CLEVER Pilot Trial: A Phase II Pilot Trial of HydroxyChLoroquine, EVErolimus or the Combination for Prevention of Recurrent Breast Cancer
Phase: PHASE2
Conditions: Breast Cancer Stage IIB
Sponsor: Abramson Cancer Center at Penn Medicine

UPCC 24116/IRB#826253 
 
A Phase II Pilot Trial of Hydroxychloroquine, Everolimus or the 
Combination for Prevention of Recurrent Breast Cancer (“CLEVER”) 
 
 
Principal Investigator 
     
 
Angela DeMichele, MD, MSCE 
 
 
 
 
 
 
 
 
Sub-Investigators:                  
 
Lewis Chodosh, MD, PhD  
Amy Clark, MD, MSCE 
Kevin Fox, MD 
Jennifer Matro, MD 
Susan Domchek, MD 
Angela Bradbury, MD 
Hayley Knollman, MD 
Payal Shah, MD 
Ravi Amaravadi, MD 
Paul Wileyto, PhD 
                                                 
 
Michael Feldman, MD, PhD 
 
 
 
 
 
 
Anupma Nayak, MD 
 
 
 
 
 
 
Brooke Goodspeed, MSN, CRNP, AOCNP 
 
Medical/Research Monitor: 
 
Tara Mitchell (Gangadhar), MD 
 
Sponsor:  
 
 
 
 
Angela DeMichele, MD, MSCE / 
Abramson Cancer Center 
  
Supplier of Study Drug:  
 
 
Novartis Pharmaceuticals Corporation.  
Novartis Study Number  
 
 
CRAD001JUS260T 
Drug Supplied:  
 
 
 
Everolimus: Novartis Pharmaceuticals Corp. 
Hydroxychloroquine: Commercially available  
IND:   
 
 
 
 
IND Exempt 
Protocol Version:  
 
 
 
Amendment #7 dated 17 June 2019 
 
 
 
 
 
 

 
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Table of Contents 
1 
STUDY SUMMARY .................................................................................................. 7 
1.1 
Study Schema ........................................................................................ 12 
2 
STUDY OBJECTIVES ............................................................................................ 13 
2.1 
Primary Clinical Objective ...................................................................... 13 
2.2 
Secondary Clinical Objectives ................................................................ 13 
2.3 
Translational Objectives ......................................................................... 13 
3 
BACKGROUND ..................................................................................................... 14 
3.1 
Disseminated tumor cells as a prognostic marker and target for therapy14 
3.2 
Biology of tumor dormancy and recurrence ............................................ 16 
3.3 
Summary: Targeting dormancy with mTOR and autophagy inhibition. ... 23 
3.4 
Hydroxychloroquine (HCQ) and cancer therapy ..................................... 25 
3.5 
Everolimus (EVE, RAD001) ................................................................... 27 
3.6 
Combination therapy with HCQ and EVE ............................................... 31 
3.7 
Rationale for the proposed proof-of-concept pilot trial ............................ 32 
3.8 
Trial Overview ........................................................................................ 32 
4 
PATIENT SELECTION ........................................................................................... 33 
4.1 
Target enrollment and subject ascertainment ........................................ 33 
4.2 
Inclusion criteria ..................................................................................... 33 
4.3 
Exclusion criteria .................................................................................... 35 
5 
STUDY ASSESSMENTS AND PROCEDURES .................................................... 36 
5.1 
Schedule of Assessments and Procedures ............................................ 36 
5.2 
Screening Phase .................................................................................... 38 
5.3 
Treatment Phase .................................................................................... 38 
5.4 
First Follow-Up and Follow-Up Phase .................................................... 39 
5.5 
Laboratory and adverse event assessment ............................................ 39 
5.6 
Disease monitoring ................................................................................. 39 
6 
ENROLLMENT PROCEDURES ............................................................................ 40 
6.1 
Guidelines for Enrollment ....................................................................... 40 
7 
TREATMENT PLAN ............................................................................................... 40 
7.1 
Study Arms ............................................................................................. 41 
7.2 
Drug Administration ................................................................................ 41 
 
 

 
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7.3 
Adjuvant Endocrine Therapy .................................................................. 42 
7.4 
Concomitant Treatment and Supportive Care Guidelines ...................... 42 
7.5 
Concomitant Medications ....................................................................... 43 
7.6 
Drug-Drug Interactions ........................................................................... 44 
7.7 
Criteria for Removal from Study ............................................................. 45 
7.8 
Discontinuation of Treatment ................................................................. 46 
7.9 
Withdrawal from Study ........................................................................... 46 
7.10 
Contraceptive Measures and Reproductive Risks............................... 46 
8 
EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS ........ 48 
8.1 
Toxicity Criteria ....................................................................................... 48 
8.2 
Anticipated Toxicities of Hydroxychloroquine ......................................... 48 
8.3 
Anticipated Toxicities of Everolimus ....................................................... 48 
8.4 
Monitoring of EVE suspected toxicities .................................................. 49 
8.5 
Special Toxicity Considerations for EVE ................................................ 50 
8.6 
Management of Treatment-Related Toxicities ........................................ 52 
8.7 
Dose Delays ........................................................................................... 57 
8.8 
Dose Modifications ................................................................................. 57 
9 
DRUG FORMULATION AND STORAGE .............................................................. 61 
9.1 
Agent #1:  Hydroxychloroquine .............................................................. 61 
9.2 
Agent #2: Everolimus ............................................................................. 61 
9.3 
Drug Accountability ................................................................................ 62 
10 
SAFETY ASSESSMENTS .................................................................................. 62 
10.1 
Adverse Events Reporting .................................................................. 62 
10.2 
Definitions ........................................................................................... 62 
10.3 
Abnormal Laboratory Values ............................................................... 63 
10.4 
Hospitalization, Prolonged Hospitalization or Surgery......................... 63 
10.5 
Recording of Adverse Events .............................................................. 64 
10.6 
Reporting of Serious Adverse Events ................................................. 64 
10.7 
Internal Safety and Compliance Entities ............................................. 67 
10.8 
Medical Monitoring .............................................................................. 67 
10.9 
Study Monitoring Plan ......................................................................... 68 
10.10 Auditing and Inspecting ...................................................................... 68 
10.11 Data Safety Monitoring Board ............................................................. 68 

 
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10.12 Protocol Deviations ............................................................................. 68 
11 
MEASUREMENT OF EFFECT ........................................................................... 69 
11.1 
Definitions ........................................................................................... 69 
11.2 
Guidelines for Evaluation of Disease .................................................. 70 
12 
STATISTICAL CONSIDERATIONS AND METHODOLOGY .............................. 70 
12.1 
Sample Size ........................................................................................ 70 
12.2 
Analysis Plan ...................................................................................... 70 
13 
CORRELATIVE SCIENCES ............................................................................... 72 
13.1 
Biospecimens ...................................................................................... 72 
13.2 
Laboratory Assays .............................................................................. 73 
13.3 
Genetic Testing ................................................................................... 76 
14 
DATA HANDLING AND RECORD KEEPING..................................................... 76 
14.1 
Confidentiality ..................................................................................... 76 
14.2 
Source Documents .............................................................................. 77 
14.3 
Case Report Forms ............................................................................. 77 
15 
STUDY FINANCES ............................................................................................ 77 
15.1 
Funding Source ................................................................................... 77 
15.2 
Conflict of Interest ............................................................................... 77 
15.3 
Patient Stipends or Payments ............................................................. 77 
16 
GENERAL REGULATORY CONSIDERATIONS AND CREDENTIALING ......... 77 
16.1 
Publication of results ........................................................................... 77 
16.2 
Disclosure and confidentiality .............................................................. 78 
16.3 
Ethics and Good Clinical Practice ....................................................... 78 
16.4 
Institutional Review Board/Independent Ethics Committee ................. 78 
16.5 
Informed consent ................................................................................ 78 
16.6 
Declaration of Helsinki ........................................................................ 79 
17 
References ......................................................................................................... 79 
18 
Appendices ......................................................................................................... 83 
 
 
 

 
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List of abbreviations 
HCQ: Hydroxychloroquine 
RCC: renal cell carcinoma 
AV: autophagic vesicles 
mTOR: mammalian target of rapamycin  
PFS: progression-free survival  
CTEP: Cancer Therapy and Evaluation Program  
PK: Pharmacokinetic  
PD: Phramocdynamic 
ASCO: American Society of Clinical Oncology  
GBM: Glioblastoma multiforme 
PI3K: phosphatidyl inositol 3-phosphate kinase 
AUC: Area under the curve  
VEGFR: vascular endothelial growth factor receptor  
CQ: chloroquine 
DLT: Dose limiting toxicity  
MTD: Maximal Tolerated Dose 
EM: Electron Microscopy  
ECOG: Eastern Cooperative Oncology Group  
ANC: Absolute neutrophil count 
ALT: alanine transaminase 
AST: Aspartate aminotrasnferase 
RECIST: Response Evaluation Criteria in Solid Tumors 
INR: International Normalized ratio  
PTT: Partial thromboplastin time 
HIV: Human Immunodeficiency virus 
HBV: Hepatitis B Virus 
HCV: Hepatitis C Virus 
NCI: National Cancer Institute 
CTCAE: Common Terminology Criteria for Adverse Events 
AE: Adverse Event 
CT: Computed tomography  
DLCO: Diffusion capacity of the lung for carbon monoxide 
HBsAg: Hepatitis B surface antigen 
HBsAb: Hepatitis B surface antibody  
HBcAb: Hepatitis B core antibody  
DNA: deoxyribonucleic acid  
RNA: ribonucleic acid 
 MRI: Magnetic resonance imaging 
ECG: electrocardiogram  
H&E: hematoxylin and eosin  
IHC: immunohistochemistry  
PBMC: peripheral blood mononuclear cell 
PBS: phosphate buffered saline  

 
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IND: investigational new drug  
IRB: institutional review board  
FDA: federal drug administration  
SAE: significant adverse event 
DSMC: data safety and monitoring committee 
CTRMSC: Clinical trial review and monitoring scientific committee 
HIPAA: Health Insurance Portability and Accountability Act 
CRF: Case report form  
 
 

 
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1 
STUDY SUMMARY 
Title 
A Phase II Pilot Trial of Hydroxychloroquine, Everolimus or the 
Combination for Prevention of Recurrent Breast Cancer 
Short Title 
CLEVER Pilot Trial 
Protocol 
Number 
IRB #826253; UPCC 24116 
Methodology 
This is a randomized, controlled, open label pilot trial. A 
total of 60 patients will be enrolled, with 15 patients allocated to 
each treatment arm. Patients will be evaluated at the initiation of 
every cycle for adverse events, and dose reduction will be 
employed for toxicity.  Bone marrow aspiration will be performed 
at the end of cycle 3, cycle 6, and if patient continued on second 
treatment phase, at the end of cycle 12.  The initial six-month 
study period is designed to assess the effects of HCQ, EVE and 
their combination.  After three months of observation, control 
patients will be offered HCQ/EVE investigational therapy for six 
cycles; thus, the control group is actually a delayed treatment 
group and all patients will receive treatment.  Patients who 
continue to demonstrate positive bone marrow DTCs after six 
cycles will be offered the opportunity to continue on combination 
therapy for an additional 6 cycles.  Patients will continue to be 
followed for recurrence events for up to 3 years after completion 
of treatment. 
Study Duration 
Active Enrollment:  3 years; Follow-up:  3 years 
Study Center(s) 
The University of Pennsylvania and affiliated sites 
Objectives 
0B0B0BPrimary Clinical Objective:  To evaluate the feasibility of 
administering HCQ, EVE or the combination in patients who 
have completed primary therapy for breast cancer and 
harbor bone marrow disseminated tumor cells 
UEndpoint:  
>75% of patients in each arm reaching the 6-month evaluation 
time point without grade 3 or 4 toxicity.  
1B1B1BSecondary Clinical Objectives:   
3B3B3B1. To determine the safety of administering HCQ + EVE in 
combination with adjuvant endocrine therapy 
UEndpoints 
Incidence of adverse events within each treatment stratum and 
dose level by NCI CTCAE v4 criteria.   
4B4B4B2.  To evaluate preliminary efficacy of HCQ, EVE or the 
combination in reducing or eliminating DTCs 
UEndpoint 

 
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 Percent change in bone marrow DTCs at 6 and 12 
months of treatment relative to baseline (pre-treatment) 
 Incremental benefit of an additional 6 months of therapy 
in patients who have DTCs after initial 6 months of 
therapy 
 Incremental benefit of combination therapy compared to 
single-agent therapy 
5B5B5B3.  To estimate the risk of recurrence after treatment with 
HCQ, EVE or the combination 
UEndpoint 
 3-year recurrence free survival (RFS) 
2B2B2BTranslational Objectives: 
6B6B6B1.  To assess the utility of a novel DTC assay, DTC-Flow, as 
a pharmacodynamic marker in trial participants to detect 
DTCs and their response to study therapy, compared to 
DTC-IHC.  
UEndpoints:  
 Measurement of the number and type of DTCs by DTC-
FLOW before, during and at the conclusion of study 
therapy 
 Comparison to measurement of DTCs by IHC 
7B7B7B2.  To determine whether patient DTCs biologically reflect 
primary tumor and predict response to study therapy.  
UEndpoints:  
 WES, RNA-seq, and CNV analysis will be performed on 
primary breast tumors and DTCs isolated by DTC-Flow 
to:  
 Identify similarities in molecular features between primary 
tumors and DTCs 
 Define genomic markers in DTCs that predict response to 
HCQ, EVE or their combination; 3) Identify similarities in 
molecular features between DTCs, CTCs and ptDNA. 
8B8B8B3.  To determine whether patient circulating tumor cells 
(CTCs) biologically reflect primary tumor, accurately 
identify patients with bone marrow DTCs at baseline, 
predict for the elimination of DTCs with therapy, and/or 
predict for improved clinical outcomes relative to therapy 
UEndpointsU:   
 Associations between biological and genomic features of 

 
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CTCs and primary tumor 
 Measurement of CTCs before, during and at the 
conclusion of study therapy, compared to DTC and 
clinical outcomes. 
9B9B9B4.  To determine whether plasma tumor DNA (ptDNA) 
biologically reflects primary tumor, accurately identifies 
patients with bone marrow DTCs, or predicts response to 
therapy 
UEndpoints:   
Concordance of mutations between ptDNA and primary tumor 
Measurement of ptDNA before, during and at the conclusion of 
study therapy, compared to DTC outcomes. 
Number of 
Subjects 
The target number of study participants is 60 
Diagnosis and 
Main Inclusion 
Criteria 
 Histologically-confirmed, primary, invasive breast cancer 
diagnosed within 5 years of study entry. 
 Qualifying risk status, at diagnosis, meeting one of the 
following: 
o 
43T43THistologically positive axillary lymph nodes 
o 
43T43TPrimary tumor that is ER/PR<10%/Her2 negative 
o 
43T43TPrimary tumor with Breast Cancer Recurrence 
Score of ≥ 25 per the Genomic Health Oncotype 
DX breast cancer test and/or high risk MammaPrint 
o 
43T43TEvidence of residual disease in the breast on 
pathological assessment after neoadjuvant 
chemotherapy.  
 Patients must have completed all primary therapy (definitive 
surgery, (neo)adjuvant chemotherapy adjuvant radiation and/or 
Her2-directed therapy) for the index malignancy at least 4 
weeks prior to study entry. All prior treatment-related toxicity 
must be resolved prior to study enrollment.  Concurrent receipt 

 
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of adjuvant endocrine and bone modifying agents is allowed 
per standard of care guidelines.  
 Bone marrow aspirate after completion of therapy 
demonstrates detectable DTCs (via IHC) 
 No evidence of recurrent local or distant breast cancer by 
physical examination, blood tests (CBC, LFTs, Alk Phos), or 
symptom-directed imaging, per NCCN guidelines. 
 Age ≥ 18 years 
 ECOG performance status  2 
 No contraindications to the study medications or uncontrolled 
medical illness. 
 Adequate bone marrow function as shown by: ANC ≥ 1.5 x 
10P9
P/L, Platelets ≥ 100 x 10P9
P/L, Hb >9 g/dL 
 Adequate liver function as shown by:  Serum bilirubin ≤ 1.5 x 
ULN, ALT and AST ≤ 2.5 x ULN, and INR ≤1.5   
 Anticoagulation is allowed if target INR ≤ 1.5 on a stable dose 
of warfarin or on a stable dose of anticoagulant for >2 weeks at 
time of randomization.  For patients on therapeutic anti-
coagulants, medication must be clinically held peri-procedure 
(bone marrow aspirate) per standard clinical management.  
 Adequate renal function: serum creatinine ≤ 2.0 x ULN or 
creatinine clearance (CrCl) ≥ 30mL/min obtained within 28 
days prior to registration.  A calculated creatinine clearance by 
Cockcroft-Gault Formula is acceptable in lieu of a measured 
value. 
 Serum cholesterol ≤300 mg/dL OR ≤7.75 mmol/L AND 
triglycerides ≤ 2.5 x ULN. NOTE: In case one or both of these 
thresholds are exceeded, a fasting lipid assessment will be 
ordered. If one or both of these thresholds are still exceeded in 
a fasting state, the patient can only be included after initiation 
of appropriate lipid lowering medication. 
 Ability to provide informed consent 
 
Statistical 
Methodology 
The primary endpoint of this pilot study is feasibility.  The 
secondary endpoints are tolerability and the measured effect of 
the investigational therapy on DTCs in patients with measureable 
DTCs at baseline and the comparison of this effect to that in a 
control population.  Because this is proof-of-concept, sample 
sizes are not sufficient to address long-term recurrence risk.  
Randomization is being performed to minimize uncontrolled 
confounding.   
The trial endpoint “feasibility” will be defined as Uat least 11U 
of 15 patients within each treatment regimen completing 6 cycles 
of therapy without grade 3 or 4 toxicity.  While arms may be 

 
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discontinued for lack of feasibility, the trial may continue enrolling 
to those arms that remain feasible.   
Toxicity will be assessed using NCI CTCAE v4 criteria.  
Patients who drop out before completing cycle 6 for reasons 
other than grade 3/4 toxicity will be replaced.  With 15 patients 
per group, an exact 95% confidence interval around the feasibility 
and toxicity rates will be no more than 0.52 units wide.   
We will define “response” for each patient as a 50% or 
greater decrease in DTC number at each treatment time point 
compared to baseline.  A treatment arm/regimen will be 
considered successful if at least 3 of 15 of patients experience 
this predefined response endpoint.   
We will also estimate, within each treatment group, the % 
change in DTC number.  While the trial is powered for feasibility, 
rather than detection of change in DTCs across treatment group, 
15 patients per group will provide 80% power (using a two-group 
t-test with a two-sided alpha level of 0.05) to detect a decrease of 
approximately 1 standard deviation and will provide important 
preliminary data for designing future confirmatory trials. 

 
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1.1 
Study Schema 
 
 
 

 
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2 
STUDY OBJECTIVES 
2.1 Primary Clinical Objective:  To evaluate the feasibility of administering HCQ, 
EVE or the combination in patients who have completed primary therapy for 
breast cancer and harbor bone marrow disseminated tumor cells 
 
UEndpoint:  
 >75% of patients overall completing 6 cycles of therapy without 
grade 3 or 4 toxicity 
2.2 Secondary Clinical Objectives   
2.2.1 To determine the safety of administering HCQ + EVE in combination with 
adjuvant endocrine therapy 
UEndpoints 
• 
Incidence of adverse events within each treatment stratum and 
dose level by NCI CTCAE v4 criteria.   
2.2.2 To evaluate preliminary efficacy of HCQ, EVE or the combination in reducing 
or eliminating DTCs 
UEndpoints 
• 
Change in bone marrow DTCs at 3, 6 and 12 months of treatment 
relative to baseline (pre-treatment) 
• 
Change in bone marrow DTCs at 3 months with no treatment (Arm 
D Control) compared to any treatment (Arms A, B or C) 
• 
Incremental benefit of an additional 6 months of therapy in patients 
who have DTCs after initial 6 months of therapy 
• 
Incremental benefit of combination therapy compared to single-
agent therapy 
2.2.3 To estimate the risk of recurrence after treatment with HCQ, EVE, or the 
combination 
UEndpoint 
• 
3-year recurrence free survival (RFS) 
2.3 Translational Objectives 
2.3.1 To assess the utility of a novel DTC assay, DTC-Flow, as a 
pharmacodynamic marker in trial participants to detect DTCs and their 
response to study therapy, compared to DTC-IHC.  
UEndpoints:  
 Measurement of the number and type of DTCs by DTC-FLOW 
before, during and at the conclusion of study therapy 
 Comparison to measurement of DTCs by IHC 
2.3.2 To determine whether patient DTCs biologically reflect primary tumor and 

 
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predict response to study therapy.  
UEndpoints:  
WES, RNA-seq, and CNV analysis will be performed on primary breast 
tumors and DTCs isolated by DTC-Flow post-NAC to:  
 Identify similarities in molecular features between primary 
tumors and DTCs 
 Define genomic markers in DTCs that predict response to 
HCQ, EVE or their combination; 3) Identify similarities in 
molecular features between DTCs, CTCs and ptDNA. 
2.3.3 To determine whether patient circulating tumor cells (CTCs) biologically 
reflect primary tumor, accurately identify patients with bone marrow DTCs at 
baseline, predict for the elimination of DTCs with therapy, and/or predict for 
improved clinical outcomes relative to therapy 
                UEndpoints:   
 Associations between biological and genomic features of CTCs and 
primary tumor 
 Measurement of CTCs before, during and at the conclusion of 
study therapy, compared to DTC and clinical outcomes. 
2.3.4 To determine whether plasma tumor DNA (ptDNA) biologically reflects 
primary tumor, accurately identifies patients with bone marrow DTCs, or 
predicts response to therapy 
UEndpoints:   
 Concordance of mutations between ptDNA and primary tumor 
 Measurement of ptDNA before, during and at the conclusion of 
study therapy, compared to DTC outcomes. 
 
3 
BACKGROUND 
Historically, cancer therapy has focused on drug targets found in the primary 
tumor.  In recent years, however, it has become increasingly clear that the molecular 
pathways underlying breast cancer dormancy and recurrence are distinct from those of 
primary tumorigenesis.  Consistent with this, our investigations in preclinical models 
indicate that dormant tumor cells possess unique vulnerabilities not observed in actively 
growing tumor cells.  These unique attributes present a wealth of new drug targets that 
could hold the key to more effective treatments for this disease by preventing tumor 
recurrence and its associated mortality. 
3.1 
Disseminated tumor cells as a prognostic marker and target for therapy 
Disseminated tumor cells (DTCs) can be detected in up to 30–40% of patients 
with early stage breast cancer(1, 2).  A growing body of evidence has established that 
the presence of DTCs in these patients is an independent prognostic factor for 
recurrence(1, 3-5).  Some of the strongest evidence for the prognostic significance of 
DTCs in breast cancer patients emerged from a pooled analysis of 4,703 patients with 

 
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stage I, II, or III breast cancer in whom DTCs were identified in 31% of patients(6).  The 
presence of DTCs in bone marrow predicted poor breast cancer-specific survival, poor 
overall survival, and a high risk of loco-regional and distant recurrence. Additional 
studies, also using an immunohistochemical (IHC) assays to identify cytokeratin-positive 
(CK+) cells in bone marrow aspirates, have confirmed this association(7-9).  Despite 
variability in the patient populations, assay methods, and antibodies used to identify 
DTCs in these studies, there is striking overall consistency in the rate at which DTCs 
were found and their prognostic significance, suggesting that DTCs are a powerful 
biomarker to identify early stage patients at significant risk of recurrence. 
Despite mounting evidence for DTCs as a prognostic biomarker for recurrence, 
remarkably little has been done to utilize the presence of these cells either to identify 
patients at risk or as a target for adjuvant therapy.  Currently, DTC assays are not used 
as standard of care, and other approaches to monitor for recurrence such as radiologic 
scanning and circulating tumor markers (e.g., CA27-29, CA15-3) are inaccurate and 
currently not recommended by the American Society of Clinical Oncology for use in the 
surveillance period(10).  This lack of an active approach to monitor for residual cancer 
cells at a point where intervention could be successful leads to enormous distress for 
patients(11-13) and limits approaches to prevent incurable metastatic disease.  This, in 
turn, has led to a paucity of data on whether targeting and eliminating DTCs would have 
a meaningful impact on recurrence and survival outcomes.  Only one published study to 
date(14) has utilized the persistence of DTCs as an indication for additional adjuvant 
therapy.  In this trial, patients who had completed 6 cycles of anthracycline-based 
adjuvant chemotherapy were tested for the presence of DTCs by IHC assay at 8 
months post-treatment.  Patients with at least 1 DTC detected were given 6 cycles of 
docetaxel.  There was no randomized control group.  Patients with persistence of DTCs 
post-docetaxel (21%) had significantly reduced disease-free interval (DFI) compared to 
those who had clearance of DTCs (HR 7.58, 95% CI 2.3-24.7).  Patients with clearance 
of DTCs after docetaxel had comparable DFI to those who had no DTCs after 
anthracycline-based therapy alone.  Though limited by the lack of randomization for 
treatment amongst those with DTCs, these important data suggest that clearance of 
DTCs may represent an effective strategy for reducing recurrence.    

