.2.3 TRC105 5.2.4 Goserelin 5.2.5 Concomitant Medications 5.3 Dose Modifications and Delays  
5.3.1 Dose modifications and delays with letrozole.  
5.3.2 Dose modifications and delays with everolimus 5.3.3 Dose modifications and delays with TRC105 5.3.4 Dose modifications and delays with goserelin 5.4 Study Procedures 5.4.1 Screening phase 5.4.2 Treatment Phase 5.4.3 Safety Follow-up Phase 5.5 Study Termination 5.6 Subject Discontinuation
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6. study drug preparation and

6.2 Drug Formulations 6.2.1 Letrozole Formulation 6.2.2 Everolimus Formulation 6.2.3 TRC105 Formulation 6.2.4 Goserelin Formulation 6.3 Study Drug Storage and Handling 6.4 Study Drug Compliance 6.5 Study Drug Accountability
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7. visit schedule and assessments

7.1 Study Flow and Visit Schedule 7.2 Measurement of Effect 7.3 Pharmacokinetic and Pharmacodynamic Studies and Research Biopsy  
8. **ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS**  
8.1 Adverse Events 8.2 Adverse Event Characteristics 8.3 Expedited Adverse Event Reporting 8.4 Routine Adverse Event Reporting  
8.5 Serious Adverse Events  
8.6 Adverse reactions with everolimus 8.7 Adverse reactions with TRC105
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9. data collection and study

9.3 Study Management  
  
10. **STATISTICAL CONSIDERATIONS**
10.1 Sample Size and Power Justification 10.2 Statistical Analysis 10.3 Stratification Factors 11. **PROTOCOL ADHERENCE**
11.1 Amendments to the Protocol
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references list of figures

| Figure A   | Genomic aberrations along the PI3K/AKT/mTOR pathway                            |
|------------|--------------------------------------------------------------------------------|
| Figure B   | Results of the TBCRC002 study                                                  |
| Figure C   | Interactions of endoglin with TGF-βR.                                          |
| Figure D   | Study design.                                                                  |
| Figure E   | Chemical structure of Everolimus                                               |
| Figure F   | Study Schema                                                                   |
| Figure G   | Timeline of the pharmacokinetic / pharmacodynamics studies and research biopsy |
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list of tables

| Table 4-1   | Cohorts and dose escalations of the phase I part of the study                                                                           |                                                           |
|-------------|-----------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|
| Table 4-2   | Dose escalation rules                                                                                                                   |                                                           |
| Table 5-1   | Everolimus - Drug substance                                                                                                             |                                                           |
| Table 5-2   | Dosing guidelines for Everolimus-related non-hematologic toxicities                                                                     |                                                           |
| Table 5-3   | Dosing guidelines for Everolimus-related hematologic toxicities                                                                         |                                                           |
| Table 5-4   | Management of oral mucositis associated with everolimus.                                                                                |                                                           |
| Table 5-5   | Management of hyperlipidemia and hypertriglycemia associated with everolimus.                                                           |                                                           |
| Table 5-6   | Recommendations for positive baseline results for hepatitis B.                                                                          |                                                           |
| Table 5-7   | Guidelines for management of hepatitis B.                                                                                               |                                                           |
| Table 5-8   | Definitions and Guidelines for the management of Hepatitis C.                                                                           |                                                           |
| Table 5-9   | Guidelines for the management of treatment-related non-infectious pneumonitis                                                           |                                                           |
| Table 5-10  | Guidelines for the management of infusion related reactions.                                                                            |                                                           |
|             | Table 5-11                                                                                                                              | Drug interactions: inducers/inhibitors of isoenzyme CYP3A |
| Table 5-12  | Drug interactions: inducers/inhibitors of P-gp and P-gp/CYP3A dual inhibitors                                                           |                                                           |
| Table 6-1   | Dosing regimen and schedule                                                                                                             |                                                           |
| Table 7-1   | Study Calendar                                                                                                                          |                                                           |
| Table 8-1   | Adverse Drug Reactions associated with everolimus                                                                                       |                                                           |
| Table 8-2   | Fatal AE reported by investigators across all RAD001 studies as having a suspected  causal relationship to everolimus as of 31-Mar-2014 |                                                           |
| Table 8-3   | Serious, unexpected, life-threatening AE considered related to study drug, and  reported on at least one occasion as of 31-Mar-2014.    |                                                           |
| Table 10.1  | Probability of early termination and observing one response or at least one response  at the first stage (N1=10)                        |                                                           |
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appendices

APPENDIX A
Performance Status Criteria APPENDIX B
Drugs with potential interactions with everolimus APPENDIX C
American College of Radiology Practice Guidelines for Imaging Studies of the Breast
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appendix d

National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 4.03
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appendix e

FDA MedWatch 3500A Form APPENDIX F
SOP for Investigator INDs Held by Physician Members of the UAB CCC
APPENDIX G Blood and Biopsy Procedures
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1. objectives and endpoints

1.1 Objectives 1.1.1 Primary Objective  
Determine the tolerability and feasibility of combining everolimus and TRC105 with letrozole administered for 24 weeks in the preoperative (neoadjuvant) setting in postmenopausal women with newly diagnosed potentially resectable local or locally advanced (T2-T4a-c, N0-2, M0; excluding patients with T4d or inflammatory breast cancer) estrogen and/or progesterone receptor positive, Her2/neu negative breast cancer.
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1.1.2. secondary objectives

a. Determine the efficacy of the combination of letrozole, everolimus, and TRC105 in postmenopausal women with newly diagnosed potentially resectable local or locally advanced estrogen and/or progesterone receptor positive, Her2/neu negative breast cancer. b. Determine the pharmacodynamic parameters of everolimus and TRC105 when used in combination with letrozole in the same patient population. c. Determine the pharmacokinetic parameters of everolimus and TRC105 when used in combination with letrozole in the same patient population.
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1.1.3 exploratory objective

Evaluate specific biomarkers of potential prognostic value and biomarkers potentially predictive of response or resistance to the combination of everolimus and TRC105 with letrozole.
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1.2 endpoints 1.2.1 primary endpoint

Determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D) and rates of adverse events associated with the combination of letrozole with everolimus and TRC105.
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1.2.2. secondary endpoints

a. Determine the rates of complete pathologic remission and downstaging achieved with the investigational treatment at the time of surgery. b. Determine the changes in tumor cell proliferation by means of changes in Ki67 expression and changes in serum concentration of markers of angiogenesis (VEGF-A, soluble VEGFR2, activin A
and activin B, angiopoietin-1 and -2, TGF-β, soluble endoglin) before and after the investigational treatment and correlate with clinical, radiographic, and pathologic response.  
c. Determine the Cmax, Tmax, AUC, T½, clearance of everolimus and TRC105 when used in combination with letrozole.
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1.2.3 exploratory endpoint

a. Correlate the levels of PTEN immunostaining with response. b. Correlate levels of phosphorylated AKT (pAKT) immunostaining with response. c. Correlate expression of endothelial (CD105 and CD31/CD34) and pericyte (NG2, and MCAM [CD146]) markers in the initial diagnostic and final surgical specimens with response. d. Determine translational (mRNA to protein) profiles by means of next-generation whole exome sequencing and sequencing of ribosome-protected mRNA associated with resistance to the investigational combination.
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2. background 2.1 rationale and

The achievement of a pathologic complete remission (pCR) with neoadjuvant (preoperative) therapy in early-stage breast cancer has an extremely favorable impact on long-term outcomes and is an excellent prognostic surrogate marker9-13,55,56. The Food and Drug Administration (FDA) has adopted the achievement of a pCR as an endpoint in neoadjuvant trials leading to accelerated approval of novel agents in breast cancer13,57. Responses to neoadjuvant therapy however, depend on the intrinsic molecular subtype14 with the immunophenotypically luminal (hormone-receptor positive and Her2 negative) breast cancers rarely achieving a pCR12. Nonetheless, in this luminal subgroup, downstaging to at least pathologic stage 1 is equally important as it has been associated with a low risk of recurrence15. In a preoperative study that compared 4 months of hormonal treatment with letrozole or tamoxifen (P024) in stage 2 or 3 hormone receptor positive breast cancer, women whose disease shrunk to stage 1 or 0 (n=30) had a favorable long term outcome (no relapse with a median follow up of 61.2 months, one death due to unknown causes) as compared to the 30% relapse incidence among women whose disease was not preoperatively downstaged (n=175)15,19,21. An approach that intends to enhance or deepen the responses in luminal breast cancer is also supported by 2 large studies with response guided neoadjuvant chemotherapy58-60. After 2 cycles of neoadjuvant chemotherapy with docetaxel at 75 mg/m 2, doxorubicin at 50 mg/m2, and cyclophosphamide at 500 mg/m2(TAC), in women whose disease responded, the treatment was extended to a total of 8 cycles (versus standard 6 cycles)59 or alternatively, in women whose disease did not respond, the treatment was changed to a non-cross resistant regimen (capecitabine and vinorelbine versus continuation of TAC)60. In subsequent exploratory analyses, both disease-free and overall survival were better with a response guided approach58 despite the fact that the rates of pCR were not significantly improved. Moreover, at least in Luminal A breast cancers, the achievement of a pCR per se was not associated improved 21  
outcomes58. **Taken together, although the achievement of pCR is not uniformly associated with**
improved clinical outcomes, a preoperative strategy that intends to enhance the responses in luminal breast cancer holds particular promise in yielding superior long term clinical outcomes. In the neoadjuvant setting for hormone receptor positive/Her2 negative breast cancer, chemotherapy and hormonal therapy have been shown to yield similar benefits in postmenopausal women16,17.  
While in one study the clinical response rates were higher (but not statistically significant) with neoadjuvant chemotherapy as opposed to hormonal therapy, by subgroup analysis, there was no difference in the efficacy of either intervention in postmenopausal women (57% vs. 52% clinical response rate with neoadjuvant chemotherapy vs. hormonal therapy, respectively)16. Of note, the pCR rate was low with either intervention (1/47 vs. 0/48 achieved a pCR with neoadjuvant chemotherapy vs. hormonal therapy, respectively)16. In another study that compared the efficacy of neoadjuvant chemotherapy vs. hormonal therapy specifically in postmenopausal women, the outcomes were highly similar: rates of complete and partial clinical response were 62%, 67%, and 63% for anastrozole, exemestane and chemotherapy, respectively17. In fact, neoadjuvant hormonal therapy was found to be associated with an increase in the proportion of patients suitable for breast conserving surgery17. Along these lines, hormonal therapy is generally preferred as it is associated with fewer adverse events and is overall better tolerated in this subgroup of patients who frequently have other comorbidities18.  
In the neoadjuvant setting, aromatase inhibitors have been consistently shown to be superior over tamoxifen19-23, making the former the agents of choice in postmenopausal women with hormonereceptor positive breast cancer. Despite the high rates of clinical and radiographic response achieved with hormonal therapy alone19-23, the rates of downstaging are still low (approximately 15%15).  
Similarly, the rates of pCR are very low (less than 5%) 4,15,16. **Collectively, there is a need to**
improve the efficacy of aromatase inhibitors in the neoadjuvant setting with minimal additional toxicities as it directly impacts clinical outcomes.
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2.1.2 rationale for incorporating everolimus

The phosphatidyl inositol-3 kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway is an intracellular signal transduction pathway downstream of receptor tyrosine kinases (**figure A**36). Class IA PI3K molecules are heterodimers composed of a regulatory subunit
(p85) and a catalytic subunit (p110), the α isoform of which (encoded by *PIK3CA*) is widely mutated or amplified in human cancers37,38, including breast cancer39,40. Comprehensive whole-genome sequencing data indicate that the frequency of *PIK3CA* mutations in hormone receptor positive breast cancer is 41.3%36 (35.8% in a randomized clinical trial with everolimus where *PIK3CA* mutations were systematically evaluated4). Deletions were identified also in *PTEN* (7%), a negative regulator of the PI3K/AKT/mTOR pathway, as well as copy number aberrations and mutations in *AKT* (52%
and 2%, respectively)36. Comprehensive genomic analyses of breast cancer samples in the context of The Cancer Genome Atlas Project also highlighted the high frequency of mutations along the PI3K/AKT/mTOR pathway41. In particular, activating mutations in the *PIK3CA,* inactivating mutations or loss of *PTEN,* and loss of *INPP4B* (a negative regulator of the PI3K/AKT pathway) were detected in 49%, 13%, and 9% (luminal A) and 32%, 24%, and 16% (luminal B), respectively. At the preclinical level, breast cancer cells with upregulated PI3K/AKT/mTOR pathway were shown 22  
to be resistant to hormonal therapy and this resistance was restored by everolimus and other mTOR  
  
inhibitors42,43. The mechanistic connection between hormone resistance and PI3K/AKT/mTOR
pathway activation relies on the ligand-independent activation of the estrogen receptor (ER) by downstream substrates of the mTOR complex5,6. **Taken together, the PI3K/AKT/mTOR pathway**
can be activated by various genetic events in hormone receptor positive **breast cancer and its** inhibition with the FDA-approved agent everolimus can restore or enhance sensitivity to endocrine therapies. Moreover, inhibiting mTOR, a downstream signaling node where multiple pathways converge as opposed to blocking upstream mediators or individual surface receptors, has the potential to abrogate pleiotropic primary and escape tumor-promoting signaling cascades that mediate resistance to endocrine therapies.
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2.1.3 background clinical studies with

