Conditions: Breast Cancer Breast Cancer, Male Breast Cancer Female HER2-positive Breast Cancer
Keywords: Histologically confirmed HER2 positive breast cancer Unresectable breast cancer Locally advanced breast cancer Metastatic breast cancer Clinical stage 2-3 early stage breast cancer
Allocation: NA | Intervention model: SEQUENTIAL
Enrollment: 51
Healthy volunteers: no

Brief summary:
This purpose of this study is to evaluate the safety and to find the optimal dose in participants with human epidermal growth factor receptor 2 (HER2) positive breast cancer who are given the combination of Interferon-gamma with paclitaxel, trastuzumab and pertuzumab. This study will also look at other effects of Interferon-gamma with paclitaxel, trastuzumab and pertuzumab, including its effect on this type of cancer.

Interferon-gamma is a biologically manufactured protein that is similar to a protein the body makes naturally. In the body, interferon gamma is produced by immune cells and helps to prevent serious infections.

Detailed description:

Arms:
arm 1: Phase 1: Participants with HER-2 positive metastatic breast cancer enrolled in groups of 3-6 or more; each group participant to be given the same dose and schedule of Interferon-gamma plus paclitaxel, trastuzumab, and pertuzumab. If group participants do not have bad side effects, the next group will be given a higher dose of Interferon-gamma. This will continue until the highest safe dose of Interferon-gamma is found. Once highest safe dose of Interferon-gamma is found, participants may be enrolled in Phase II.

Phase 2: Approximately 31 participants with Stage 2-3 HER2 positive early stage breast cancer enrolled to receive therapy with Interferon-gamma plus paclitaxel, trastuzumab, and pertuzumab. Interferon-gamma given at dose found in the Phase 1.

Phase 2: Post therapy surgery.

Interventions: intervention 1: Interferon-gamma (IFN-γ) intervention 2: Paclitaxel intervention 3: Trastuzumab intervention 4: Pertuzumab intervention 5: Post Therapy Surgery
Intervention descriptions: intervention 1: Phase 1: IFN-γ 50 or 75 mcg/m\^2 SQ x 3 days/week for 12 weeks. Phase 2: IFN-γ at Recommended Phase II Dose (RP2D) subcutaneously (SQ) x 3 days/week, for 12 weeks. intervention 2: Phase 1 and Phase 2: Paclitaxel 80 mg/m\^2/week, for 12 weeks. intervention 3: Phase 1 and Phase 2: Trastuzumab 8 mg/kg intravenous (IV) loading dose on cycle 1/day 1 (C1D1), followed by 6 mg/kg on subsequent cycles every 3 weeks, for 12 weeks. intervention 4: Phase 1 and Phase 2: Pertuzumab 840 mg IV loading dose on C1D1, followed by 420 mg on subsequent cycles every 3 weeks, for 12 weeks. intervention 5: Phase 2: Participants will be assessed for surgery following the fourth cycle of study therapy (or earlier if study treatment is cancelled due to unmanageable side effects).

Protocol document:
Prot_SAP_000.pdf: 
A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
Pertuzumab in Patients with HER-2 Positive Breast Cancer 
 
 
NCT03112590 
 
Version 8.0 
August 2019 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
Pertuzumab in Patients with HER-2 Positive Breast Cancer 
 
Version 8.0   8 AUGUST 2019                                                                                                                                        Page 1 
 
 Title:  A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
Pertuzumab in Patients with HER-2 Positive Breast Cancer 
 
 
Study Sponsor  
 
H. Lee Moffitt Cancer Center and Research Institute 
 
 
 
Principal Investigator:  
            Hyo S. Han, M.D. 
H. Lee Moffitt Cancer Center and Research Institute 
10920 N. McKinley Dr 
Tampa, FL  33612 
Tel: (813) 745-4933 
                                                                 Fax: (813) 745-7287 
hyo.han@moffitt.org 
 
Laboratory Principal Investigator: 
Brian Czerniecki, M.D. PhD 
 
                                   H. Lee Moffitt Cancer Center and Research Institute  
10920 N. McKinley Dr 
 
 
 
 
Tampa, FL 33612 
 
 
 
 
Tel: (813) 745-7569      
                                               Fax: (813) 745-5977 
 
 
 
 
brian.czerniecki@moffitt.org 
 
Statistician:                                             Brooke Fridley, PhD 
                                                                  13131 Magnolia Drive   
                                                                  Tampa, FL 33612    
                                                                  Tel: (813) 745-1461  
 brooke.fridley@moffitt.org   
 
Research Coordinator:                          Dawn Goodridge, RN  
10920 N. McKinley Drive 
Tampa, FL 33612  
Tel: (813) 745-1807 
Fax: (813) 449-8562  
dawn.goodridge@moffitt.org  
 
 
 
 
 
 
 
 
 
 
 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
Pertuzumab in Patients with HER-2 Positive Breast Cancer 
 
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TABLE OF CONTENTS 
TREATMENT SCHEMA ............................................................................................................... 4 
1. 
OBJECTIVES ........................................................................................................................ 5 
1.1 Primary Objectives ............................................................................................................... 5 
1.2 
Secondary Objectives: .................................................................................................. 5 
1.3    Exploratory Objectives: ..................................................................................................... 5 
2. 
INTRODUCTION ................................................................................................................. 5 
2.1 Background ........................................................................................................................... 5 
2.2 Preclinical data ...................................................................................................................... 6 
2.3 Summary ............................................................................................................................. 13 
2.4 Agents under Investigation ................................................................................................. 14 
2.4.1 Interferon-gamma 1b (IFN-γ 1b, Actimmune®) .................................................................. 14 
2.4.2 Paclitaxel, Trastuzumab and Pertuzumab ............................................................................ 15 
3. 
PATIENT SELECTION ..................................................................................................... 15 
1.1 
Inclusion Criteria ........................................................................................................ 15 
3.2 
Exclusion Criteria ....................................................................................................... 17 
3.3 Inclusion of Women and Minorities ................................................................................... 17 
3.4 Patient Registration ............................................................................................................. 17 
4. 
TREATMENT PLAN ......................................................................................................... 18 
4.1 
Agent Administration ................................................................................................. 18 
4.1.1 
IFN-γ + Paclitaxel + Trastuzumab + Pertuzumab ...................................................... 18 
4.1.2    Prophylactic or Supportive Medications ........................................................................... 19 
4.1.3 
Dose Escalation Schema and Definition of Dose Limiting Toxicity (DLT) (Phase I) 20 
4.1.4   Recommended Phase II Dose (RP2D) Definition ............................................................. 21 
4.1.5 
Surgery (Phase II) ....................................................................................................... 21 
4.1.6 
Post-Protocol Therapy ................................................................................................ 21 
4.1.7    Determination of the Recommended Phase II Dose ......................................................... 21 
4.2 
Duration of Therapy ................................................................................................... 21 
4.2.1 
Monitoring of Discontinued Patients .......................................................................... 22 
4.3 
Stopping Rules for Phase II Portion of the Trial ........................................................ 22 
4.4  
Duration of Follow up ................................................................................................ 22 
5. 
REPORTED ADVERSE EVENTS/DOSE MODIFICATIONS ..................................... 23 
5.1 
Reported Adverse Events ........................................................................................... 23 
5.1.1 
Paclitaxel ..................................................................................................................... 23 
5.1.2    Trastuzumab ...................................................................................................................... 24 
5.1.3    Pertuzumab ....................................................................................................................... 24 
5.1.4 
IFN-γ ........................................................................................................................... 25 
5.2 
Dosing Delays/Dose Modifications ............................................................................ 25 
5.2.1 
Criteria for Administering Chemotherapy Paclitaxel and IFN-γ ................................ 25 
5.2.2 
Dose Modifications for Toxicity ................................................................................. 26 
6. 
AGENT FORMULATION AND PROCUREMENT ...................................................... 27 
6.1 
Paclitaxel .................................................................................................................... 27 
6.2    Trastuzumab ..................................................................................................................... 27 
6.3    Pertuzumab ...................................................................................................................... 28 
6.4 nab-Paclitaxel ...................................................................................................................... 29 
6.5 IFN-γ ................................................................................................................................... 29 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
Pertuzumab in Patients with HER-2 Positive Breast Cancer 
 