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Building upon these data using therapies that specifically target unique features 
of DTCs represents a potentially transformative, innovative new approach to clinical 
trials that will reduce recurrence, as 
shown in the conceptual model in Figure 
1. We now propose to conduct human
studies that will translate to the clinic our 
findings in mouse models identifying 
unique biologic features of dormant 
minimal residual disease (MRD), 
including reliance for survival on 
autophagy and the mTOR pathway, and 
demonstrating that targeted inhibition of 
these pathways reduces the population of 
dormant residual tumor cells and
improves recurrence-free survival (RFS).
Key components of this effort are: 1) 
Novel clinical trials of agents targeting 
dormancy that are safe and tolerable in 
patients who, though harboring DTCs, may still be cured of their disease; and 2) Further 
refinement of the therapeutic approach through additional mouse preclinical studies and 
development of a flow-based DTC assay that will enable molecular and genetic 
characterization of both mouse and human DTCs to elucidate the biology of human 
tumor dormancy, identify appropriate patients and agents for intervention, and provide a 
pharmacodynamic marker of treatment effect.  This approach, if successful, will enable 
real-time surveillance and intervention for patients at risk of recurrence, provide 
effective tools for proactive secondary prevention, and improve survival outcomes for 
patients with breast cancer.  
3.2 
Biology of tumor dormancy and recurrence 
Dormant residual cancer cells likely constitute the reservoir from which recurrent 
tumors arise.  Accordingly, the lack of biological understanding and therapeutic 
approaches aimed at these cells constitute major obstacles to the successful treatment 
of this disease.  To address this critical gap, our laboratory developed a series of 
conditional genetically engineered mouse (GEM) models for breast cancer that faithfully 
recapitulate key stages of breast cancer progression, including tumor dormancy and 
recurrence, and phenocopy human breast cancers with respect to the oncogenic 
pathways activated (e.g., HER2, MYC, Wnt1, Akt), histological appearance, and tumor 
progression over time(15-24).  Such models can facilitate the in vivo analysis of breast 
cancer progression and provide a platform for assessing the efficacy of interventions 
designed to prevent recurrent disease.  Using these models, we found that virtually all 
mice bearing fully regressed tumors harbor dormant residual cancer cells in all common 
recurrent organ sites observed in patients, long after the apparently complete 
regression of their tumors.  Furthermore, mice relapse at both local and distant sites 
with overt metastases.  Accordingly, these models enable identification and functional 
analysis of the population of dormant residual cells that give rise to recurrent tumors, 
thereby permitting elucidation of the mechanisms underlying the persistence of minimal 
PEN

 
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residual disease and relapse. 
3.2.1 
GEM models recapitulate breast cancer dormancy and recurrence 
The GEM models we have developed permit the inducible expression of 
oncogenes in the mammary epithelium of transgenic mice treated with doxycycline(15-
24).  Expression of the reverse tetracycline-inducible transactivator, rtTA, is driven in the 
mammary epithelium by the mouse mammary tumor virus (MMTV) LTR in mice bearing 
the MMTV-rtTA (MTB) transgene.  Target transgenes are placed under the control of a 
tetO regulatory cassette.  Transgene expression in this system is mammary-specific, 
rapidly inducible and deinducible, undetectable in the uninduced state, and can be 
titrated over a broad range(15-24).  Using this system, we conditionally expressed the 
HER2/neu (MTB/TAN), c-MYC (MTB/TOM), Wnt1 (MTB/TWNT) and Akt (MTB/TAKT) 
oncogenes because of their demonstrated relevance to human breast cancer.  
Following oncogene induction with doxycycline, MTB/TAN, MTB/TOM, MTB/TWNT and 
MTB/TAKT bitransgenic (MTB/Tet-Onc) mice develop epithelial hyperplasias, focal 
atypical hyperplasias, and invasive mammary adenocarcinomas that spontaneously 
metastasize to the bone marrow, lungs, liver, lymphatics, , and brain – sites that are 
characteristically found in patients with breast cancer(25-28).  Tumor formation is highly 
penetrant, mammary-specific, and entirely dependent upon transgene induction by 
doxycyclineP(15, 19)
P.  
3.2.2 
Regressed tumors recur spontaneously in GEM 
Strikingly, nearly all mammary tumors in MTB/Tet-Onc mice rapidly regress to a 
non-palpable state following oncogene down-regulation induced by doxycycline 
withdrawal, indicating that the vast majority of cells within these tumors are oncogene 
“addicted”(16, 18, 19, 24).  The marked regression of primary tumors following 
oncogene down-regulation is similar to that observed in human cancer patients treated 
with targeted therapies, including trastuzumab(29, 30).  However, breast cancer 
patients treated with chemotherapy, targeted therapies, and/or hormonal therapies often 
recur.  As such, we suspected that at least some tumor cells would survive, and 
eventually resume growth, following oncogene down-regulation.  To test this possibility, 
MTB/Tet-Onc mice in which doxycycline withdrawal had led to complete regression of 
primary tumors were monitored for tumor recurrence in the absence of doxycycline 
treatment.  This revealed that ~80% of tumors recurred at their original sites over 
periods of up to one year, and that mice relapsed at distant sites as well(16, 18, 19, 31).  
Mutations detected in primary tumors were also present in recurrent tumors, indicating 
that they are bona fide recurrences(16, 18, 19).  Tumor regression and recurrence 
occurs with similar kinetics in recipient mice bearing orthotopic primary mammary 
tumors derived from cultured or uncultured primary tumor cells from MTB/TAN or 
MTB/TWNT mice(16, 32-34).  Further, we have observed a similar pattern of tumor 
regression and recurrence in mice treated with doxorubicin, cyclophosphamide and 
paclitaxel – chemotherapeutic agents that are commonly used to treat breast cancer 
patients(33).   
These findings suggested that mice bearing fully regressed tumors harbor 
minimal residual disease in which growth can spontaneously reactivate, resulting in 
cancer recurrence.  To test this hypothesis directly, MTB/TAN mice bearing fully 

 
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regressed tumors that had not recurred spontaneously within 6 months of HER2/neu 
down-regulation were reinduced with doxycycline and monitored for tumors.  All mice 
rapidly re-developed tumors at their original sites within 1-2 wk(23).  This demonstrates 
that, despite their clinical regression, all mice bearing regressed tumors harbor residual 
tumor cells in which growth can be reactivated.  
Notably, in MTB/TAN mice maintained off doxycycline for extended periods of 
time following primary tumor regression, spontaneous recurrences arose with stochastic 
kinetics(20).  Moreover, recurrent tumors grew at comparable rates regardless of 
whether they arose relatively early (i.e., 50 days) or late (i.e., 200-300 days) following 
tumor regression.  Similar recurrent tumor growth rates for early or late relapses is also 
observed in breast cancer patients and is not compatible with a constant growth model 
for surviving residual tumor cells.  Rather, this suggests that residual tumor cells may 
exist in a dormant state prior to recurrent tumor outgrowth.   
3.2.3 
Regressed tumors harbor residual neoplastic cells that are 
dormant.   
A particularly valuable aspect of this model is the ability to identify and analyze 
residual tumor cells, which in turn enables elucidation of the mechanisms by which they 
survive, as well as determination of the impact of therapeutic interventions on this 
critical reservoir of cells.  By comparing previously tumor-bearing and non-tumor-
bearing mammary glands from mice harboring fully regressed tumors, we identified a 
histopathological lesion that is found only in glands that previously harbored a 
mammary tumor.  Morphologically identical residual lesions were generated by 
orthotopically injecting cultured primary tumor cells from MTB/TAN mice into wild-type 
recipients maintained on doxycycline, allowing tumors to form, and then abrogating 
oncogene expression by doxycycline withdrawal.  Furthermore, orthotopic residual 
lesions formed from GFP-labeled tumor cells, or the use of cre-activated ROSA-YFP 
reporter mice in combination with GEM tumor models, permit visualization of residual 
tumor cells in intact mice following tumor regression as well as their unambiguous 
identification, isolation and analysis. 
To address directly whether residual tumor cells exist in a state of cellular 
dormancy, we performed BrdU labeling on tumor cells either within primary tumors in 
MTB/Tet-Onc mice maintained on doxycycline, or in residual lesions generated by 
oncogene down-regulation in tumor-bearing mice.  While primary tumors exhibited high 
rates of proliferation, tumor cells within residual lesions were quiescent and exhibited 
lower proliferation rates than those observed in the adult mammary gland, which is itself 
a relatively quiescent tissue.   
The concept of tumor dormancy implies that cells are capable of being “re-
awakened”, whereby they reactivate growth.  This contrasts with other possible 
explanations for low proliferation rates in residual tumor cells, such as terminal 
differentiation or senescence(35).  To determine whether non-proliferating residual 
tumor cells are capable of re-entering the cell cycle, we re-administered doxycycline to 
MTB/TAN or MTB/TWNT mice bearing residual lesions and labeled animals with BrdU.  
Oncogene reactivation for 48 hours resulted in high rates of tumor cell proliferation in 
residual lesions, demonstrating that residual tumor cells are reversibly growth-arrested 
and competent to re-enter the cell cycle.  These data strongly suggest that residual 

 
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tumor cells in these models exist in a state of cellular dormancy.  This provides a 
valuable in vivo model for tumor dormancy and provides a preclinical platform for testing 
our hypothesis that agents that target survival mechanisms of dormant tumor cells will 
result in a reduction in both minimal residual disease burden and risk of tumor 
recurrence. 
3.2.4 
GEM models for local recurrence provide insight into mechanisms 
of distant recurrence.   
While local recurrence is itself a critical problem to understand, more than two-
thirds of recurrent breast cancers in women occur at a distant site.  A growing body of 
evidence suggests that the processes by which tumor cells – either local or distant – 
survive in a dormant state and ultimately re-establish malignant growth may be related 
mechanistically.  Consistent with this possibility, local recurrence is strongly associated 
with an increased risk of distant relapse and mortality(25-28).  Moreover, the timing of 
local and distant tumor relapse following surgery are similar(36).  Using the above GEM 
models for tumor dormancy and local recurrence, we have identified several pathways 
that are required for mammary tumor recurrence in mice and that are also strongly 
associated with the risk of distant relapse in breast cancer patients.  These include our 
findings that activation of the Notch(32) or c-MET(34) signaling pathways, up-regulation 
of Snail(20), or down-regulation of the tumor suppressor Par-4(33), each promote local 
recurrence in mice.  Importantly, interrogation of gene expression data sets 
representing >4,000 primary human breast cancers revealed that elevated Notch 
activity, c-MET activity and Snail expression – and decreased Par-4 expression – each 
predict an increased risk of distant relapse in patients(20, 32-34).  These and other 
observations demonstrate that GEM models for local recurrence are informative for 
mechanisms underlying distant, as well as local, relapse in patients. 
3.2.5 
Preclinical studies identify autophagy and mTOR as critical 
vulnerabilities of dormant tumor cells.   
As described below, interrogation of our GEM models has led to the identification 
of autophagy and mTOR pathway activity as key mechanisms of dormant tumor cell 
survival and preclinical studies have demonstrated that targeted pharmacological 
inhibition of autophagy or mTOR depletes the reservoir of dormant residual tumor cells 
in mice and prevents tumor recurrence. 
Autophagy inhibition prevents breast cancer recurrence by impairing the survival 
of dormant residual tumor cells.  We tested the role of autophagy in dormant tumor cell 
survival in the above GEM models.  Using MTB/TAN mammary tumor cells stably 
expressing the autophagy marker eGFP-LC3, we demonstrated that HER2 inhibition in 
vitro results in the induction of autophagy in residual tumor cells, as revealed by 
increased levels of endogenous LC3-II, decreased levels of p62, an increased number 
of double-membraned autophagosomes observed by electron microscopy, and 
relocalization of LC3 to autophagosomes.  Importantly, fluorescence microscopy 
performed on mice harboring fully regressed eGFP-LC3-labeled orthotopic tumors 
revealed an increase in the number of autophagic eGFP-positive punctae per tumor cell 
compared to actively growing primary tumors.  These results suggest that dormant 
residual tumor cells undergo autophagy in vivo. 

 
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To determine whether autophagy functionally contributes to mammary tumor 
recurrence, primary MTB/TAN tumor cells were generated expressing shRNAs targeting 
either Atg5 or Atg7, each of which are required for autophagy.  These cells were then 
injected orthotopically into the mammary glands of nu/nu mice on doxycycline, as were 
cells transduced with a vector control, to form primary tumors.  Doxycycline was 
withdrawn to induce HER2/neu down-regulation and regression of tumors to a non-
palpable state.  Mice were then monitored for recurrence.  This revealed that genetic 
inhibition of autophagy by knocking down either Atg5 or Atg7 dramatically impaired 
tumor recurrence (HR 5.35, 95% CI 1.72-16.62, p = 0.004; HR 7.56, 95% CI 2.33-24.58, 
p < 0.001, respectively).  To extend these results, we determined the effects of Beclin 1 
loss on mammary tumor recurrence, since Beclin 1 is required for formation of the 
autophagosome.  We crossed Beclin 1+/- mice to MTB/TAN mice to generate cohorts of 
MTB/TAN;Beclin 1+/+ and MTB/TAN;Beclin 1+/- mice. Primary mammary tumors were 
induced by activation of HER2/neu via doxycycline administration.  The incidence, 
latency, multiplicity and growth rate of primary HER2/neu-induced tumors did not differ 
between Beclin 1+/+ and Beclin 1+/- mice.  Tumor-bearing mice were then deinduced 
and mice bearing fully regressed tumors were monitored for recurrence.  Deletion of 
one allele of Beclin 1 markedly delayed tumor recurrence with the median latency for 
tumor recurrence increasing from 77 days to 131 days (HR 2.38, 95% CI 1.15-4.94, p = 
0.019).  In aggregate, these results strongly suggest that autophagy is required for 
tumor recurrence. 
To address the cellular basis for this effect, we next asked what stage(s) of 
mammary tumor recurrence requires autophagy.  MTB/TAN primary tumor cells 
expressing an shRNA targeting Atg5 were labeled with an H2B-mCherry reporter.  
Control MTB/TAN primary tumor cells transduced with an empty vector were labeled 
with an H2B-eGFP reporter.  These two isogenic fluorescent cell populations were 
admixed in equal parts, injected into the mammary glands of nu/nu mice maintained on 
doxycycline, and allowed to generate orthotopic primary tumors.  Doxycycline was then 
withdrawn to initiate tumor regression and fluorescence microscopy was used to 
determine the ratio of mCherry-labeled Atg5 knockdown cells to eGFP-labeled control 
cells in primary tumors, residual tumor lesions 14 d or 28 d following HER2/neu down-
regulation, and recurrent tumors.  As predicted based on the unperturbed kinetics of 
primary tumorigenesis in Beclin 1+/- mice, we observed no selection for or against cells 
with Atg5 knockdown in primary tumors or residual lesions 14 d post-HER2/neu down-
regulation, a time point roughly corresponding to the completion of tumor regression 
and onset of tumor dormancy.  In contrast, Atg5 knockdown cells were markedly 
selected against in residual lesions 28 d following HER2/neu down-regulation, a time 
point at which residual tumor cells have been in a dormant state for ~2 wk.  This 
competitive disadvantage was even more pronounced in recurrent tumors, in which only 
1% of tumor cells were mCherry-positive.  Together, these data indicate that residual 
tumor cells in which autophagy is impaired are at a strong, cell-intrinsic selective 
disadvantage once they enter the dormant state.   

 
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Our findings to this point suggested that genetic inhibition of autophagy impairs 
dormant tumor cell survival.  To determine whether this was true for pharmacological 
inhibition of autophagy, we tested whether the anti-malarial drug chloroquine (CQ) – a 
known autophagy inhibitor – would phenocopy these effects and demonstrate activity 
against dormant tumor cells.  Consistent with this possibility, dormant MTB/TAN tumor 
cells maintained in the absence of HER2/neu expression in vitro were markedly more 
sensitive to chloroquine treatment than proliferating cells, suggesting that dormant 
tumor cells are preferentially dependent upon autophagy for survival.  We next tested 
whether dormant mammary tumors cells in vivo are dependent upon autophagy for 
survival.  Mice harboring orthotopic eGFP-labeled dormant residual MTB/TAN tumor 
cells were treated with CQ for 2 wks beginning 21 days after HER2/neu down-
regulation, at which time tumor cells are dormant.  Mammary glands were then 
harvested, digested to form a single-cell suspension, and the number of eGFP-positive 
residual tumor cells was determined by flow cytometry.  This analysis revealed that 
mice treated with CQ for 2 wks within the period of dormancy harbored ~40% fewer 
dormant tumor cells (p < 0.05) than mice treated with a vehicle control (Fig. 2A).  Given 
the absence of tumor cell proliferation at these time points, these data strongly suggest 
that autophagy is required for the survival of dormant mammary tumor cells in vivo. 
Our findings to this point suggested that dormant tumor cells might be uniquely 
dependent upon autophagy for their survival.  If correct, our data would predict that 
inhibitors of autophagy would be more effective against dormant residual tumor cells 
than actively growing tumors.  If so, CQ could potentially be used to reduce the pool of 
dormant residual tumor cells and thereby prevent or delay tumor recurrence.  To test 
this hypothesis, CQ treatment was initiated in mice bearing dormant residual disease 28 
d following HER2/neu down-regulation in orthotopic tumors.  Mice were then monitored 
for tumor recurrence.  This revealed that autophagy inhibition Urestricted specifically to 
the period of tumor dormancyU markedly delayed tumor recurrence (H.R. = 2.88, 95% CI 
1.39-5.98, p = 0.005) (Fig. 2B).  Notably, the magnitude of the effect of CQ 
administration beginning 28 d after HER2/neu down-regulation was nearly identical to 
that observed for CQ administration beginning immediately at the time of HER2/neu 
down-regulation.  This finding was consistent with results from the fluorescent cell 
competition assay indicating that cells with impaired autophagy are not selected against 
during primary tumor formation or within the first 14 d following HER2/neu down-
regulation, during which time the vast majority of tumor cells are eliminated.  This 
 
Fig. 2: (A, B) Transient chloroquine administration reduces the number of surviving dormant residual 
tumor cells (A), and prevents spontaneous mammary tumor recurrence when administered to mice bearing dormant 
minimal residual disease (B).  (C, D) Rapamycin prevents spontaneous mammary tumor recurrence when 
administered to mice bearing dormant minimal residual disease (C), and transient administration reduces the 
number of surviving dormant residual tumor cells (D). 

 
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finding was also consistent with our observations that CQ did not affect the rate of 
growth of primary or recurrent tumors.  Together, these observations provide further 
support for a model in which autophagy is required for mammary tumor recurrence and 
in which the effects of autophagy inhibition on tumor cell survival are largely confined to 
the dormant state. 
In summary, using genetic and pharmacological approaches, we have 
demonstrated that inhibition of autophagy by CQ administration, Atg5 or Atg7 
knockdown, or deletion of one allele of Beclin 1, is sufficient to delay or prevent 
spontaneous tumor recurrence in mice bearing dormant minimal residual disease.  The 
requirement for autophagy during tumor recurrence is attributable to its ability to serve 
as a survival mechanism for dormant residual tumor cells.  Notably, we found that 
dormant mammary tumor cells are uniquely reliant upon autophagy for their survival 
compared to actively growing primary or recurrent tumor cells.  Consistent with this 
cellular mechanism of action, pharmacological inhibition of autophagy in mice bearing 
dormant minimal residual disease significantly decreased the number of surviving 
dormant tumor cells and substantially delayed mammary tumor recurrence.  As CQ and 
hydroxychloroquine (HCQ) have been used safely in millions of people worldwide for 
the prevention and treatment of malaria and have a favorable therapeutic index, these 
drugs represent an attractive approach to inhibiting autophagy in vivo. 
3.2.6 
mTOR inhibition prevents breast cancer recurrence by impairing 
the survival of dormant residual tumor cells.   
Based on our findings that autophagy inhibition delays mammary tumor 
recurrence, coupled with the fact that rapamycin induces autophagy by inhibiting the 
mTORC1 complex, we hypothesized that rapamycin treatment would increase the 
number of dormant mammary tumor cells surviving HER2/neu down-regulation and 
thereby accelerate tumor recurrence.  To determine the effect of rapamycin treatment 
on recurrence, eGFP-labeled MTB/TAN primary tumor cells were injected orthotopically 
into nu/nu mice maintained on doxycycline.  Once primary tumors formed, HER2/neu 
was down-regulated to induce tumor regression.  Treatment with 4 mg/kg rapamycin or 
vehicle control began at 28 d post-deinduction, when primary tumors had regressed to a 
non-palpable state and a small population of dormant residual tumor cells remains.  
Surprisingly, treatment with rapamycin dramatically delayed the latency of tumor 
recurrence compared to treatment with a vehicle control (HR 23.26, 95% CI 8.06-67.19, 
p < 0.0001) (Fig. 2C).   
Although the finding that rapamycin delays recurrence was unexpected, given 
the magnitude of this effect we sought to determine its cellular mechanism.  To address 
the possibility that rapamycin delayed the time to detection of recurrent tumors by 
decreasing their growth rate, rapamycin or vehicle was administered to mice harboring 
orthotopic MTB/TAN recurrent tumors.  The mean growth rate of recurrent tumors was 
not affected by treatment with rapamycin at any time point examined, suggesting that 
actively growing MTB/TAN recurrent tumors are not affected by rapamycin treatment. 
Therefore, we next asked whether rapamycin delayed mammary tumor 
recurrence by decreasing the survival of dormant tumor cells in vivo.  To do so, we 
treated mice bearing dormant residual disease with rapamycin or vehicle control for 2 
wk.  Mammary glands from these mice were then harvested, digested to form a single 