The elucidation of the mechanisms of resistance to endocrine therapies61 set the stage for many successful clinical trials where responses to endocrine therapies and aromatase inhibitors in particular were enhanced4or restored3 with the addition of everolimus. **Everolimus has been the prototypic**
agent which, in combination with aromatase inhibitors led to enhancement or restoration of responses to endocrine therapies in luminal breast cancer. Furthermore, its success in luminal breast cancer demonstrated that the strategy with the highest potential to successfully address the limitations of preoperative endocrine therapy is with rational combinations of novel agents capitalizing on the knowledge garnered in the preclinical arena. An upfront dual ER and mTOR co-targeting approach to enhance the responses of hormonal therapy  
and/or circumvent *de novo* endocrine resistance62 is supported by a randomized placebo-controlled neoadjuvant clinical trial in postmenopausal women with ER-positive breast cancer, whereby the addition of everolimus to letrozole led to an increase in the clinical response rate (68.1% vs 59.1%)4.  
Although the benefit was greater in patients harboring *PIK3CA* mutations (especially in exon 9),
patients with wild type *PIK3CA* still benefitted from the combination4. This observation suggests that everolimus inhibits alternative operational mechanisms of hormone resistance either within or outside the PI3K/AKT pathway, consistent with mTOR lying at a downstream signaling node where multiple pathways converge4. Two clinical trials evaluated the addition of everolimus to exemestane3and tamoxifen63 in restoring resistance to hormone therapy in hormone-receptor positive, Her2 negative advanced or metastatic breast cancer. In both studies the clinical outcomes were significantly improved with the addition of everolimus. In the former study3(which led to the approval of everolimus in postmenopausal women with advanced hormone receptor-positive, Her2-negative breast cancer in combination with exemestane after failure of treatment with letrozole or anastrozole), the addition of everolimus to exemestane led to a 6.5 month incremental improvement in the progression free survival (10.6 months in the combination arm vs 4.1 months in the exemestane alone arm)3. There was however an increased incidence of adverse events in the combination arm with stomatitis (8% in the combination arm vs. 1% in the exemestane alone arm), anemia (6% vs <1%), dyspnea (4% vs. 1%), hyperglycemia (4% vs. <1%), fatigue (4% vs. 1%), and pneumonitis (3% vs. 0%) constituting the most frequent adverse events associated with the addition of everolimus3.  
Taken **together, these clinical studies support the addition of everolimus to letrozole in** enhancing the responses of hormonal therapy and/or circumventing *de novo* **or acquired** resistance to endocrine therapies.
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2.1.4 rationale for incorporating anti-angiogenic

Angiogenesis is an integral step in tumor progression24. Specifically in hormone-receptor positive breast cancer, enhanced angiogenesis accelerated tumor growth25, and overexpression of the Vascular Endothelial Growth Factor (VEGF) either by the tumor26,64 or stromal cells27 was associated with hormone-independent growth and resistance to endocrine therapies. An interplay between estrogens and angiogenic activity exists65,66 and *VEGF* is one of the genes whose expression is modulated by estrogens. Indeed, the presence of functional estrogen response elements in the *VEGF*
gene67-69 underpin the transcriptional activation of *VEGF* by estrogens70. In mouse models of luminal breast cancer, overexpression of VEGF by human MCF-7 breast cancers not only enhanced estrogen dependent tumor growth, but also enabled estrogen independent tumor formation *in vivo*26. The stimulation of MCF-7 tumor growth by VEGF is through both a paracrine effect on tumor angiogenesis leading to neovascularization and an autocrine effect on tumor cell proliferation26.  
Tumor-released VEGF recruits stromal cells and promotes a desmoplastic microenvironment; stromal cells in turn, provide mitogenic and angiogenic growth factors stimulating both tumor and stromal cell growth64. Beside the breast cancer cells themselves, VEGF may be secreted by the activated stroma. In mouse models of xenografted ER-positive tumors, VEGF secreted by the stroma and acting cooperatively with other factors can substitute for estrogens and foster hormoneindependent growth of luminal tumors27. At the clinical level, in hormone-receptor positive breast cancer, elevated intratumoral levels of VEGF have been associated with suboptimal responses to 24  
hormonal therapies and poorer clinical outcomes71-73 lending support to the hypothesis that VEGF  
  
and angiogenesis lie in the core of intrinsic resistance to endocrine therapies. Our studies in the UAB Breast Cancer Spore using the MCF-7 tamoxifen murine xenografts model by Dr. Fran Kern and collaborators indicate that paracrine effects of VEGF over-expression by ER+
breast cancer cells can reverse the effectiveness of tamoxifen as a cytostatic or cytotoxic agent64. In this MCF-7 model, they demonstrated that increased expression of VEGF causes an acquired Tamoxifen resistance and tumor loss of estrogen dependence *in vivo*. MCF-7 cells with VEGF coexpression (stably transfected or regulated expression) had *in vitro* tumor cell proliferation and estrogen dependence identical to non-transfected MCF-7, while MCF-7/VEGF cells *in vivo* displayed increased tumor growth rates, higher rates of metastases, and estrogen independence.  
These survival and proliferation signals may be an indirect effect of VEGF presumably reflecting the enhanced vascularity of tumors with secondary growth factor and signal pathway modulation. Reversal of VEGF over-expression *in vivo* returned tumors to estrogen dependent growth. Dr. Zhican Qu, Ph.D., a recipient of a career development award of the UAB-Breast SPORE in our group, conducted preclinical efficacy studies of tamoxifen and bevacizumab in combination for the treatment of breast cancer using a novel mouse model. In her previous studies she showed that elevated levels of VEGF increases tumor growth, and promotes metastasis and tamoxifen resistance; thus, she hypothesized that a combination of agents targeting both ER and VEGF signaling may be a more effective treatment than the use of either drug alone. She tested this hypothesis using a combination of tamoxifen and bevacizumab in a xenograft nude mouse model with regulated-VEGF expression. The efficacy of the treatment was tested in two groups of mice, one with early and one with late stage ER-positive breast cancer. All mice received orthotopic implantation of 107 tumor cells at mammary fat pat and a 17β-estradiol pellet supplement on Day 1. Doxycycline was given to all mice to induce expression and secretion of VEGF from tumor cells. Animals in the early stage group started therapy (tamoxifen or bevacizumab or placebo or tamoxifen-bevacizumab in combination) on Day 7 when the tumor size reached about 100 mm3. The bevacizumab-tamoxifen combined treatment demonstrated better control of primary tumor growth than therapy with tamoxifen or bevacizumab alone. The survival rate was superior in the group of mice treated with combination therapy compared with the groups treated with single agents or placebo. Animals in the late stage group started therapy on Day 32 when the tumor size reached about 1000 mm3. The combined treatment showed again a better reduction of tumor growth rate and a significant better survival. 83% of mice survived in the combined treatment group, while only about 50% of mice survived in the monotherapy groups. These results demonstrated that the Avastin-tamoxifen combination is more effective on inhibiting tumor growth of ER positive breast cancer and increasing survival rate than either drug treatment alone (2005 AACR Annual Meeting, abstract \#1091).
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2.1.5 background clinical studies with

These preclinical studies that support the role of angiogenesis in hormone-independent growth of luminal breast cancer set the stage for a pilot, single-institution (UAB) study of preoperative letrozole in combination with bevacizumab (a monoclonal antibody targeting VEGF, the prototypic proangiogenic factor and a crucial regulator of both normal and pathologic angiogenesis74) in postmenopausal women with hormone-receptor positive breast cancer2. In our study (n=25),
preoperative letrozole and bevacizumab for 24 weeks resulted in a pCR rate of 12% (n=3), while the overall objective response rate was 68% (n=17). Additionally, 4% (n=1) achieved pCR in the breast but not in the lymph nodes and the rate of stage 0/1 attainment was 32%2. The treatment was overall well tolerated with a rate of discontinuation due to adverse events of 8% (n=2). With a median followup of 55 months and 2 responders lost to follow up, none of the other 15 patients with objective response have relapsed. So far, 2 patients experienced a recurrence despite postoperative chemotherapy and hormonal therapy; these patients had achieved stable (SD) and progressive (PD) disease, respectively as their best response while on the investigational preoperative regimen. Our results are consistent with the concept that downstaging to at least pathologic stage 1 is equally important as achievement of a pCR and it has been associated with an extremely low risk of recurrence15.  
We conducted a separate, independent randomized open-label phase II study was conducted by the Translational Breast Cancer Research Consortium (TBCRC) comparing preoperative letrozole with or without bevacizumab in postmenopausal women with operable hormone receptor positive breast cancer1(trial TBCRC002). The study showed that bevacizumab "deepened" the responses obtained with letrozole alone (**figure B**). Downstaging from stage 2/3 to 0/1 was achieved in 45% of patients in the letrozole-bevacizumab arm as compared to 25% in the letrozole-alone group. Remarkably, the combination induced a pCR in 11.1% of patients1, comparable to the 12% pCR rate reported in a prior pilot single-institution study2; no complete or near-complete remissions were observed in the letrozole-alone group. Of note, the addition of everolimus to letrozole has not led to such a high pCR rate: of the 246 patients who received letrozole +/- everolimus preoperatively for 4 months with available surgical excision specimens, only 3 achieved a pCR (2 in the combination arm versus 1 in the letrozole alone arm)4.  
  
Our study sets a successful clinical precedence of rationally combining an antiangiogenic agent with an aromatase inhibitor in postmenopausal women with hormone receptor-positive and Her2 negative **breast cancer; this combination not only enhanced the response rate but even** led to an unprecedented pCR rate in an intrinsic subgroup where pCRs are rarely seen.
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2.1.6 rationale for targeting endoglin

Endoglin (CD105) is essential for normal vascular development75, and heterozygous expression of endoglin is associated with hereditary hemorrhagic telangiectasia type 1 (HHT-1,Rendu–Osler–
Webber syndrome), a human disease characterized by ectatic blood vessel formation76. In adults, endoglin is a marker of activated angiogenesis that is predominantly or exclusively expressed in de novo tumor vessels32,33, as opposed to the minimal expression in the normal quiescent endothelium which has a very low turnover28. Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-β) receptor (TGF-βR) and diverts TGF-β downstream signaling toward proliferation and migration as well as transcription of proangiogenic genes including endoglin itself29-31. As TGF-β signaling can mediate diverse and opposing processes in endothelial cells, endoglin inhibits TGF-β-mediated endothelial quiescence (**figure C** 77,78). Indeed, in the absence of endoglin, TGF-β mediated endothelial cell quiescence is significantly enhanced28,79. The clinical implications of this observation is that targeting endoglin will selectively inhibit tumor angiogenesis and at levels **below baseline**.  
  