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7. 
CORRELATIVE/SPECIAL STUDIES ............................................................................. 30 
8. 
STUDY CALENDAR .......................................................................................................... 33 
9. 
MEASUREMENT OF EFFECT ........................................................................................ 34 
9.1  
Definitions .................................................................................................................. 34 
9.1.1 
Pathological Response ................................................................................................ 34 
9.1.2 
Clinical Response........................................................................................................ 34 
9.1.3   Radiologic Response .......................................................................................................... 35 
10. REGULATORY AND REPORTING REQUIREMENTS .............................................. 35 
10.1 
Adverse Event............................................................................................................. 35 
10.2 
Serious Adverse Event................................................................................................ 36 
10.3 
Laboratory Test Abnormalities ................................................................................... 37 
10.4 
Pregnancy ................................................................................................................... 38 
10.5 
Adverse Drug Reactions with Concomitant Medication ............................................ 38 
10.6 
Adverse Events Updates / IND Safety Reports .......................................................... 38 
11. DATA MANAGEMENT .................................................................................................... 39 
11.1 
Data Collection ........................................................................................................... 39 
11.2 
Protocol Monitoring Committee ................................................................................. 39 
11.3 
Study Monitoring and Auditing .................................................................................. 39 
11.3.1 
Investigator Responsibilities ....................................................................................... 39 
11.3.2 
Site Responsibilities .................................................................................................... 39 
11.3.3 
Monitoring .................................................................................................................. 39 
11.4    Interim Analyses ................................................................................................................ 40 
12.  STATISTICAL CONSIDERATIONS ............................................................................... 40 
12.1 
Sample Size Determination ........................................................................................ 40 
12.2 
Reporting and Exclusions ........................................................................................... 42 
13.   REFERENCES .................................................................................................................... 44 
APPENDIX A - Page 1 of 2 ........................................................................................................ 47 
Correlative Studies: Paraffin Tissue ......................................................................................... 47 
Pathologic Response Form – Submission of Pathologic Materials for Review ....................... 48 
 
 
 
 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
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TREATMENT SCHEMA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phase I  
HER2 positive metastatic breast cancer 
 
IFN-γ 50 or 75 mcg/m2 SQ x3 days/week  
 
plus  
 
Paclitaxel 80 mg/m2/week 
Trastuzumab 8mg/kg IV loading dose on 
C1D1, followed by 6mg/kg on subsequent 
cycles every 3 weeks 
Pertuzumab 840 mg IV loading dose on 
C1D1, followed by 420 mg on subsequent 
cycles every 3 weeks 
 
x 12 weeks 
Phase II – clinical stage 1-3 HER2 
positive early stage breast cancer  
 
IFN-γ RP2D SQ x3 days/week  
 
plus  
 
Paclitaxel 80 mg/m2/week 
Trastuzumab 8mg/kg IV loading dose on 
C1D1, followed by 6mg/kg on subsequent 
cycles every 3 weeks 
Pertuzumab 840 mg IV loading dose on 
C1D1, followed by 420 mg on subsequent 
cycles every 3 weeks 
 
x 12 weeks 
 
 
Phase II           Surgery 
 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
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1. 
OBJECTIVES 
1.1 Primary Objectives 
• Phase I 
To evaluate the safety and tolerability of the combination of IFN-γ with 
paclitaxel,  trastuzumab and pertuzumab for 12 weeks and determine the 
recommended phase II dose (RP2D) in patients with metastatic breast cancer  
• Phase II 
To evaluate the pathologic complete response rate (pCR) after combination of  
IFN-γ with paclitaxel,  trastuzumab and pertuzumab for 12 weeks of neoadjuvant 
therapy in patients with clinical stage 1-3(primary tumor size ≥10mm)) HER2 
positive breast cancer 
 
  1.2 
Secondary Objectives: 
• Phase I 
To evaluate overall response rate (OR, CR+PR) using standard response 
evaluation criteria in solid tumors (RECIST) version 1.1 
To evaluate median progression free survival (PFS) 
• Phase II 
To evaluate safety and tolerability of the combination therapy  
To evaluate the PFS 
 
  1.3    Exploratory Objectives: 
                  (A) Determine whether IFN-γ combination therapies restore anti-HER-2 CD4 Th1                 
response,  
(B) Change the tumor associated immune response, or  
(C) Change the known tumor biomarkers that predict response in the HER-2 positive 
breast cancer 
 
2. 
INTRODUCTION 
2.1 Background  
 
About 500,000 patients worldwide will die each year from breast cancer (1). This comes with 
considerable cost burdens to all nations, not to mention associated morbidities to individual 
breast cancer survivors. Patients with HER-2-expressing (HER-2pos) breast cancer have 
significant risk of developing systemic disease (2, 3).  HER-2 targeted therapies have improved 
survival in many patients (4-6) but resistance often develops, resulting in relapse. There are also 
increased central nervous system failures (7). Additional therapies are therefore needed to 
complete the eradication of HER-2posbreast cancer.  Addition of immune based therapies against 
HER-2pos breast cancer offers an alternative approach from antibody and kinase-inhibitor 
approaches. Our goal is to place this disease in the category of Hodgkin’s Lymphoma, anal 
carcinoma, and testicular cancer; diseases once causing significant mortalities that today are 


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extremely curable. We have made a series of observations that support the hypothesis that 
restoring a deficit in Th1 immunity will render HER-2 targeted therapies more effective, increase 
pathologic complete responses (pCR), and reduce mortality in patients with HER-2pos breast 
cancer. 
 