 
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cell suspension, and the number of eGFP-labeled dormant tumor cells was determined 
using flow cytometry.  After 2 wk of treatment, rapamycin resulted in a 30% decrease in 
the number of dormant residual cells present compared to vehicle control (Fig. 2D).  
Since dormant MTB/TAN tumor cells exist in the absence of proliferation, the decrease 
in dormant tumor cell number observed following rapamycin treatment suggests that 
mTOR activity is required for the survival of dormant tumor cells.   
We next wished to determine if transient rapamycin treatment would exert 
persistent effects, as would be predicted if it reduces the number of dormant residual 
tumor cells.  Therefore, we treated mice that harbored dormant residual disease with 
rapamycin for 2 wk.  Treatment was then stopped and the formation of recurrent tumors 
was monitored.  As observed with chronic rapamycin treatment, treatment with 
rapamycin for 2 wk significantly delayed tumor recurrence (H.R. = 2.29, 95% CI 1.27-
5.17, p = 0.0088).  This was consistent with our findings above that rapamycin treatment 
decreases the survival of dormant tumor cells.  Together, these results strongly suggest 
that mTOR pathway inhibition by rapamycin potently prevents mammary tumor 
recurrence by impairing the survival of dormant residual mammary tumor cells. 
3.3 
Summary: Targeting dormancy with mTOR and autophagy inhibition.   
Most anti-neoplastic therapeutic modalities preferentially target actively 
proliferating cancer cells.  In light of our identification of cellular dormancy as a stage of 
tumor progression that is particularly reliant upon both autophagy and mTOR pathway 
activity for survival, our findings suggest that dormant residual tumor cells may be 
uniquely susceptible to agents such as HCQ and rapamycin analogues that inhibit these 
pathways.  To date however, pre-clinical and clinical trials have principally, if not 
exclusively, employed autophagy inhibitors and mTOR inhibitors to target actively 
proliferating stages of disease, which our findings predict may be intrinsically resistant 
to autophagy inhibition.  Accordingly, our results suggest that autophagy and/or mTOR 
inhibition in clinical contexts characterized by tumor dormancy may be more effective, 
and support autophagy and mTOR inhibition as tractable therapeutic targets for 
preventing breast cancer recurrence by depleting the reservoir of residual tumor cells. 
3.3.1 
Detection of minimal residual disease in bone marrow.   
IHC analysis for CK+ cells in the bone marrow is currently the “gold standard” for 
identifying patients with DTCs following therapy and this assay is clinically validated 
insofar as it is strongly associated with risk of relapse.  However, there are at least four 
major shortcomings of this assay: 1) it is likely to be relatively insensitive due to cell loss 
during the cell isolation phase of the assay; 2) it is likely to be relatively insensitive due 
to the inherently lower sensitivity of IHC compared to flow cytometry; 3) only CK+ cells 
are assayed for, neglecting the possibility that some DTCs may not express classical 
epithelial markers, or may express them at low levels below the limit of detection for 
IHC; and 4) there is limited ability to molecularly interrogate isolated cell populations of 
interest.  Since bone marrow DTCs are strongly associated with risk of recurrent 
disease, and little is known about the phenotype of bone marrow DTCs in breast cancer 
patients, we have pursued proof-of-principle approaches in mice and human bone 
marrow samples to optimize approaches for detecting and isolating small numbers of 
DTCs with the ultimate goal of more sensitive and specific detection, improved 

 
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quantitative enumeration and the ability to interrogate DTC phenotypic and genetic 
properties.   
To accomplish this, we have used a new in-line magnetic depletion and acoustic 
focusing system, the BD Biosciences FACSFocus that, combined with the BD Influx cell 
sorter, allows a streamlined workflow for detection and isolation of rare events to high 
purity.  The Abramson Cancer Center Flow Cytometry and Cell Sorting Shared 
Resource has worked closely with BD Biosciences in the development of this system, 
which is currently available exclusively at Penn.  The system consists of a magnetic cell 
separation whereby cells stained with CD45 BD IMag™ reagent are passed by a 
magnet placed in line with the sample injection tubing on the Influx, resulting in 
depletion of unwanted CD45+ cells from the marrow sample.  Upon leaving the magnet, 
the sample then flows to a microfluidic chip with inlets for sample and wash buffer, and 
outlets for waste and sample.  Vibrating at ~2 MHz, the chip acts as an acoustic 
focuser, simultaneously washing and concentrating the sample about 4-5 fold.  After 
leaving the chip, the sample flows to the nozzle of the Influx, with electronics optimized 
for cell recovery.  The system permits streamlined sample preparation, with minimal cell 
loss, and no centrifugation if directly conjugated antibodies are used.  Target cell 
recovery is on the order of 60-80%.  To test this approach in our GEM models, we 
crossed MTB/TAN mice with TetO-TurboCre (TTC);Rosa26-lox-stop-lox-YFP (rYFP) 
mice to generate MTB/TAN;TTC;rYFP mice in which doxycycline administration results 
in inducible expression of both HER2/neu and the cre recombinase in mammary 
epithelial cells.  Consequently, doxycycline treated mice harbor fluorescently-labeled 
HER2/neu-induced mammary adenocarcinomas due to cre-mediated excision of the 
stop cassette preceding rYFP.  Fluorescent primary tumors from doxycycline-induced 
MTB/TAN;TTC;rYFP mice were enzymatically dissociated and injected orthotopically 
into nu/nu mice on doxycycline to permit primary tumor outgrowth.  Bone marrow cell 
suspensions were prepared from tumor-bearing mice by flushing the marrow cavity of 
tibiae and femurs, while single cell suspensions were prepared from primary tumors, 
lung and other tissues by enzymatic digestion and mechanical dissociation.  Cell 
suspensions were stained with an APC-conjugated anti-CD45 antibody, followed by 
incubation with anti-APC magnetic particles (BD IMag).  DAPI was used to identify 
live/dead cell populations.  Primary tumor and DTC populations were isolated by FACS 
using the BD Influx cell sorter, in combination with novel in-line pre-enrichment 
technology (BD FACSFocus).  
Viable DTCs were identified as YFP+/CD45-/DAPI-.  Primary tumor cells and 
CD45P+
P hematopoietic cells were isolated as controls.  RNA was isolated, whole 
transcriptome amplification (WTA) performed, and expression of selected genes was 
examined by qPCR using TaqMan Gene Expression Assays.  This analysis revealed 
that, as expected, DTCs isolated from mouse bone marrow or lung, as well as primary 
tumor cells, express YFP but are negative for CD45 expression (Fig. 3A).  Conversely, 
isolated CD45+ cells were positive for CD45 expression, but negative for YFP 
expression (Fig. 3B).  These results confirm our ability to identify and isolate small 
numbers of DTCs in bone marrow and tissues of mice.  Additional experiments have 
confirmed our ability to perform WTA and whole genome amplification on as few as 10 
cells, as well as perform expression profiling and genomic testing for 47 oncogenic 

 
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mutations occurring in human cancers. 
To pilot this approach in patient samples, we spiked MCF-7 breast cancer cells 
into normal human bone marrow, performed RBC lysis, CD45 magnetic depletion, 
staining for EpCAM and HER2, and analyzed on the FACSFocus sorter.  This revealed 
the ability to detect spiked human breast cancer cells as EpCAM+/HER2+ and CD45– 
(Fig. 3C). 
3.4 
Hydroxychloroquine (HCQ) and cancer therapy 
Chloroquine (CQ) is a synthetic 4-aminoquinoline that has been used for 60 
years in humans for malaria prophylaxis and treatment(58), rheumatoid arthritis(59), 
and human immunodeficiency virus (HIV)(60). It is an inexpensive orally available drug 
that has CNS penetration. It has a large therapeutic index, and its most predictable 
cumulative toxicity is retinopathy, which can be prevented by discontinuation of the 
drug(61).  It is this toxicity and worldwide malarial resistance to CQ that lead to 
discontinuation of extensive research into CQ’s non-malarial applications.  Chloroquine 
derivatives such as HCQ are still used extensively in rheumatoid arthritis and lupus 
erythematosis and have a larger therapeutic index.  The chemical structure of CQ 
derivatives allows them to serve as a weak base which is trapped in acidic cellular 
compartments(62). Thus chloroquine deacidifies lysosomes, inhibiting the last step in 
autophagy. With this last step blocked, a cell reliant on autophagy will increase the 
generation of autophagosomes and will eventually undergo either apoptotic or non-
apoptotic cell death. Evidence in mouse models and human cancer cell lines suggest 
CQ may have significant anti-tumor activity by inhibiting autophagy induced by cancer 
therapy(46).  
Adding chloroquine to improve the efficacy of anticancer therapy has already 
been tested in a randomized clinical trial. A small single institution placebo controlled 
phase III trial testing the addition of CQ at an oral daily dose of 150 mg to RT and 
carmustine in patients with newly diagnosed glioblastoma multiforme (GBM) yielded 
surprising results(49). Median overall survival was significantly longer in the CQ-treated 
patients (24 months) than in controls (11 months). At the end of the observation period, 
six patients (40%) treated with CQ were alive at 59, 45, 30, 20 (1 each) and 27 (2 
patients) months after surgery. In contrast, patients in the control group survived 32, 25, 
and 22 months. Although not statistically significantly different, the rate of death over 
time was almost half as large in the CQ group compared to the placebo group (hazard 
ratio, 0.52 [95% CI, 0.21 to 1.26]; p= 0.139). 
Hydroxychloroquine (HCQ) is commonly prescribed for rheumatoid arthritis and 
lupus at a dose of 400 mg po daily. A pharmacokinetic/pharmacodynamic study of 
escalating doses of HCQ at 400 mg/800 mg/1200 mg po daily in patients with 
rheumatoid arthritis followed by maintenance doses of 400 mg po daily found that doses 
of up to 1200 mg po daily were well tolerated(63). Dose limiting toxicities of nausea, 
vomiting and abdominal pain were observed at 800 and 1200 mg po daily. This toxicity 
correlated with blood HCQ levels, but not with blood levels of the other active 
metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), or 
bisdesethylchloroquine (BDCQ).  Improvement of symptoms in rheumatoid arthritis 
correlated with blood DHCQ levels, suggesting a dose-response relationship. 
Chloroquine derivatives are metabolized through the p450 enzyme system and CQ may 

 
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inhibit the metabolism of CYP2D6-metabolized drugs(64, 65).  
A predictable cumulative toxicity associated with CQ is retinopathy, and this is 
another reason why dose escalation with CQ would be limited.  While a link between 
HCQ and retinopathy has also been made, it occurs infrequently and only after a 
prolonged exposure. A study using multifocal electroretinography to detect early pre-
clinical retinal changes in long-term HCQ users found that 10 out of 11 patients that 
developed early pre-clinical changes had been taking HCQ at doses of 400 mg po daily 
for greater than 5 years.(66) No overt retinopathy was noted in the 19 patients followed.  
This suggests that at a cumulative dose of 730 g, the risk of retinal changes increases, 
but techniques such as multifocal electroretinography can detect early changes and 
prevent overt visual loss. 
3.4.1 
Trials of autophagy inhibition in cancer 
Autophagy is an intracellular process characterized by the formation of 
autophagic vesicles (AV) that sequester cytoplasmic contents and target them for 
degradation in lysosomes. This process is activated by metabolic stress and also by 
most cancer therapies (39, 45). Autophagy is used by cancer cells to remove damaged 
organelles and recycle macromolecules that serve as an internal reservoir of fuel in the 
face of a crisis of nutrient availability.  Inhibition of mTOR is one of the most potent 
inducers of autophagy. Therapeutic activation of autophagy may be a key resistance 
mechanism to mTOR inhibition with Everolimus (EVE, RAD001). Dr. Amaravadi has 
reported in vivo studies that demonstrate that inhibition of therapy-induced autophagy 
with chloroquine derivatives enhances cell death and tumor regression (46)(5). While 
these studies combined chemotherapy with hydroxychloroquine (HCQ), Dr. Amaravadi 
has unpublished data that rapamycin combined with HCQ results in enhanced cell 
death compared to rapamycin or HCQ alone. Dr. Eileen White at UMDNJ has 
compelling evidence of synergistic cell death when CCI-779 is combined with HCQ in 
renal cell carcinoma cell lines and in an orthotopic mouse model of renal cell 
carcinoma(39) (unpublished data presented at CTEP early drug discovery meeting 
2010). A number of other investigators have found that cell death induced by 
therapeutic interruption of growth factor cell signaling is enhanced by combination with 
HCQ (47, 48)(6-9). There are now 14 clinical trials registered with the NCI combining 
HCQ with approved or investigational anticancer agents. Investigators from multiple 
disciplines within cancer therapy research have become convinced that autophagy is 
the next important target in cancer therapy (39), and this has lead to the formation of an 
Autophagy in Clinical Cancer Consortium which will be conducting some of the 
correlative studies that are common to many of the HCQ trials.    
There are 5 phase I/II clinical trials through the University of Pennsylvania 
involving HCQ. The most mature trial is a 15 center phase I/II trial of 
temozolomide/radiation/and HCQ in patients with newly diagnosed (adjuvant) GBM.  
This first HCQ trial was launched in 2007 after a publication of a randomized phase III 
trial that demonstrated a trend towards doubling of overall survival in GBM patients 
treated with radiation chemotherapy and chloroquine compared to patients treated with 
radiation and chemotherapy alone in the adjuvant setting(49).  We have reported the 
results of our phase I/II trial in GBM at ASCO 2010 annual meeting. We found an MTD 
for HCQ in this combination, and also found a PK-PD interaction that demonstrated 

 
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effective HCQ-dose-dependent autophagy inhibition in patients (50).  The phase II 
portion of the trial has completed accrual of 76 patients in 4 months.   
In addition to this trial there is a phase I trial of temsirolimus and HCQ in patients 
with advanced solid tumors. This trial is partially sponsored by Wyeth/Pfizer. Although 
preliminary data cannot be included here, they are encouraging and support the 
preclinical findings.  
3.5 
Everolimus (EVE, RAD001) 
Everolimus is a novel oral derivative of rapamycin. Everolimus has been in 
clinical development since 1996 as an immunosuppressant in solid organ 
transplantation and has obtained marketing authorization (Certican P®
P) for prophylaxis of 
rejection in renal and cardiac transplantation in a number of countries, including the 
majority of the European Union. Everolimus has been in development for patients with 
various malignancies since 2002. Everolimus is being investigated as an anticancer 
agent based on its potential to inhibit tumor cell growth, proliferation, and angiogenesis. 
At weekly and daily schedules and at various doses explored, RAD0001 is generally 
well tolerated. The most frequent adverse events (rash, mucositis, fatigue and 
headache) associated with Everolimus therapy are manageable. Non-infectious 
pneumonitis has been reported with mTOR inhibitors but is commonly low-grade and 
reversible. 
3.5.1 
Everolimus mechanism of action: mTOR inhibition  
Everolimus selectively inhibits mTOR a highly conserved serine-threonine 
kinase, which is present in all cells and is a central regulator of protein synthesis and 
ultimately cell growth, cell proliferation, angiogenesis and cell survival. TOR is the only 
currently known target of everolimus (51).TOR is downstream of PI3K/AKT pathway, a 
pathway known to be dysregulated in a wide spectrum of human cancers. In addition, 
activation of the PI3K/AKT/mTOR pathway is frequently a characteristic of worsening 
prognosis through increased aggressiveness, resistance to treatment and progression.  
The main known functions of mTOR include the following (51, 52) 1) mTOR 
functions as a sensor of mitogens, growth factors and energy and nutrient levels, 
facilitating cell-cycle progression from G1 to S phase in appropriate growth conditions; 
2) Through inactivating eukaryotic initiation factor 4E binding proteins (4E-BP1) and 
activating the 40S ribosomal S6 kinases (i.e., p70S6K1), mTOR regulates translation of 
important massages, including those encoding the HIF-1 proteins, c-myc, ornithine 
decarboxylase, and cyclin D1, as well as ribosomal proteins themselves; 3) The 
activation of mTOR pathway leads to the increased production of pro-angiogenic factors 
(i.e., VEGF) in tumors and to tumor, endothelial and smooth muscle cell growth and 
proliferation; 4) The regulation of mTOR signaling is complex and involves positive 
regulators, such as AKT that phosphorylate and inactivate negative regulators such as 
the Tuberous Sclerosis Complex (TSC1/TSC2). 
Recent evidence indicates that mTOR is represented by two distinct multiprotein 
signaling complexes, mTORC1 (mTOR complex 1, rapamycin sensitive) and mTORC2 
(mTOR complex 2, rapamycin insensitive)(53). mTORC1 is mainly activated via the PI3 
kinase pathway through AKT (also known as PKB, protein kinase B) and the tuberous 
sclerosis complex (TSC1/TSC2) (52). Activated AKT phosphorylates TSC2, which lead 

 
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to the dissociation of TSC1/TSC2 complex, thus inhibiting the ability of TSC2 to act as a 
GTPase activating protein. This allows Rheb, a small G-protein, to remain in a GTP 
bound state and to activate mTORC1. AKT can also activate mTORC1 by PRAS40 
phosphorylation, thereby relieving the PRAS40-mediated inhibition of mTORC1 (54). 
The second complex, mTORC2 (mTOR complex 2) is activated through a currently 
unknown mechanism, possibly by receptor tyrosine kinase (RTK) signaling. It has been 
suggested that mTORC2 phosphorylates and activates a different pool of AKT that is 
not upstream of mTORC1. PHLPP phosphatase plays a role of a negative regulator. 
mTORC2 is rapamycin insensitive and is required for the organization of the actin 
cytoskeleton. 
The macrocyclic lactone rapamycin and its derivatives such as Everolimus 
modulates mTORC1-mediated signaling by binding to the 12 kDa cytosolic FK506-
binding protein immunophilin FKBP12, the resulting rapamycin-FKBP12 complexes bind 
to a specific site near the catalytic domain of mTORC1 and inhibit phosphorylation of 
mTOR substrates. As a consequence, downstream signaling events involved in 
regulation of the G1 to S-phase transition are inhibited. This mechanism is thought to be 
responsible for the immunosuppressive effects of rapamycin as well as its putative 
antineoplastic activity (55). As many cancers are characterized by dysregulation of G1 
transit (for example, overexpression of cyclin or cyclin-dependent kinases), inhibition of 
mTOR becomes an intriguing target for cancer therapy (52).  
 
3.5.2 
Everolimus Pharmacokinetics (PK) 
Everolimus is rapidly absorbed with a median t RRmaxRR of 1-2 hours. The 
bioavailability of the drug is believed to be 11% or greater. The AUC RR0-τRR is dose-
proportional over the dose range between 5 and 70 mg in the weekly regimen and 5 
and 10 mg in the daily regimen. C RRmaxRR is dose-proportional between 5 and 10 mg for 
both the weekly and daily regimens. At doses of 20 mg/week and higher, the increase in 
CRRmaxRR is less than dose-proportional. The coefficient of variation between patients is 
approximately 50%. Trough levels (24 hour post-dose) correlate well with AUC RR0-τRR at 
steady-state during daily administration. In whole blood, at a daily dose of 10 mg, about 
20% of Everolimus is confined in plasma with 26% being unbound. The remaining 80% 
is sequestered in blood cells.  
Everolimus is extensively metabolized in the liver and eliminated in the bile. 
Major metabolites are inactive. Elimination half-life is approximately 30 hours. The 
clearance of Everolimus is approximately halved in patients with mild-moderate hepatic 
impairment (Child-Pugh Class A or B), while renal impairment has little or no impact on 
the pharmacokinetics of Everolimus. 
Age, weight and gender in the adult population do not affect the 
pharmacokinetics of Everolimus to a clinically relevant extent. The clearance of 
Everolimus is reduced in children. Pharmacokinetic characteristics are not notably 
different between Caucasian and Japanese subjects, whereas in Black patients, 
population pharmacokinetic studies have shown an average 20% higher clearance. 
A high-fat meal altered the absorption of Everolimus with 1.3 hour delay in t RRmaxRR, a 
60% reduction in C RRmaxRR and a 16% reduction in AUC. Everolimus is a substrate of 
CYP3A4 and a substrate and a moderate inhibitor of the multi-drug efflux pump P-

 
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glycoprotein (P-gP, MDR1, ABCB1). Hence, its metabolism is sensitive to drugs that 
modify these enzymes (substrates, inducers, or inhibitors of these enzymes). 
Competitive inhibition could occur when Everolimus is combined with drugs which are 
also CYP3A4 or P-glycoprotein substrates. Table 10 (Section 7.3) lists examples of 
clinically relevant CYP3A inhibitors and inducers. Please refer to Section 7.3 for more 
information on the concomitant use of CYP3A4 inhibitors/inducers and other 
medications. More information on Everolimus pharmacokinetics is provided in the 
[Investigator’s Brochure]. 
3.5.3 
Everolimus pharmacodynamic studies 
PK/pharmacodynamic modeling based on inhibition of the biomarker p70S6 
kinase 1 [S6K1] in peripheral blood mononuclear cells [PBMC] suggests that 5-10 mg 
daily should be an adequate dose to produce a high-degree of sustained target 
inhibition ([Study C2101] / [Study 2102], Lane, et al 2003). Furthermore, molecular 
pharmacodynamic (MPD) studies, using immunocytochemistry (IHC) in biopsied tumor 
tissue, assessed the degree of inhibition and its duration for pS6, p4E-BP1 and pAKT 
expression with the daily and weekly dosing. There was high inhibition of the 
downstream markers S6K1 and 4E-BP1 at 5 mg/day, which was complete at 10 
mg/day, while preliminary results suggest increase in pAKT expression with maximal 
effect at 10 mg daily ([Study C2107]).  More information is provided in the [Investigator’s 
Brochure]. 
3.5.4 
Clinical trials of Everolimus in cancer patients  
Everolimus has been in development for patients with cancer since 2002. 
Everolimus (Afinitor®) was approved by the United States Food and Drug 
Administration in March 2009 for the treatment of advanced renal cell carcinoma (RCC) 
after failure of treatment with sunitinib or sorafenib. In 2010, Afinitor® was received 
United States (US) approval for the treatment of 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 treatment of postmenopausal women with advanced 
hormone receptor-positive, HER2- negative breast cancer (advanced HR+ BC) 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. In the US, Afinitor® 
was approved in 2016 for advanced non-functional NET of gastrointestinal (GI) or lung 
origin. 
 Approximately 112,317 cancer patients have been enrolled in studies with everolimus as 
of 
 
31-Mar-2017(this 
number 
excludes 
patients 
who 
received 
marketed 
Afinitor®/Votubia®/Afinitor DISPERZ, those on planned and roll over studies as well as 
those on investigator-sponsored studies: 
 110,217  patients in Novartis-sponsored clinical trials 

 
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 2,100 patients in the individual patient supply program 
Approximately 47,349 patients were enrolled globally in all investigator-sponsored studies 
as of 31-Mar-2017. 
 