In line with the premise that enhanced tumor vasculature correlates with adverse prognosis and the fact that endoglin is a marker of activated angiogenic endothelium, endoglin expression has been consistently associated with adverse clinical outcomes across multiple malignancies31. VEGF inhibition leads to reactive upregulation of endoglin7and large established CD105(+) vessels lying in the core of the tumors have been shown to be insensitive to VEGF inhibitors8. In those studies, CD105 expression was increased following inhibition of the VEGF pathway. CD105 expression increased more than two-fold in human pancreatic cancers grown in mice treated with an antibody that binds VEGF7. Similarly, treatment of human bladder cancers grown in mice with an antibody that blocks activation of the VEGF receptor increased CD105 expression within the core tumor vasculature8. Finally, a study of spontaneously occurring neuroendocrine tumors in RIP-Tag2 mice treated with antibody to VEGF indicated that TGF- β was the most highly upregulated factor, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)47. Taken together, these studies indicate that the **CD105 pathway is a significant mechanism of escape from**
VEGF inhibitor therapy.  
Endoglin is extensively expressed in breast cancer80 and increased endoglin expression at baseline has been associated with poorer responses to neoadjuvant treatment81. In preclinical murine xenograft models of HR(+) breast cancer, anti-endoglin monoclonal antibodies inhibited selectively tumor angiogenesis and delayed the growth of established tumors, while sparing normal vasculature34. Of note, in these preclinical "proof-of-concept" studies, the antitumor efficacy of the monoclonal antibody may have been obscured by the fact that almost 60% of the malignant vasculature was murine to which the human investigational antibody had little affinity34. **Collectively, endoglin is a**
rational target of tumor angiogenesis as it is exclusively expressed in the activated endothelium and plays a central pathophysiologic role in promoting angiogenesis. Endoglin is cleaved near the plasma membrane by membrane-anchored matrix metalloproteinase  
(MMP)-14 to release a soluble form of endoglin (sEng) into the circulation82. Although membranebound endoglin promotes angiogenesis, sEng antagonizes this process82 through multiple mechanisms including cell surface receptor downregulation and ligand sequestration83,84. The mechanism by which the novel monoclonal antibody intended to be used in the proposed study (TRC105) exerts its antiangiogenic effect is not clear in its details; however one of the proposed mechanisms involves enhanced endoglin shedding by stabilizing preformed endoglin/MMP-14 complexes83. Indeed, results from the phase I trial reveal that there is a dramatic dose-dependent increase in sEng levels in patients treated with TRC10583. Enhanced shedding not only reduces the overall cell surface level of endoglin, but also the resulting product (sEng) exerts an antiangiogenic effect by itself83.
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2.1.7 clinical studies with trc105

TRC105 was administered to 50 patients with solid tumors in the context of a phase 1 first-in-human dose escalation study following a 3+3 design35. The maximum tolerated dose was determined at 10 mg/kg weekly; 15 mg/kg every two weeks was equally well tolerated and also resulted in continuous serum concentrations known to saturate CD105 binding sites on proliferating endothelium. Hypoproliferative anemia was the dose-limiting toxicity: above the maximum tolerated dose, at 15 mg/kg weekly, all 3 patients that remained on study through cycle 2 developed dose-limiting grade 3 anemia, and one of the three progressed to grade 4 anemia by cycle 3. Infusion reactions, including some that were serious, were observed following TRC105 administration, usually with the initial dose. Other adverse events most commonly observed with TRC105, either alone or in combination, include fatigue, mucocutaneous telangiectasia resulting in epistaxis and gingival bleeding, flushing, and headache. Classic toxicities associated with VEGF inhibition, including hypertension, proteinuria, and thrombosis, have been seen in isolation. In fact, 3 clinical trials combining TRC105 with bevacizumab are currently ongoing (NCT01564914, NCT01648348, and NCT01332721). Multiple other phase Ib and phase II studies of TRC105 in combination with chemotherapy, with VEGF inhibitors, and as a single agent in patients with advanced prostate, sarcoma, renal cell carcinoma, choriocarcinoma, glioblastoma multiforme, and hepatocellular cancer are currently ongoing35,85.  
Collectively, **TRC105 allows for the rational therapeutic targeting of malignant angiogenesis** while sparing the cardiovascular toxicities of most contemporaneous antiangiogenic agents to which **postmenopausal women with preexistent conditions may be particularly predisposed.** Although the completed clinical trials with TRC105 were not designed to assess efficacy, several patients benefitted from the investigational drug. In the phase I study where TRC105 was administered as a single agent in patients with advanced metastatic malignancies and having received a median of 4 prior lines of therapy, 6/44 patients remained progression-free for longer than 4 months35.
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2.1.8 synergism between trc105 and

In animal models, mTOR inhibition was shown to reduce the levels of VEGF and downregulate significantly the response of endothelial cells to stimulation by VEGF45. mTOR signaling was also shown to play a key role in hypoxia-triggered smooth muscle and endothelial cell proliferation and 29  
angiogenesis in vitro; this response (triggered by growth factors other than VEGF) was abrogated with mTOR inhibition46. On the other hand, VEGF inhibition leads to reactive upregulation of endoglin7and large established CD105(+) vessels lying in the core of the tumors have been shown to be insensitive to VEGF inhibitors8. Other preclinical studies have also demonstrated that endoglin/CD105 represents a principal escape pathway from VEGF inhibition7,47. **Collectively,**
mTOR inhibition with anti-CD105 targeted therapy is anticipated to represent a more inclusive antiangiogenic approach as opposed to anti-VEGF therapy alone as it inhibits signaling from alternative proangiogenic factors and targets malignant angiogenesis with inherent (preexistent CD105 expression) or acquired (reactive CD105 upregulation) resistance to VEGF inhibition.
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2.1.9 duration of neoadjuvant treatment

Most studies that prospectively evaluated preoperative hormonal therapy have limited the duration of treatment to 3-4 months14,15,19-21,23. Several studies however, suggest that extending neoadjuvant hormonal therapy beyond 3-4 months is safe and associated with further and sustained reductions in the tumor size leading to a higher proportion of breast conserving surgeries14,86,87. Indeed, in a prospective phase II clinical trial with preoperative letrozole treatment to maximum response, the mean time to first response was 3.9 months while the median time to maximum response was 4.2 months87. It is noteworthy that approximately one third of the responders achieved maximum response beyond 6 months; however, very few responders were identified after 6 months and none from 8 to 12 months. The overall clinical response rate with preoperative letrozole was 76.8%87.  
Similarly, in a retrospective study of 182 patients treated preoperatively with letrozole, the rate of clinical complete or partial response rose from 69.8% at 3 months to 83.5% by extending letrozole treatment up to 24 months86. Moreover, continuing letrozole beyond 3 months increased the number of women who initially required mastectomy or had locally advanced breast cancer and subsequently became suitable for breast conserving surgery from 60% at 3 months to 72%86.  
Taken together, neoadjuvant endocrine therapy requires longer time to achieve responses as compared to cytotoxic chemotherapy. The majority of responders can be identified within the first 4 months of treatment; however, some patients require longer durations of treatment to achieve their maximum response14. In luminal breast cancer, such sustained and increased reductions in the tumor size are associated with better long-term clinical outcomes.
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2.1.10 rationale for ovarian suppression

The IMPACT trial (Immediate Preoperative Anastrozole, Tamoxifen or Combined with Tamoxifen) established the superiority of aromatase inhibition over tamoxifen or tamoxifen + aromatase inhibition in the preoperative setting. Although the overall clinical response rate was not significantly different between the 3 arms, more patients classified as requiring mastectomy were eligible for breast conserving surgery on the anastrozole treatment arm48 . In addition, suppression of the proliferation marker Ki67 (which in itself has been shown to be predictive of favorable outcome) was statistically greater with anastrozole as compared with tamoxifen or the combination 49. Aromarase inhibitors in premenopausal women are contraindicated as they may increase gonadotropin secretion because of the reduced feedback of estrogen to the hypothalamus and 30  
pituitary50. To that end, in order for premenopausal women to benefit from aromatase inhibition, they should become postmenopausal through ovarian suppression with gonadotropin-releasing hormone analogs. Furthermore, ovarian suppression in combination with aromatase inhibition has been shown to be superior over tamoxifen in the adjuvant setting in premenopausal women 88 For this clinical trial goserelin subcutaneously q 4 weeks will be used to suppress ovarian function.
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2.2 correlative studies background 2.2.1

As outlined in section 2.1.2 and 2.1.3, the PI3K/AKT/mTOR pathway can be activated by various genetic aberrations at multiple nodes along the pathway leading to resistance to or evasion from hormone blockade. Loss of PTEN increases PI3K activity and increased phosphorylation, activation, or membrane translocation of signal transducers downstream of the PI3K including Akt, mTOR, S6 kinase, and 4-EBP-1 89. Immunohistochemistry for the phosphorylated AKT (pAKT) is a surrogate for activation of the PI3K/AKT/mTOR pathway irrespective of the underlying genetic aberration; activating point mutations of signal transducers along the pathway can be captured by whole-exome sequencing.
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2.2.2 regulation of gene expression

Over the recent years, significant progress has been made in understanding the mechanisms of transcriptional (DNA to RNA) regulation; however, little is known about the regulation of gene expression at the **translational (mRNA to protein)** level. Comprehensive analyses have shown that while mRNA and respective protein levels may correlate better than previously thought (still only at the level of R
2=0.41), the abundance of a protein in a cell is predominantly determined at the level of translation90,91. Moreover, changes in translation have been shown to precede and be considerably more dramatic than changes in transcription in response to growth factor signaling modulation92.  
While mass spectrometry allows us to investigate translation at the protein level, it has its limitations
(limitations in quantitation, incomplete and biased coverage93) and small changes in the expression of critical genes that cast a large shadow at multiple cellular levels may be missed. The advent of ribosomal profiling94-100 (deep sequencing of the ribosome protected mRNA fragments in parallel with mRNA-sequencing) harnesses the comprehensive and unbiased nature of massively parallel deep sequencing to provide a global genomic view of ribosome occupancy across the entire exome and is a good proxy of active protein production. There is a strong rationale in investigating ribosome occupancy across the exome as genes exert their functions by the protein products they encode. Ribosomal profiling provides "snapshots" of which mRNAs are being actively translated and how efficiently. It serves as a proxy of protein synthesis and currently, no other technology can capture which mRNAs are being actively translated as close as ribosomal profiling. Which mRNAs are actively translated and at what efficiency is a layer of complexity in gene expression which has not be captured in the currently completed comprehensive genomic analyses in breast cancer.
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3. patient selection 3.1 study

Patients with newly diagnosed, potentially resectable (stage 2 or 3), local or locally advanced estrogen and/or progesterone receptor positive and HER2/neu negative breast cancer will be enrolled in this study
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3.2 eligibility criteria

Patients must have baseline evaluations performed prior to the first dose of study drug and must meet all inclusion and exclusion criteria. Results of all baseline evaluations, which assure that all inclusion and exclusion criteria have been satisfied must be reviewed by the Principal Investigator or his/her designee prior to enrollment of that patient. In addition, the patient must be thoroughly informed about all aspects of the study, including the study visit schedule and required evaluations and all regulatory requirements for informed consent. The written informed consent must be obtained from the patient prior to enrollment. The following criteria apply to all patients enrolled onto the study unless otherwise specified. 3.2.1 Newly diagnosed, histologically confirmed breast cancer.  
3.2.2 hormone receptor positive and HER2 negative breast cancer 12,58; patients with Her2/neu positive tumors irrespective of their hormone receptor status will be excluded. At least 10% of tumor cell nuclei should be immunoreactive for hormone receptors (ER and/or PR) to be deemed eligible for the study101. Her2/neu negative is defined as102:
a. Immunohistochemistry (IHC) score of 0 (no staining is observed or membrane staining that is incomplete and is faint/barely perceptible and within ≤ 10% of tumor cells) OR
b. IHC score of 1 (incomplete membrane staining that is faint/barely perceptible and within <10%
of tumor cells) OR
c. Fluorescence *in situ* hybridization (FISH) HER2/CEP17 ratio of <1.8 or average HER2 gene copy number of <4 signals/nucleus for test systems without an internal control probe.  
d. Equivocal results for Her2/neu (defined as: IHC 2+ or FISH HER2/CEP17 ratio of 1.8-2.2 or average HER2 gene copy number 4-6 HER2 signals/nucleus for test systems without an internal control probe) should prompt reflex test (same specimen using an alternative method) or order a new test (new specimen if available, using IHC or FISH).  
3.2.3 Histological grade I, II or III according to the modified Bloom Richardson scale52.  
3.2.4 Stage at diagnosis T2 through T4a-c, N0 through N2, and M0. Patients with inflammatory breast cancer or metastatic disease at diagnosis will be excluded. Patients with multicentric, multifocal, and/or bilateral disease are allowed to participate so long as all tumors meet the histologic criteria of the study. 3.2.5 Determined menopausal status. Postmenopausal status is defined according to the National Comprehensive Cancer Network103 a. age >60 years old OR
b. prior bilateral oophorectomy regardless of age c. if patient <60 years old and amenorrheic for >12 months in the absence of ovarian suppression, FSH and estradiol have to be in the postmenopausal range women who do not meet these criteria will be determined as non-postmenopausal and will require goserelin subcutaneously q 4 weeks to achieve ovarian suppression.  
3.2.6 No prior treatment with therapeutic intent for breast cancer. 3.2.7 No life threatening parenchymal disease or rapidly progressing disease warranting cytotoxic chemotherapy. 3.2.8 Patients must have disease that can be measured and followed by mammogram and/or breast ultrasound (in special cases a dedicated breast MRI may be clinically indicated). The target lesion must not have been previously irradiated. 3.2.9 Resectable/Operable or potentially resectable/operable breast cancer as determined by the treating surgical oncologist. 3.2.10 ECOG performance status <2 (Karnofsky >60%, see **Appendix A**) 3.2.11 Life expectancy of greater than 6 months 3.2.12 Patients must have normal organ and marrow function as defined below:  
- leukocytes >3,000/mcL - absolute neutrophil count >1,500/mcL - hemoglobin >10 gr/dL - platelets >100,000/mcL  
- total bilirubin <2 X institutional ULN (except for known  
Gilbert's syndrome/familial non-hemolytic  
jaundice)  
- AST(SGOT)/ALT(SGPT) <2.5 X institutional upper limit of normal - creatinine Up to 1.5 UNL, or  
- creatinine clearance >40 mL/min/1.73 m2for patients with  
creatinine levels above institutional normal  
- INR < 2  
3.2.13 Fasting serum cholesterol ≤300 mg/dL OR ≤7.75 mmol/L AND fasting triglycerides ≤2.5x ULN. NOTE: In case one or both of these thresholds are exceeded, the patient can only be included after initiation of appropriate lipid lowering medication; 3.2.14 Age >19 years. Because no dosing or adverse event data are currently available on the use of TRC105 in patients <18 years of age85, children are excluded from this study.  
3.2.15 No bleeding from gastrointestinal ulcers or other sites of bleeding. 3.2.16 Ability to understand and willingness to sign a written informed consent document. 3.2.17 Signed informed consent obtained prior to any screening procedures.
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3.3 exclusion criteria