2.2 Preclinical data  
 
Evidence for Loss of anti-HER-2 Th1 Response during Tumorigenesis: We have made the 
observation that healthy adult women possess unusually high, pre-existing Th1 immunity against 
HER-2 in the peripheral blood, and that these Th1 cells are progressively lost during HER-2pos 
tumorigenesis (8). This deficit is first detectable at the ductal carcinoma in situ (DCIS) stage, and 
becomes profoundly suppressed in early invasion, even by stage I (Fig 1). There is no such loss 
of HER-2 Th1 immune responses during HER-2neg tumorigenesis, neither are there losses of 
responsiveness against other (non-tumor) control antigens, suggesting this suppression is 
antigen-selective and not a product of global anergy (Fig 1).  Standard therapy of surgery, 
radiation, and chemotherapy with trastuzumab does not routinely correct this anti-HER-2 Th1 
loss, but vaccination with HER-2-pulsed type I dendritic cells (DC1) can dramatically increase 
peripheral anti-HER-2-CD4 Th1, suggesting this is not a fixed deficit, but instead one that can be 
corrected by appropriate immunization (8).   
 
The anti-HER-2 Th1 Response Correlates with Clinical Outcomes: HER-2pos Breast cancer 
patients achieving a pCR to neoadjuvant chemotherapy demonstrate improved survival while 
those with residual disease at the time of surgery demonstrate increased risk of recurrence (9). 
Although as a group, invasive HER-2pos breast cancer patients show depressed anti-HER-2 Th1 
immunity, some individuals are profoundly suppressed while others retain moderate 
responsiveness. Those patients that regain or partially retain anti-HER-2 Th1 demonstrate higher 
pCR rates to neoadjuvant chemo/trastuzumab therapy, while those with residual disease display 
the lowest anti-HER-2 CD4 Th1 responses (Fig 2A). Shown is the loss of repertoire but there is 
also significant difference in overall and cumulative responses (9). The anti-HER-2 CD4 
immune response represents an increase interferon gamma (IFN-γ) production in the Tbetpos Th1 
population and not GATA 3pos Th2 population (Fig 2B), suggesting the Th1 immune response 
may be critical to mediating anti-HER-2 breast cancer responses. Indeed, recent studies suggest 
elevated immune gene expression in the tumor is associated with improved outcomes (10). We 
have also investigated the relationship between anti-HER-2 immunity and disease-free survival. 
Patients with most diminished anti-HER-2 Th1 immune responses show decreased disease free 
survival (DFS) (Fig 2C) while those with retained anti-HER-2 Th1 responses have substantially 
greater DFS (Fig 2C) (11).  This deficit of anti-HER-2 CD4 fits with the clinical picture of those 
with metastatic breast cancers who initially respond for a period of time to HER-2 targeted 
therapies but later fail, become resistant to therapy and recur. Correcting this response is 
paramount to preventing this clinical scenario. These results also suggest that the anti-HER-2 
Th1 response can be used to determine if pCR is achieved before and after neoadjuvant 
immunologic therapies proposed. Since the oncodriver is the critical tumor antigen involved in 
tumor growth and invasion, the anti-HER-2 CD4 Th1 response can be both a novel monitor of 


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risk as well as a therapeutic rescue. The current gold standard is MRI, which is neither sensitive 
nor specific enough to personalize therapy. We have submitted a preliminary manuscript 
demonstrating anti-HER-2 CD4 Th1 response has 98% accuracy rate in predicting pCR to 
neoadjuvant therapy.   
Correcting the Anti-HER-2 CD4 Th1 may be Essential for Clinical Response in HER-2pos 
Breast Cancer: We and others have shown that there is a significant loss of MHC class I 
expression with increasing HER-2 expression (12). Hence CD8 T cells do not readily recognize 
these tumor cells.  Interestingly the MHC class I peptide HER-2 vaccine Neuvax (E75) had its 
greatest activity in low HER-2 1+ and 0 expressing patients, which aligns with our findings of 
MHC class I expression being highest in HER-2 low expressing breast cells vs HER-2 high and 
intermediate cells with low Class I expression (12, 13).  However, IFN- γ and TNF-α treatment 
of HER-2 expressing breast cancer cells increases MHC class I expression and also enhances 
CD8 T cell recognition (12).  Hence, as we have shown ex vivo that correction of the anti-HER-2 
CD4 Th1 response is critical to allowing anti-HER-2 CD8 T cells to recognize and kill, or 
release cytokines in HER-2 intermediate and high expressing cells (HER-2 2+ and HER-2 3+). 
Restoring this anti-HER-2 CD4 Th1 response or providing Th1 cytokines may be critical for 
effective CD8 T cell activity, which has been shown to be critical for response to HER-2 targeted 
therapies, chemotherapy and radiation (14-16). With the increase in MHC class I caused by IFN-
γ is a simultaneous increase in PD1L on the breast cancer cells, which may afford the 
opportunity to add checkpoint inhibitors to therapy when restoring anti-HER-2 CD4 Th1 (12).  
 
Th1 Cytokines Cause Induction of Tumor Senescence and Apoptosis in HER-2 Expressing 
Breast Cancer: Since there is a specific loss of anti-HER-2 CD4 Th1 during breast 
tumorigenesis, and the Th1 cytokines increase MHC class I expression on HER-2pos breast 
cancer cells, we further explored whether Th1 cytokines had other effects on HER-2 expressing 
tumor cells.  We have investigated the role of Th1 cytokines IFN-γ and TNF-α and found the 
combination of these two cytokines induce tumor senescence and apoptosis in breast cancer cells 
expressing HER-2 (Fig 3) as measure by β gal or p15 expression. There is a dose dependent 
increase in tumor senescence  with these cytokines (Fig 3C ), as well as a sensitivity of HER-2 
expressing cells, with high HER-2 expressing cells being more sensitive to lower concentrations 
of Th1 cytokines than intermediate expressing cells (Fig 3 D). These two cytokines work in 
concert and only impact HER-2 expressing cell lines, as there is no significant impact in triple 
negative breast cancer (TNBC) (Fig 3D). More recently, we have investigated the use of 
combination therapy against HER-2 using trastuzumab and pertuzumab with Th1 cytokines IFN-
γ and TNF-α (Fig 4), and noted a synergistic effect on the induction of apoptosis and tumor 
senescence in HER-2 expressing breast cancer, suggesting IFN-γ and TNF-α in combination with 
HER-2 targeted therapies can have dramatic impact on HER-2 expressing breast cancer.  
We have also investigated the impact of single agent IFN-γ with trastuzumab and pertuzumab, 
and identified that at relatively low concentrations, there is an increase in tumor senescence of 
HER-2 expressing breast cancer cells (Fig 5). In addition, in HER-2 expressing cells that have 
become resistant to trastuzumab, IFN-γ and trastuzumab and pertuzumab dramatically induce 
tumor senescence (Fig 5).  Shown is an example of the HCC 1419 cell line that is resistant to the 