Eight single-agent Novartis sponsored trials have or are being conducted in 
various advanced malignancies. Five Phase I studies evaluated several escalating 
doses with either weekly or daily administration (Studies C2101/02, C2106, C2107, 
C1101) of Everolimus determined that the 10 mg/day and 50-70 mg/week dosages 
were proposed for further studies, when using Everolimus as a single agent, and as a 
target maximum dose in combination studies. Two Phase II monotherapy studies were 
designed to evaluate the safety and efficacy of a single dose of 10 mg administered 
daily including Study C2235 in advanced NSCLC (n=81) and Study C2239 in advanced 
pancreatic neuroendocrine tumors (n=160). 
A Phase III, randomized, double blind, placebo controlled study in patients with 
mRCC who progressed on a VEGFr TKI demonstrated that everolimus 10 mg daily 
(Motzer, 2008) prolonged median PFS from 1.87 months for patients receiving placebo 
to 4.01 months for everolimus-treated patients, assessed by central independent review 
blinded to clinical data (hazard ratio 0.30, 95% CI 0.22-0.40, p<0·0001)(56). Updated 
results presented at the American Society of Clinical Oncology 2009 Genitourinary 
Cancers Symposium (Kay et al, 2009) demonstrated everolimus as having even greater 
superiority to placebo in the primary endpoint of PFS. Median PFS was prolonged from 
1.9 months for patients receiving placebo 
to 4.9 months for everolimus-treated 
patients, assessed by central 
independent review blinded to clinical data (hazard ratio 0.33, 95% CI 0.25-0.43, 
Table 1:  Adverse Events for Combination HCQ/EVE 
in Advanced Renal Cell Carcinoma 

 
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p<0.001)(57).  
Overall, the most frequent adverse effects suspected to be related to Everolimus 
have been stomatitis, rash, anemia, fatigue, asthenia, diarrhea, anorexia, nausea, 
hypercholesterolemia, mucosal inflammation, vomiting, hypertriglyceridemia, cough, 
peripheral edema, dry skin, epistaxis, pruritus and dyspnea. The most common Grade 3 
or 4 adverse reactions suspected to be related to treatment were anemia, infections, 
hyperglycemia, stomatitis, fatigue, lymphopenia, hypercholesterolemia, pneumonitis, 
and elevated gamma-glutamyl transferase concentrations. 
Non-infectious low-Grade (Grade 1/2) pneumonitis has led to development of 
treatment guidelines for the disorder 
(Table 4). The primary DLT has been 
severe (Grade 3) stomatitis, and 
occasionally fatigue, hyperglycemia, and 
neutropenia. For more information on 
known undesirable effects of Everolimus 
refer to Section 7.2.  
Further detailed information 
regarding Everolimus clinical 
development, safety and efficacy is 
provided in the [Investigator’s Brochure]. 
3.6 
Combination therapy with HCQ 
and EVE 
Translation from mouse to human 
studies required selection of agents for 
inhibition of autophagy and mTOR that 
are safe and specific.  Though 
chloroquine was used in our mouse 
preclinical studies, in humans it produces 
sudden death in infants and severe 
retinopathy. Hydroxychloroquine (HCQ) 
is a safe alternative that is an 
established anti-malarial and anti-
inflammatory used in rheumatoid arthritis 
and lupus erythematosis.  It is orally 
administered and has rapid 
gastrointestinal absorption and renal 
excretion.  Dr. Ravi Amaravadi, (co-investigator on this proposal), has demonstrated 
autophagy inhibition by HCQ by EM and several studies by Amaravadi and others have 
shown that HCQ augments the cytotoxicity of a number of chemotherapies and targeted 
therapiesP54
P.  Thus, HCQ would be expected to inhibit autophagy, thereby killing BM-
DTCs that are relying on autophagy for survival.  Temsirolimus (TEM) and Everolimus 
(EVE) each inhibit mTOR through allosteric binding to mTORC1P55
P.  Further, mTORC1-
ULK1 complex interactions can directly upregulate autophagy, an effect that is blocked 
by suppression of mTORC1 activityP56
P, thus potentially enhancing HCQ effects on 

 
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autophagy inhibition.  We collaborated with Dr. Amaravadi on a phase I trial of the 
TEM/HCQ combination in advanced solid tumors, which also demonstrated autophagy 
inhibitionP57
P.  But while TEM/HCQ was tolerable, EVE has the advantage of an all-oral 
regimen, and is already FDA approved in the metastatic setting P58,59
P. Dr. Amaravadi has 
now completed accrual to a phase I/II trial in advanced renal cell carcinoma 
(NCT01510119), demonstrating the safety and feasibility of the HCQ/EVE combination 
Three patients received HCQ at 400 mg twice daily, followed by an additional 31 
patients who received the target HCQ dose of 600 mg twice daily and concurrent EVE 
at 10 mg daily.  The combination was well tolerated.  The only grade 3/4 AEs were 
anorexia (1 patient), and edema, hyperglycemia and hypertriglyceridemia (each in 2 
patients). 
3.7 
Rationale for the proposed proof-of-concept pilot trial   
The data presented above provide compelling evidence that the source of 
relapse in some breast cancer patients may be cells that have entered a state of 
dormancy, and exist in a state distinct from the primary tumor from which they 
originated, both biologically and with respect to their response to therapies.  Currently, 
there are no adjuvant therapies that specifically target dormancy or take advantage of 
the unique vulnerabilities of minimal residual disease, despite the fact that the vast 
majority of breast cancer patients reside in this state.  The proposed investigations take 
our novel preclinical findings to the critical next stage: redefining the paradigm of 
adjuvant therapy by targeting residual dormant disease with agents specifically 
identified to block key survival mechanisms in these cells, mTOR and autophagy.   
Our preclinical studies in mice showing prevention or delay of recurrence with 
CQ and rapamycin provide a convincing rationale for translating these findings to the 
clinic.  Furthermore, the existence of a clinically validated IHC assay for DTCs to identify 
at-risk patients, our initial work to develop an improved DTC assay, and phase I/II data 
demonstrating the safety and feasibility of treating cancer patients with HCQ and EVE, 
have together laid the groundwork needed to conduct this novel, innovative clinical 
trial.  Our multidisciplinary team will work closely to translate findings bi-directionally 
from concurrent human and mouse studies and, in doing so, will further understanding 
of the biology of dormancy, develop improved methods for identifying and analyzing 
human DTCs, and operationalize our platform to bring the next generation of clinical 
trials targeting dormancy to the clinic.  This approach has the potential to provide the 
“breakthrough” needed to eradicate recurrent breast cancer and provide the tools and 
evidence needed to give every breast cancer patient the ability to be monitored actively 
and to take proactive measures for secondary prevention. 
3.8 
Trial Overview 
To translate findings from our mouse preclinical studies to humans, we 
developed a regimen targeting autophagy and mTOR signaling that is safe, effective 
and feasible in a potentially curative setting.  HCQ is an established anti-malarial that is 
also used in chronic inflammatory diseases such as rheumatoid arthritis, lupus 
erythematosis and others.  It is orally administered and has rapid gastrointestinal 
absorption and elimination by the kidney.  Autophagy inhibition with HCQ has been 
shown to augment the cytotoxicity of a number of chemotherapies and targeted 

 
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therapies(39).  Temsirolimus (TEM) and Everolimus (EVE) are rapamycin derivatives 
that inhibit mTOR through allosteric binding to mTORC1(40).  Further, mTORC1-ULK1 
complex interactions can directly regulate autophagy, an effect that is blocked through 
the suppression of mTORC1 activity(41), thus potentially enhancing the effect of HCQ in 
inhibiting autophagy.  To generate initial safety data and assess target inhibition, we 
performed a phase I trial of TEM and HCQ in advanced solid tumors(42).  Patients 
received oral HCQ at 200 – 1200 mg daily in divided doses and temsirolimus, 25 mg 
intravenously on a weekly schedule.  In the 1200 mg HCQ cohort (n=16), the most 
common grade 3/4 events were nausea (n=4), anorexia (n=2), and dysguesia, elevated 
creatinine, fatigue, anemia and lymphopenia, each in 1 patient.  Other common grade 
1/2 toxicities seen in the overall study cohort included constipation (13%), diarrhea 
(18%), rash (38%), stomatitis (41%) and weight loss (18%).  Thus, this was a tolerable 
regimen with no severe safety signals.  In addition, tissue studies demonstrated that this 
combination effectively inhibited target.  However, moving into the surveillance setting in 
breast cancer, an all-oral regimen is more feasible, appealing to patients, and titratable 
in a potentially curative setting.  Moreover, EVE has been studied extensively in breast 
cancer patients, demonstrating safety and activity in combination with both endocrine 
and HER2-directed therapies(43, 44).  Thus we sought data on the safety and efficacy 
of HCQ with EVE.  Dr. Amaravadi (sub-investigator on this proposal) has now 
completed dose escalation on a phase I/II study of HCQ and EVE in patients with 
advanced renal cell carcinoma (NCT01510119).  In 29 patients, there were no SAEs or 
grade 4 toxicities, two grade 3 episodes each of nausea and edema, and one episode 
elevated triglycerides.  These data confirm that HCQ at 600 mg twice daily and EVE at 
10 mg daily are safe and tolerable.  
 
4 
PATIENT SELECTION 
4.1 
Target enrollment and subject ascertainment 
A total of 60 patients in the post-treatment surveillance period with bone marrow 
positive for DTCs will be ascertained from the ongoing PENN-SURMOUNT Screening 
Study (UPCC 28115; IRB# 824098) or other studies in which they have the DTC-IHC 
assay performed in the Clinical Laboratory of the Department of Pathology at the 
University of Pennsylvania.  Patients on UPCC 28115, the ongoing screening study, 
meet the same clinical eligibility criteria as outlined for the clinical trial below. Screening 
DTC results (IHC assay) will be made available to the participant’s study doctor, who 
will discuss them with the participant. If the screening DTC result is positive, the 
proposed clinical trial will be discussed.  
4.2 
Inclusion criteria  
4.2.1 
Histologically-confirmed, primary, invasive breast cancer diagnosed 
within 5 years of entry into a companion DTC screening protocol. 
4.2.2 
Qualifying risk status, at diagnosis utilizing receptor testing by 
ASCO/CAP guidelines, meeting one of the following: 
4.2.2.1 
Histologically positive axillary lymph nodes 

 
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4.2.2.2 
Primary tumor with triple negative subtype: estrogen 
receptor (ER) <10%, progesterone receptor (PR) <10% and negative 
Her2-overexpression by ASCO-CAP guidelines 
4.2.2.3 
Primary tumor with Breast Cancer Recurrence Score of ≥ 25 
per the Genomic Health Oncotype DX breast cancer test and/or high risk 
MammaPrint 
4.2.2.4 
Evidence of residual disease in the breast on pathological 
assessment after neoadjuvant chemotherapy.  
4.2.3 
Patients must have completed all primary therapy (definitive surgery, 
(neo)adjuvant chemotherapy adjuvant radiation and/or Her2-directed 
therapy) for the index malignancy at least 4 weeks prior to study entry. 
All prior treatment-related toxicity must be resolved prior to study 
enrollment.  Concurrent receipt of adjuvant endocrine and bone 
modifying agents is allowed per standard of care guidelines. Tamoxifen 
is not allowed due to drug-drug interactions with HCQ (see Section 7.6 
and Table 3). 
4.2.4 
Bone marrow aspirate after completion of therapy demonstrates 
detectable DTCs (via IHC) 
4.2.5 
No evidence of recurrent local or distant breast cancer by physical 
examination, blood tests (CBC, LFTs, Alk Phos), or symptom-directed 
imaging, per NCCN guidelines. 
4.2.6 
 Age ≥ 18 years 
4.2.7 
ECOG performance status  2 
4.2.8 
No contraindications to the study medications or uncontrolled medical 
illness. 
4.2.9 
Adequate bone marrow function as shown by: ANC ≥ 1.5 x 109/L, 
Platelets ≥ 100 x 109/L, Hb >9 g/dL 
4.2.10 Adequate liver function as shown by:  Serum bilirubin ≤ 1.5 x ULN, ALT 
and AST ≤ 2.5 x ULN, and INR ≤1.5   
4.2.11 Anticoagulation is allowed if target INR ≤ 1.5 on a stable dose of warfarin 
or on a stable dose of anticoagulant for >2 weeks at time of 
randomization.  For patients on therapeutic anti-coagulants, medication 
must be clinically held peri-procedure (bone marrow aspirate) per 
standard clinical management.  
4.2.12 Adequate renal function: serum creatinine ≤ 2.0 x ULN or creatinine 
clearance (CrCl) ≥ 30mL/min obtained within 28 days prior to registration.  
A calculated creatinine clearance by Cockcroft-Gault Formula is 
acceptable in lieu of a measured value. 

 
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4.2.13 Serum cholesterol ≤300 mg/dL OR ≤7.75 mmol/L AND triglycerides ≤ 2.5 
x ULN. NOTE: In case one or both of these thresholds are exceeded, a 
fasting lipid assessment will be ordered. If one or both of these 
thresholds are still exceeded in a fasting state, the patient can only be 
included after initiation of appropriate lipid lowering medication. 
4.2.14 Ability to provide informed consent 
4.3 
Exclusion criteria 
4.3.1 
Concurrent enrollment on another investigational therapy 
4.3.2 
Prior treatment with an mTOR inhibitor (sirolimus, temsirolimus, 
everolimus).   
4.3.3 
Known hypersensitivity to Everolimus or other rapamycins (sirolimus, 
temsirolimus) or to its excipients. 
4.3.4 
Known allergies or hypersensitivity to corticosteroids 
4.3.5 
Patients receiving chronic, high dose systemic treatment with 
corticosteroids defined as:  chronic use of cortisone >50mg; 
hydrocortisone >40mg, prednisone >10mg, methylprednisone >8mg or 
dexamethasone > 1.5mg; or another immunosuppressive agent. Topical 
or inhaled corticosteroids are allowed. 
4.3.6 
Patients should not receive immunization with attenuated live vaccines 
within one week of study entry or during study period. 
4.3.7 
Patients who have any severe and/or uncontrolled medical conditions or 
other conditions that could affect their participation in the study including: 
 Symptomatic congestive heart failure of New York heart 
Association Class III or IV  
 Unstable angina pectoris, myocardial infarction within 6 months of 
start of study drug, serious uncontrolled cardiac arrhythmia or any 
other clinically significant cardiac disease 
 Severely impaired lung function with a previously documented 
spirometry and DLCO that is 50% of the normal predicted value 
and/or 02 saturation that is 88% or less at rest on room air 
 Uncontrolled diabetes as defined by fasting serum glucose >1.5 x 
ULN. For eligibility assessment, if non-fasting serum glucose ≤ 1.5 
x ULN, then a fasting serum glucose does not need to be 
obtained. 
 Active (acute or chronic) or uncontrolled severe infections 
 Liver disease such as cirrhosis, chronic active hepatitis or chronic 
persistent hepatitis 
 A known history of HIV seropositivity as reported by the patient 

 
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 Impairment of gastrointestinal function or gastrointestinal disease 
that may significantly alter the absorption of EVE (e.g., ulcerative 
disease, uncontrolled nausea, vomiting, diarrhea, malabsorption 
syndrome or small bowel resection) 
 Patients with an active, bleeding diathesis  
 Active or latent, untreated Hepatitis B or C.  A detailed 
assessment of Hepatitis B/C medical history and risk factors must 
be done at screening for all patients.  HBV DNA and HCV RNA 
PCR testing are required at screening for all patients with a 
positive medical history based on risk factors and/or confirmation 
of prior HBV/HCV infection.  
4.3.8 
Female patients who are pregnant or breast feeding, or adults of 
reproductive potential who are not using effective birth control methods. 
If barrier contraceptives are being used, these must be continued 
throughout the trial and for 8 weeks after stopping study drug, by both 
sexes. Hormonal contraceptives are not acceptable as a sole method of 
contraception.  (Women of childbearing potential must have a negative 
urine or serum pregnancy test within 7 days prior to administration of 
EVE. Pregnancy testing is required unless age ≥ 60, prior hysterectomy, 
prior oophorectomy, ≥ 24 months amenorrhea, or patient is between 12-
24 months amenorrhea and on anti-estrogen therapy with estradiol < 30 
pg/mL and FSH in post-menopausal range) 
 
5 
STUDY ASSESSMENTS AND PROCEDURES 
5.1 
Schedule of Assessments and Procedures 
The table below lists the procedures and assessments that patients must 
undergo as part of this pilot clinical trial.   
 

 
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Table 2:  Study Calendar 
 
Screening 
Phase1 
Treatment 
Phase 
First 
Follow-Up 
Follow-Up 
Phase 
Cycle (C) 
1 cycle = 28 days 
0 (baseline) 
Each Cycle 
30 days 
after last 
dose IP 
therapy 
Q6 month 
x 3 years 
Day (D) of Cycle 
 
D14 
 
 
Time Window (days) 
-30 to 0 
+/- 3 
+/- 15 
+/- 35 
SCREENING PHASE: 
Informed Consent 
X 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
Demographics 
X 
 
 
 
CLINICAL EVALUATIONS: 
Medical History 
X 
 
 
 
Physical Exam/Vital Signs 
X 
X 
X 
X 
ECOG Performance status 
X 
X 
X 
 
LABORATORY/RADIOLOGIC EVALUATIONS: 
Hematology  (CBC/diff, plt) 
X 
X 
X 
X 
Comprehensive Metabolic Panel2 
X 
X 
X 
X 
Coagulation Studies (PT/PTT) 
X 
 
 
 
Serum Pregnancy Test3 
X 
 
 
 
TREATMENT ADMINISTRATION: 
IP Dispensing, Drug Compliance 
 
X 
 
 
CORRELATIVE STUDIES: 
Breast tumor specimen (primary 
tumor) 
X 
 
 
 
Bone Marrow Aspirate for DTCs 
X7 
X5 
 
X8 
Blood sample for patient tumor 
DNA and CTCs 
X 
X6 
 
X 
ADDITIONAL INFORMATION: 
Concomitant Medications Review 
X 
X 
X 
X 
Adverse Events/Toxicity 
Assessment 
X 
X4 
X 
 

 
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1. 
Within 30 days prior to starting treatment, unless otherwise noted. 
2. 
Serum chemistry includes measurement of sodium, potassium, chloride, bicarbonate, BUN, creatinine, 
glucose, total bilirubin, calcium, total protein, albumin, AST, ALT, and alkaline phosphatase.
P 
PIn addition 
to the standard blood chemistry (complete metabolic panel), a lipid panel will be done to measure serum 
cholesterol and triglycerides. If any lipid assessment exceeds the threshold identified in eligibility criterion 
4.2.13, then a fasting lipid panel will be ordered. Magnesium and phosphorous levels will be evaluated at 
screening for all subjects, and subsequently during the treatment phase and at first follow-up for those 
patients receiving everolimus. 
3. 
For women of childbearing potential only, within 7 days of initiating treatment. 
4. 
Participants randomized to Arm D will be monitored remotely by phone during the 12 week observation 
period. Lab assessments are not required during the observation period. 
5. 
Bone marrow aspirate performed at the end observation (before the start of therapy), at the end of C3 
(between D23-28), at the end of C6 (between D23-21 days after last dose IP therapy), and, if patient 
continued on additional treatment, at the end of C12, (between D23-21 days after last dose IP therapy).  
6. 
Blood collected for ptDNA and CTCs pretreatment and at the completion of observation (before the 
start of therapy), at the completion of C3 (before the start of C4), at the completion of C6 (within 21 
days of last dose), and at completion of C12 (within 21 days of last dose). 
7. 
Baseline bone marrow aspirate positive for DTCs must have been done within 6 months of the 
screening phase.    
8. 
Participants who clear DTCs from the bone marrow (i.e., negative DTC-IHC test) after 6 or 12 cycles 
of therapy will have optional bone marrow aspirate collected for retesting at the first 6 month follow up 
visit. 
 
Note:  Additional tests may be performed at the discretion of the treating 
investigator as clinically indicated.  The sample collection schedules outlined 
above are based on an ideal subject. The sample schedule should be followed 
as closely as is realistically possible; however, the schedule may be modified 
due to problems such as scheduling delays or conflicts (e.g., clinic closure, 
poor weather conditions, vacations, etc.) 
 
5.2 
Screening Phase 
Patients found to have detectable DTCs by immunohistochemistry, through 
the PENN-SURMOUNT Screening study (UPCC 28115; IRB# 824098) or other 
studies will be screened for trial participation during the screening phase.  This will 
include assessments of medical history, overall physical condition, physical exam, 
baseline laboratory studies, and symptom-directed imaging (if applicable) to rule out 
the presence of overt metastatic disease, per NCCN guidelines.  Patients will 
undergo a baseline bone marrow aspirate if their prior bone marrow aspirate 
detecting DTCs occurred > 6 months prior to the screening phase.  If a bone marrow 
aspirate positive for DTCs occurred ≤ 6 months prior to screening, this will not be 
repeated, and the prior DTC count will provide the baseline DTC assessment. 
 
5.3 
Treatment Phase 
Patients who have signed informed consent will be randomized to start the 
treatment phase.  The study arms are described below.  Patients are assessed for 
adverse events, toxicity and laboratory abnormalities at each study visit. Bone 
marrow aspirate will be performed at the end of the third cycle of observation, 
between D23-28 (Arm D), at the end of 3 cycles of therapy, between D23-D28 (all 
arms), after 6 cycles of therapy, between D23 of C6 and within 21 days of the last 
dose of C6 (all arms), and, if patient continues on treatment, at the end of C12, 
between D23 of C12 and within 21 days of the last dose of C12. After the first 6 
cycles of study therapy, patients will stop all study medication and proceed with 

 
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bone marrow evaluation per protocol. When the results are available it will be 
determined whether or not the patient will continue on study therapy. If continuing 
on study therapy, Cycle 7 must be initiated within 45 days after the last dose of 
cycle 6. Results from the end of C3 and end of observation bone marrow tests will 
not be returned to patients. Patients, however, will receive the results of the end of 
C6 and, if applicable and if desired, end of C12 bone marrow tests. End of C6 and, 
if applicable, end of C12 bone marrow DTC results disclosure can be done 
remotely over the phone if deemed appropriate by the PI or treating physician. 
 
5.4 
First Follow-Up and Follow-Up Phase 
Patients will be seen in follow-up 30 days after their last dose of study 
treatment (“first follow-up”) and then at 6 month intervals x 3 years.  At these visits, 
symptoms related to residual side effects of study treatment or potentially reflecting 
recurrent disease will be queried and physical examination will be performed. AEs 
will be collected up until 30 days after the last dose of study treatment.  Any 
unresolved AEs will be followed until resolved to grade ≤1 or baseline, or can be 
otherwise attributed. Collection of new AEs (other than those related to study 
procedures) beyond the first follow up visit is not required. Blood will be collected for 
ptDNA and CTC at 6 month intervals x 3 years. There is an optional bone marrow 
aspirate collection for DTC testing at the first 6 month follow up visit for patients who 
had a negative DTC-IHC test after 6 or 12 cycles of therapy. Patients will have the 
opportunity to receive the results of this bone marrow test. Patients may also choose 
not to receive the results of this bone marrow test, in which case the results will not 
be placed in the EMR.  
To decrease the burden as much as possible on subjects, subjects may go to 
their local oncologist for all follow up visits beyond the 30 day “first follow up.” 
Records will be requested from local care providers so that the study team can 
continue to track the subjects for safety and outcome. To minimize inconvenience 
and burden, and to improve compliance, subjects have the option of having their 
clinical labs drawn locally. For those subjects who are approved for remote follow up 
with their local providers, the research blood requirement and any other labs that are 
billed to research and not considered SOC will be waived. However, all subjects will 
retain the option of returning to UPenn if they would like to undergo study research 
blood collection. 
 
5.5 
Laboratory and adverse event assessment 
Patients randomized to Arms A, B or C will be evaluated at the start of each 
treatment cycle for both toxicity assessment and laboratory evaluation.  Patients 
who are randomized to control (Arm D) will be contacted by phone at the start of 
each observation cycle as a check-in and to monitor for adverse events. Laboratory 
evaluation is not necessary for patients on Arm D during the observation period. 
When Arm D patients return to start combination treatment, they will undergo toxicity 
assessment and laboratory evaluation at the start of each treatment cycle. 
 
5.6 
Disease monitoring 
The goal of the proposed trial is to pharmacologically target dormant tumor 

 
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cells to prevent recurrent breast cancer.  To enter the trial, patients must have no 
evidence of recurrent local or distant breast cancer by physical examination, blood 
tests (CBC, LFTs, Alk Phos), or symptom-directed imaging, per NCCN guidelines.  
This testing is repeated 6 months after the last dose of study treatment to determine 
whether the patient has experienced a recurrence during the study period or follow 
up period.  No further imaging will be done as part of study procedures during or 
after the trial, as detection of overt metastatic disease and time-to-event analyses 
are beyond the scope of this pilot trial. 
6 
ENROLLMENT PROCEDURES 
6.1 Guidelines for Enrollment 
The following are required to enroll a patient on the trial: 
1) Copy of the patient’s signed and dated Informed Consent including documentation 
of the consent process  
2) Patient Enrollment Form  
3) Complete provided protocol-specific eligibility checklist using the eligibility 
assessment documented in the participant’s medical/research record. To be eligible 
for registration to the study, the participant must meet each inclusion and exclusion 
criterion listed in the eligibility checklist.  
4) Tissue Block Registration  
The Study Coordinator or designate will assign sequence numbers to all 
patients in screening.  Only patients deemed eligible will be registered to 
investigational treatment. Sequence numbers will not be re-used if a patient screen 
fails. Following registration, eligible participants should begin study treatment 
consistent with the protocol no later than 30 days after registration by the Study 
Coordinator.  If a participant does not receive protocol therapy following registration 
within the allowed time period, the participant’s registration on the study may be 
canceled.  The Project Manager should be notified of cancellations as soon as 
possible. 
Issues that would cause treatment delays should be discussed with the 
Principal Investigator. If a participant does not receive protocol therapy following 
enrollment within allowed time period, the participant will become ineligible and will 
be removed from the study. Such patients will have to undergo re-screening in order 
to participate in the study. Any requests for eligibility exceptions and/or deviations 
must be approved in writing by the Principal Investigator, the Medical Monitor and 
the IRB.  
As is generally accepted, standard of care procedures performed prior to 
consent, but within the protocol defined screening window for each assessment, can 
be used for study purposes.  All research-only procedures must be performed after 
the consent date. 
 