3.3.1 Patients currently receiving anticancer therapies or who have received anticancer therapies within 4 weeks of the start of the investigational therapy (including chemotherapy, radiation therapy, antibody based therapy).  
3.3.2 Her2/neu positive102 or estrogen and progesterone receptor negative101 breast cancer.  
Patients with triple-negative breast cancer are also excluded. 3.3.3 Metastatic breast cancer. 3.3.4 Inflammatory (T4d) breast cancer. 3.3.5 Patients may not be receiving any other investigational agents. If patients are currently part of or have participated in any clinical investigation with an investigational drug, the last administration of the investigational study should be at least 1 month prior to dosing. 3.3.6 Inoperable breast cancer even after neoadjuvant treatment as assessed by the treating surgical oncologist. 3.3.7 Undetermined or unknown menopausal status. 3.3.8 Disease that cannot be measured and/or accurately followed by mammogram and/or breast ultrasound and/or dedicated breast MRI. 3.3.9 Target lesion that has been previously irradiated. 3.3.10 History of allergic reactions, intolerance or hypersensitivity attributed to compounds of similar chemical or biologic composition to TRC105 and/or everolimus or other rapamycin analogs (e.g. sirolimus, temsirolimus). 3.3.11 Known impairment of gastrointestinal (GI) function or GI disease that may significantly alter the absorption of oral Everolimus 3.3.12 Patients must have normal organ and marrow function. Patients with any of the following abnormal parameters will be excluded:  
| -   | leukocytes                | <3,000/mcL   |
|-----|---------------------------|--------------|
| -   | absolute neutrophil count | <1,500/mcL   |
| -   | hemoglobin                | <10 gr/dL    |
| -   | platelets                 | <100,000/mcL |  
- total bilirubin >2 X institutional ULN (except for known  
  
Gilbert's syndrome/familial non-hemolytic jaundice)
- AST(SGOT)/ALT(SGPT) >2.5 X institutional upper limit of normal - creatinine > 1.5 UNL, or
- creatinine clearance <40 mL/min/1.73 m2for patients with creatinine levels above institutional normal.  
- INR ≥2 3.3.13 Uncontrolled diabetes mellitus as defined by HbA1c >8% despite adequate therapy. Patients with a known history of impaired fasting glucose or diabetes mellitus (DM) may be included, however blood glucose and antidiabetic treatment must be monitored closely throughout the trial and adjusted as necessary. 3.3.14 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements. Examples of uncontrolled medical conditions include:
a. Unstable angina pectoris, symptomatic congestive heart failure, myocardial infarction ≤ 6 months prior to start of Everolimus, serious uncontrolled cardiac arrhythmia, or any other clinically significant cardiac disease b. Symptomatic congestive heart failure of New York Heart Association Class III or IV c. Active (acute or chronic) or uncontrolled severe infection, liver disease such as cirrhosis, decompensated liver disease, and active and chronic hepatitis (i.e. quantifiable HBV-DNA and/or positive HbsAg, quantifiable HCV-RNA),
d. Known severely impaired lung function (spirometry and DLCO 50% or less of normal and O2 saturation 88% or less at rest on room air),
e. Active, bleeding diathesis.  
3.3.15 Chronic treatment with corticosteroids or other immunosuppressive agents. Topical or inhaled corticosteroids are allowed. 3.3.16 Patients who have received live attenuated vaccines within 1 week of start of Everolimus and during the study. Patient should also avoid close contact with others who have received live attenuated vaccines. Examples of live attenuated vaccines include intranasal influenza, measles, mumps, rubella, oral polio, BCG, yellow fever, varicella and TY21a typhoid vaccines 3.3.17 ECOG performance status >2 (Karnofsky <60%, see Appendix A) 3.3.18 Pregnancy. Women of child-bearing potential, defined as all women physiologically capable of becoming pregnant, (including female pediatric patients who are menarcheal or who become menarcheal during the treatment) must use highly effective contraception during the study and for 8 weeks after stopping treatment. Highly effective contraception is defined as either:  
- Total abstinence: When this is in line with the preferred and usual lifestyle of the subject.  
[Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and withdrawal are not acceptable methods of contraception]
- Female Sterilization: have had surgical bilateral oophorectomy (with or without hysterectomy),
total hysterectomy or tubal ligation at least six weeks before taking study treatment. In case of oophorectomy alone, only when the reproductive status of the woman has been confirmed by follow up hormone level assessment
- Male partner sterilization: The vasectomized male partner should be the sole partner for that subject with the appropriate post-vasectomy documentation of the absence of sperm in the ejaculate.  
- Use of oral, injected or implanted hormonal methods of contraception or placement of an intrauterine device (IUD) or intrauterine system (IUS) or other forms of hormonal contraception that have comparable efficacy (failure rate <1%), for example, hormone vaginal ring or transdermal hormone contraception. In case of use of oral contraception women should have been stable on the oral agent for a minimum of 3 months before taking everolimus
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3.3.19 known history of hiv

3.3.20 Need for concurrent treatment with medications that strongly interact with everolimus (CYP3A4 inducers or inhibitors, see **Appendix B**). 3.3.21 History of another malignancy within the last five years except non-melanoma skin cancer, carcinoma in–situ of uterine cervix, uteri, and breast from which the patient has been disease free for at least 3 years. Second primary breast cancers are allowed regardless of the number of years since they were first diagnosed. 3.3.22 Major surgical procedure, open biopsy, or significant traumatic injury within 28 days prior to Day 0, anticipation of need for major surgical procedure during the course of the study. As an example, port placement or core biopsies are not considered major surgical procedures.
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3.4 inclusion of women and

Both men and women and members of all races and ethnic groups are eligible for this trial. Minority patients are particularly encouraged to participate.
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3.5 subjects who fail to

A subject may fail to meet entrance criteria because of an abnormal laboratory value, vital sign(s), or concurrent medications with strong interactions with the investigational combination. The abnormal screening test(s) may be repeated as soon as the investigator anticipates that it will be within acceptable range to meet eligibility criteria, but must be completed within the 4-week screening phase. Rescreening can be performed only once and the subject is not required to sign a new informed consent form. If the rescreening test(s) cannot be performed within the 4-week screening phase or return abnormal not meeting the entrance criteria, or the medical condition of the subject deteriorates so that she no  
longer meets the eligibility criteria, the subject will be deemed a screen failure and withdrawn from the study.
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4. study design

The principal investigator will hold the IND for the trial (**Appendix F**). This is a single-institution openlabel phase I/II study of preoperative treatment with the combination of letrozole, everolimus, and TRC105 in postmenopausal women with newly diagnosed potentially resectable local or locally advanced (T2-T4a-c, N0-2, M0; excluding patients with T4d or inflammatory breast cancer) estrogen and/or progesterone receptor positive, Her2/neu negative breast cancer. Accrual will be halted as a result of any grade ≥3 hemorrhage, cerebrovascular and vasovagal adverse events; FDA will be contacted to discuss further accrual or potential need for protocol amendment. Premenopausal women at the time of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression. The study consists of 2 parts: a phase I and a phase II part (**figure D**).
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4.1 phase i dose escalation

The first part of the study will follow the conventional 3+3 dose escalation design. Up to 18 patients will be enrolled. During the first part (phase I, **dose escalation**), the Maximum Tolerated Dose
(MTD) of the combination in terms of safety will be determined. The dose of letrozole will be kept the same for all cohorts (2.5 mg PO daily) (**Table 4-1**). The dose of everolimus will be escalated from 5 mg PO daily (cohort 1) to 10 mg PO daily (cohort 2). The dose of everolimus will be deescalated to 5 mg PO daily (cohort 1) if the dose of 10 mg PO daily is not well tolerated. The dose of TRC105 will be 15 mg/kg q 2 weeks (cohorts 1 and 2). The dose of TRC105 will be de-escalated to 10 mg/kg q 2 weeks (cohort -1) if 15 mg/kg q 2 weeks is not well tolerated. All doses that will be used in this trial have shown to be safe and active in clinical trials. Premenopausal women at the time  
of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression.  
| Cohort   | TRC105 (mg/kg IV q 2 weeks)   | Everolimus (mg PO daily)   |
|----------|-------------------------------|----------------------------|
| 1        | 15                            | 5                          |
| 2        | 15                            | 10                         |
| -1       | 10                            | 5                          |  
Table 4-1. Cohorts and dose escalations of the phase I part of the study
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4.1.1 definition of a dose

a) Any grade 3 or 4 non-hematologic toxicity except anorexia, alopecia, nausea (which is not refractory to antiemetics), fatigue, fever without neutropenia; b) Failure to recover to baseline (except alopecia) after delaying the next dose by more than 14 days; c) Grade 3 or 4 neutropenia complicated by fever >38.5°C or infection, or grade 4 neutropenia of
≥7 days duration; or d) Grade 4 thrombocytopenia, or grade 3 thrombocytopenia complicated by hemorrhage.  
In addition, accrual will be halted as a result of any grade ≥3 hemorrhage, cerebrovascular and vasovagal adverse events; FDA will be contacted to discuss further accrual or potential need for protocol amendment.
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4.1.2 dose escalation rule

Dose escalation will follow the decision rules shown in table 4-2.  
Table 4-2. Dose escalation rules  
| Number of Patients with DLT at a  Given Dose Level   | Escalation Decision Rule                                                                                                                                                                                                                                                                                       |
|------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0 out of 3                                           | Enter 3 patients at the next dose level.                                                                                                                                                                                                                                                                       |
| 1 out of 3                                           | Enter 3 additional patients in the same dose level (for a  total of 6 patients at this dose level). If 0 of the 3 additional patients experience DLT, then  proceed to the next dose level. If 1 or more of these additional patients experience DLT  (i.e., ≥ 2 of 6 total patients), dose escalation will be |  
| stopped.           |                                    |
|--------------------|------------------------------------|
| >2 out 3           | Dose escalation will be stopped.   |
| If MTD not reached | Cohort 2 will be selected for RP2D |
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4.1.3 definition of maximum tolerated

The **maximum tolerated dose** (MTD) is defined as the highest dose at which 0 out of first 3 or 1 out of total of 6 patients experience DLT during the first cycle of therapy; this dose level will be recommended as RP2D in the second portion of the study. If accrual to cohort 2 (highest dose of both everolimus and TRC105) is completed without reaching the MTD, this dose will be selected as the RP2D. In every cohort, patients withdrawn without receiving study treatment will be replaced, as will patients withdrawn before completing the first cycle of treatment for any reason other than DLT.
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4.2 phase ii expansion phase

The second part of the study (phase II, **expansion phase**), will ensue once the MTD and RP2D have been determined. The objectives of the dose expansion phase is to estimate the rate of complete pathologic remission (pCR) or downstaging in addition to continue evaluation of the safety. In order to minimize exposure of patients to ineffective therapy, Gehan's two-stage design will be used51.  
The study will enroll 10 patients first, and if no patient has pCR or downstaging, the study will close on the basis that it is unlikely (p=0.1074) that 0/10 responses would occur if the true response rate were >20%. If at least one patient has pCR or downstaging, an additional 10 patients will be treated (total of 20). For patients enrolled in Phase II, dose of everolimus can be de-escalated down to 2.5mg if needed. No further de-escalation is allowed. If 2.5mg not tolerated then the patient will be removed from study.
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4.3 study accrual and treatment