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
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effects of trastuzumab, however the combination of IFN-γ, trastuzumab, and pertuzumab induces 
tumor senescence and increased tumor apoptosis. Other resistant cell lines have not shown have 
similar effects.   
Pre-clinical Data: IFN-γ synergizes with Taxanes and anti-HER-2 Blockade:  Mark Green’s 
group at University of Pennsylvania has documented that combining IFN-γ, HER-2 targeted 
therapy, and taxane chemotherapy results in enhanced response of HER-2 expressing breast 
cancers (Fig 6) (17).  We propose that combinations of systemic IFN-γ combined with 
trastuzumab and pertuzumab, alone and in combination with single agent taxanes, will enhance  
sensitivity of HER-2 expressing breast cancer cells to HER-2 directed therapies, inducing more 
complete responses and paving the way for correcting the anti-HER-2 Th1 response for long 
term cures and persistent disease free survival. Since IFN-γ is already approved and a well-
tolerated therapy, we propose to conduct a Phase I study in metastatic HER-2 expressing breast 
cancer receiving taxanes with trastuzumab and pertuzumab.  These patients generally become 
resistant to HER-2 targeted therapies and fail therapy, often dying of disease. This trial would 
serve an unmet need in this group of patients that have no good alternatives except salvage 
palliative chemotherapy. If IFN-γ in combination with taxanes with trastuzumab and pertuzumab 
is well tolerated in phase 1 clinical trial, a subsequent phase 2 neoadjuvant study of this 
combination will be conducted to assess the efficacy and safety. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Healthy
Benign lesion
n=22
n=31
n=11
n=11
n=10
n=21
HER-2neg DCIS
HER-2neg IBC
HER-2pos DCIS
HER-2pos IBC
0
50
100 0
1
2
3
4
5
6
0
100
200
300
Responsivity
(% responsive to at least one peptide)
Repertoire
(# peptides recognized)
Cumulative response
(total # spots per 106 cells)
Negative correlation of natural anti-HER-2 Th1 immunity with 
progression of HER-2pos breast disease (IFN-γ ELISPOT)
Patient status
Figure 1. Fresh PBMCs from unvaccinated subjects (either healthy or experiencing a
variety of defined breast lesions) were individually stimulated with each  of 6 MHC class 
II promiscuous-binding peptides based on the HER-2 primary sequence and subjected to 
IFN-γ ELISPOT analysis. Results are displayed as the percentage of subjects responding 
to at least one of the 6 peptides (“responsivity; left panel), the absolute number of peptides
responded to (“repertoire”; center panel) and the total number of IFN-γ spots  from all
6 peptides (“cumulative response; right panel). In each instance it was noted that healthy
individuals had unusually high natural immunity to HER-2 peptides that waned 
significantly with the development of HER-2pos DCIS and further diminished with 
HER-2pos invasive cancer (IBC). Significant suppression of anti-HER-2 Th1 responses 
were not observed for any HER-2neg disease state.
1


A Phase I-II Study of Interferon-gamma Plus Weekly Paclitaxel, Trastuzumab and 
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6
5
4
3
2
1
0
# reactive peptides
p<.0001
pCR
yes      no
n=16   n=24
Tbet+/IFN+
GATA3+/IFN+
GATA3+/IFN-
0
0.1
0.2 0.3 0.4
% CD4+ PBMC
pCR
no pCR
1.0
0.8
0.6
0.4
0.2
0.0
disease-free survival
months post Tx
Th1 response
(anti-HER-2)
no Th1 response
20
0
40 60 80 100 120
p<.001
A
B
C
Th1 molecular phenotype correlates
with pCR after chemo/ Herceptin 
therapy (flow cytometry)
Anti-HER-2 Th1 immunity
correlates with disease-free 
survival after chemo/ Herceptin 
therapy
Figure 2. Retained natural anti-HER-2 Th1 immunity correlates with 
complete pathologic response (pCR) to chemo/herceptin therapy. A) Fresh 
unexpanded PBMCs from HER-2pos breast cancer patients that received standard 
chemo- herceptin therapy were stimulated individually by 6 HER-2 peptide 
epitopes in IFN-γ ELISPOT assay. Plot displays number of epitopes recognized 
by each individual tested. B) Flow cytometry analysis of anti-HER-2 T cells 
shows disparity between pCR and non-pCR subjects lies in the Tbetpos/IFN-γ
compartment and not with GATA3pos Th2 cells. C) Plot of disease-free survival 
of HER-2pos breast cancer patients with retained (upper trace) versus lost (lower 
trace) anti-HER-2 TH1 immunity suggests strong advantage for retained anti-
HER-2 Th1 immunity. 
pCR correlates 
with retained 
HER-2Th1 immunity


A P h ase I -II St u d y of I nterf er o n-g a m m a Pl us Wee kl y P a clit a xel , Tr ast uz u m a b a n d 
Pert uz u m a b i n P atie nts wit h H E R - 2 P ositi ve B re ast C a ncer 
 
V e r si o n 8. 0   8 A U G U S T 2 0 1 9                                                                                                                                        P a g e 1 0  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fi g ure 3. T h 1 c yt o ki nes, T N F -α a n d  I F N-γ s y ner gize t o i n d uce se nesce nce i n breast ca nc er 
cells i n a d ose de pe n de nt ma n ner i n versel y c orrelate d wit h H E R 2 e x pressi o n. A. S K -B R -3 
breast ca ncer li ne u ntreate d or i nc u bate d wit h 1 0 n g/ ml T N F -αal o ne, 1 0 0 U/ ml I F N -γ al o ne, 
or T N F αa n d I F N -γ i n c o m b i nati o n. O nl y paire d c yt o ki nes i n d uce d se nesce nce. T o p pa nel: 
re prese ntati ve  data  fr o m  1  of  3  i n de pe n de nt  e x peri me nts.  B ott o m  pa nel: de nsit o metric 
a nal ysis  prese nte d  as  %  of  S A --gal -p ositi ve  cells,  mea n ± S D  ( n = 3),  * p  <  0. 0 5. B. 
p 1 5I N K b a n d p 1 6I N K 4a e x pressi o n of cells descri be d i n A were a nal yze d b y wester n bl ot. 
Vi nc uli n  was  use d  as  l oa di n g  c o ntr ol. C. T- 4 7 D  breast  ca ncer  cells  were  u ntreate d  ( 1), 
treate d wit h et o p osi de ( 6), or i nc u bate d wit h i ncreasi n g c o nce ntrati o ns of T N F- an d  I F N-
: 1 0 n g/ ml a n d 1 0 0 U/ ml ( 2), 5 0 n g/ ml a n d 5 0 0 U/ ml ( 3), 7 5 n g/ ml a n d 7 5 0 U/ ml ( 5), 1 0 0 
n g/ ml  a n d  1 0 0 0  U/ ml  ( 5). T o p  p a nel :  re prese ntati ve  d ata  fr o m  1  of  3  i n de pe n de nt 
e x peri me nts. B ott o m  p a nel :  de nsit o metric  a nal ysis  prese nte d  as  %  of  S A--gal -p osi ti ve 
cells,  mea n ± S D  ( n = 3),  * p  <  0. 0 5,  * * p  <  0. 0 1,  * * * p  <  0. 0 0 1. D. C o m bi nati o n  treat me nt 
wit h T h 1 c yt o ki nes, T N F -α a n d I F N -γ, res ulte d i n greater se nesce nce i n S K -B R -3 ( 1 0 n g/ ml 
T N F -α + 1 0 0 U/ ml I F N -γ)  a n d  T -4 7 D  ( 1 0 0  n g/ ml  T N F -α  +  1 0 0 0  U/ ml  I F N -γ)  cells,  
c o m pare d  wit h  u ntreate d  c o ntr ols;  M D A -M B -2 3 1  cells  ( 2 0 0  n g/ ml  T N F -α  +  2 0 0 0  U/ ml 
I F N-γ) re mai ne d lar gel y u naffecte d b y d ual T N F -α + I F N -γ treat me nt. Data prese nte d as % 
of S A --gal -p ositi ve cells, mea n ± S D ( n = 3), * * p < 0. 0 1, * * * p < 0. 0 0 1.  
 