7 
TREATMENT PLAN 
This is a randomized, controlled, open label pilot trial. A total of 60 patients 

 
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will be enrolled, with at least 15 patients allocated to each treatment group (Arms C 
and D constituting the same treatment group). The initial treatment period is 6 
months (24 weeks).  During the initial treatment period, patients will be randomized 
to one of 4 arms:  HCQ alone (Arm A), EVE alone (Arm B), combination HCQ and 
EVE (Arm C) – Arm C is closing to accrual with Amendment 7 – or control/delayed 
combination HCQ and EVE (Arm D). Starting with Amendment 7, patients who 
would have been allocated to Arm C will be redirected to Arm D in order to increase 
the number of patients with repeat DTC assessments to aid assay reproducibility 
analysis   At the conclusion of 3 cycles of therapy (Arms A, B, and C) or 3 cycles of 
observation (Arm D), patients will undergo repeat bone marrow aspirate. Patients on 
Arm D will then commence combination therapy with HCQ + EVE for 6 cycles (24 
weeks). After 3 cycles of therapy, patients on Arm D will undergo another bone 
marrow aspirate. After 6 cycles of therapy, patients on all arms will undergo another 
bone marrow aspirate.  Patients with a negative bone marrow aspirate for DTCs at 
after cycle 6 will have all therapy discontinued and enter the follow up phase.  
Patients who have persistently positive bone marrow DTCs will be continued on 
combination HCQ + EVE for an additional 6 cycles (24 weeks).  At the conclusion of 
this combination treatment period (cycle 12), all treatment will be discontinued and a 
bone marrow aspirate will be performed.  
Patients will be evaluated at the initiation of every cycle for adverse events, 
and dose reductions will be employed for toxicity. Patients will continue to be 
followed for recurrence events for 3 years after completion of treatment. 
 
7.1 
Study Arms 
Patients will be randomized during the Screening Phase to one of four 
investigational arms.  These are shown in the schema above and detailed as follows 
(Note that Arm C is closing with Amendment 7): 
Arm A:   Patients will receive HCQ, 600 mg BID, for 24 weeks.  If they are 
determined to have detectable DTCs after week 24, they will be crossed over to 
combination therapy, in which they will continue HCQ, 600 mg BID and add EVE, 10 
mg daily for an additional 24 weeks. 
Arm B:   Patients will receive EVE, 10 mg daily, for 24 weeks.  If they are 
determined to have detectable DTCs after week 24, they will be crossed over to 
combination therapy, in which they will continue EVE, 10 mg daily and add HCQ, 
600 mg BID, for an additional 24 weeks. 
Arm C:   Patients will receive the combination of HCQ, 600 mg BID, and 
EVE, 10 mg daily, for 24 weeks.  If they are determined to have detectable DTCs 
after week 24, they will continue combination HCQ/EVE therapy for an additional 24 
weeks. Arm C is closing with Amendment 7. Patients radnomized after Amendment 
7 who would have been assigned to Arm C will be redirected to Arm D. 
Arm D:   Patients will be observed for 12 weeks without study therapy.  At the 
completion of the observation period, they will receive combination therapy of HCQ, 
600 mg BID and EVE, 10 mg daily for 24 weeks.  If they are determined to have 
detectable DTCs after week 24, they will continue combination HCQ/EVE therapy for 
an additional 24 weeks. 
 
7.2 
Drug Administration 

 
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Patients will be randomized to receive HCQ, EVE, the combination, or 
observation (control)/delayed combination. A “cycle” is 28 days (4 weeks) in 
duration.  Both agents will be given continuously (28 of 28 days), without break, 
unless necessary due to toxicity.  Patients will initially be treated on the assigned 
arm for 6 cycles (24 weeks), or observed for 3 cycles (Arm D), followed by delayed 
treatment on the combination.  At that point, patients who are persistently positive 
for bone marrow DTCs will be allowed to crossover as outlined in the study schema 
and below.  The crossover therapy will be given for 6 cycles (24 weeks) as well. 
Because EVE and HCQ are oral medications, pill counts and treatment diaries will 
be used to monitor adherence to therapy.  Patients are required to bring the pill 
bottles and treatment diaries to each visit.    
7.2.1 
Hydroxychloroquine   
Patients randomized to an HCQ-containing arm will receive HCQ at a dose of 
600 mg orally twice daily. Capsules of HCQ are available in 200 mg strength, thus 
patients will initially start with 3 capsules twice daily for a total of 6 capsules per day.  
HCQ will be administered in divided doses (BID). When taking HCQ twice daily, the 
two daily doses should be taken 12 hours apart as close to 9am and 9pm as 
possible and documented clearly on the patient calendar.  Patients receiving 
antacids, sucralfate, cholestyramine, and/or bicarbonate should have the HCQ drug 
dose administered at least 1 hour before or 2 hours after these medications.  
Hydroxychloroquine will be obtained through a written prescription by study MD as 
an initial 28- day supply and refilled every cycle.  Patients will be required to bring 
the prescription to their visit on day 1 of each cycle, along with previous months’ pill 
container so that pill counts can be performed. In addition, treatment diaries will be 
used to monitor adherence to therapy.  Patients are required to bring the pill bottles 
and treatment diaries to each visit.  Missed or vomited doses should not be made 
up; patient should resume planned dose at next dosing interval. 
7.2.2 
Everolimus   
Patients randomized to an EVE-containing arm will receive EVE as a dose of 
10 mg orally daily, on a continuous basis.  Patients will be instructed to take EVE in 
the morning, at the same time each day. EVE may be taken with or without food. If 
vomiting occurs, no attempt should be made to replace the vomited dose.  All 
dosages prescribed and dispensed to the patient and all dose changes during the 
study must be recorded.  EVE will be provided by Novartis Pharmaceuticals 
Corporation. Antiemetic prophylaxis is not required.  However, control of nausea and 
vomiting may require institution of multiple anti-emetic medication including 
phenothiazines or 5-HT3 antagonists.  Due to its interaction with P450 enzymes, 
aprepitant use should be avoided. 
7.3 
Adjuvant Endocrine Therapy 
Patients with hormone receptor positive primary or residual tumor will 
continue on their assigned adjuvant endocrine therapy throughout the duration of the 
trial. Note that tamoxifen is not allowed during the study due to drug-drug 
interactions with HCQ (see Section 7.6 and Table 3). 
7.4 
Concomitant Treatment and Supportive Care Guidelines 
Necessary supportive measures for optimal medical care will be provided 
during the study (including administration of prophylactic antiemetic medication if 

 
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considered appropriate by the Investigator). 
7.4.1 Antiepileptics:   
Because HCQ has known effects on P450 enzymes, patients requiring anti-
convulsants may be treated with any of the non-enzyme inducing anti-convulsants 
which include: felbamate, valproic acid, gabapentin, lamotrigine, tiagibine, 
topiramate, zonisamide, or levetiracetam.  
7.4.2 Hematologic Growth Factors 
The use of hematologic growth factors (filgrastim, pegfilgrastim, epoetin alfa) 
is permitted to provide optimal care for patients with severe myelosuppression. 
7.4.3 Bone targeting therapies  
Bone targeting therapies such as bisphosphonates and RANK lignad 
inhibitors may be administered per standard of care guideliens. Zometa or 
denosumab may be given if clinically indicated. 
7.5 
Concomitant Medications 
The case report form must capture the concurrent use of all other drugs, 
over-the-counter medications, or alternative therapies.  Patients must adhere to the 
following: 
 Patients will be instructed not to take any additional medications (including 
over-the-counter products) during the course of the study without prior 
consultation with the investigator. At each visit, the investigator will ask the 
patient about any new medications he/she is or has taken after the start of the 
study drug. 
 All Concomitant medications/Significant non-drug therapies taken within 28 
days of starting study treatment through the 30- day safety follow up visit, 
including physical therapy and blood transfusions, should be recorded. 
 Patients on chronic medications that can be given concomitantly with the study 
agents should be continued.  The investigator should instruct the patient to 
notify the study site about any new medications he/she takes after the start of 
the study drug. All medications (other than study drug) and significant non-drug 
therapies (including physical therapy and blood transfusions) administered 
after the patient starts treatment with study drug, and any changes in dosing 
should be recorded. 
 All supportive measures consistent with optimal patient care can be given 
throughout the study at the discretion of the treating physician, as long as they 
are not part of the list of prohibited medications. In general, the use of any 
concomitant medication/therapies deemed necessary for the care of the patient 
is permitted with the following considerations: 
 Prophylactic anti-emetics should be started only once the patient experienced 
nausea or vomiting at the discretion of the investigator. It is recommended that 
patients use drugs that do not cause QT prolongation.  
 Patients may not receive radiotherapy while on this trial. 
 Other investigational therapies must not be used while the patient is on the 
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 Anticancer systemic therapy other than the study treatments must not be given 
to patients while on the study. If such agents are required for a patient then the 
patient must be discontinued from the study. 
 Herbal preparations are not allowed throughout the study. These herbal 
preparations include, but are not limited to: St. John’s wort, Kava, ephedra (ma 
huang), gingko biloba, dehydroepiandrosterone (DHEA), yohimbe, saw 
palmetto, and ginseng. Patients should stop using these herbal preparations 
14 days prior to first dose of study drug 
7.6 
Drug-Drug Interactions 
The following restrictions apply during the entire duration of the study: 
 No other investigational therapy should be given to patients. 
 No anticancer agents other than the study medication should be given to 
patients. If such agents are required for a patient then the patient must first be 
withdrawn from the study. 
 Hydroxychloroquine interacts with CYP enzymes, causing potential drug-drug 
Interations. CYP3A4 modifiers are described below (Table 3) and must be 
evaluated carefully.  Examples are shown in the table. A comprehensive list of 
cytochrome P450 isoenzymes and CYP3A4 inhibitors, inducers, and 
substrates can be found at http://medicine.iupui.edu/flockhart.  This website is 
continually revised and should be checked frequently for updates.   
 Co-administration with strong inhibitors of CYP3A4 (e.g., ketoconazole, 
itraconazole, ritonavir) or P-glycoprotein (PgP) should be avoided.  Seville 
orange, star fruit, grapefruit and their juices affect P450 and PgP activity. 
Concomitant use should be avoided 
 Co-administration with moderate CYP3A4 inhibitors (e.g., erythromycin, 
fluconazole) or PgP inhibitors should be used with caution.  If patient requires 
co-administration of moderate CYP3A4 inhibitors or PgP inhibitors, reduce the 
dose of EVE to 5 mg daily. Additional dose reductions to 5 mg every other day 
may be required to manage toxicities. If the inhibitor is discontinued the EVE 
dose should be returned to the dose used prior to initiation of the moderate 
CYP3A4/PgP inhibitor. 
 No chronic treatment with systemic steroids or other immunosuppressive 
agents (at a dose equivalent of greater than 20 mg prednisone per day) or other 
immunosuppressive agents) is allowed. Topical or inhaled corticosteroids are 
allowed. 
 For hydroxychloroquine, there are known drug interactions with the following 
medications:  penicillamine, telbivudine, botulinum toxins, digoxin, metoprolol, 
aurothioglucose, and propafenone.  Depending on the specific drug, HCQ 
increases the levels of these other drugs, or interferes with the activity of the 
agent (specifically with botulinum toxin) and the combination should be used 
with caution. 
 
Table 3:  Partial List of Prohibited Medications Based on CYP Metabolism 
Substrates (competitive inhibition) 
 
AntibioticsP1
P: 
 
clarithromycin* 
 
erythromycin  
Calcium Channel Blockers: 
 
amlodipine 
 
diltiazem 

 
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telithromycin* 
Anti-arrhythmics: 
 
quinidine 
Benzodiazepines: 
 
alprazolam 
 
diazepam 
 
midazolam 
 
triazolam 
Immune Modulators: 
 
cyclosporine 
 
tacrolimus (FK506) 
HIV Protease Inhibitors: 
 
indinavir* 
 
ritonavir* 
 
saquinavir* 
Prokinetic: 
 
cisapride 
Antihistamines: 
 
astemizole 
 
chlorpheniramine45 
 
felodipine 
 
nifedipine 
 
nisoldipine 
 
nitrendipine 
 
verapamil 
HMG CoA Reductase Inhibitors P2
P:  
 
atorvastatin 
 
cerivastatin 
 
lovastatin 
 
simvastatin 
Miscellaneous: 
 
aprepitant 
 
buspirone 
 
haloperidol 
 
methadone 
 
pimozide  
 
quinine 
 
sildenafil 
 
tamoxifen 
 
trazodone 
 
vincristine 
Inducers 
Carbamazepine 
Phenobarbital 
Phenytoin* 
Rifabutin* 
Rifampin* 
St John’s wort 
Troglitazone 
Inhibitors 
Amiodarone 
Cimetidine 
Clarithromycin 
Delaviridine 
Diltiazem 
Erythromycin 
Fluvoxamine* 
Grapefruit juice 
Sevilla orange 
Indinavir 
Itraconazole* 
Ketoconazole* 
Voriconazole* 
Posaconazole* 
Mibefradil 
Nefazodone* 
Nelfinavir* 
Troleandomycin 
Verapamil 
Based on: Ingelman-Sundberg M, Human drug metabolising cytochrome P450 
enzymes: properties and polymorphisms, Naunyn Schmiedebergs Arch Pharmacol. 2004 
Jan;369(1):89-104.  and [http://www.medicine.iupui.edu/flockhart/clinlist.htm as of July 13, 
2006] 
* asterisk denotes strong inhibition/ induction  
Please note: 
strong inhibitor implies that it can cause ≥5-fold increase in AUC or ≥80% decrease in clearance 
of sensitive CYP substrates 
moderate inhibitor implies that it can cause 2 to 5-fold increase in AUC values or 50-80% 
decrease in clearance  of sensitive CYP substrates. 
(Distinction is not always categorical as interaction can vary according to conditions). 
Macrolide antibiotics: 
Azithromycin is not a CYP3A substrate. It may therefore be employed 
where antibiotherapy with a macrolide is desirable in a patient being treated with EVE 
Statins: Atorvastatin and pravastatin may be associated with EVE, since a PK interaction study 
has shown that there is no relevant PK interaction.  
7.7 Criteria for Removal from Study 
Patients may be removed from the study before completing the full follow-up 

 
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period in the event of unacceptable toxicity, or either physician or patient preference.    
7.8 Discontinuation of Treatment 
Patients who discontinue study treatment prior the final planned dose will 
enter the follow up phase, and complete all procedures and follow-up dictated by the 
protocol.  Patients who choose to discontinue all study participation, including follow-
up, may do so at any time without penalty, by following the procedures outlined in 
section 7.11.  At the time of any treatment discontinuation or withdrawal, site 
research staff should document the reasons and conditions under which 
discontinuation of treatment occurred and whether the patient is willing to continue 
follow up or will be requesting full withdrawal from the study. Each subject has the 
right to discontinue study treatment or withdraw from the overall study at any time 
without prejudice.  The investigator may discontinue any subject’s participation for 
any reason, including adverse event or failure to comply with the protocol.  Because 
this is a randomized pilot trial following intent-to-treat principals, patients who 
discontinue study treatment will be followed for study outcomes as outlined in the 
protocol. Reasons for withdrawal from study treatment include the following:  
 
 Delay in treatment > 28 days due to treatment-related toxicity or major 
event (if due to treatment-related toxicity, patient will be taken off the 
treatment that is most likely to be related to the toxicity). 
 Disease Recurrence:  Remove patient from protocol therapy at the time 
progressive disease is documented. 
 Extraordinary Medical Circumstances: If at any time the treating 
physician feels constraints of this protocol are detrimental to the 
patient’s health remove the patient from protocol therapy. 
 Patient’s refusal to continue treatment: In this event, document the 
reason(s) for withdrawal. 
 Failure to comply with protocol (as judged by the investigator such as 
compliance below 80%, failure to maintain appointments, etc.). 
7.9 Withdrawal from Study 
Patients who choose to withdraw from study participation (including any 
additional treatment, follow-up, or study procedures) may do so, without penalty, by 
sending a request to be withdrawn from the study, in writing, to the Principal 
Investigator.   In addition, this request should specifically address whether research 
samples that remain in the possession of the study staff can be used for future 
research per the previously signed consent form.  Should the patient request 
withdrawal from the study, the PI and project manager should be alerted 
immediately, and updated with the conditions of the request. 
7.10 Contraceptive Measures and Reproductive Risks 
UFemale Participants 
Female participants should not become pregnant while on this study and for 8 
weeks after their last dose of study treatment because the study drugs could have a 
negative effect on an unborn baby.  In addition, female participant should not 
breastfeed while on this study during treatment and for 2 weeks after the last dose 

 
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as these drugs may also affect a breast-feeding child.  Pregnant women and women 
who are breast-feeding are not allowed to participate in this study. If a participant 
becomes pregnant, the participant will no longer be able to participate in this study. 
 
If a participant is of child bearing potential, she must agree to use two 
medically accepted forms of birth control including condoms, diaphragms, cervical 
cap, an intra-uterine device (IUD), surgical sterility (tubal ligation or a partner that 
has undergone a vasectomy), or oral contraceptives, OR she must agree to 
completely abstain from intercourse during participation in this study and for 8 weeks 
after her last dose of study treatment.  Abstinence at certain times of the cycle only, 
such as during the days of ovulation, after ovulation and withdrawal are UnotU 
acceptable methods of birth control. The participant’s form of birth control must be 
approved by the study doctor. 
 
Even when approved contraceptive methods are used, there is always a 
small risk that a participant could still become pregnant. If a participant becomes 
pregnant during the course of this study or up to 8 weeks after the last dose of study 
treatment, the participant must discontinue study treatment, tell the investigator 
immediately, and consult an obstetrician or maternal-fetal specialist. If a participant 
becomes pregnant while on this study, the study staff will ask permission to collect 
information about the participant’s pregnancy. 
 
UMale Participants 
Male participants should not father a child while on this study and for 8 weeks 
after the last dose of study treatment, because the drug involved could have a 
negative effect on an unborn baby.  If a participant’s spouse or partner has the 
potential to become pregnant, both the participant and partner must use two 
medically accepted forms of birth control including condoms, diaphragms, cervical 
cap, an intra-uterine device (IUD), surgical sterility (vasectomy or a partner that has 
undergone a tubal ligation), or oral contraceptives, OR the participant must agree to 
completely abstain from intercourse during participation in this study and for 8 weeks 
after the last dose of study treatment.  Abstinence at certain times of the cycle only, 
such as during the days of ovulation, after ovulation and withdrawal are not 
acceptable methods of birth control. The participant’s form of birth control must be 
approved by the study doctor.  
 
Male participants should also inform their partner of the potential harm to an 
unborn child.  If a participant’s partner should become pregnant during the course of 
this study or up to 8 weeks after the participant’s last dose of study treatment, the 
pregnancy will need to be reported to the study doctor immediately, and the 
participant’s partner should promptly notify her doctor.  The study doctor will also 
ask to follow-up on the pregnancy. 
 
Fertility 
The potential for everolimus to cause infertility in male and female patients is 
unknown. However, menstrual irregularities, secondary amenorrhea and associated 
luteinizing hormone (LH)/follicle stimulating hormone (FSH) imbalance has been 

 
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observed in female patients receiving everolimus. Blood levels of FSH and LH 
increased, blood levels of testosterone decreased, and azoospermia have been 
observed in male patients receiving everolimus. 
 
8 
EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS 
8.1 
Toxicity Criteria 
This study will utilize the CTCAE version 4.0 for toxicity  and Adverse Event 
Reporting.  A copy  of the CTCAE version 4.0 can be downloaded from the CTEP 
home page (http://ctep.cancer.gov). All appropriate treatment areas should have 
access to a copy of the CTCAE version 4.0 
8.2 
Anticipated Toxicities of Hydroxychloroquine 
 
Toxicities that can be seen with hydroxychloroquine include: 
 
 Central nervous system: Irritability, nervousness, emotional changes, 
nightmares, psychosis, headache, dizziness, vertigo, seizure, ataxia, lassitude. 
 
 Dermatologic: Bleaching of hair, alopecia, pigmentation changes (skin and 
mucosal; 
black-blue 
color), 
rash 
(urticarial, 
morbilliform, 
lichenoid, 
maculopapular, purpuric, erythema annulare centrifugum, Stevens-Johnson 
syndrome, acute generalized exanthematous pustulosis, and exfoliative 
dermatitis).  
 
 Gastrointestinal: Anorexia, nausea, vomiting, diarrhea, abdominal cramping.  
 
 Hematologic: 
Aplastic 
anemia, 
agranulocytosis, 
leukopenia, 
thrombocytopenia, 
hemolysis 
(in 
patients 
with 
glucose-6-phosphate 
deficiency).  
 
 Hepatic: Abnormal liver function/hepatic failure (isolated cases). 
 
 Neuromuscular & skeletal: Myopathy leading to progressive weakness and 
atrophy of proximal muscle groups (may be associated with mild sensory 
changes, loss of deep tendon reflexes, and abnormal nerve conduction). 
 
 Ocular: 
Disturbance 
in 
accommodation, 
keratopathy, 
corneal 
changes/deposits (visual disturbances, blurred vision, photophobia - reversible 
on discontinuation), macular edema, atrophy, abnormal pigmentation, 
retinopathy (early changes reversible - may progress despite discontinuation if 
advanced), optic disc pallor/atrophy, attenuation of retinal arterioles, 
pigmentary retinopathy, scotoma, decreased visual acuity, nystagmus. 
 
8.3 
Anticipated Toxicities of Everolimus 
 
Toxicities that can be seen with Everolimus include: 

 
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 Cardiovascular: Edema, peripheral edema, chest pain, congestive cardiac 
failure 
 
 Central nervous system: Pain, fever, headache, insomnia, dysgeusia, 
ageusia 
 
 Dermatologic: Rash, pruritus, nail disorder/thinning, dry skin, acne, 
erythema, hand-foot syndrome, angioedema 
 
 Endocrine & metabolic: decreased appetite, Hyperglycemia, 
hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, 
hypophosphatemia, hypokalemia, diabetes mellitus, dehydration 
 
 Gastrointestinal: Mucositis, nausea, anorexia, diarrhea, abdominal pain, 
constipation , stomatitis, taste disturbance, vomiting, weight loss, dry 
mouth, dyspepsia, dysphagia 
 
 Genitourinary: Urinary tract infection 
 
 Hematologic: Anemia, lymphopenia, thrombocytopenia (DLT), leukopenia, 
neutropenia, pancytopenia, pure red cell aplasia  
 
 Vascular disorders: hemorrhage, hypertension, deep vein thrombosis 
 
 Hepatic: Alkaline phosphatase increased, AST increased  
 
 Neuromuscular & skeletal: Weakness, back pain, arthralgia 
 
 Renal: Creatinine increased, proteinuria, renal failure 
 
 Respiratory: Dyspnea, cough, epistaxis, pharyngitis, pneumonitis, 
hemoptysis, pulmonary embolism, acute respiratory distress syndrome 
 
 Miscellaneous: Infection (includes abscess, bronchitis, cellulitis, herpes 
simplex, herpes zoster), hypersensitivity, amenorrhea, aspartate 
aminotransferase increased alanine aminotransferase increased 
 
 
 
 
 Other Medical Problems: The presence of other medical problems may 
affect the use of this medicine. For example bowel problems, diabetes, 
hyperlipidemia (increased blood cholesterol or fats), infection, kidney 
disease, lung disease (This medicine may worsen these conditions, or 
brain tumor (Use with caution). This medicine may increase the risk of 
having serious bleeding in the head. 
8.4 
Monitoring of EVE suspected toxicities  
Patients whose treatment is interrupted or permanently discontinued due to 

 
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an adverse event or abnormal laboratory value suspected to be related to EVE must 
be followed at least weekly until the adverse event or abnormal laboratory resolves 
or returns to grade ≤1. If a patient requires a dose delay of > 28 days  from the 
intended day of the next scheduled dose, then the patient must be discontinued from 
the study or continued on HCQ alone if there is evidence of clinical benefit and 
toxicities resolve to < grade 2.  
8.5 
Special Toxicity Considerations for EVE 
8.5.1 
Hyperlipidemia  
Hyperlipidemia was reported as a serious adverse reaction. It is a recognized 
side-effect of rapamycins. Use of lipid-lowering drugs should be associated with 
dietary recommendations. If blood lipid levels exceed the thresholds identified in 
eligibility criterion 4.2.13, then fasting blood lipid levels will be assessed. As long as 
blood lipid levels remain below the thresholds in 4.2.13, monitoring of blood lipid 
levels can continue with patient in a non-fasting state. However, if at any time blood 
lipid levels exceed these thresholds, further monitoring of blood lipid levels requires 
patients to be fasting. Fasting status must be verified when interpreting results. For 
guidance on management of hyperlipidemia refer to Section 8.6.5. 
 