It is anticipated that 26 - 38 patients will be enrolled in the study and accrual is expected to be completed in 24 months (see Section 10. **STATISTICAL CONSIDERATIONS**). Patients meeting the eligibility criteria and who have signed the consent form will start Letrozole 2.5 mg PO daily, Everolimus 5 or 10 mg PO daily, and TRC105 15 or 10 mg/kg IV every two weeks with the first infusion administered in 2 split doses on days 1 (3 mg/kg) and 4 (12 mg/kg or 7 mg/kg), respectively. Premenopausal women at the time of study enrollment will receive goserelin 3.6 mg subcutaneously q 4 weeks to achieve ovarian suppression. A cycle is defined as 4 weeks of treatment. Breast ultrasound, will be conducted upon completion of each cycle to complement clinical assessments of the size of the tumor(s). The duration of neoadjuvant therapy is 24 weeks or 6 cycles. No extensions of the neoadjuvant therapy beyond 6 months are allowed. After neoadjuvant therapy, patients will undergo surgical treatment and will receive adjuvant therapy (radiation therapy, chemotherapy, hormonal therapy) according to the standard of care or at the discretion of the treating physician. The accrual rate is projected to be approximately 1-2 patients per month. Accrual will be halted as a result of any grade ≥3 hemorrhage, cerebrovascular and vasovagal adverse events; FDA will be contacted to discuss further accrual or potential need for protocol amendment.
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4.4 rationale for the study

The safety and efficacy of letrozole and everolimus for 4 months in the preoperative setting have been well established4, allowing the use of these agents in combination at their approved therapeutic doses. Nonetheless, the addition of everolimus at 10 mg PO daily was associated with an increase in the adverse events of any grade4suggesting that extension of treatment to 6 months without dose reductions, delays, or interruptions may not be possible. To that end, a dose of everolimus at 5 mg PO daily is selected for cohort 1. The addition of an antiangiogenic agent with a non-overlapping pharmacokinetic and toxicity profile is anticipated to increase the proportion as well as the magnitude of responses achieved without a significant increment in the rate of adverse events. However, TRC105 has not been administered in combination with letrozole and everolimus before. To that end, the phase I portion of the study is designed to determine the MTD and RP2D. Because of a concern that a dose of 10 mg/kg every two weeks will not allow for continuous serum levels of TRC105 placing patients at risk for infusion reactions each time they receive TRC105, the starting dose for the latter was selected at 15 mg/kg q 2 weeks. Provisions are made for a cohort -1 with a dose of TRC105 of 10 mg/kg in case the dose of 15 mg/kg is not well tolerated.  
Preoperative treatment with an aromatase inhibitor has been shown to be superior over tamoxifen48. Premenopausal women cannot receive aromatase inhibitors50 and in order to benefit from the latter they should become postmenopausal. GnRH analogs can safely achieve ovarian suppression and in fact ovarian suppression in combination with an aromatase inhibitor has been shown already to be superior to tamoxifen in the adjuvant setting88.  
The primary objective of the study is to establish the safety and tolerability of combining everolimus and TRC105 with letrozole in the preoperative setting for 24 weeks. During the phase II part of the study we will continue to evaluate the safety and tolerability of the investigational combination as well as collect preliminary data about the activity of the regimen.
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4.5 rationale for the preoperative

The preoperative setting is increasingly utilized in the evaluation and development of novel investigational therapies that can potentially provide meaningful clinical benefits over the current standard of care in breast cancer104. Although the long term outcomes with adjuvant and neoadjuvant therapy may be similar, the most pertinent advantages of neoadjuvant therapy are summarized as follows:
a. Neoadjuvant therapy allows for the clinical and radiographic evaluation of a regimen realtime. In doing so, an ineffective therapy can be promptly discontinued sparing toxicities and time.  
b. The neoadjuvant setting allows for the evaluation of tumor tissue biomarkers predictive of response from the time of biopsy to the time of definitive breast surgery.  
c. The neoadjuvant setting allows for the utilization of endpoints that are indicative of clinical benefit and can be readily assessed (rates of pCR and downstaging) as opposed to endpoints 40  
such as progression-free and overall survival which require considerably longer duration of follow-ups for their definitive assessment.  
d. Preoperative treatments permit breast conserving surgeries in patients who would otherwise require mastectomy (ies) and, therefore, may be more acceptable among patients potentially eligible for the proposed study.
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4.6 rationale for the drug

The proposed combination relies on preclinical evidence that has placed aberrant angiogenesis in the core of resistance to endocrine therapies and has elucidated the pathways that are operational in luminal breast cancer and can mitigate or completely abrogate the effect of aromatase inhibitors (see section **2. Background**). In the clinical arena, the concept of targeting angiogenesis in the preoperative setting in luminal breast cancer was successfully translated into 2 independent clinical trials: a single-institution pilot study2and a randomized, open-label phase 2 study conducted by the Translational Breast Cancer Research Consortium (TBCRC). In both studies, antiangiogenic therapies have been shown to significantly enhance the antitumor response to endocrine therapies1,2. Targeting the pathways that mediate *de novo* or acquired resistance to aromatase inhibitors in luminal breast cancer in the preoperative setting with everolimus has also been clinically validated in a large randomized phase II clinical trial4. The addition of everolimus to letrozole led to higher clinical response rates irrespective of the *PIK3CA* mutational status4. The clinical precedence set by these studies along with the preclinical evidence of potential synergism between everolimus and TRC105 provide the rationale of the proposed investigational therapy.
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4.7 rationale for tumor assessments

It is anticipated that some tumors may not respond to the investigational therapy. With letrozole alone in the neoadjuvant setting, the rate of progressive disease has ranged between 2.2 and 12%1,17,86. In addition, certain tumors may not be well visualized or cannot be followed accurately with successive imaging studies. It is important to ensure accurate clinical and imaging assessment of the tumor to safeguard against progressive disease. In equivocal cases as well as cases of discrepancy between imaging modalities and physical examination, provisions will be made for the use of MRI. Altogether though, patients whose disease cannot be measured and/or accurately followed by mammogram and/or breast ultrasound and/or exceptionally dedicated breast MRI are not eligible for the study. As most responders to neoadjuvant hormonal therapy can be identified within the first 4 months14,87, patients with stable disease after 4 cycles or progressive disease at any time point will be promptly diverted to surgery or cytotoxic chemotherapy at the discretion of the treating physician.
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4.8 rationale for the duration

The rationale for extending the preoperative investigational therapy beyond 3-4 months relies on 2 prior clinical studies that have shown a sustained and continued benefit by extending neoadjuvant hormonal therapy to 6 months and beyond14,86,87. The efficacy and tolerability of letrozole in combination with bevacizumab has been established in 2 prior clinical trials1,2. Extension of preoperative treatment beyond 6 months is not allowed by the protocol.
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5. treatment plan 5.1 subject

At the time of study entry, the patient will be evaluated by a multidisciplinary team of medical oncologists, radiation oncologists, and surgical oncologists. Before registration, investigators are required to indicate the type of operation they intend to perform. Patients will be consented by the investigator and the coordinator of the trial. Patients will be registered in the Clinical Trials Network and Monitoring Office; the office will verify the inclusion/exclusion criteria in real time.
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5.2 study medications 5.2.1 letrozole

For this trial, patients will receive 2.5 mg PO daily until 1 day before surgery. The hormonal agent is commercially available and will not be provided by the study. Description of the drug: Letrozole (Femara®) is a nonsteroidal competitive inhibitor of the aromatase enzyme system, which is responsible for the synthesis of estrogens from androgenic substrates (specifically, the synthesis of estrone from the preferred substrate androstenedione and estradiol from testosterone)50. It is an oral agent with a half-life approximating 48 hours allowing for a once-daily administration schedule50.  
Pharmacokinetic properties: Letrozole is completely and rapidly absorbed from the gastrointestinal tract105. Absorption is not affected by food. Once absorbed, it is rapidly and extensively distributed into tissues (volume of distribution at steady state is ∼2 liters/kg). Letrozole is eliminated mainly by metabolism. The major metabolite, which does not inhibit aromatase, is 4′methanobisbenzonitrile.  
This metabolite is glucuronidated and excreted primarily in the urine. Neither renal impairment (creatinine clearance < 9 ml/min) nor moderate hepatic impairment (Child-Pugh classification A and B) significantly influences letrozole pharmacokinetic parameters. Letrozole pharmacokinetics are not altered by age (range 35 to >80 years); the effects of race have not been studied105.  
Pharmacodynamic properties: In postmenopausal patients with advanced breast cancer, daily letrozole doses of 0.1–5 mg suppress plasma concentrations of estradiol, estrone, and estrone sulfate by 75–95%, with maximal suppression within 2–3 days. Suppression is dose related, with doses of ≥0.5 mg often reducing estrone and estrone sulfate levels to below the limit of detection. Estrogen suppression was maintained throughout treatment in all patients given ≥0.5 mg. At a clinically used dosage (2.5 mg PO daily), letrozole does not impair adrenal synthesis of glucocorticoids or aldosterone105.  
Adverse events: Adverse events associated with these medications are provided in the package insert (http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/020726s027lbl.pdf). The most frequent adverse events associated with letrozole include hot flashes, arthralgia/arthritis, night sweats, nausea, fatigue, myalgias, and edemas. Hypercholesterolemia grade 1 and 2 is frequent but grade 3 and 4 is rare (0.4%). Also, all aromatase inhibitors have been associated with osteoporosis. Formal assessment of bone density by means of DEXA scan relies at the discretion of the treating physician, as treatment for 6 months alone is not anticipated to incur clinically significant decreases in bone density. Following surgery, if patients proceed to adjuvant hormonal therapy with an aromatase inhibitor for 5 years, a DEXA scan is recommended as well as bone-strengthening medication(s) per institutional standards.  
5.2.2 **Everolimus** (Further details can be found in the Everolimus Investigator's Brochure)
table of contents protocol synopsis
for this trial the dose

Everolimus is a novel derivative of rapamycin. It has been in clinical development since 1996 as an immunosuppressant in solid organ transplantation. Everolimus is approved in Europe and other global markets (trade name: Certican®) for cardiac and renal transplantation, and in the United States (trade name: Zortress®) for the prevention of organ rejection of kidney transplantation. Afinitor® was approved for adults with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafinib in 2009. In 2010, Afinitor® received United States (US) approval for patients with subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC). Everolimus is also available as Votubia® in the European Union (EU) for patients with SEGA associated with TSC who require therapeutic intervention but are not candidates for curative surgical resection. Afinitor® was approved for "progressive pancreatic neuroendocrine tumor (PNET) in patients with unresectable, locally advanced, or metastatic disease" in 2011 in various countries, including the US and Europe. In 2012 Afinitor® received approval for the 43  
treatment of postmenopausal women with advanced hormone receptor-positive, HER2- negative breast cancer in combination with exemestane, after failure of treatment with letrozole or anastrozole. Furthermore in 2012, Afinitor® received approval for the treatment of patients with TSC who have renal angiomyolipoma not requiring immediate surgery. Approximately 35,982 cancer patients have been treated with everolimus as of 31-Mar-2014 (19,668 patients in Novartis-sponsored clinical trials, 2,394 patients in the individual patient supply program, and more than 13,930 patients in investigator-sponsored studies). In addition, healthy volunteer subjects and non-oncology hepatically impaired subjects have participated in the clinical pharmacology studies. The following is a brief summary of the main characteristics of Everolimus. More complete information can be obtained from the Everolimus Investigator's Brochure (IB). Description of the drug: Everolimus is an orally bioavailable derivative of rapamycin that acts as a signal transduction inhibitor (Table 5-1, **Figure E)**. Everolimus selectively inhibits mTOR (mammalian target of rapamycin), specifically targeting the mTOR-raptor signal transduction complex. mTOR is a key serine-threonine kinase in the PI3K/AKT signaling cascade, which is known to be dysregulated in a wide spectrum of human cancers106.  
| (vascular endothelial growth factor) production and VEGF-induced proliferation of endothelial  cells). Table 5-1: Everolimus - Drug substance Chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,2 8E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2- [(1S,3R,4R)-4-(2-hydroxyethoxy)-3- methoxycyclohexyl]-1-methylethyl}-19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-11,36- dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta16,24, 26,28-tetraene-2,3,10,14,20-pentaone International non-proprietary name Everolimus   |
|---|  
F**igure E: Chemical structure of Everolimus**  
  