A P h ase I -II St u d y of I nterf er o n-g a m m a Pl us Wee kl y P a clit a xel , Tr ast uz u m a b a n d 
Pert uz u m a b i n P atie nts wit h H E R - 2 P ositi ve B re ast C a ncer 
 
V e r si o n 8. 0   8 A U G U S T 2 0 1 9                                                                                                                                        P a g e 1 1  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fi g ure 4  
Fi g ure 4. C o m bi ne d H E R 2 i n hi biti o n a n d H E R 2 di merizati o n i n hi biti o n e n ha nces T h 1 
c yt o ki ne -me diate d se nesce nce a n d a p o pt osis i n breast ca ncer cells. A. S K -B R -3 cells 
u ntreate d ( 1), treate d wit h 1 0 n g/ ml T N F - a nd 1 0 0 U/ ml I F N - ( 2), treate d wit h 1 0 
u g/ ml of trast uz u ma b ( T z m), a n d pert uz u ma b ( P er) ( 3), or treate d wit h t he 
c o m bi nati o n of b ot h T N F - a n d I F N- a n d T z m a n d P er treat me nts ( 4). T o p p a nel:  
de nsit o metric a nal ysis prese nt e d as % of S A --gal -p ositi ve cells, mea n ± S D ( n = 3), 
* * * p < 0. 0 0 1.  B ott o m p a nel:  re prese ntati ve data fr o m 1 of 3 i n de pe n de nt e x peri me nts. 
B. p 1 5I N K b or  clea ve d cas pase -3 e x pressi o n of cells descri b e d i n A. Vi nc uli n was 
use d as l oa di n g c o ntr ol. Si mil ar res ults were o bser ve d i n 3 i n de pe n de nt e x peri me nts. 
C. I n d ucti o n of a p o pt osis of S K -B R -3 cells treate d as descri be d a b o ve was perf or me d 
b y stai ni n g f or a n ne xi n V a n d PI e x pressi o n of cells descri be d i n A a n d a nal yze d b y 
fl o w c yt o metr y. T o p p a nel:  de nsit o metric a nal ysis prese nte d as % of a n ne xi n V +  PI +  
cells, mea n ± S E M ( n = 3), * * p < 0. 0 1. B ott o m p a nel : pl ots are re prese ntati ve data fr o m 
1 of 3 i n de pe n de nt e x peri me nts.  
 


A P h ase I -II St u d y of I nterf er o n-g a m m a Pl us Wee kl y P a clit a xel , Tr ast uz u m a b a n d 
Pert uz u m a b i n P atie nts wit h H E R - 2 P ositi ve B re ast C a ncer 
 
V e r si o n 8. 0   8 A U G U S T 2 0 1 9                                                                                                                                        P a g e 1 2  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fi g ure 5  
Fi g ure 3.  I F N-γ wit h tr ast uz u m a b a n d pert uz u m a b i n d u ce se nesce nce a n d 
a p o pt osis i n bre ast c a ncer cells. A.  S K -B R - 3 a n d H C C-1 4 1 9 cells, u ntreate d 
( 1), or treate d wit h 1 0 u g/ ml of trast uz u ma b ( T z m) a n d pert uz u ma b ( P er) ( 2 -6), 
a n d  i nc u bate d wit h i ncreasi n g c o nce ntrati o ns I F N-
f or SK-B R -3 a n d H C C -
1 4 1 9 res pecti vel y: 5 0 a n d 2 5 0 U/ ml ( 3), 1 0 0 a n d 5 0 0 U/ ml ( 4), 2 5 0 a n d 7 5 0 
U/ ml ( 5), 5 0 0 a n d 1 0 0 0 U/ ml ( 6) . Left p a nel , de nsit o metric a nal ysis prese nte d as 
% of S A --gal -p ositi ve cells, mea n ± S D ( n = 3), * p < 0. 0 5, * * p < 0. 0 1, * * * p < 
0. 0 0 1; ns, n ot si g nifica nt. Ri g ht p a nel : re prese ntati ve data fr o m 1 of 3 
i n de pe n de nt e x peri me nts i n S K-B R -3 cells ( t o p p a nel) a n d H C C-1 4 1 9  cells 
(b ott o m p a nel ). B  a n d C.  p 1 5I N K b a n d clea ve d cas pase -3 e x pressi o n of S K -
B R -3 ( B ) a n d H C C-1 4 1 9 ( C ) cells descri be d i n A. Vi nc uli n was use d as l oa di n g 
c o ntr ol. Si milar res ults were o bser ve d i n 3 i n de pe n de nt e x peri me nts. 
 


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2.3 Summary  
 
There is an identified deficit in the anti-HER-2 CD4 Th1 response that occurs early in HER-2 
breast tumorigenesis. The loss of anti-HER-2 Th1 response is predictive of an incomplete 
response to neoadjuvant therapy and shortened DFS. The Th1 cytokine IFN-γ has tremendous 
impact on HER-2 expressing breast cancer cells and can make them more susceptible to taxane 
chemotherapy and HER-2 directed therapy. We will leverage these findings and translate this 
into clinical trials to determine the effects of combining IFN-γ with taxane and HER-2 directed 
therapies, first in those with metastatic HER-2 positive breast cancer, and then in a neoadjuvant 
Figure 6 
Figure 6. Murine models of HER-2 expressing breast cancer inhibited by 
combinations of anti-HER-2 and INF-γ and taxanes.  
(A) Implanted H2N113 tumors were treated with control IgG, IFN- γ (three times 
per week), 7.16.4 (twice per week), or the combination of IFN- γ and 
7.16.4. 
(B) IFN- γ receptor was knocked down by shRNA in H2N113. The resulting 
tumor cells (1x106) were injected subcutaneously into MMTV-neu mice and 
treated similarly as in (A). Data represent mean + SEM. A Student’s t test was 
performed to compare the difference in the tumor size of different treatment 
groups. *p < 0.05 and **p < 0.01 compared with control; #p < 0.05 and ##p < 
0.01 compared with the 7.16.4 group; &p < 0.05 and &&p < 0.01 compared with 
the IFN-g group. 
(C) H2N113 tumors from each group of mice treated as indicated were obtained 
after treatment for Figure 4A was finished. Tumor-infiltrated 
MDSCs were isolated and compared using CD11b, Gr-1, and CD45 antibody by 
FACS. *p < 0.05 and **p < 0.01 as compared with the IgG treated group. 
(D) In vitro migration assay. H2N113 cells were seeded on 12-well plate and 
cultured until subconfluent. Cells were then treated with control IgG 
(10 mg/ml), 7.16.4 (10 mg/ml), IFN- γ (10 IU/ml), and 7.16.4 and IFN- γ, and 
conditioned media were collected at day 3 of culture. Migration of MDSC was 
measured by the Transwell system (pore size: 4 mm). MDSCs were isolated from 
spleens of tumor-bearing mice and then seeded in the apical chamber. 
Conditioned media were then placed in the basolateral chamber and incubated for 
3 hr. The cells that migrated to the bottom chamber were collected and counted. 
Fresh medium was used as control (medium). #p < 0.05 (compared with either 
7.16.4- or IFN-g-treated group). 
(E) In vivo co-treatment significantly reduced the expression of ALDH1 in the 
tumor. Tumors from each group of mice treated as indicated were obtained after 
treatment for Figure 4A was finished. Each lysate was adjusted to 10 mg/lane and 
examined for Snail and ALDH1 expression by 
western blot. b-actin was used as the loading control. Shown are representative 
western blots of typical experiments. 
(F) H2N113 tumor cells (0.25 x 106) were injected subcutaneously into both sides 
of the back of 6- to 10-week-old MMTV-neu mice at day 0. Mice were 
treated with control PBS, 7.9.5, and 7.16.4 (0.625 mg/kg each) or the combination 
of IFN- γ, 7.9.5, and 7.16.4 twice per week from day 1. 
(G) H2N113 tumor cells (1 x 106) were injected subcutaneously into MMTV-neu 
mice similarly as in (A). Mice were treated with control PBS, 7.16.4 (1.5 mg/kg 
twice per week), and docetaxel (5.5 mg/kg twice per week), 7.16.4 and IFN- γ (5 
x 105 IU/kg three times per week), or the combination of IFN- γ, 7.16.4, and 
docetaxel. Data represent mean + SEM. A Student’s t test was performed to 
compare the difference in the tumor size of different treatment groups. *p < 0.05, 
**p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with control; #p < 0.05 
and ##p < 0.01 compared with the 7.16.4 and docetaxel group; &p < 0.05 
and &&p < 0.01 compared with the 7.16.4 and IFN-g group. 