8.5.2 
Hyperglycemia  
Hyperglycemia was reported as a serious adverse reaction. Similarly, the 
fasting state of patients should be verified when interpreting results. For guidance on 
management of hyperglycemia refer to Section 8.6.5. 
 
8.5.3 
Pneumonitis  
Pneumonitis is a recognized adverse effect of rapamycins (sirolimus, 
temsirolimus, and everolimus). Numerous case reports in the literature suggest that 
rapamycin-associated pneumonitis is relatively unaggressive, limited in extent, and 
reversible upon drug discontinuation. The term ‘pneumonitis’ is used here to 
describe non-infectious, non-malignant infiltration in the lungs which is evident 
radiologically. More precise diagnosis should follow histocytological examination 
following lung biopsy, generally during bronchoscopy which may or may not be 
symptomatic. Advice on the management of pneumonitis has been provided in 
Section 8.6.6.  
In oncology studies with EVE, severe pneumonitis suspected as drug-related 
has been reported as a serious adverse event on 13 occasions and additionally in 
the following associated preferred terms including acute respiratory distress 
syndrome (n=2), alveolitis (n=1) and allergic alveolitis (n=1), interstitial lung disease 
(n=10), lung infiltration (n=23), cryptogenic organizing pneumonia, lung 
consolidation, pulmonary alvealoar haemorrhage, pulmonary toxicity and pulmonary 
fibrosis (n=1, each). One fatal case of drug-related pneumonitis was reported for a 
patient with metastatic infiltrating ductal carcinoma of the breast treated with 10 
mg/day, which developed approximately two months after starting EVE. Cytology for 
both the pleural and pericardial fluids was positive for malignancy.  The death was 
considered possibly related to the underlying late stage tumor and study drug. 
Additionally, one patient treated with 10 mg/day died due to severe acute respiratory 
distress syndrome and septic shock. Thoracic CT scan demonstrated condensation 

 
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in the majority of the left lower lobe and frosted glass appearance in the left upper 
lobe, lingula, and right lung. 
Along with the cases of non-infectious pneumonitis, serious opportunistic 
infections have also been reported in cancer patients treated with EVE: 
mycobactrium, aspergillus, and fatal candidal sepsis, and fatal pneumocystis carnii 
in particular. Because EVE, as other rapamycins, inhibits proliferation of activated 
lymphocytes and reduces neutrophil counts, treatment with EVE must be considered 
as predisposing patients to the risk of infection. This risk will be higher in patients 
severely immunocompromised because of their underlying disease and/or co-
medications. Outcome may be fatal in case of serious infections.  
 
8.5.4 
Myelosuppression 
Myelosuppresion is frequent when EVE therapy is initiated. Without clinical 
significance and infrequently, anemia and thrombocytopenia have been reported. In 
heavily pretreated patients with aggressive lymphoma, the incidence of grade 3 
anemia, neutropenia, and thrombocytopenia was reported to be 11%, 16%, and 
30%, respectively. Serious, suspected drug-related hemorrhages have been 
exceptional. Nevertheless, EVE should be considered as predisposing patients to 
hemorrhage, potentially fatal, should they develop severe drug-related 
thrombocytopenia. 
 
8.5.5 
Liver Function Abnormalities 
Discrete, reversible changes in liver enzymes have been found to occur in 
numerous patients during treatment with EVE in oncology clinical studies, and in a 
study in rheumatoid arthritis. In oncology studies, these changes may be evident 
only in patients without severe underlying morbidity. The increase in transaminase’s 
(AST and ALT) generally appears after 4 weeks of treatment. In all but a few cases it 
does not exceed Grade 1 (≤ 3.0 x ULN). Similarly, mild increases in alkaline 
phosphatases can coexist. Spontaneous corrections or intermittent correction with 
continued treatment can occur. Serum bilirubin is not increased.  In studies of 
patients with advanced cancers, clinically relevant changes in liver enzymes have 
been invariably associated with the presence of liver metastases and/or progression 
of the underlying cancer.  
 
8.5.6 
Renal Insufficiency 
Renal failure has been reported in five suspected cases to date. One patient 
with no alternative explanation made a complete recovery following study drug 
adjustment and no treatment/therapy for the event. The rest or the patients had 
concurrent morbidities, which might have contributed to the reported events. 
 
8.5.7 
Hypophosphatemia, 
hypomagnesemia, 
hyponatremia 
and 
hypocalcemia  
These electrolyte abnormalities have been reported as serious adverse 
reactions. Electrolytes should be monitored in patients treated with EVE. 
 

 
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8.6 
Management of Treatment-Related Toxicities 
8.6.1 
Mucosal Inflammation and Stomatitis: 
Stomatitis mostly occurs within the first 8 weeks of treatment.  In a single arm 
study in 92 postmenopausal breast cancer patients, a topical alcohol-free 
corticosteroid oral solution was administered as a mouthwash during the initial 8 
weeks of starting treatment with Afinitor ® plus exemestane.  In this study, a clinically 
meaningful reduction in the incidence and severity of stomatitis was observed. 
 
Stomatitis/oral mucositis/mouth ulcers due to EVE should be treated using 
local supportive care. Please note that investigators in earlier trials have described 
the oral toxicities associated with EVE as mouth ulcers, rather than mucositis or 
stomatitis. If your 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: 
 
All patients randomized to EVE should be instructed to do the following, 
starting one day prior to first dose: 
o Maintain good oral hygiene, avoidance of spicy, acidic, hard and hot 
food and beverages 
o Use mild-flavored toothpastes 
o Use Alcohol-free steroid-based Mouthwash 10 mL (0.5 mg/5 mL 
dexamethasone oral solution) swish for 2 mins and spit QID 
 
For mild toxicity (Grade 1), use conservative measures such as non-
alcoholic mouth wash or salt water (0.9%) mouth wash several times a day until 
resolution.  
 
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 
OrabaseP®
P). 
 
Agents containing hydrogen peroxide, iodine, and thyme derivatives may tend 
to worsen mouth ulcers. It is preferable to avoid these agents.  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 EVE metabolism, thereby 
leading to higher EVE 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. 
Note:  Stomatitis/oral mucositis should be appropriately graded using the 
functional grading given on the NCI-CTC for adverse events, version 4.0. 
8.6.2 
Rash 
UFor >Grade 1: 

 
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 Use fragrance free moisturizing cream or ointment BID over entire body. 
 Use ammonium lactate 12% cream or salicylic acid 6% cream BID over 
dry/scaly/hyperkeratotic areas such as palms and soles. 
 Use zinc oxide 13-40% at night for areas with fissures. 
 
UFor > Grade 2: 
 Use topical steroid ointment or cream (Clobetasol 0.05%, Betamethasone 
0.05%, Fluocinonide 0.05%) BID and zinc oxide 13-40% at night for areas with 
fissures. 
8.6.3 
Nausea/Vomiting 
Routine use of prophylactic anti-emetics is NOT recommended. 
 
For Grade > 2:   
 Begin maximum supportive care and adequate anti-emetic treatments, 
including ondansetron and aprepitant. May opt to omit if grade I. 
8.6.4 
Diarrhea 
Antidiarrheal medication per standard practice is recommended at the first 
sign of abdominal cramping, loose stools or overt diarrhea. 
UAt first bout of diarrhea: 
 Initiate loperamide: initial dose of 4 mg (2 tablets/capsules) with the first bout 
of diarrhea followed by 2 mg (1 tablet/capsule) every 4 hours or after every 
unformed stool (maximum 16 mg a day) and continue loperamide at this 
frequency until diarrhea free for 12 hours. 
UFor persistent Grade 1U diarrhea on loperamide: 
 Add Lomotil (diphenoxylate hydrochloride and atropine sulfate) 1 tablet (2.5 
mg) every 6 hours to 8 hours  
UFor persistent Grade 2:  
 Consider adding octreotide (short-acting) 150 μg SC TID; or after initial dose 
of short-acting octreotide, if well tolerated, a single dose of octreotide LAR 20 
mg IM 
UFor Grade 3:  
 Administer octreotide (100-150 μg SC BID or IV (25-50 μg/h) if dehydration is 
severe, with dose escalation up to 500 μg SC TID). 
 Use IV fluids as appropriate. 
 Consider prophylactic antibiotics as needed (eg, fluoroquinolones) especially if 
diarrhea is persistent beyond 24 hours or there is fever or Grade 3-4 
neutropenia. 
8.6.5 
Management of hyperlipidemia and hyperglycemia  
Treatment of hyperlipidemia should take into account the pre-treatment status 
and dietary habits. If blood lipid levels exceed the thresholds identified in eligibility 

 
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criterion 4.2.13 and treatment of hyperlipidemia is being considered, further blood 
tests to monitor hyperlipidemia must be taken in the fasting state. 
Hypercholesterolemia (> 300 mg/dL or 7.75 mmol/L) or hypertriglyceridemia (>2.5 x 
ULN) should be treated with a 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase 
inhibitor (e.g., atorvastatin, pravastatin) or appropriate lipid-lowering medication, in 
addition to diet. 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. 
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 kinase (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. 
Grade 3 hyperglycemia has been observed in patients receiving EVE 
therapy. In many cases in study EVE C2222, the affected patients had an abnormal 
fasting glucose at baseline. Based on this finding, it is suggested that optimal 
glucose control should be achieved before starting a patient on EVE and should be 
monitored during EVE therapy. 
8.6.6 
Management of non-infectious pneumonitis 
Both asymptomatic radiological changes (grade 1) and symptomatic non-
infectious pneumonitis (grade 2 = not interfering with activities of daily living or grade 
3 = interfering with activities of daily living and oxygen indicated) have been noted in 
patients receiving EVE therapy. Non-infectious pneumonitis has been associated 
with EVE and other mTOR inhibitors (Atkins 2004). In order to monitor for 
asymptomatic (grade 1) pulmonary infiltrates, a chest X-ray is required if a CT scan 
of chest is not used for bi-monthly disease evaluations. Additional chest CT scans 
may be performed, when clinically necessary. If non-infectious pneumonitis 
develops, a consultation with a pulmonologist should be considered. If the patient 
develops grade 3 pneumonitis, treatment with EVE should be interrupted and the 
patient should be treated as medically indicated (short course corticosteroids, 
oxygen, etc). 
Management of non-infectious pneumonitis suspected to be associated with 
EVE and dose modifications instructions are provided below. 
 
Table 4:  Management of non-infectious pneumonitis 
Worst Grade 
Pneumonitis 
Required Investigations 
Management of 
Pneumonitis  
EVE Dose Adjustment 
Grade 1 
CT scans with lung windows 
and pulmonary function 
testing including: spirometry, 
DLCO, and room air ORR2 
RRsaturation at rest. Repeat 
chest x-ray/CT scan every 2 
Cycles until return to baseline. 
No specific 
therapy is 
required 
Administer 100% of EVE 
dose. 
Grade 2 
CT scan with lung windows 
and pulmonary function 
testing including: spirometry, 
DLCO, and room air ORR2 
RRsaturation at rest. Repeat each 
subsequent Cycle until return 
Symptomatic 
only. Prescribe 
corticosteroids 
if cough is 
troublesome. 
Reduce EVE dose until 
recovery to ≤ Grade 1. EVE 
may also be interrupted if 
symptoms are troublesome. 
Patients will be withdrawn 
from the study if they fail to 

 
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Worst Grade 
Pneumonitis 
Required Investigations 
Management of 
Pneumonitis  
EVE Dose Adjustment 
to baseline. Consider 
bronchoscopy *  
recover to ≤ Grade 1 within 3 
weeks. 
Grade 3 
CT scan with lung windows 
and pulmonary function 
testing including: spirometry, 
DLCO, and room air ORR2 
RRsaturation at rest.; Repeat 
each subsequent Cycle until 
return to baseline. 
Bronchoscopy is 
recommended * 
Prescribe 
corticosteroids 
if infective 
origin is ruled 
out. Taper as 
medically 
indicated. 
Hold treatment until 
recovery to ≤ Grade 1. May 
restart protocol 
treatment within 2 weeks at 
a reduced dose (by one 
level) if evidence of clinical 
benefit. 
Patients will be withdrawn 
from the study if they fail to 
recover to ≤ Grade 1 within 2 
weeks.  
Grade 4 
CT scan with lung windows 
and required pulmonary 
function testing includes: 
spirometry, DLCO, and room 
air ORR2 RRsaturation at rest. 
Repeat each subsequent Cycle 
until return to baseline. 
Bronchoscopy is 
recommended *. 
Prescribe 
corticosteroids 
if infective 
origin is ruled 
out. Taper as 
medically 
indicated. 
Discontinue treatment. 
*A bronchoscopy with biopsy and/or bronchoalveolar lavage is 
recommended. 
8.6.7 
Wound healing complications 
Impaired wound healing is a class effect of rapamycin derivatives, including 
everolimus.  Caution should therefore be exercised with the use of Afinitor in the 
peri-surgical period. 
8.6.8 
Management of hypophosphatemia 
Hypophosphatemia is a known side effect of EVE and will be monitored for 
patients receiving EVE. The CTCAE v4 grading of hypophosphatemia appears to be 
incompatible with the HUP laboratory’s reference range.  The HUP lab’s LLN is 2.4 
mg/dL. Grade 1 is <LLN – 2.5 mg/dL (which is N/A), Grade 2 is <2.5 – 2.0 mg/dL, 
Grade 3 is <2.0 – 1.0 mg/dL.  Thus, a subject's phosphorus could be just 0.1 mg/dL 
below normal be considered a Grade 2 by the CTCAE v4.  Therefore, while this 
protocol will still use the CTCAE v4 for grading purposes, we have instituted the 
guidelines outlined below for the management of hypophosphatemia.  (It is noted 
that the CTCAE v5 has revised the hypophosphatemia grading guidelines.) 
Hypophosphatemia should be managed with supplementation/oral 
replacement therapy as clinically indicated, at the discretion of the PI/treating 
physician. Events of hypophosphatemia, even if Grade 3 by the CTCAE v4 will not 
be subject to the EVE dose modification guidelines for non-hematological toxicity 
outlined in Table 11. EVE should be interrupted only if the hypophosphatemia event 
is severe or medically significant as determined by the PI/treating physician and/or if 
hospitalization or prolongation of existing hospitalization is indicated (these are the 
Grade 3 criteria in CTCAE v5). 
8.6.9 
Management of Hepatitis reactivation/flare 
In cancer patients with hepatitis B, whether carriers or in chronic state, use of 
antivirals during anticancer therapy has been shown to reduce the risk of hepatitis B 
virus (HBV) reactivation and associated HBV morbidity and mortality (Loomba et al. 

 
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2008). 
8.6.9.1 
Monitoring and prophylactic treatment for hepatitis B 
reactivation 
Table 5 provides details of monitoring and prophylactic therapy according to 
the baseline results of viral load and serologic markers testing. 
 
Table 5:  Action to be taken for positive baseline hepatitis B results 
Test 
Result 
Result 
Result 
Result 
Result 
HBV-DNA 
+ 
+ or - 
- 
- 
- 
HBsAg 
+ or - 
+ 
- 
- 
- 
HBs Ab 
+ or - 
+ or - 
+ 
and no prior HBV 
vaccination 
+ or - 
- 
or + with 
prior 
HBV 
vaccinati
on 
HBc Ab 
+ or - 
+ or - 
+ or - 
+ 
- 
Recom-
mendation 
Prophylaxis treatment should 
be started 1-2 weeks prior to 
first dose of  study drug 
Monitor HBV-DNA 
approximately every 6 weeks 
 
No prophylaxis 
Monitor HBV-DNA 
approximately every  3 weeks 
 
No 
specific 
action 
 
Antiviral prophylaxis therapy should continue for at least 4 weeks after last 
dose of study drug. For hepatitis B reactivation, definition and management 
guidelines, see Table 6.   
 
Table 6:  Action to be taken in event of Hepatitis Reactivation 
HBV reactivation  (with or without clinical signs and symptoms)* 
For patients with baseline results:  
Positive HBV-DNA 
OR 
positive HBsAg 
-------------------------------------- 
reactivation is defined as:   
[Increase of 1 log in HBV-DNA 
relative to baseline HBV-DNA 
value OR new appearance of 
measurable HBV-DNA] 
 
Treat: Start a second antiviral  
AND 
Interrupt study drug administration until resolution:  
  ≤ baseline HBV-DNA levels 
If resolution occurs within ≤ 28 days, study drug should be 
re-started at one dose lower, if available. If the patient is 
already receiving the lowest dose of study drug 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 
study drug but continue both antiviral therapies at least 4 
weeks after last dose of study drug. 
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]  
------------------------------------- 
reactivation is defined as: 
New appearance of measurable 
HBV-DNA 
Treat : Start first antiviral medication  
AND 
Interrupt study drug administration until resolution:  
  ≤ baseline HBV-DNA levels 
If resolution occurs within ≤ 28 days study drug should be 
re-started at one dose lower, if available. If the patient is 
already receiving the lowest dose of study drug 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.  

 
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* All reactivations of hepatitis B are to be recorded as grade 3 (CTCAE v 3.0 
Metabolic Laboratory/Other: Viral Re-activation), unless considered life threatening 
by the investigator; in which case they should be recorded as grade 4 (CTCAE v 3.0 
Metabolic Laboratory/Other: Viral Re-activation). Date of viral reactivation is the date 
on which both DNA and ALT criteria were met (e.g. for a patient who was HBV-DNA 
positive on 01-JAN-10 and whose ALT reached ≥ 5 × ULN on 01-APR-10, the date 
of viral reactivation is 01-APR-10). 
8.6.9.2 
Monitoring for Hepatitis C Flare 
The following two categories of patients should be monitored every 6 weeks 
for HCV reactivation: 
1.  Patients with detectable HCV RNA-PCR test at baseline. 
2.  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’) 
For definition of hepatitis C reactivation and the management guidelines, see 
Table 3-4 Guidelines for management of hepatitis C. 
 
 
 
 
Table 7:  Guidelines for management of hepatitis C 
HCV flare* 
For patients with baseline results:  
Detectable HCV-RNA, HCV flare is defined as: 
2 log10 IU/mL increase in HCV-RNA 
AND 
ALT elevation x 5 ULN or 3 baseline level, whichever is higher 
Discontinue study drug  
 
 
For patients with baseline results: 
Knowledge of past hepatitis C infection with no detectable HCV-RNA,   
 HCV flare is defined as: 
New appearance of detectable HCV-RNA  
AND 
ALT elevation x 5 ULN or 3 baseline level, whichever is higher 
Discontinue study drug 
 
* All flares of hepatitis C are to be recorded as grade 3 (CTCAE v3.0 
Metabolic Laboratory/Other: Viral Flare), unless considered life threatening by the 
investigator; in which case they should be recorded as grade 4 (CTCAE v 3.0 
Metabolic Laboratory/Other: ViralFlare). 
8.7 
Dose Delays 
   .   Treatment should be delayed for major events if HCQ or EVE may further 
complicate the major event.  “Major events” are non treatment-related grade 3 and 4 
hematologic and non-hematologic toxicities. If a major event requires a delay of 
treatment, treatment must be delayed until toxicity is resolved (≤ Grade 1 or ≤ 
Baseline).  If toxicity is not resolved in ≤ 4 weeks (28 days), treatment will be 
discontinued (see Section 7.8).  
8.8 
Dose Modifications 
For patients who are unable to tolerate the protocol-specified dosing 
If resolution occurs > 28 days, patients should discontinue 
study drug but continue antiviral therapy at least 4 weeks 
after last dose of study drug. 

 
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schedule, dose adjustments are permitted in order to keep the patient on study drug.  
Dose modification guidelines are found in the sections below.  Any consideration to 
deviate from these protocol-defined dose modification guidelines should be 
discussed with the Principal Investigator and treating physician for approval or 
disapproval in advance. Since this is a pilot study, this protocol allows physician 
discretion surrounding dose adjustment decisions. Dose adjustment decisions based 
on PI/physician discretion must be documented as such. 
Because nausea, vomiting, diarrhea or rash may be attributable to either 
HCQ or EVE, Table 8 outlines the order in which to dose reduce one or the other 
agent.  All other toxicities should be managed with dose modification of EVE alone. 
 
Table 8:  Guidelines for Selecting Agent for Dose Reduction if Overlapping Toxicities 
Overlapping 
Toxicity  
Dose Reduce First   Dose Reduce Second  
Rash  
EVE 
HCQ 
Nausea 
HCQ  
EVE 
Anorexia  
HCQ  
EVE 
Diarrhea 
EVE 
HCQ  
 
8.8.1 Hydroxychloroquine Dose Modification 
Any AE of ≥ Grade 3 and attributed as possibly, probably or definitely related 
to HCQ will result in the dose being held until the AE has resolved to ≤ grade 1 or 
baseline while EVE dosing may continue uninterrupted. If the AE resolves, 
reinstitution of treatment can occur but at a reduced dose as described in Table 9. If 
the AE recurs at the reduced dose, treatment will be held until the AE has resolved 
to ≤ grade 1 and when resolved treatment can be reinstituted at the next lower dose 
level. No more than 2 dose reductions are allowed during the maintenance cycles.     
 
Table 9: Hydroxychloroquine Dose Reduction Schema 
Dose Level 
Dose mg/day 
Reduce to 
-2 
400 mg twice daily 
400 mg daily 
-1 
600 mg am and 400 mg pm 
400 mg twice daily 
1 
600 mg twice daily 
600 mg am and 400mg pm 
 
If any AEs occur at grade ≤ 2 that are attributed as possibly, probably or 
definitely related to HCQ, HCQ may be continued and the AE managed with 
supportive care, with the exception of visual field deficit. UVisual field deficit is a 
potential symptom of peripheral retinal toxicity should prompt drug 
discontinuation and ophthalmologic evaluation immediately.  With particular 
regard to visual field deficits patients should be cautioned to report any visual 
symptoms, particularly difficulty seeing entire words or faces, intolerance to 
glare, decreased night vision, or loss of peripheral vision U.U 
If any AEs occur at grade ≤ 2 that are attributed as possibly, probably or 
definitely related to HCQ, HCQ may be interrupted if the toxicity is either intolerable 
to the patient or PI/physician discretion determines it is in the best interest of the 
patient.  In such cases, HCQ should be interrupted until recovery to grade 1. If 

 
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resolution occurs in ≤ 7 days HCQ may be reintroduced at the same dose. If 
resolution takes > 7 days HCQ should be reintroduced at the lower dose level.  If the 
event returns to grade 2, then HCQ should be interrupted until recovery to grade 1, 
after which HCQ should be reintroduce at the lower dose level. 
 
 
8.8.1.1 
Management of suspected HCQ induced myopathy 
Myopathy leading to muscle weakness is a rare but potentially serious side effect of 
HCQ. In our experience, this toxicity resolves with holding drug and can be reduced by 
reducing dose. With close attention to early signs of this toxicity, the goal is for it to be 
managed before it becomes serious and reaches a higher grade. Therefore, in the instance 
of grade >1 muscle weakness and/or elevated CPK or at MD discretion (even if the related 
AE is still only grade 1), HCQ should be held.  When myopathy/muscle weakness returns to 
grade ≤1 (or after holding for a period per MD discretion, as long as symptoms and lab 
values are trending back to normal), HCQ may be reintroduced at the next lower dose 
level.  It is not necessary that CPK return to grade ≤1 to restart drug, so long as myopathy 
has.  It is intended that close monitoring and implementing HCQ dose reduction at early 
signs of myopathy will prevent subjects progressively worsening to the point that they have 
to come off study drug (HCQ) altogether. 
8.8.2 
Everolimus (EVE) Dose Reduction Guidelines 
Known EVE hematological toxicities will not be attributed to HCQ but will only 
be attributed to EVE and will result in the following EVE dose modifications, as 
shown in Table 11. Hematologic toxicity of unexpected severity may be attributed to 
HCQ (in addition to EVE at investigator discretion).  Non-hematological toxicities 
having an attribution to EVE of possible, probable or definite will result in the 
following EVE dose modifications, as shown in Table 11.  For EVE dosing, the 
patient must have an ANC ≥ 1.0 x 10P9
P/L and platelet count > 75 x 10P9
P/L. All non-
hematological toxicity grade ≥ 3 (except for alopecia, nausea and vomiting) must 
have resolved to CTCAE grade ≤ 1.  If toxicity persists, treatment should be delayed 
by one week for up to 4 consecutive weeks. If after 4 weeks (28 days) of delay all 
toxicity has still not resolved, any further treatment with EVE should be stopped. 
HCQ may be continued after EVE has been stopped on the combination arm. 
 