mTOR pathway and cancer: At the cellular and molecular level, Everolimus acts as a signal transduction inhibitor. It selectively inhibits mTOR (mammalian target of rapamycin), a key protein kinase which regulates cell growth, proliferation and survival. The mTOR kinase is mainly activated via the phosphatidylinositol 3-kinase (PI3-Kinase) pathway through AKT/PKB and the tuberous sclerosis complex (TSC1/2). Mutations in these components or in PTEN, a negative regulator of PI3kinase, may result in their dysregulation. Abnormal functioning of various components of the signaling pathways contributes to the pathophysiology of numerous human cancers. Various preclinical models have confirmed the role of this pathway in tumor development107. The main known functions of mTOR include the following44:
mTOR functions as a sensor of mitogens, growth factors and energy and nutrient levels; Facilitating cell-cycle progression from G1-S phase in appropriate growth conditions; The PI3K/mTOR pathway itself is frequently dysregulated in many human cancers, and oncogenic transformation may sensitize tumor cells to mTOR inhibitors; PI3-kinase mutations have been reported in the primary tumor in 10-20% of human colorectal cancers108,109; The loss of PTEN protein, either through gene deletion or functional silencing (promoter hypermethylation), is reported in approximately 60% of primary human colorectal cancers110; The mTOR pathway is involved in the production of pro-angiogenic factors (i.e., VEGF) and inhibition of endothelial cell growth and proliferation; Through inactivating eukaryotic initiation factor 4E binding proteins and activating the 40S
ribosomal S6 kinases (i.e., p70S6K1), mTOR regulates protein translation, including the HIF-1 proteins. Inhibition of mTOR is expected to lead to decreased expression of HIF-1.  
Non-clinical experience: Everolimus inhibits the proliferation of a range of human tumor cell lines in vitro including lines originating from lung, breast, prostate, colon, melanoma and glioblastoma. IC50s range from sub/low nM to µM. Everolimus also inhibits the proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, with particular potency against VEGF-induced proliferation suggesting that Everolimus may also act as an anti-angiogenic agent. The antiangiogenic activity of Everolimus was confirmed in vivo. Everolimus selectively inhibited VEGFdependent angiogenic response at well tolerated doses. Mice with primary and metastatic tumors treated with Everolimus showed a significant reduction in blood vessel density when compared to controls. The potential of Everolimus as an anti-cancer agent was shown in rodent models. Everolimus is orally bioavailable, residing longer in tumor tissue than in plasma in a subcutaneous mouse xenograft model, and demonstrating high tumor penetration in a rat pancreatic tumor model. The pharmacokinetic profile of Everolimus indicates sufficient tumor penetration, above that needed to inhibit the proliferation of endothelial cells and tumor cell lines deemed sensitive to Everolimus in vitro. Everolimus administered orally daily was a potent inhibitor of tumor growth, at well tolerated doses, in 11 different mouse xenograft models (including breast, pancreatic, colon, epidermoid, lung and melanoma) and two syngeneic models (rat pancreatic, mouse orthotopic melanoma). These models included tumor lines considered sensitive and "relatively resistant" in vitro. In general, Everolimus was better tolerated in mouse xenograft models than standard cytotoxic agents (i.e., doxorubicin and 5-fluorouracil), while possessing similar anti-tumor activity. Additionally, activity in a VEGFimpregnated subcutaneous implant model of angiogenesis and reduced vascularity (vessel density)
of Everolimus-treated tumors (murine melanoma) provided evidence of in vivo effects of angiogenesis. It is not clear which molecular determinants predict responsiveness of tumor cells to Everolimus. Molecular analysis has revealed that relative sensitivity to Everolimus in vitro correlates with the degree of phosphorylation (activation) of the AKT/PKB protein kinase and the S6 ribosomal protein; in some cases (i.e., glioblastoma) there is also a correlation with PTEN status. In vivo studies investigating the anti-tumor activity of Everolimus in experimental animal tumor models showed that Everolimus monotherapy typically reduced tumor cell growth rates rather than produced regressions. These effects occurred within the dose range of 2.5 mg to 10 mg/kg, orally once a day. In preclinical models, the administration of Everolimus is associated with reduction of protein phosphorylation in target proteins downstream of mTOR, notably phosphorylated S6 (p-S6) and p4E-BP1, and occasionally with an increase in phosphorylated AKT, a protein upstream of mTOR
signaling pathway. All significant adverse events observed in toxicology studies with Everolimus in mice, rats, monkeys and mini-pigs were consistent with its anticipated pharmacological action as an anti-proliferative and immunosuppressant and at least in part reversible after a 2 or 4-week recovery period with the exception of the changes in male reproductive organs, most notably testes. In vitro genotoxicity studies covering relevant genotoxicity end-points showed no evidence of clastogenic or mutagenic activity. In male fertility studies in rats, testicular morphology was affected at 0.5 mg/kg and above, and sperm motility, sperm head count and plasma testosterone levels were diminished at 5 mg/kg which corresponded to 0.7 times the estimated clinical exposure at 10 mg/day, and caused a decrease in male fertility. There was evidence of reversibility. Female fertility was not affected, but everolimus caused an increase of pre-implantation loss in female rats at doses > 0.1 mg/kg, suggesting it could 46  
also potentially impact fertility in females. Everolimus crossed the placenta and was toxic to the conceptus. In rats, everolimus caused embryo/fetotoxicity at systemic exposure below the planned therapeutic level comprising mortality and reduced fetal weight. The incidence of skeletal variations and malformations at 0.3 and 0.9 mg/kg (e.g. sternal cleft) was increased. In rabbits, embryo toxicity was evident by an increase in late resorptions. Effects of everolimus on the pre- and postnatal development of rats were limited to slightly affected body weight and survival in the F1-generation at ≥0.1 mg/kg, and did not indicate a specific toxic potential. The potential reproductive risk for humans is unknown. However, due to the observed malformations in rats, everolimus should be considered potentially teratogenic. Everolimus should not be given to pregnant women unless the potential benefit outweighs the potential risk for the fetus. Women of childbearing potential should be advised to use highly effective contraception methods while they are receiving everolimus and up to 8 weeks after treatment has been stopped. It is not known whether everolimus is excreted in human milk. In animal studies, everolimus and/or its metabolites were readily transferred into the milk of lactating rats. Therefore women who are taking everolimus should not breastfeed. Pharmacokinetic properties: Everolimus is rapidly absorbed after oral administration, with a median time to peak blood levels (tmax) of 1.3-1.8 hours post dose111. The extent of absorption is estimated at above 11%. The area under the blood concentration-time curve (AUC) is dose-proportional over the dose range tested while maximum blood concentration (Cmax) appears to plateau at dose levels higher than 20 mg. The elimination half-life in cancer patients averaged 30 hours, which is similar to that in healthy subjects. Inter-patient variability is moderate with the coefficient of variation of approximately 50%. In healthy subjects, high fat meals reduced systemic exposure to everolimus 10 mg (as measured by AUC0-∞) by 22% and the peak plasma concentration Cmax by 54%. Light fat meals reduced AUC0-∞ by 32% and Cmax by 42%. Food, however, had no apparent effect on the post absorption phase concentration-time profile. Steady-state trough levels are highly predictive of AUC, with a coefficient of determination of 0.96, as has been reported in renal transplantation patients. Everolimus is mainly metabolized by the cytochrome P450 isoenzyme 3A4 (CYP3A4) in the liver and to some extent in the intestinal wall111. Everolimus is also a substrate of P-glycoprotein
(P-gp). Therefore, absorption and subsequent elimination of systematically absorbed everolimus may be influenced by medicinal products that interact with CYP3A4 and/or P-gp. Strong CYP3A inhibitors (such as ketoconazole, itraconazole, ritonavir; **Appendix B**) and inducers (such as rifampicin, rifabutin; **Appendix B**) should be avoided. Everolimus, at a daily dose of either 5 mg or 10 mg, was added to the daily letrozole regimen of 2.5 mg in breast cancer patients4. Letrozole is mainly metabolized by CYP3A4 and CYP2A6 with minor contribution of renal clearance. Due to the low affinity of letrozole to CYP3A4, the potential for pharmacokinetic interaction is remote. Pharmacokinetic profiles of letrozole were investigated before addition of everolimus and after everolimus reached steady state (day 15). Data suggested that, indeed, co-administration of everolimus with 2.5 mg/day letrozole did not influence the pharmacokinetics of letrozole. Pharmacokinetic characteristics are similar for Caucasian and Japanese subjects. Pharmacokinetic studies in Black transplant patients have shown an average 20% higher clearance. No dose adjustments are required for renal impairment or in geriatric populations but are required in hepatic impairment (see section 5.3.2).  
Pharmacodynamic properties: The pharmacodynamic properties of everolimus have been investigated in a phase I study in patients with advanced solid tumors (including breast cancer) in peripheral blood mononuclear cells112. S6 kinase 1 activity (a substrate of mTOR) in peripheral-blood mononuclear cells was inhibited for at least 7 days at doses ≥20 mg/week. Duration of suppression lengthened with increasing dosage. 20 mg was presumed to be the minimum dose ensuring target inhibition over 7 days112.  
Adverse events. The addition of everolimus to letrozole has been associated with a higher rate of dose reductions or interruptions due to adverse events (52.9%) as well as discontinuations (18.8%)
compared with letrozole alone (7.6% and 9.1%, respectively) in the neoadjuvant setting4. Grade 1 and 2 adverse events were also more frequent in the combination arm (89.8% vs. 63.6% in the letrozole alone arm)4. The most frequent adverse events seen in higher frequency in the combination arm as compared to the letrozole alone group include stomatitis (36.5% vs. 6.1%), rash (20.4% vs. 7.6%), asthenia (17.5% vs. 9.8%), fatigue (12.4% vs. 4.5%), anorexia (12.4% vs. 3.8%), headache 10.9% vs. 5.3%), pruritus (13.1% vs. none), dyspnea (7.3% vs. 1.5%), pneumonitis (2.9% vs. none). Laboratory abnormalities seen in higher frequency in the combination arm as compared with the control arm include hypercholesterolemia (16.1% vs. 6.1%), thrombocytopenia (18.2% vs. 0.8%), hyperglycemia (13.1% vs. 3%), increased ALT (11.7% vs. 3.8%), anemia (11.7% vs 0.8%), and neutropenia (9.5% vs. 1.5%). Grade 3 and 4 adverse events were also more frequent in the combination arm (22.6%) as opposed to the letrozole alone arm (3.8%). Stomatitis (2.2%), fatigue (1.5%), and pneumonitis (2.2%) were the most frequent grade 3 and 4 adverse events seen in more than 1 patients (n=137) associated with the addition of everolimus, while hyperglycemia (5.1%), increased ALT (1.5%), hypokalemia (1.5%), thrombocytopenia (1.5%) constituted the most frequent biochemical grade 3 and 4 adverse events. Serious adverse events associated with everolimus occurred in 7/137 patients: pneumonitis in three patients; pneumonia and mouth ulcers/stomatitis each in two patients; and dyspnea, neutropenia, and thrombocytopenia in one patient each. All three patient cases of pneumonitis resolved within 15 days of everolimus discontinuation. Among the placebo-treated patients, there was one serious adverse event of myocardial ischemia4.  
Overall, safety data available from completed, controlled and uncontrolled studies indicate that everolimus is generally well tolerated at weekly or daily dose schedules. The safety profile is characterized by manageable adverse events (AEs). These AEs are generally reversible and noncumulative.  
Adverse events most frequently observed with everolimus are stomatitis, rash, diarrhea, fatigue, infections, asthenia, nausea, peripheral edema, decreased appetite, headache, dysgeusia, epistaxis, mucosal inflammation, pneumonitis, weight decreased, vomiting, pruritus, cough, dyspnea, dry skin, nail disorder, and pyrexia. Overall, the most frequently observed laboratory abnormalities include: decreased hematology parameters including hemoglobin, lymphocytes, platelets, and neutrophils (or collectively as pancytopenia).; increased clinical chemistry parameters including cholesterol, triglycerides, glucose, aspartate transaminases, creatinine, alanine transaminases, and bilirubin; and decreased clinical chemistry parameters including phosphate and potassium. The majority of these AEs have been of mild to moderate severity. See section 8.6 **Adverse reactions with everolimus** for details regarding the reported adverse events associated with everolimus so far. The proceeding 48  
sections summarize recommendations for dose interruption, reduction, or discontinuation of Afinitor in the management of adverse drug reactions (**Table 5-2** and **Table 5-3**). General management recommendations are also provided as applicable. Clinical judgment of the treating physician should guide the management plan of each patient based on individual benefit/risk assessment.  
| Table 5-2: Dosing guidelines for Everolimus-related non-hematologic toxicities( see 4.2)                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Toxicity                                                                                                                                                                                                                                             | Action                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| Non-Infectious Pneumonitis                                                                                                                                                                                                                           | Please refer to Table 5-9.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Reactivation of HBV or HCV flare                                                                                                                                                                                                                     | Please refer to Table 5-7 and Table 5-8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| AST or ALT elevation Grade 1 (> ULN - 3.0 x ULN) Grade 2 (> 3.0 - 5.0 x ULN)                                                                                                                                                                         | Maintain current dose level                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| AST or ALT elevation                                                                                                                                                                                                                                 | - Interrupt Everolimus administration until resolution to ≤ grade 1 (or ≤  grade 2 if baseline values were within the range of grade 2). If                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Grade 3 (> 5.0 - 20.0 ULN)*                                                                                                                                                                                                                          | resolution occurs ≤ 7 days, Everolimus should be re-started at the dose  level prior to interruption. - If resolution takes > 7 days, or if event recurs within 28 days, hold  Everolimus until recovery to ≤ grade 1 or baseline grade / value and  reintroduce Everolimus at one dose level lower, if available (for this  trial if it was 2.5 mgs, then therapy will be discontinued).                                                                                                                                                                                                                                      |
| AST or ALT elevation Grade 4 (> 20 x ULN)*                                                                                                                                                                                                           | Interrupt Everolimus administration until resolution to ≤ grade 1 (or ≤  grade 2 if baseline values were within the range of grade 2). If  resolution occurs ≤ 7 days, Everolimus should be re-started at one dose  level lower. If resolution takes > 7 days, discontinue Everolimus. Discontinue Everolimus.                                                                                                                                                                                                                                                                                                                 |
| Recurrence of grade 4 after dose  reduction or toxicity requiring  Everolimus interruption for > 28 days Intolerable grade 2 mucositis, or grade  3 AE, except hyperglycemia or  hypertriglyceridemia or  hypercholesterolemia (see  Section5.2.2.1) | - Interrupt Everolimus administration until resolution to ≤ grade 1 or  baseline grade / value. If resolution occurs within ≤ 7 days, Everolimus  should be re-started at the dose level prior to interruption. - If resolution takes > 7 days, or if event recurs within 28 days, hold  Everolimus until recovery to ≤ grade 1 or baseline grade / value and  reintroduce Everolimus at one dose level lower, if available (for this  trial if it was 2.5 mgs, then therapy will be discontinued). - Patients will be withdrawn from the study if they fail to recover to  grade 1 or baseline grade / value within 28 days. |
| Any other grade 4                                                                                                                                                                                                                                    | Hold Everolimus until recovery to grade  1 or baseline value Reintroduce Everolimus at one dose level lower, if available (for this  trial if it was 2.5 mgs, then therapy will be discontinued).                                                                                                                                                                                                                                                                                                                                                                                                                             |
| Grade 3 or 4 clinical liver failure                                                                                                                                                                                                                  | Discontinue Everolimus                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| (asterixis or encephalopathy/coma) Recurrence of intolerable grade 2  mucositis or grade 3 event after dose  reduction                                                                                                                               | Reduce dose to the next lower dose level, if available. The lowest  possible dose level of Everolimus is 2.5 mg daily. Below this level,  Everolimus must be discontinued. If toxicity recurs at Grade 3,  consider discontinuation                                                                                                                                                                                                                                                                                                                                                                                            |
| Recurrence of grade 4 after dose                                                                                                                                                                                                                     | Discontinue Everolimus                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| reduction Any non-hematologic toxicity  requiring Everolimus interruption for  > 28 days                                                                                                                                                             | Discontinue Everolimus                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| * Should HCV flare be confirmed, the guidelines for flare must take precedence (please refer to Tables 5-7 and  5-8)                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |  
| Table 5-3: Dosing guidelines for Everolimus-related hematologic toxicities (see 4.2)   |                                                                                                                                                                                                                                                                                                         |
|----------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Toxicity                                                                               | Action                                                                                                                                                                                                                                                                                                  |
| Grade 2 thrombocytopenia (platelets <75,                                               | No action                                                                                                                                                                                                                                                                                               |
| ≥ 50x109/L) Grade 3 thrombocytopenia (platelets <50,                                   | - Interrupt Everolimus until resolution to grade 1                                                                                                                                                                                                                                                     |
| ≥ 25 x109/L)                                                                           | - If resolution occurs ≤ 7 days, reintroduce Everolimus at the dose  level prior to interruption. - If resolution occurs > 7 days, or event occurs within 28 days,  reintroduce Everolimus at one dose level lower, if available (for  this trial if it was 2.5mgs, then therapy will be discontinued). |
| Grade 4 thrombocytopenia (platelets < 25                                               | Interrupt Everolimus until recovery to grade  1. Then reintroduce                                                                                                                                                                                                                                      |
| x109/L)                                                                                | Everolimus at one dose level lower, if available (for this trial if it  was 2.5mgs, then therapy will be discontinued).                                                                                                                                                                                 |
| Grade 3 neutropenia or anemia                                                          | - Interrupt Everolimus until resolution to grade ≤1 or baseline value                                                                                                                                                                                                                                   |
| (neutrophil <1, ≥0.5 x109/L)                                                           | - If AE resolution occurs ≤ 7 days, reintroduce Everolimus at the  same dose level. - If AE resolution occurs > 7 days, or event occurs within 28 days,  reintroduce Everolimus at one dose level lower, if available (for  this trial if it was 2.5mgs, then therapy will be discontinued).            |
| Grade 4 neutropenia or anemia                                                          | Interrupt Everolimus until recovery to grade  1 or baseline value.  Reintroduce Everolimus at one dose level lower, if available (for  this trial if it was 2.5mgs, then therapy will be discontinued).*                                                                                               |
| Febrile neutropenia                                                                    | Interrupt Everolimus until resolution to grade  1 (or baseline  value) and no fever. Reintroduce Everolimus at one dose level  lower, if available (for this trial if it was 2.5mgs, then therapy  will be discontinued).*                                                                             |
| Recurrence of grade 3 toxicity after dose                                              | Reduce dose to the next lower dose level, if available. The lowest                                                                                                                                                                                                                                      |
| reduction                                                                              | possible dose level of Everolimus is 2.5 mg every day. Below this  level, Everolimus must be discontinued.                                                                                                                                                                                              |
| *Recurrence of grade 4 toxicity  (including febrile neutropenia) after  dose reduction | Discontinue Everolimus                                                                                                                                                                                                                                                                                  |
| *Any hematologic toxicity requiring                                                    | Discontinue Everolimus                                                                                                                                                                                                                                                                                  |
| Everolimus interruption for > 28 days                                                  |                                                                                                                                                                                                                                                                                                         |
table of contents protocol synopsis
5.2.2.1 management of stomatitis oral