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trial with the same combination therapy to increase the pCR rates of patients with HER-2 
expressing locally advanced breast cancer. The significance of enhanced pCR in HER-2 
expressing breast cancer would translate into increased survival and decreased morbidity. In 
addition, we would develop the anti-HER-2 CD4 Th1 response as a biomarker to predict 
response to neoadjuvant therapy, systemic therapy in metastatic breast cancer, and ultimately to 
identify those at risk of recurrence, since we will collect blood specimens in all patients in these 
studies. This would be a significant advance to personalizing therapy and a useful tool for both 
patients and oncologists treating these patients.   
 
2.4 Agents under Investigation  
2.4.1 Interferon-gamma 1b (IFN-γ 1b, Actimmune®) 
IFN-γ is a cytokine and is the only type II interferon.  It is naturally produced mainly by natural 
killer cells and T-lymphocytes and works as an immunomodulator in various capacities.  In its 
role as an immunomodulator it is 100-10,000 times more potent than the type I interferons.  IFN-
γ has many functions physiologically, including but not limited to regulating major 
histocompatibility complex (MHC) expression, activating the differentiation and function of 
phagocytes, augments interactions between macrophages and T-cells, and plays a key role in 
regulating T-cell subsets to determine the type of immune effector function during a specific 
immune response. (18)   
Recombinant IFN-γ 1b (Actimmune®) is commercially available and approved for the treatment 
of the rare pediatric conditions chronic granulomatous disease (CGD) and severe malignant 
osteopetrosis (SMO). (18)  It has, however, previously been evaluated in patients with advanced 
malignancies and thus has a known and proven safety and toxicity profile.  Previous phase III 
trials of IFN-γ as a single agent in advanced RCC and melanoma did not improve outcomes 
compared to their respective comparator arms, but it was safe and reasonably well tolerated. (19, 
20)  Additionally, Devane and colleagues performed a dose titration study in healthy volunteers 
to establish the best tolerated dosing and schedule in adults in regards to the onset of flu-like 
symptoms. (21) 
IFN-γ will be self-administered as a subcutaneous (SQ) injection at a starting dose of 50 mcg/m2 
on a three times weekly basis.  This starting dose was selected based on data from a phase I dose-
finding study of patients with resected melanomas performed by Maluish and colleagues which 
demonstrated evidence of enhanced immunologic activity (as determined by repeated 
measurements of hydrogen peroxide levels from monocytes and natural killer cell activity) at 
various doses and routes of administration.(22)   Based on their results and conclusions, IFN-γ 
dosing from 10-100 mcg/m2 achieved consistently high immunologic effects with a tolerable side 
effect profile.  The 50 mcg/m2 is the approved dose in CGD and SMO and was deemed the 
appropriate starting dose level.   
The recombinant form of IFN-γ is rapidly cleared after intravenous administration, but is 
absorbed more slowly by the SQ route. (23)  Following SQ injection, greater than 89% of the 
dose given is absorbed.  As measured at a 100 mcg/m2 dose, the mean elimination half-life is 5.9 
hours.  Peak plasma concentration is achieved at seven hours following SQ dosing.  No drug 
accumulation has been reported after twelve consecutive daily injections using a 100 mcg/m2 
dose.  While studies in healthy volunteers have failed to document detection of IFN-γ in the 


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urine of healthy subjects, animal studies have suggested some urinary excretion may be present, 
but predominant elimination is via the liver.   
2.4.2 Paclitaxel, Trastuzumab and Pertuzumab 
Paclitaxel promotes the assembly of microtubule formation and stabilizes them by preventing 
depolymerization. It is insoluble in water and, therefore, is formulated in cremophor. Paclitaxel is 
approved by the FDA for the treatment of breast cancer and is widely used in the adjuvant, 
neoadjuvant and metastatic setting. It is administered as an intravenous (IV) infusion and can be 
used either on a 3-weekly schedule, weekly or every 2 week schedule. Main toxicities associated 
with the use of paclitaxel are myelosuppression and neuropathy. Hypersensitivity reactions 
requiring treatment have occurred in 2% to 4% of patients receiving paclitaxel in clinical trials; 
thus, patients should be premedicated with corticosteroids, diphenhydramine, and H2 antagonists. 
Combination of taxanes (docetaxel or paclitaxel) with HER2 targeted agents of trastuzumab and 
pertuzumab is routinely used for the treatment of patients with HER2 positive breast cancer as 
neoadjuvant therapy for locally advanced stage (24) and upfront line of therapy for metastatic 
stage (25).  Paclitaxel 80 mg/m2 weekly regimen will be used in combination with trastuzumab 
(8mg/kg loading dose, then 6mg/m2 q 21 days) and pertuzumab (840 mg loading dose, then 420 
mg q 21 days) for this proposed clinical trial. The study protocol is looking at the safety/efficacy 
of the combination of standard therapy (paclitaxel, trastuzumab and pertuzumab) and the study 
drug, interferon-gamma. Known potential adverse effects from the standard therapy (paclitaxel, 
trastuzumab and pertuzumab) include but are not limited to neutropenia, anemia, 
thrombocytopenia, neutropenic fever, sepsis, nausea, vomiting, diarrhea, mucositis, allergic 
reaction, neuropathy, alopecia and liver enzyme elevation and ejection fraction decline/ 
congestive heart failure.  In addition, known adverse reactions from Interferon-gamma include 
flu-like symptoms such as fever/chills, headache, nausea, vomiting, diarrhea, rash, myalgia and 
arthralgia and less commonly increased liver enzymes, neutropenia, thrombocytopenia, allergic 
reactions.  
3. 
PATIENT SELECTION 
1.1 
Inclusion Criteria 
 