If any non-hematological toxicity observed Grade ≥ 3 (except for alopecia, 
nausea and vomiting) and/or if platelets < 75 x 10P9
P/L and/or ANC < 1 x 10P9
P/L, 
causing delay of two weeks of EVE then the dose should be reduced by one dose 
level (see Table 10). For patients who would require dose >2 dose reductions EVE 
will be stopped. If any non-hematological toxicity observed was CTCAE Grade 4 
(except for alopecia, nausea and vomiting) then EVE should be stopped. 
 
 
Table 10:  EVE Dose Reduction Guidelines 
Dose Level  
Dose 
Reduce to 
1 
10 mg daily 
5 mg daily 
-1 
5 mg daily 
5 mg every other day 
-2 
5 mg every other day 
Discontinue EVE 

 
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Table 11: Criteria for dose-modification for suspected EVE toxicity and re-initiation of EVE 
treatment 
 
Toxicity 
Actions 
Non-hematological toxicity 
 
Grade 2  
(except pneumonitis – refer to 
Table 6) 
If the toxicity is tolerable to the patient, maintain the same dose. If the 
toxicity is intolerable to patient, interrupt EVE until recovery to grade 
1. If resolution occurs ≤ 7 days reintroduce EVE at same dose. If 
resolution takes > 7 days or if the event returns to grade 2, then 
interrupt EVE until recovery to grade 1. Then reintroduce EVE at 
lower dose level. 
Grade 3 
(except hyperlipidemia*) 
(except pneumonitis – refer to 
Table 6) 
(except hypophosphatemia, 
refer to Section 8.6.8) 
Interrupt EVE until recovery to grade 1. Then reintroduce EVE at the 
lower dose level. For pneumonitis consider the use of a short course of 
corticosteroids. 
Grade 4 
Discontinue EVE 
Hematological toxicity 
 
Grade 2 Thrombocytopenia  
(<75,000 – 50,000/mm^3 ; <75 – 
50.0 x 10^9/L) 
Temporary dose interruption until recovery to Grade < 1. Re-initiate 
treatment at the same dose. 
 
Grade 3 Thrombocytopenia  
(<50,000 – 25,000/mm^3 ; <50.0 
– 25.0 x 10^9/L) 
Interrupt EVE until resolution to grade 1  
Re-initiate treatment at a lower dose. 
Grade 4 Thrombocytopenia  
(<25,000/mm^3 ; <25.0 x 
10^9/L) 
Interrupt EVE until recovery to grade  1. Then reintroduce Everolimus 
at one dose level lower, if available. 
Grade 3 Neutropenia  (<1,000 – 
500/mm^3 ; <1.0 – 0.5 x 10^9/L) 
or anemia 
Temporary dose interruption until recovery to Grade < 2. Re-initiate 
treatment at the same dose 
Grade 4 Neutropenia  
(<500/mm^3 ; <0.5 x 10^9/L) or 
anemia 
Interrupt EVE until recovery to grade  2. Reintroduce EVE at one 
dose level lower, if available.** 
Grade 3 Febrile Neutropenia 
(ANC <1,000/mm^3 with a single 
temperature of > 38.3 °C 
(101°F) or a sustained 
temperature of > 38 °C (100.4 
°F) for more than one hour) 
Hold further dosing until Grade < 2 ANC and no fever. Then resume 
dosing at the next lower dose level if available. 
Grade 4 Febrile Neutropenia 
(life-threatening consequences ; 
urgent intervention indicated)) 
Discontinue EVE. 
Recurrence of grade 3 toxicity 
after dose reduction 
Reduce dose to the next lower dose level, if available. The lowest 
possible dose level of EVE is 5 mg every other day (2.5 mg daily). 
Below this level, EVE must be discontinued. 
**Recurrence of grade 4 toxicity 
(including febrile neutropenia) 
after dose reduction 
Discontinue EVE 
**Any toxicity requiring 
interruption for > 28 days  
Discontinue EVE 

 
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*Grade 3 hyperlipidemia (hypercholesterolemia and/or hypertriglyceridemia) 
should be managed using medical therapies. 
 
9 
DRUG FORMULATION AND STORAGE 
9.1 
Agent #1:  Hydroxychloroquine  
 
Generic name:  Hydroxychloroquine sulfate 
 
Commercial name:  Plaquenil  
 
Chemical name: 7-Chloro-4-[4-[ethyl-(2-hydroxyethyl)amino]-1-
methylbutylamino] quinolone 
 
Hydroxychloroquine sulfate is a colorless crystalline solid, soluble in water to 
at least 20 
percent; chemically the drug is 2-[[4-[(7-Chloro-4-quinolyl) amino]pentyl] 
ethylamino] ethanol 
sulfate (1:1). 
9.1.1 Supply 
As described above, hydroxychloroquine will be obtained through a written 
prescription by study MD as an initial 28- day supply and refilled every cycle.  
Patients will be required to bring the prescription to their visit on day 1 of each cycle, 
along with previous months’ pill container so that pill counts can be performed. In 
addition,  treatment diaries will be used to monitor adherence to therapy.  Patients 
are required to bring the pill bottles and treatment diaries to each visit.    
9.1.2 Formulation 
PLAQUENIL (hydroxychloroquine sulfate) tablets contain 200 mg 
hydroxychloroquine 
sulfate, equivalent to 155 mg base, and are for oral administration. 
Inactive Ingredients: Dibasic Calcium Phosphate, Hydroxypropyl 
Methylcellulose, Magnesium Stearate, Polyethylene glycol 400, Polysorbate 80, 
Starch, Titanium Dioxide. 
9.1.3 Dosing, Administration and Storage 
When taking HCQ twice daily, the two daily doses should be taken 12 hours 
apart as close to 9am and 9pm as possible and documented clearly on the patient 
calendar.  Patients receiving antacids, sucralfate, cholestyramine, and/or 
bicarbonate should have the HCQ drug dose administered at least 1 hour before or 
2 hours after these medications. Missed doses should not be made up; patient 
should resume planned dose at next dosing interval. 
 
9.2 
Agent #2: Everolimus 
 
Generic name: Everolimus 

 
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Commercial name: AfinitorPTM 
 
Chemical name: dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-
methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-
11,36-dioxa-4-azatricyclo[30.3.1.0P4,9
P]hexatriaconta-16,24,26,28-tetraene-
2,3,10,14,20-pentone 
 
9.2.1 
Supply 
Everolimus will be supplied by Novartis Pharmaceuticals Corporation for this 
trial and dispensed by the Oncologic Investigational Pharmacy at the Abramson 
Cancer Center of the University of Pennyslvania. 
9.2.2 
Formulation 
EVE is formulated as tablets for oral administration of 5mg strength. Tablets 
are blister-packed under aluminum foil in units of 10 tablets, which should be 
opened only at the time of administration as drug is both hygroscopic and light-
sensitive. 
9.2.3 Dosing, Administration and Storage 
Patients randomized to an EVE-containing arm will receive EVE as a dose of 
10 mg orally daily, on a continuous basis.  Patients will be instructed to take EVE in 
the morning, at the same time each day. EVE may be taken with or without food. 
9.3 Drug Accountability 
Clinical drug supply must be accounted for and patients will be asked to 
return all unused study drug and packaging on a regular basis, at the end of the 
study or at the time of study drug discontinuation. Accountability for the drug at all 
study sites is the responsibility of the principal investigator and designated 
Pharmacy representative. The investigator will ensure that the investigational drug is 
used only in accordance with this protocol. Drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and 
destruction/disposal per institutional policy will be maintained by the clinical site. 
Accountability records will include dates, quantities, lot numbers, expiration dates, 
and patient numbers. 
At the conclusion of the study, and, as appropriate during the course of the 
study, the investigator will return all used and unused study drug, packaging, drug 
labels, and a copy of the completed drug accountability ledger to Novartis. 
 
10 SAFETY ASSESSMENTS 
10.1 Adverse Events Reporting Period 
Patients will be evaluated for evidence of toxicity from the time of the first 
dose of HCQ/EVE to 30 days after last study drug or longer in the event of residual 
toxicity. 
10.2 Definitions 
Adverse Event:  An adverse event (AE) is any symptom, sign, illness or 

 
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experience that develops or worsens in severity during the course of the study. 
Intercurrent illnesses or injuries should be regarded as adverse events. Abnormal 
results of diagnostic procedures are considered to be adverse events if the 
abnormality: 
.. results in study withdrawal 
.. is associated with a serious adverse event 
.. is associated with clinical signs or symptoms 
.. leads to additional treatment or to further diagnostic tests 
.. is considered by the investigator to be of clinical significance 
 
Serious Adverse Event: A serious adverse event (SAE) is any adverse 
event, without regard to causality, that is life-threatening or that results in any of the 
following outcomes: death; in-patient hospitalization or prolongation of existing 
hospitalization; persistent or significant disability or incapacity; or a congenital 
anomaly or birth defect.  Any other medical event that, in the medical judgment of 
the Principal Investigator, may jeopardize the subject or may require medical or 
surgical intervention to prevent one of the outcomes listed above is also considered 
an SAE.  A planned medical or surgical procedure is not, in itself, an SAE. Also 
specifically excluded from this definition of SAE is any event judged by the Principal 
Investigator to represent progression of the malignancy under study, unless it results 
in death within the SAE Reporting Period. 
 
Post-study Adverse Event:  All unresolved adverse events should be 
followed by the investigator until the events are resolved, the patient is lost to follow-
up, or the adverse event is otherwise explained. At the last scheduled visit, the 
investigator should instruct each patient to report any subsequent event(s) that the 
patient, or the patient’s personal physician, believes might reasonably be related to 
participation in this study.  
10.3 Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event 
if the abnormality is of a degree, typically at least grade 2 and not present as grade 
1 or better at baseline, that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation. 
10.4 Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization 
should be documented and reported as a serious adverse event unless specifically 
instructed otherwise in this protocol. Any condition responsible for surgery should be 
documented as an adverse event if the condition meets the criteria for an adverse 
event. 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery 
are reported as an adverse event in the following circumstances: 
-Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition. Surgery should not be reported as an 
outcome of an adverse event if the purpose of the surgery was elective or diagnostic 
and the outcome was uneventful. 
-Hospitalization or prolonged hospitalization required to allow efficacy 

 
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measurement for the study. 
-Hospitalization or prolonged hospitalization for therapy of the target disease 
of the study, unless it is a worsening or increase in frequency of hospital admissions 
as judged by the clinical investigator. 
 
10.5 Recording of Adverse Events 
At each contact with the patient, the investigator must seek information on 
adverse events by specific questioning and, as appropriate, by examination. 
Information on all adverse events should be recorded immediately in the source 
document, and also in the appropriate adverse event module of the case report form 
(CRF). All clearly related signs, symptoms, and abnormal diagnostic procedures 
results should recorded in the source document, though should be grouped under 
one diagnosis. All adverse events occurring during the study period must be 
recorded. The clinical course of each event should be followed until resolution, 
stabilization, or until it has been determined that the study treatment or participation 
is not the cause. Serious adverse events that are still ongoing at the end of the study 
period must be followed up to determine the final outcome. Any serious adverse 
event that occurs after the study period and is considered to be possibly related to 
the study treatment or study participation should be recorded and reported 
immediately. 
10.6 Reporting of Adverse Events and Serious Adverse Events 
10.6.1 Study Chair Notification by Investigator 
A serious adverse event must be reported to the Study Chair within 24 hours 
of a member of the study team becoming aware of the event.  
Report serious adverse events by phone and facsimile to: 
 
UAngela DeMichele, MD, MSCE, or via the HUP page operator 
 
At the time of the initial report, the following information should be provided: 
• Study identifier 
• Study Center 
• Patient number 
• A description of the event 
• Date of onset 
• Current status 
• Whether study treatment was discontinued 
• The reason why the event is classified as serious 
• Investigator assessment of the association between the event and study 
treatment 
 
FAX#: 215-662-7865 
 
10.6.2 IRB Notification by Investigator-sponsor 
Reports of all serious adverse events (including follow-up information) must 
be submitted to the IRB and CTSRMC (Penn) within 10 working days, according to 
IRB guidelines. 

 
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UReporting Process to IRB at PennU:  Principal Investigators are encouraged 
to submit reports of unanticipated problems posing risks to subjects or others using 
the form: “Unanticipated Problems Posing Risks to Subjects or Others Including 
Reportable Adverse Events” via HS-ERA or a written report of the event (including a 
description of the event with information regarding its fulfillment of the above criteria, 
follow-up/resolution and need for revision to consent form and/or other study 
documentation) within 7 working days and the PI or her designee will enter the event 
into HS-ERA and VELOS for Penn and participating sites.  Participating sites should 
follow local, institutional guidelines on Event Reporting. 
For reportable deaths, the initial submission to the IRB may be made by 
contacting the IRB Director or designee at 215.573.1206.  All deaths will be reported 
via HS-ERA, as well.   
10.6.3 FDA Notification by Investigator-sponsor 
This study is IND exempt and reporting to the FDA is voluntary using a 
Medwatch 3500 or via the FDA’s website for voluntary reporting. 
 
10.6.4 Abramson Cancer Center DSMC notification 
All adverse events meeting the ACC’S DSMC reporting requirements outlined 
below will be reported to the ACC DSMC through the PennCTMS (Velos) Clinical Trial 
Management System AE/SAE Form. 
 All Grade 3 or higher adverse events regardless of attribution or 
expectedness must be submitted to the ACC’S DSMC within 
10 business days of knowledge. 
 All unexpected deaths must be reported within two business days of 
knowledge. Deaths related to disease progression must clearly state that fact. 
 All others deaths must be reported within 30 days of knowledge. Deaths of 
subjects greater than 90 days from the last study treatment/intervention are 
not reportable. 
All AE/SAEs should include grade, attribution and expectedness as determined by 
the PI or sub-I. Only an investigator may determine the attribution and 
expectedness. 
For attributions of "unrelated", an alternative explanation must be provided to explain 
to what the event is being attributed. 
For studies using multiple agents in a single study, the agent to which the event is 
being attributed should be identified. 
Every effort should be made to report an event as a diagnosis, not as a list of 
symptoms. Symptoms that led to the diagnosis should be included in the event 
description, but should not be the actual event. 
 
10.6.5 IND SAFETY UPDATES/ALERTS  
All IND Safety Updates/Alerts (sent by investigators) with a grade 3 or higher, 
regardless of attribution or expectedness must be submitted to the DSMC within 30 
days. Reports should be sent to the DSMC for 6 months from the date the last Penn 

 
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subject was treated.  
Although this protocol is IND-exempt, it is the responsibility of each PI to 
notify all participating investigators, in a written safety report using an IND safety 
report template, of any adverse event associated with the use of the drug that is 
both serious and unexpected, as well as any finding from tests in laboratory animals 
that suggest a significant risk for human subjects.  Additionally, investigators are 
also required to identify in IND safety reports all previous reports concerning similar 
adverse events and to analyze the significance of the current event in light of the 
previous reports.  Reports will be sent, via e-mail to participating sites, for review 
and forwarding to their internal committees, as appropriate.   
 
 
USAE Definition.U   
10.6.6 Novartis instructions for rapid notification of serious adverse 
events 
1. The principal investigator has the obligation to report all serious adverse 
events to the FDA, IRB, and Novartis Pharmaceuticals Clinical Safety and 
Epidemiology Department (CS&E). 
2.  All events reported to the FDA by the investigator are to be filed utilizing 
the Form FDA 3500A (MedWatch Form).  
3.  All events must be reported, by FAX (888-299-4565), to Novartis 
Pharmaceuticals CS&E Department within 24 hours of learning of its occurrence. 
This includes serious, related, labeled (expected) and serious, related, unlabeled 
(unexpected) adverse experiences. All deaths during treatment or within 30 days 
following completion of active protocol therapy must be reported within 5 working 
days. 
4.  Any serious adverse event occurring after the patient has been 
administered one dose of study drug until 4 weeks after the patient has stopped 
study participation must be reported. This includes the period in which the study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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protocol interferes with the standard medical treatment given to a patient (e.g. 
treatment withdrawal during washout period, change in treatment to a fixed dose of 
concomitant medication). 
5.  Serious adverse events occurring more than 30 days after study 
discontinuation need only be reported if a relationship to the Novartis study drug (or 
therapy) is suspected.  
10.7 Internal Safety and Compliance Entities  
The Data Safety and Monitoring Committee (DSMC) of the University of 
Pennsylvania’s Abramson Cancer Center will monitor the data quality and 
adherence to safety rules. Additionally, the DSMC will review all safety/toxicity data 
for the trial and recommend trial suspension or termination as needed. Specific 
details of monitoring and audit frequency will be included in the Monitoring Plan, but 
will be at least every 6 months.  
 
Patients will be evaluated for toxicity if they have received at least one dose 
of HCQ. 
 
 
  
The timely reporting of adverse events (including toxic deaths) is required by 
the Food and Drug Administration.  The reporting of toxicities is part of the data 
reporting for this study. 
 
 
 
10.8 Medical and Research Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the 
study at his/her site.  This safety monitoring will include careful assessment and 
appropriate reporting of adverse events, as noted above, as well as the construction 
and implementation of a site data and safety monitoring plan.  Medical monitoring by 
an independent clinician within the Department of Medicine, Division of Hematology-
Oncology will include a regular assessment of the number and type of serious 
adverse events on a periodic basis. Dr. Tara (Gangadhar) Mitchell is a board 
certified oncologist, and a member of the solid malignancies clinical trials group at 
UPENN.  She is the PI on multiple phase I/II trials. The medical monitor will meet 
with Dr. DeMichele and clinical research staff quarterly. She will review adverse 
events, response assessments, and decisions related to modifications. 
 Furthermore, Dr. Mitchell will serve in the role of independent research 
monitor.  The roles/responsibilities of the independent research monitor are outlined 
below.  It is noted that Dr. Mitchell has no apparent conflict of interest and as the 
independent research monitor, she is not under the supervision of the PI or other 
investigators or research staff.   
 
The research monitor: 
 
 may discuss the research protocol with the investigators, interview human 
subjects, and consult with others outside of the study about the research; 
 shall have authority to stop a research protocol in progress, remove individual 
human subjects from a research protocol, and take whatever steps are 

 
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necessary to protect the safety and well-being of human subjects until the 
IRB can assess the monitor's report; 
 shall have the responsibility to promptly report their observations and findings 
to the IRB or other designated official and the Human Research Protection 
Office (HRPO) of the US Army Medical Research and Material Command 
(USAMRMC) 
 
10.9 Study Monitoring Plan 
This study will be monitored by the Principal Investigator and sub-
investigators, as appropriate.  Such monitoring will include at least weekly meetings 
of the study team to review accrual, toxicity, SAEs, dose escalations and study 
finding.  In addition, the PI will ensure that data are completed in a timely manner 
and he or his designee will periodically review the data for accuracy, completeness 
and integrity.   
10.10 Auditing and Inspecting 
The investigators will permit the Cancer Center’s Administrative Director of 
Compliance and Auditing or her designee to review records, data and facilities at 
mutually agreeable times.   
This study will be audited by the Department of Compliance and Monitoring 
(DOCM) as a high-risk study. Auditing will occur 6 months after the first subject 
enrolls and every six months thereafter until the study terminates. Audits will be 
scheduled approximately 5 weeks in advance of the audit. Lack of cooperation with 
scheduling will result in the study being put on hold at the participating sites until an 
audit is conducted. The DOCM may at its discretion, modify the audit schedule or 
the audit process.  
10.11 Data Safety Monitoring Board 
In addition to the above safeguards, an independent data safety monitoring 
committee, consisting of 2 medical oncologists and a member of the patient 
advocate team will meet with the PI and the study team monthly to review safety 
data, to assure that no unanticipated safety signals or risks are arising in the 
conduct of the study.  In addition, the DSMB will review interim safety analyses for 
each arm after every 3 patients have been enrolled.  We will implement a Bayesian 
toxicity monitoring approach with a target protocol-defined DLT rate of 30% as 
outlined in the statistical plan.  This additional safeguard is to ensure that patients on 
this trial, with potentially curable disease, are being monitored closely. 
10.12 Protocol Deviations and Exception Requests 
A protocol deviation is a one time, unintentional action or process that departs 
from the IRB and DSMC approved study protocol, involving one incident and 
identified retrospectively, after the event occurred. If the impact on the protocol 
disrupts the study design, may affect the outcome (endpoints) or compromises the 
safety and welfare of the subjects, the deviation is considered “major” and must be 
reported to the DSMC and the IRB within 10 business days.  
Deviations must be submitted if they:  
 have the potential to adversely affect subject safety 
 increase risks to participants 

 
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 adversely affect the integrity of the data 
 violate the rights and welfare of participants 
 affect the subject’s willingness to participate in research 
 
Other minor deviations should be explained in a memo to file or on a 
deviation log and summarized at continuing review.   
If there is a planned deviation from the protocol, the situation will be 
submitted to the IRB and DSMC PRIOR to the event as an exception request, and 
approval will be sought before implementation.  Exceptions to eligibility, 
treatment/dosing, contraindicated treatment/therapies/ interventions or safety tests 
for investigator-initiated treatment studies may be rejected by the ACC DSMC.  
Exception requests must provide strong and compelling scientific and clinical 
information as well as a statement explaining whether or not the protocol will be 
amended.  If the protocol will not be amended a rationale must be provided.  A 
protocol amendment is required if this situation is likely to recur.  Study PI and 
Medical Monitor approval must be obtained prior to submitting an exception request. 
 
11 MEASUREMENT OF EFFECT 
For the purposes of this study, patients should be re-evaluated for response 
per the study calendar.  
 
11.1 Definitions  
11.1.1 Evaluable for toxicity   
All patients will be evaluable for toxicity from the time of their first treatment 
with HCQ, EVE or both. 
 
11.1.2 Evaluable for objective response  
Only those patients who have measureable DTCs present at baseline, have 
received at least one cycle of therapy, and have had their disease re-evaluated at 
the 24 week timepoint will be considered evaluable for response.  These patients will 
have their response classified according to the definitions stated below.  We will 
report on both the “intent-to-treat” population and the evaluable population per the 
above criteria in order to fully determine whether the interventions are both feasible 
and showing sufficient evidence of activity to take forward to a larger phase III trial 
powered for relapse-free survival (RFS). 
 
11.1.3 Measurable Disease 
Patients enrolled on the CLEVER trial will have completed surgery for their 
primary breast tumor and be free of overt metastatic disease.  Thus there is no 
tumor to measure for the effect of the study therapy.  Rather, the goal of this trial is 
to eradicate minimal residual disease, in the form of disseminated tumor cells 
(DTCs) that are present in the bone marrow.  Therefore, bone marrow DTCs 
constitute the “measureable” disease in this trial.  A patient will be classified as 
having “measureable disease” if at least 1 disseminated tumor cell (DTC) is detected 

 
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in 5 ml of bone marrow aspirate by immunohistochemistry, as described in section 
13.2.1. 
 