Patients with a clinical history of stomatitis/mucositis/mouth ulcers and those with gastrointestinal morbidity associated with mouth/dental infections, irritation of esophageal mucosa e.g. gastroesophageal reflux disease (GERD) and pre-existing stomatitis/mucositis must be monitored even more closely. Patients should be instructed to report the first onset of buccal mucosa irritation/reddening to their study physician immediately. Mouth ulcers, stomatitis and oral mucositis have been seen in patients treated with everolimus. Stomatitis/oral mucositis/mouth ulcers due to everolimus should be treated using local supportive care. If examination reveals mouth ulcers rather than a more general inflammation of the mouth,  
please classify the adverse event as such. Please follow the paradigm below for treatment of stomatitis/oral mucositis/mouth ulcers (**Table 5-4**):
1. For mild toxicity (grade 1) no dose adjustment required; use conservative measures such as **nonalcoholic mouth wash or salt water (0.9%) mouth wash** several times a day until resolution.  
2. For more severe toxicity (grade 2 in which case patients have pain but are able to maintain adequate oral alimentation, or grade 3 in which case patients cannot maintain adequate oral alimentation), the suggested treatments are topical analgesic mouth treatments (i.e., local anesthetics such as benzocaine, butyl aminobenzoate, tetracaine hydrochloride, menthol, or phenol) with or without topical corticosteroids, such as triamcinolone oral paste 0.1% (Kenalog in Orabase®).  
3. Agents containing hydrogen peroxide, iodine, and thyme derivatives may tend to worsen mouth ulcers. It is preferable to avoid these agents. 4. Antifungal agents must be avoided unless a fungal infection is diagnosed. In particular, systemic imidazole antifungal agents (ketoconazole, fluconazole, itraconazole, etc.) should be avoided in all patients due to their strong inhibition of everolimus metabolism, therefore leading to higher everolimus exposures. Therefore, topical antifungal agents are preferred if an infection is diagnosed. Similarly, antiviral agents such as acyclovir should be avoided unless a viral infection is diagnosed.  
| Severity                           | Everolimus Dose Adjustment and Management Recommendations (see 4.2)                                                                                                                                                                                                                                                      |
|------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 (Minimal symptoms,         | No dose adjustment required.                                                                                                                                                                                                                                                                                             |
| normal diet)                       | Manage with non-alcoholic or salt water (0.9%) mouth wash several times a  day.                                                                                                                                                                                                                                          |
| Grade 2 (Symptomatic but can eat   | - Temporary dose interruption until recovery to grade ≤1. Re-initiate                                                                                                                                                                                                                                                    |
| and swallow modified diet)         | everolimus at the same dose. - If stomatitis recurs at grade 2, interrupt dose until recovery to grade ≤1. Reinitiate everolimus at a lower dose (for this trial if it was 2.5mgs, then  therapy will be discontinued). Manage with topical analgesic mouth treatments (e.g. benzocaine,butyl  aminobenzoate, tetracaine hydrochloride, menthol or phenol)with or without  topical corticosteroids (i.e. triamcinolone oral paste).                                                                                                                                                                                                                                                                                                                          |
| Grade 3 (Symptomatic and unable to | Temporary dose interruption until recovery to grade ≤1.                                                                                                                                                                                                                                                                  |
| adequately eat or hydrate orally)  | Re-initiate everolimus at lower dose (for this trial if it was 2.5mgs, then  therapy will be discontinued). Manage with topical analgesic mouth treatments (i.e. benzocaine,butyl  aminobenzoate, tetracaine hydrochloride, menthol or phenol) with or without  topical corticosteroids (i.e. triamcinolone oral paste)* |
| Grade 4 (Symptoms associated with  | Discontinue everolimus and treat with appropriate medical therapy.                                                                                                                                                                                                                                                       |
| life-threatening consequences)     |                                                                                                                                                                                                                                                                                                                          |
table of contents protocol synopsis
table 5-4 management of oral

5.2.2.2 **Management of Hyperlipidemia and Hyperglycemia** Treatment of hyperlipidemia should take into account the pre-treatment status and dietary habits. Blood tests to monitor hyperlipidemia must be taken in the fasting state. Grade 2 or higher hypercholesterolemia (>300 mg/dL or 7.75 mmol/L) or grade 2 hypertriglyceridemia or higher (>2.5x upper normal limit) should be treated with a 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitor (e.g. atorvastatin, pravastatin, fluvastatin) or appropriate triglyceride-lowering medication, in addition to diet. Note: Concomitant therapy with fibrates and an HMG-CoA reductase inhibitor is associated with an increased risk of a rare but serious skeletal muscle toxicity manifested by rhabdomyolysis, markedly elevated creatine phosphokinase (CPK) levels and myoglobinuria, acute renal failure and sometimes death. The risk versus benefit of using this therapy should be determined for individual patients based on their risk of cardiovascular complications of hyperlipidemia. Dyslipidemia (including hypercholesterolemia and hypertriglyceridemia) has been reported in patients taking everolimus. Monitoring of blood cholesterol and triglycerides prior to the start of everolimus therapy and periodically thereafter as well as management with appropriate medical therapy is recommended. Hyperglycemia has been reported in patients taking everolimus. Monitoring of fasting serum glucose is recommended prior to the start of Everolimus and periodically thereafter. More frequent monitoring is recommended when everolimus is co-administered with other drugs that may induce hyperglycemia. Optimal glycemic control should be achieved before starting a patient on Everolimus. Hyperlipidemia and hypertriglyceridemia should be treated according to local best clinical practice (**Table 5-5**). Patients should be monitored clinically and through serum biochemistry for the development of rhabdomyolysis and other adverse events as required in the product label/data sheets for HMG-CoA reductase inhibitors (statins).  
| Severity   | Everolimus Dose Adjustment and Management Recommendations                                                                                                                                     |
|------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1    | No dose adjustment required. Initiate appropriate medical therapy and monitor.                                                                                                                |
| Grade 2    | No dose adjustment required. Manage with appropriate medical therapy and monitor.                                                                                                             |
| Grade 3    | Temporary dose interruption. Re-initiate everolimus at lower dose (for this trial if it was 2.5mgs, then therapy will be  discontinued). Manage with appropriate medical therapy and monitor. |
| Grade 4    | Discontinue everolimus and treat with appropriate medical therapy.                                                                                                                            |
table of contents protocol synopsis
table 5-5. management of hyperlipidemia/hypertriglycemia