3.1.1  Patients must have a histologically confirmed HER2 positive breast cancer (by 
IHC 3+ or FISH ratio ≥ 2.0)  
Phase I: unresectable locally advanced or metastatic breast cancer              
Phase II: clinical stage 1-3 early stage breast cancer with primary tumor is at 
least 1 cm measured by clinical exam or by radiologic breast imaging tests   
 
3.1.2     Prior therapy 
           Phase I: patients must be candidates to receive paclitaxel  chemotherapy in   
           combination with trastuzumab and pertuzumab 
 
            Phase II: No prior chemotherapy, radiation, or definitive therapeutic surgery  
            (e.g., mastectomy, lumpectomy or axillary dissection) for this malignancy.  
            Patients who have had a prior sentinel lymph node biopsy for this malignancy   
            are eligible. 


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Note: Patients who receive equal to or less than 1 cycle of therapy (up to 4 
weeks) will be allowed to enroll on this trial. 
 
3.1.3 Patients who received tamoxifen or another selective estrogen receptor 
modulator (SERM) for the prevention or treatment of breast cancer or for other 
indications (e.g., osteoporosis, prior ductal carcinoma in situ [DCIS]), or who 
receive aromatase inhibitors for prevention or treatment of breast cancer, are 
eligible. Patients who are hormone-receptor positive and who have received 
other hormonal agents for the treatment of breast cancer (e.g., Fulvestrant®) are 
also eligible. Tamoxifen therapy or other hormonal agents should be 
discontinued at least 1 week before the patient is started on study therapy.  
 
3.1.4  Age ≥ 18 years.  
 
3.1.5  ECOG performance status 0 or 1.   
 
3.1.6 Patients must have normal organ and marrow function as defined below within 
2 weeks of registration (except where specified otherwise): 
- 
Absolute neutrophil count (ANC) ≥ 1,500/ µL  
- 
Platelets ≥ 100,000/ µL  
- 
Total bilirubin ≤ 1.5 x institutional upper limit of normal (ULN), except 
patients with Gilbert’s syndrome in whom total bilirubin must be  
      <3.0 mg/dL 
- 
AST/ALT ≤ 3 x institutional upper limit of normal 
-  Left ventricular ejection fraction (LVEF) EF ≥ of 50% (by 
echocardiogram or MUGA scan within 12 weeks of registration) 
- 
Creatinine ≤ 1.5 x institutional upper limit of normal (ULN) 
- 
ECG - QTc < 480 msec 
 
3.1.7 Women of childbearing potential must agree to use adequate contraception 
(hormonal or barrier method of birth control) prior to study entry and for the 
duration of study participation. Should a woman become pregnant or suspect 
she is pregnant while participating in this study, she should inform her treating 
physician immediately. 
 
3.1.8 Ability to understand and willingness to sign a written informed consent 
document. 
 
 


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3.2 
Exclusion Criteria 
 
3.2.1 Patients may not be receiving any other investigational agents during protocol 
therapy, or up to 14 days or 5 half-lives (whichever is longer) prior to 
beginning protocol therapy. There should be a least a 1-week interval between 
last dose of endocrine therapy and protocol therapy. 
 
3.2.2 Patients who have had chemotherapy or radiation therapy within 2 weeks prior 
to beginning protocol therapy.  
 
3.2.3 Uncontrolled intercurrent illness including, but not limited to, ongoing or 
active infection, congenital prolonged QT syndrome, symptomatic congestive 
heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situations that would limit compliance with study requirements. 
 
3.2.4 Current use of corticosteroid therapy > 5 mg/day of prednisone or equivalent 
doses of other corticosteroids (topical, intranasal, and inhaled corticosteroids in 
standard doses and physiologic replacement for subjects with adrenal 
insufficiency are allowed). 
 
3.2.5 Patients with known active or symptomatic central nervous system (CNS) 
metastases and/or carcinomatous meningitis. Asymptomatic, treated, and/or 
stable brain metastases, as measured by subsequent radiologic evaluations at 
least two months apart, are permitted. 
 
3.2.6 Pregnant or breast feeding.  
 
3.2.7 Known HIV-positive  
 
3.2.8 Known current or a history of hepatitis B or C virus, including chronic and 
dormant states, unless disease has been treated and confirmed cleared.  
 
3.2.9 Major surgery within 4 weeks of initiation of study drug 
 
3.2.10 Second invasive malignancy requiring active treatment  
 
 
3.3 Inclusion of Women and Minorities  
 
Both men and women of all races and ethnic groups are eligible for this trial. 
 
 
3.4 Patient Registration  
 


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Once clinical eligibility is confirmed, patients must sign an informed consent prior to registration 
indicating awareness of the investigative nature of the study and its inherent risks in keeping 
with the policies of the hospital and federal regulations. 
 
4. 
TREATMENT PLAN 
4.1 
Agent Administration 
 
Treatment will be administered on an outpatient basis. Reported adverse events and appropriate 
dose modifications for all agents are described in Section 5.1. No investigational or commercial 
agents or therapies other than those described below may be administered with the intent to treat 
the patient’s malignancy. 
 
The portions of the treatment regimen that must be administered as per protocol guidelines 
include the following. Each cycle will be consisted of 21 days. 
• Cycles  1 to 4: IFN-γ + paclitaxel + trastuzumab + pertuzumab  (phase I and II 
portions of study)  
• Biopsies and/or tissue acquisition (phase I or II portion) 
• Surgery (phase II portion only)  
 
4.1.1 IFN-γ + Paclitaxel + Trastuzumab + Pertuzumab  
• 
IFN-γ (50, 75 mcg/m2), three times weekly for 12 weeks. Patients and/or 
caregivers will be trained on self-administration of subcutaneous IFN-γ on 
Cycle 1 Day 1 (C1D1).. Patients will document each dose of 
administration in a diary provided to them by the study coordinator.  
• 
On C1D1, patients will perform supervised self-administration of IFN-γ at 
Moffitt Cancer Center (MCC) clinical research unit (CRU) and will 
receive their first dose of paclitaxel at least 1 hour after the IFN-γ 
injection. 
• 
Paclitaxel 80 mg/m2 IV infusion over 1 hour weekly x 12 weeks (days 1, 
8, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 79) 
• 
Trastuzumab will be given using an 8 mg/kg loading dose on C1D1, 
followed by 6 mg/kg IV on subsequent cycles q21 days.  
• 
Pertuzumab will be given using an 840 mg IV loading dose on C1D1, 
followed by 420 mg IV on subsequent cycles q21 days.  
• 
Repeat paclitaxel weekly x 12 doses if: (a) ANC ≥ 1000/µL, platelets 
≥ 50,000/µL, (b) recovered from non-hematologic toxicity to grade 1 or 
less (except alopecia), or grade 2 or less neuropathy. G-CSF may be added 
if there is febrile neutropenia or neutropenia preventing treatment on 
schedule (see Sections 5.2.1 and 5.2.2 for guidelines). If treatment cycles 
are delayed, a minimum of 12 paclitaxel ± IFN-γ injection cycles must be 