11.2 Guidelines for Evaluation of Disease 
11.2.1 Measurement Methods  
Bone marrow aspirate will be used to obtain sample for assessment of DTCs.  
This will be performed in a standard fashion as outlined in section 13.  Bone marrow 
will be assayed for DTCs by standard immunohistochemical assay performed under 
CLIA conditions in the Clinical Pathology Laboratory at the University of 
Pennsylvania as described in section 13. 
11.2.2 Response Criteria 
Response in this trial will be performed in all patients, and is defined by the 
change in detectable DTC number (# cell/5 ml marrow aspirate) from baseline to 
each timepoint.  We will define “response” for each patient as a 50% or greater 
decrease in DTC number at each treatment time point compared to baseline.  The 
interventional arm will be considered successful if at least 20% of patients 
experience this predefined response endpoint.  We anticipate that the baseline 
variability in DTC measurement will lead to no more than a 4% response rate in the 
absence of treatment (null rate).The efficacy objective of this trial (a secondary 
objective) is to evaluate the preliminary efficacy of EVE, HCQ or the combination in 
reducing or eliminating breast cancer DTCs, and provide estimates of effect for 
future definitive trials.  The primary measure of efficacy in this trial is change in the 
number of detectable disseminated tumor cells (DTCs) in the bone marrow of 
patients on study.  DTCs will be detected from 5 ml of bone marrow extracted from 
the posterior iliac crest at baseline (pre-treatment), after 3 cycles, 6 cycles, and 12 
cycles (if applicable) of study treatment, and at 6 months after the last dose of study 
treatment (optional).   
 
12 STATISTICAL CONSIDERATIONS AND METHODOLOGY 
12.1 Sample Size 
The primary endpoint of this pilot study is feasibility. 60 patients will be 
enrolled on the trial.  Based on the literature and our preliminary experience, we 
anticipate detecting BM-DTCs in 20 - 25% of the patients we screen on the PENN-
SURMOUNT study (IRB# 824098/UPCC 28115), thus have planned to screen 250 - 
300 patients over three years, allowing for refusals and drop-out.   
12.2 Analysis Plan 
12.2.1 Primary Endpoint 
The phase II trial endpoint “feasibility” is based upon the overall safety and 
tolerability of the treatment arms, and will be defined as Uat least U11 of 15 patients 
within each treatment group completing 6 cycles of therapy without grade 3 or 4 
toxicity.  When evaluating feasibility of the combination therapy, Arm C and D may 
be combined for analysis, since both arms receive the same study treatment (Arm D 
is just a delayed start).  Toxicity will be determined by NCI CTCAE v4 criteria, and 
categorized by system and grade.  Patients who drop out before the 6-month time 

 
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point for reasons other than toxicity and not related to feasibility will be replaced.  
With 15 patients per group, an exact 95% confidence interval around the feasibility 
and toxicity rates will be no more than 0.52 units wide.  Arms that are discontinued 
for lack of feasibility will be dropped from the trial, but others may continue, so long 
as they remain “feasible” by this definition.   
 
Because this is a potentially curable patient population, we will also institute 
an early stopping rule, should the toxicity rates exceed those expected or acceptable 
in this setting.  To do this, we will implement a Bayesian toxicity monitoring approach 
with a target protocol-defined DLT rate of 30%.  We assume a Beta(1,2) prior, 
equivalent to one DLT observed in 3 treated patients.  Early termination for toxicity 
will be considered based on a posterior probability that the toxicity rate exceeds 
30%.  Early termination will be considered if DLT is observed in 2 of 3 patients (0.84 
posterior probability), 3 of 6 patients (0.81), 4 of 9 patients (0.79), 5 of 12 patients 
(0.78), 6 of 15 patients (0.78).  
12.2.2 Secondary Endpoints 
The secondary endpoints are tolerability/toxicity and the measured effect of 
the investigational therapy on DTCs in patients with measureable DTCs at baseline 
and the comparison of this effect to that in a control population.  Because this is 
proof-of-concept, sample sizes are not sufficient to address long-term recurrence 
risk.  Randomization is being performed to minimize uncontrolled confounding.  
  
We will define “response” for each patient as a 50% or greater decrease in 
DTC number at each treatment time point compared to baseline.  A treatment arm 
will be considered successful if at least 3 of 15 patients experience this predefined 
response endpoint.  This strategy is being used to provide preliminary estimates of 
effect on DTCs and to assess whether one or more treatment approaches has 
sufficient activity to be considered in a larger study powered for relapse-free 
survival.  We anticipate that the baseline variability in DTC measurement will lead to 
no more than a 4% response rate in the absence of treatment (null rate).  While the 
trial is powered for feasibility, 15 patients provide approximately 80% power to 
detect a response rate of 20%, warranting further study of the treatment strategy 
evaluated in this patient population and clinical setting.   
 
We will also estimate, within each treatment group, the % change in BM-DTC 
number, defined as the difference between BM-DTCs enumerated at 6 months and 
at baseline, divided by the number at baseline; we will compare this quantity across 
treatment groups using simple regression analyses with indicator variables for the 
three active treatments compared to the delayed treatment.  While the trial is 
powered for feasibility, rather than detection of change in BM-DTCs across 
treatment group, 15 patients per group will provide 80% power (using a two-group t-
test with a two-sided alpha level of 0.05) to detect a decrease in BM-DTCs of 
approximately 1 standard deviation between any two groups and will provide 
important preliminary data for designing future trials.   
 
Because of tumor heterogeneity, the natural history of recurrence varies 
substantially between breast cancer subtypes.  We have elected not to expand the 

 
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sample size to evaluate subtypes individually in this feasibility pilot, but expect that 
we will enroll a mix of patients across subtypes that will shed light on variability in 
DTCs by subtype in an exploratory fashion.  
 
 In addition, we will estimate the 3-year relapse-free survival (RFS) of patients 
enrolled on the trial.  Since this is a proof-of-concept and safety trial, we have 
elected not to power the trial for this endpoint, but anticipate collecting valuable 
information to inform and power a subsequent definitive trial powered for this 
endpoint.  
12.2.3 Translational Endpoints 
We expect these studies to inform whether gene expression features, 
mutations, or copy number alterations predict response of DTC numbers to 
treatment with HCQ, GED or their combination, including alterations in the 
PI3K/mTOR pathway.  In addition, we anticipate that these studies will identify 
markers in residual tumors that could serve as therapeutic targets for future trials.  In 
addition, all analyses involving DTC counts determined by DTC-IHC will be repeated 
using DTC counts from DTC-Flow and results compared between the two assays. 
 
13 CORRELATIVE SCIENCES 
Patients enrolled on the CLEVER Trial will have bone marrow aspirates and 
blood collected serially to perform correlative studies of DTCs, CTCs and ptDNA as 
per the study calendar. Tumor tissue from the primary tumor (or from residual 
disease after neoadjuvant therapy, if applicable) will also be obtained and subjected 
to whole exome sequencing (WES) to obtain mutational profiles and driver 
mutations to be used for ptDNA biomarkers.  
13.1 Biospecimens 
13.1.1 Blood samples 
Blood collections will be performed at the time points depicted in the study 
calendar, including at the time of bone marrow aspirates and then at 6-month 
intervals during the follow up phase for subjects who return to Penn for their follow 
up visits. 
13.1.2 Tissue Samples 
Serial sections of FFPE-fixed primary tumors will be obtained.  Samples from 
other institutions will be shipped to our main site.  A representative top section will 
be H&E stained by a pathologist (Dr. Michael Feldman) to identify regions of tumor 
and estimate tumor cellularity.  Ten unstained sections of 10um thickness will be 
used as starting material for simultaneous extraction of DNA and RNA.  Samples 
with cellularity lower than 25% will undergo macro-dissection to remove non-tumor 
tissue prior to extraction of nucleic acid.  Following deparaffinization in xylene, 
nucleic acids will be extracted using the AllPrep DNA/RNA FFPE kit.  To assess 
germline variation in these patients, which is required for interpretation of NGS 
analyses, DNA will be extracted from buffy coat samples using Gentra Puregene 
Blood Kit.  Nucleic acid concentrations and yield will be determined using a Qubit 

 
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fluorometer, A260/A280 ratios will be assessed using a NanoDrop 
spectrophotometer, and fragment length distribution and RNA Integrity Numbers 
(RIN) will be determined using an Agilent Bioanalyzer.   
13.1.3 Bone marrow aspirates 
Bone marrow aspirates will be obtained clinically within the outpatient setting, 
by qualified personnel.  Only liquid aspirate will be collected, bone marrow core 
biopsies are not needed.  The clinician obtaining the bone marrow sample will 
assess the anatomic landmarks before the area is prepped and draped in sterile 
fashion.  The patient will be turned to a decubitus position and local anesthetic will 
be administered to the posterior ileac crest.  Using a Jamshedi needle, 
approximately 10 ml of bone marrow fluid will be aspirated into two EDTA-containing 
tubes.  5 ml will be used for the standard immunohistochemical (IHC) assay which 
will be performed in the setting of the Pathology Department of the University of 
Pennsylvania.   Additional 5 ml will be sent to the research laboratory of Dr. Lewis 
Chodosh at the University of Pennsylvania for DTC-FLOW analysis. Bone marrow 
aspirates will be divided into equal aliquots that will be used to enumerate DTCs 
using either DTC-Flow or DTC-IHC, and for downstream analysis.  Samples will be 
stored at room temperature and run within 24-48 hours of aspiration. 
13.2 Laboratory Assays 
13.2.1 DTC Assessment by Immunohistochemistry 
The presence of DTCs in the marrow by IHC is an integral biomarker required 
on this study for eligibility.  In addition, this assay will be used to quantitate DTC 
response to therapy.  The gold standard method for DTC assessment is 
immunohistochemistry (IHC), and all samples obtained during this trial will be 
subjected to IHC assessment.  IHC to detect DTCs will be performed using the 
method adapted from JanniP4 and NaumeP14
P.  This well-established assay, which has 
been used both in prior prognostic studies and to determine trial eligibility for 
additional adjuvant chemotherapyP14
P, has been replicated within the Research 
Pathology Laboratory of the University of Pennsylvania under the direction of Dr. 
Michael Feldman, a co-investigator on this proposal, and subsequently implemented 
in our CLIA laboratory for clinical use.  Briefly, 5 ml of a bone marrow aspirate 
sample drawn from the iliac crest under anesthesia and collected in an anti-
coagulated tube containing EDTA will be stored at room temperature and processed 
within 24 hours.  Density centrifugation using Lymphoprep will be performed and 
mononuclear cells (MNCs) will be harvested, washed and resuspended to 2.5x10*6 
cells/ml.  Cytospin slides will then be prepared from the cell suspension using a 
cytocentrifuge and slides will be fixed in 95% alcohol before immunostaining on a 
Leica Bond III autostainer.  An anti-pancytokeratin monoclonal antibody will be used 
to screen for the presence of DTCs in the bone marrow aspirate sample.  Cytospin 
slide containing only Hs578T cancer cell line will be used as a negative control, and 
a positive control containing pure MCF7 cancer cell line will be included in each run 
together with an antibody isotype negative control.  Each cytospin will be prepared 
with 1x10*6 MNCs and a total of 5x10*6 cells will be evaluated per patient.  Cytospin 
slides will be reviewed by Dr. Feldman and Dr. Nayak and reported.  The presence 

 
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of one or more pancytokeratin positive cells with appropriate morphology of a tumor 
cell will be reported as positive according to the Consensus Committee 
morphological criteria published by FehmP8
P and the Naume trialP14
P.  Additional 
cytospin slides will be prepared and stained in cases displaying atypical or 
indeterminate morphology. 
13.2.2 Primary Tumor Analyses 
RNA-seq, WES and copy number analysis (CNV) will be performed on all 
residual tumor specimens passing QC metrics.  WES and CNV will be performed on 
DNA isolated from buffy coat samples.  
 
RNA-seq: Libraries will be constructed using TrueSeq Stranded Total RNA 
Sample Prep followed by ribosomal RNA depletion using RiboZero (Gold).  Libraries 
will be sequenced on the HiSeq4000 with 100 bp paired-end reads (100PE) for a 
total of 50M reads after the removal of low-quality reads.  Quality of raw data will be 
confirmed by FASTQC.  Reads will be aligned to the latest version of the human 
reference genome (hg38) with STAR P109
P and quantified using featureCounts. D 
ESeq2P110,111
P will be used for subsequent normalization and differential gene 
expression analysis. 
 
WES: Libraries will be constructed using SureSelect v5 Library Preparation 
Kit.  Libraries will be sequenced on the HiSeq4000 with 150 bp paired-end reads 
(150PE) for a total of 100M reads after the removal of low-quality reads.  Quality of 
raw data will be confirmed by FASTQC.  Reads will be aligned to the latest version 
of the human reference genome (hg38) with BWA-MEMP112
P.  SNVs and Indels will be 
called using both MuTect2P113
P and VarScan2P114
P using tumor-normal pairs as input.  
Cross-comparison of calls between the tools will be used to collapse variants and 
reduce false-positives from germline variation. 
 
CNV: DNA from residual lesions will be submitted for array genomic 
competitive hybridization (aCGH) using the Agilent SurePrint G3 4x180k array using 
corresponding germline DNA as reference.  Quality of the array data will be 
assessed based on the standard QC metrics from Agilent’s Feature Extraction 
software.  Raw log ratios of copy numbers between matching tumor and buffy coat 
samples will be segmented using Circular Binary Segmentation in the ADaCGH2 
Bioconductor package,P115
P followed by the mergeLevels postprocessing step in the 
same package.  Copy number gains and losses will be called by fixed thresholds 
determined based on the distribution of the segmented and merged copy number 
data. 
13.2.3 DTC Assessment by Fluorescence-Activated Cell Sorting (“DTC-
Flow”) 
For the DTC-Flow assay, a bone marrow aspirate sample drawn from the iliac 
crest under anesthesia will be collected in an anti-coagulated tube containing EDTA 
and stored at room temperature until DTC-Flow processing (within 48 hours of 
sample collection).  Bone marrow aspirates will be processed for flow cytometric 
analysis using a stain-lyse-no wash protocol in which samples are first stained with 

 
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fluorescently-conjugated antibodies recognizing a panel of DTC markers, along with 
the hematopoietic cell marker CD45.  Bone marrow aspirates will be incubated with 
magnetic particles conjugated to antibodies against CD45, enabling the depletion of 
CD45-positive bone marrow cells, and then subjected to red blood cell lysis and 
DAPI staining to distinguish live versus dead cells.  DTCs will be detected by flow 
cytometry on a fluorescence-activated cell sorter (FACS).  DTCs will be identified as 
DAPI-negative (live), CD45-negative (non-hematopoietic) cells that are positive for 
expression of one or more DTC markers, and sorted for downstream genomic 
analyses.  Standardization of cytometry and sorting parameters will be performed 
using defined fluorescent particles, allowing the use of consistent sorting gates 
established through the analysis of multiple stained bone marrow aspirates collected 
from healthy donors. 
 
A variety of methods have been developed for single cell genomic analysis, 
and this field is rapidly evolving.  Below, we have prioritized technical approaches, 
however these choices would be modified and updated according to technical 
developments in the fieldP116-129
P. 
 
DTC allocation to produce pooled biological replicates: To mitigate the 
effect of amplification on library complexity resulting from sampling drop-out, DTCs 
isolated from patients by DTC-Flow will be allocated to 9 different pools of DTCs (3 
pools for RNA-seq; 3 pools for WES, and 3 pools for aCGH).  Each pool will be 
amplified and sequenced separately.  For patients with fewer than 9 DTCs, priority 
will be given to pools allocated for WES and aCGH. 
 
RNA-seq: The three pools of DTCs allocated to RNA-seq will be sequenced 
using SMART-seq2 methodologyP130
P, which enriches, amplifies, and processes 
polyadenylated transcripts into libraries for RNA-sequencing in one pipeline.  
SMART-Seq v4 Ultra Low Input RNA Kits are commercially available (Illumina) and 
are compatible with HiSeq 4000 sequencing.  Resulting non-zero normalized read 
counts will be averaged across pools and used as features for gene expression 
analyses. 
 
WES: DTC pools allocated to WES will be whole-genome amplified using 
multiple-displacement-amplification (MDA), which has been demonstrated to 
produce libraries with high coverage (>90%) across the targeted exome, with fewer 
false positives resulting from amplification errors than other methodsP131
P.  Following 
amplification, libraries will be constructed using the SureSelect v5 Library 
Preparation kit.  Libraries will be sequenced on the HiSeq 4000 with 150 bp paired-
end reads (150PE).  Only SNV calls that are present in at least two pools will be 
considered as true positives. 
CNV: DTCs pools allocated to aCGH will be whole-genome amplified using 
multiple annealing- and looping-based amplification cycles (MALBAC).  MALBAC 
produces a more uniform distribution of coverage across the genome, thus making it 
more suitable for CNV assays than amplification from MDA P132
P.  DNA will be 
hybridized to SurePrint G3 Human 4 x 180k aCGH arrays.  Resulting signals will be 
segmented and compared among the three pools to identify true positive CNAs. 

 
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Residual tumor genomics: RNA-seq, WES and aCGH on residual tumor 
specimens will be performed as described in 2.3.1. 
 
Circulating tumor genomics: Genomic analysis of CTCs and ptDNA will be 
performed as previously described. 
13.2.4 ptDNA Analysis 
2 10 ml blood samples will be obtained for ptDNA at each time point per the 
study calendar.  Plasma will be processed within 2 hours of collection using our 
standard protocol and stored until ready for use. Preparation of plasma within 1 to 2 
hours of venipuncture is a critical parameter for maintaining high quality plasma 
DNA P133
P. Streck tubes (Cell-Free DNA BCT) for blood collection will be used, as we 
have recently published the ability of this tube to store blood samples at room 
temperature for up to 7 days without loss of plasma DNA integrity P134
P.  
13.2.5 CTC analysis 
Two 10 mL whole blood samples will be collected in EDTA Blood Collection 
Tubes at each time point per the study calendar. Buffy coat will be processed within 
2 hours of collection using our standard protocol. Cytospin slides will be prepared 
with 1 million cells per slide for immunofluorescent staining. The remaining cells will 
be frozen as indicated in the standard processing protocol.  . The markers used for 
CTC identification will include CD49c, CK, EpCAM, and HER2. CD45 will be used to 
distinguish WBCs (CD45 positive) from CTCs (CD45 negative).  
13.3 Genetic Testing 
Participants will be given information as part of the informed consent process 
that samples will be used for research tests that will include genetic studies and 
testing.  The intent is not to give participants (or his/her medical providers) the 
results of any testing done for research purposes; however, incidental germline 
(heritable) mutations may be identified of which a participant may or may not already 
be aware.  In the case that an incidental genetic finding is identified, the Protocol 
Chair of this project will be notified.  The possible decisions for handling incidental 
findings may include notification of the participant (and provider); recommendation 
for genetic counseling, which may or may not include genetic testing (e.g., if the 
finding was not done in a CLIA certified laboratory); or, neither.  In general, a 
member of the participant’s treating team will be given the information to help with 
notification.  In all cases, the current policy of the University of Pennsylvania and 
local/participating site IRB, as applicable, will be followed and any additional 
approvals that may be required prior to participant notification will be secured in 
advance. 
  
14 DATA HANDLING AND RECORD KEEPING 
14.1 Confidentiality 
Information about study patients will be kept confidential and managed 
according to the requirements of the Health Insurance Portability and Accountability 

 
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Act (HIPAA). Those regulations require a signed patient authorization informing the 
patient of the following: 
- 
What protected health information (PHI) will be collected from patients 
in this study 
- 
Who will have access to that information and why 
- 
Who will use or disclose that information 
- 
The rights of a research patient to revoke their authorization for use of 
their PHI. 
In the event that a patient revokes authorization to collect or use PHI, the 
investigator, by regulation, retains the ability to use all information collected prior to 
the revocation of patient authorization. For patients that have revoked authorization 
to collect or use PHI, attempts should be made to obtain permission to collect at 
least vital status (i.e. that the patient is alive) at the end of their scheduled study 
period. 
14.2 Source Documents 
Source data is all information, original records of clinical findings, 
observations, or other activities in a clinical trial necessary for the reconstruction and 
evaluation of the trial. Source data are contained in source documents. Examples of 
these original documents, and data records include: hospital records, clinical and 
office charts, laboratory notes, memoranda, evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate and complete, x-rays, and 
patient files. 
14.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for 
the study. All data requested on the CRF must be recorded. All entries will be 
entered into an electronic data capture system (EDC) via Oracle Clinical.  Mandatory 
CRFs used for reporting to the ACC DSMC will be entered into Penn CTMS/VELOS. 
15 STUDY FINANCES 
15.1 Funding Source 
This trial will be funded via the 2-PREVENT Breast Cancer Translational 
Center of Excellence and the NIH Research Project Grant: R01-CA208273.  
Novartis will provide commercial EVE to study participants. The DOD will support 
some of the correlative research using samples collected as part of this study. 
15.2 Conflict of Interest 
None to declare.  
15.3 Patient Stipends or Payments 
Patients will not be paid for participating in this clinical trial. However, 
assistance may be provided with parking and transportation and the costs of non-
standard tests. 
16 GENERAL REGULATORY CONSIDERATIONS AND CREDENTIALING 
16.1 Publication of results 
Any formal presentation or publication of data from this trial may be published 
after review and comment by Novartis and prior to any outside submission.  Novartis 

 
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must receive copies of any intended communication in advance of publication (at 
least fifteen working days for presentational materials and abstracts and thirty 
working days for manuscripts).   These requirements acknowledge Novartis’ 
responsibility to provide peer input regarding the scientific content and conclusions 
of such publications or presentations.  Principal Investigation/Institution shall have 
the final authority to determine the scope and content of its publications, provided 
such authority shall be exercised with reasonable regard for the interests of Novartis 
and, in accord with the trial contract and shall not permit disclosure of Novartis 
confidential or proprietary information. 
16.2 Disclosure and confidentiality 
The investigator agrees to keep all information provided by Novartis in strict 
confidence and to request similar confidentiality from his/her staff and the 
IRB/IEC/REB. Study documents provided by Novartis (investigators' brochures and 
other material) will be stored appropriately to ensure their confidentiality. The 
information provided by Novartis to the investigator may not be disclosed to others 
without direct written authorization from Novartis, except to the extent necessary to 
obtain informed consent from patients who wish to participate in the trial. 
16.3 Ethics and Good Clinical Practice 
This study must be carried out in compliance with the protocol and the 
principles of Good Clinical Practice, as described in Novartis standard operating 
procedures and: 
 ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
Directive 91/507/EEC, The Rules Governing Medicinal Products in the 
European Community. 
 US 21 Code of Federal Regulations dealing with clinical studies 
(including parts 50 and 56 concerning informed consent and IRB 
regulations).  Declaration of Helsinki and amendments, concerning 
medical research in humans (Recommendations Guiding Physicians in 
Biomedical Research Involving Human Subjects).  
The investigator agrees to adhere to the instructions and procedures 
described in it and thereby to adhere to the principles of Good Clinical 
Practice that it conforms to. 
16.4 Institutional Review Board/Independent Ethics Committee 
Before implementing this study, the protocol, the proposed informed consent 
form and other information to subjects, must be reviewed by a properly constituted 
Institutional Review Board/Independent Ethics Committee/Research Ethics Board 
(IRB/IEC/REB). A signed and dated statement that the protocol and informed 
consent have been approved by the IRB/IEC/REB must be given to Novartis before 
study initiation. Any amendments to the protocol, other than administrative ones, 
must be reviewed by Novartis approved by this committee. 
16.5 Informed consent 
The investigator must explain to each subject (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the 
expected duration, the potential risks and benefits involved and any discomfort it 
may entail. Each subject must be informed that participation in the study is voluntary 
and that he/she may withdraw from the study at any time and that withdrawal of 

 
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consent will not affect his/her subsequent medical treatment or relationship with the 
treating physician. 
This informed consent should be given by means of a standard written 
statement, written in non-technical language. The subject should read and consider 
the statement before signing and dating it, and should be given a copy of the signed 
document. If the subject cannot read or sign the documents, oral presentation may 
be made or signature given by the subject’s legally appointed representative, if 
witnessed by a person not involved in the study, mentioning that the patient could 
not read or sign the documents. No patient can enter the study before his/her 
informed consent has been obtained. 
The informed consent form is considered to be part of the protocol, and must 
be submitted by the investigator with it for IRB/IEC/REB approval. 
16.6 Declaration of Helsinki 
The investigator must conduct the trial in accordance with the principles of the 
Declaration of Helsinki. Copies of the Declaration of Helsinki and amendments will 
be provided upon request or can be accessed via the website of the World Medical 
Association at http://www.wma.net/e/policy/17-c_e.html. 
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