Reactivation of Hepatitis B (HBV) has been observed in patients with cancer receiving chemotherapy113. Sporadic cases of Hepatitis B reactivation have also been seen in this setting with everolimus. Use of antivirals during anti-cancer therapy has been shown to reduce the risk of Hepatitis B virus reactivation and associated morbidity and mortality114. A detailed assessment of Hepatitis B/C medical history and risk factors must be done for all patients at screening, with testing performed prior to the first dose of everolimus. **Hepatitis B. Table 5-6** provides details of monitoring and prophylactic therapy according to the baseline results of viral load and serologic markers testing.  
| Test                             | Result                                                                                                                                  | Result   | Result             | Result   | Result   |
|----------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------|----------|--------------------|----------|----------|
| HBV-DNA                          | +                                                                                                                                       | + or -   | -                  | -        | -        |
| HBsAg                            | + or -                                                                                                                                  | +        | -                  | -        | -        |
| HBsAb                            | + or -                                                                                                                                  | + or -   | +                  |          |          |
| and no prior                     |                                                                                                                                         |          |                    |          |          |
| HBV vaccination                  | + or -                                                                                                                                  | -        |                    |          |          |
| or + with prior HBV  vaccination |                                                                                                                                         |          |                    |          |          |
| HBcAb                            | + or -                                                                                                                                  | + or -   | + or -             | +        | -        |
| Recommendation                   | Prophylaxis treatment should  be started 1-2 weeks prior to  first dose of Everolimus.  Monitor HBV-DNA  approximately every 4-8  weeks | No prophylaxis. Monitor HBVDNA approximately every 3-4  weeks          | No specific action |          |          |
table of contents protocol synopsis
table 5-6. recommendations for positive

Antiviral prophylaxis therapy should continue for at least 4 weeks after last dose of study drug. Table 5-7 provides guidelines for definition and management of hepatitis B reactivation.  
| HBV reactivation (with or without clinical signs and symptoms): All reactivations of HBV are to be recorded as grade  3 (e.g. CTCAE Version 3.0 - Investigations/Other: Viral Reactivation), unless considered life threatening by the  investigator, in which case they should be recorded as grade 4. Date of viral reactivation is the date on which the rise  or reappearance of HBV-DNA was recorded. For patients with baseline results: positive  Treat: Start a second antiviral AND interrupt everolimus  HBV-DNA, OR positive HBsAg administration until resolution: - ≤ grade 1 ALT (or baseline ALT, if > grade 1) and - ≤ baseline HBV-DNA levels - If resolution occurs within ≤ 28 days everolimus should be  restarted at one dose lower, if available. (see 5.2.5.2) If the patient is  already receiving the lowest dose of everolimus according to the  protocol, the patient should restart at the same dose after resolution.  Both antiviral therapies should continue at least 4 weeks after last  dose of study drug. - If resolution occurs > 28 days patients should discontinue  everolimus but continue both antiviral therapies at least 4 weeks after  last dose of study drug. Reactivation is defined as: Increase of 1  log in HBV-DNA relative to baseline,  HBV-DNA value, OR new appearance of  measurable HBV-DNA For patients with baseline results: Negative  HBV-DNA and HBsAg AND Positive HBs  Ab (with no prior history of vaccination against HBV), OR Positive HBc Ab Treat : Start first antiviral medication AND interrupt study drug  administration until resolution: ≤ baseline HBV-DNA levels - If resolution occurs within ≤ 28 days everolimus should be  restarted at one dose lower, if available. (see 5.2.5.2) If the patient is  already receiving the lowest dose of everolimus according to the  protocol, the patient should restart at the same dose after resolution.  Antiviral therapy should continue at least 4 weeks after last dose of  study drug. - If resolution occurs > 28 days Patients should discontinue study  drug but continue antiviral therapy at least 4 weeks after last dose of  everolimus. Reactivation is defined as: New appearance of measurable HBV-DNA   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
table of contents protocol synopsis
table 5-7 guidelines for management

Hepatitis C. The following two categories of patients should be monitored every 6 weeks for HCV
reactivation: patients with detectable HCV RNA-PCR test at baseline, and patients known to have a history of HCV infection, despite a negative viral load test at baseline (including those that were treated and are considered 'cured'). **Table 5-8** provides definition and guidelines for the management of hepatitis C.  
| Baseline results                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | HCV flare definition*                                                                                         | HCV flare management   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------|------------------------|
| Detectable HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | > 2 log10 IU/mL increase in HCV-RNA AND ALT elevation > 5 x ULN or 3 x baseline  level, whichever is higher.  | Discontinue Everolimus |
| Knowledge of past hepatitis C  infection with no detectable  HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                         | New appearance of detectable HCV-RNA AND ALT elevation > 5 x ULN or 3 x baseline  level, whichever is higher. | Discontinue Everolimus |
| * All flares of HCV are to be recorded as grade 3 (e.g. CTCAE Version 3.0 - Investigations - Other: Viral Flare),  unless considered life threatening by the investigator; in which case they should be recorded as grade 4. Date of  viral flare is the date on which both the clinical criteria described above were met. (e.g., for a patient whose  HCV-RNA increased by 2 logs on 01 JAN 2011 and whose ALT reached > 5 x ULN on 22 JAN 2011, the date  of viral flare is 22 JAN 2011). |                                                                                                               |                        |  
Table 5-8: Definitions and Guidelines for the management of Hepatitis C
All reactivations of hepatitis B or C are considered grade 3 adverse events according to the NCI CTCAE version 4.03 unless associated with decompensated liver dysfunction in which case they are deemed grade 4. (http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf)
table of contents protocol synopsis
5.2.2.4 management of non-infectious pneumonitis

Non-infectious pneumonitis is a class effect of rapamycin derivatives. Cases of non-infectious pneumonitis (including interstitial lung disease) have also been described in patients taking everolimus. Some of these have been severe and on rare occasions, a fatal outcome was observed.  
A diagnosis of non-infectious pneumonitis should be considered in patients presenting with nonspecific respiratory signs and symptoms such as hypoxia, pleural effusion, cough or dyspnea, and in whom infectious, neoplastic and other non-medicinal causes have been excluded by means of appropriate investigations. Opportunistic infections such as PJP should be ruled out in the differential diagnosis of non-infectious pneumonitis. Patients should be advised to report promptly any new or worsening respiratory symptoms. Diagnosis is generally suspected in individuals receiving mTOR inhibitors who develop these symptoms or in asymptomatic individuals in whom a routine chest CT scan reveals a new ground glass or alveolar infiltrate. The frequency of symptomatic pulmonary toxicity (all grades) was approximately 13% in a phase III
study of everolimus in patients with metastatic renal cell carcinoma115. Severe (CTCAE grade 3)
pneumonitis occurred in 4% of patients, and an occasional fatality was reported. Similarly, in the  
phase III study of everolimus in patients with advanced pancreatic neuroendocrine tumors symptomatic pulmonary toxicity was seen in 17% of patients with grade 3 or 4 pneumonitis seen in only 2%116. Lastly, in the clinical study that compared letrozole +/- everolimus in the neoadjuvant setting, grade 3 or 4 pneumonitis was seen in 2.2% of the patients in the everolimus (vs. none in the placebo) arm4. The lung toxicity was partly or completely reversible in the majority of cases with interventions including drug interruption, discontinuation and the use of corticosteroids. Patients who develop radiological changes suggestive of non-infectious pneumonitis and have few or no symptoms may continue Afinitor therapy without dose alteration. If symptoms are moderate (grade 2), consideration should be given to interruption of therapy until symptoms improve. The use of corticosteroids may be indicated. Afinitor may be reintroduced at a daily dose approximately 50% lower than the dose previously administered. For cases of grade 3 non-infectious pneumonitis, interrupt Afinitor until resolution to less than or equal to grade 1. Afinitor may be re-initiated at a daily dose approximately 50% lower than the dose previously administered depending on the individual clinical circumstances. If toxicity recurs at grade 3, consider discontinuation of Afinitor. For cases of grade 4 non-infectious pneumonitis, Afinitor therapy should be discontinued. Corticosteroids may be indicated until clinical symptoms resolve. For patients who require use of corticosteroids for treatment of non-infectious pneumonitis, prophylaxis for pneumocystis jirovecii pneumonia (PJP) may be considered. The two compounds studied most extensively for prophylaxis against PJP have been trimethoprim-sulfamethoxazole, given orally, and pentamidine, given as an aerosol. Individuals participating in this trial will be routinely questioned as to the presence of new or changed pulmonary symptoms consistent with lung toxicity. CT scans and pulmonary function test should be done, as clinically indicated, if there are symptoms that indicate that the patient has developed non-infectious pneumonitis. If non-infectious pneumonitis develops, the guidelines in Table 5-9 should be followed. Dose modification should be done as outlined in 5.2.5.2. Consultation with a pulmonologist is recommended for any case of pneumonitis that develops during the study.  
Table 5-9. Guidelines for the management of treatment-related non-infectious pneumonitis  
|                                                                                                                                                                                                                                                                                                   | UAB 1514 / Version 7-25-19                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |
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| Worst grade                                                                                                                                                                                                                                                                                       | Suggested investigations                                                                                                                                                                                                                                                                                                                                                                                                                                          | Management of                                                                                               | Everolimus dose adjustment                                                                                                                                                                                                                                                                |     |           |     |             |
| pneumonitis                                                                                                                                                                                                                                                                                       | pneumonitis                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |
| Grade 1  (Asymptomatic,  radiographic  findings only)                                                                                                                                                                                                                                             | CT scans with lung windows.                                                                                                                                                                                                                                                                                                                                                                                                                                       | No specific therapy is                                                                                      | No dose adjustment required.                                                                                                                                                                                                                                                              |     |           |     |             |
| required                                                                                                                                                                                                                                                                                          | Initiate appropriate monitoring.                                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |
| Grade 2  (Symptomatic,  not interfering  with Activities  of Daily Living)                                                                                                                                                                                                                        | CT scan with lung windows.  Consider pulmonary function  testing includes: spirometry,  DLCO, and room air O2 saturation  at rest. Consider a bronchoscopy  with biopsy and/or BAL.  Monitoring at each visit until return  to ≤ grade 1. Return to initial  monitoring frequency if no  recurrence.                                                                                                                                                              | Symptomatic only.  Consider corticosteroids  and/or other supportive  therapy if symptoms are  troublesome. | Rule out infection and consider  interruption of Everolimus until  symptoms improve to Grade ≤ 1. Re-initiate Everolimus at one dose level  lower (for this trial if it was 2.5 mgs,  then therapy will be discontinued). Discontinue Everolimus if failure to  recover within ≤ 28 days. |     |           |     |             |
| Grade 3  (Symptomatic,  Interfering with  Activities of  Daily Living. O2 indicated                                                                                                                                                                                                               | Rule out infection and interrupt  Everolimus until symptoms improve to  Grade ≤ 1. Consider re-initiating Everolimus at one  dose level lower (approximately 50%  lower than the dose previously  administered depending on individual  clinical circumstances) (for this trial if it  was 2.5 mgs, then therapy will be  discontinued). Discontinue Everolimus if  failure to recover within ≤ 28 days. If toxicity recurs at Grade 3, consider  discontinuation |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |
| CT scan with lung windows and  pulmonary function testing includes:  spirometry, DLCO, and room air O2  saturation at rest. Monitoring at each  visit until return to ≤ grade 1. Return  to initial monitoring frequency if no  recurrence. Bronchoscopy with  biopsy and/or BAL is  recommended. | Consider corticosteroids  if infective origin is ruled  out. Taper as medically  indicated.                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |
| Grade 4 (Lifethreatening,  ventlatory  support  indicated)                                                                                                                                                                                                                                                                                                   | CT scan with lung windows and  required pulmonary function testing,  if possible, includes: spirometry,  DLCO, and room air O2 saturation  at rest. Monitoring at each visit until  return to ≤ grade 1. Return to initial  monitoring frequency if no  recurrence. Bronchoscopy with  biopsy and/or BAL is recommended  if possible.                                                                                                                             | Consider corticosteroids  if infective origin is ruled  out. Taper as medically  indicated.                 | Rule                                                                                                                                                                                                                                                                                      | out | infection | and | discontinue |
| Everolimus.                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                             |                                                                                                                                                                                                                                                                                           |     |           |     |             |