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completed within 16 weeks. 
• 
Patients will be assessed for DLTs during the first 3 week period of cycle 
1 (Phase I). 
• 
Tumor assessments with computed tomography of the chest, abdomen, 
and pelvis (CT CAP), as well as optional nuclear medicine bone scan per 
physician discretion, will occur after four cycles (at the conclusion of the 
study treatment) on or about C5D1, then per standard of therapy thereafter 
at the treating physician’s discretion(Phase I).   
• 
Study treatment will conclude after four 21-day cycles at which point IFN-
γ will be stopped and patients who are clinically benefitting will continue 
to receive the standard therapy per the treating physician’s discretion. 
• 
Tumor assessments with clinical breast examination will occur on each 
cycle during neoadjuvant therapy (a total of 4 cycles) and patients will go 
for definitive surgery. Adjuvant therapy will be determined further per 
treating physician’s discretion. (Phase II) 
 
       4.1.2    Prophylactic or Supportive Medications 
            IFN-γ 
• Acetaminophen 325 mg orally PRN can be taken 30-60 minutes prior to 
injection for fevers and myalgias and may be used supportively at a dose of 
325-650 mg orally Q6H PRN (≤ 3 gm/day). 
• Anti-depressant medication in the form of a selective serotonin re-uptake 
inhibitor (SSRI) or next generation anti-depressant medication may be taken 
orally by patient to start up to two weeks prior or at any point during therapy 
to treat or protect against the development of depression.  This should be 
decided between the patient and treating clinician. 
• Ibuprofen 200 mg orally to 400 mg orally Q4-6H PRN (≤ 1200 mg/day) may 
be taken prophylactically or for supportive purposes for fevers and myalgias 
assuming adequate renal function as viewed by the treating clinician 
• Diphenhydramine 25 to 50 mg orally Q4-6H  prn (</= 300 mg/day) 
             Paclitaxel 
• Premedication for paclitaxel: administer dexamethasone 10 mg, 30-60 
minutes prior to C1D1 paclitaxel infusion. Dexamethasone should be reduced 
to 4 mg dose on C1D8 then omitted on subsequent cycles if there are no 
hypersensitivity reactions in the previous cycle. This is due to the potential 
concern of steroid inducing reduced immune response.  
 
            Trastuzumab and Pertuzumab  


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• Premedication for pertuzumab: administer diphenhydramine 25-50 mg by IVP 
over 2-5 minutes/PO, and an H2 blocker (e.g. ranitidine 50 mg or equivalent) 
30-60 minutes prior to pertuzumab infusion.   
• If the patient is on weekly trastuzumab, pre-medications will be given prior to 
pertuzumab on Day 1 (i.e. Week 1) and prior to trastuzumab on Days 8 and 15 
(i.e. Weeks 2 and 3) of each cycle. 
• Otherwise, follow institution’s guidelines. Additional pre-medications may be 
added per protocol at the discretion of the physician. 
4.1.3 Dose Escalation Schema and Definition of Dose Limiting Toxicity (DLT) 
(Phase I) 
 
The phase I will use a 3+3 design with two dose levels of IFN-γ. The first dose level will 
be 50 mcg/m2 and the second dose level will be 75 mcg/m2. The DLT evaluation period 
for dose escalation will be during the first three weeks. The MTD dose level is defined as 
the highest dose level with ≤1 out of 6 patients experiencing a DLT. If the first dose level 
experience two or more DLTs, then dose de-escalation will occur to 30 mcg/m2 dose (see 
table 2).  
 
Dose-limiting toxicity (DLT) during cycle one (C1) is defined as follows:  
• 
Non-hematologic or hematologic toxicities that are ≥ grade 3 in severity 
and probably or definitely related to study therapy which leads to 
chemotherapy treatment delays > 14 days are considered DLT. 
 
Table 1: Dose Escalation Schema  
 
Cycle 1 DLT 
Action 
0 of 3 
Escalate to next dose level 
1 of 3 
Treat up to 3 additional patients at same dose level 
1 of 6 
Escalate to next dose  level 
≥ 2 of 3 or  
≥ 2 of 6  
Consider this dose unacceptable. De-escalate dose level 
per below. 
 
 
     
Table 2: Dose Levels of IFN-γ 
 
Dose Level 
Dose (TIW) 
-2 
             15 mcg/m2 
-1 
30 mcg/m2 
1 (starting dose) 
50 mcg/m2 


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2 
75 mcg/m2 
 
 
4.1.4   Recommended Phase II Dose (RP2D) Definition  
The final dose level during the dose escalation phase that yields ≤ 1 DLTs will define the 
RP2D selection for phase II study. Optimum dosing level can be decided further with input 
from scientific advisory board. 
 
 
4.1.5 Surgery (Phase II) 
Patients with early breast cancer will be assessed for surgery following the fourth 
cycle of study therapy (or earlier if treatment is discontinued due to toxicity).  
 
4.1.6 Post-Protocol Therapy 
The following portions of the treatment are recommended after surgery in 
accordance with the standard of care:  
• Adjuvant chest wall/breast irradiation if standard indications met  
• Adjuvant chemotherapy if indicated  
• Adjuvant hormonal therapy for patients with ER and/or PR-positive disease 
4.1.7    Determination of the Recommended Phase II Dose 
As of January 2, 2018, a total of 9 patients have been enrolled (3 patients on dose 
level (DL) 1 and 6 patients on DL2). No DLT has been observed out of all 9   
patients enrolled. For DL1, no SAE or significant AEs have been observed for 3 
patients who completed 12 weeks of treatment. However, for DL2, there have 
been 2 out of 6 patients had SAEs including grade 3 pneumonitis (at week 8 
therapy and treatment was discontinued subsequently) and grade 3 non-
neutropenic fever (at week 6 therapy) respectively, possibly related to study 
treatment. These toxicities however did not meet the definition of dose limiting 
toxicities. Based on these findings suggesting an improved tolerability of DL1, 
the investigators have selected DL1 (50 mcg/m2) as the recommended phase 2 
dose.  
 
4.2 
Duration of Therapy 
Patients should continue protocol treatment unless one of the following criteria applies, at which 
time protocol therapy should be discontinued: 
• disease progression  
• intercurrent illness that prevents further administration of treatment 
• unacceptable adverse events(s) 


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• patient decides to withdraw from the study 
• general or specific changes in the patient’s condition render the patient unacceptable 
for further treatment in the judgment of the investigator 
• completion of protocol therapy (12 weeks) 
• patients who require a delay in the administration of paclitaxel for more than 3 weeks, 
or more than one dose reduction of paclitaxel  
 
4.2.1 Monitoring of Discontinued Patients 
Patients who discontinue IFN-γ due to adverse events related to the study drug 
must be followed until resolution or stabilization of the event.  This condition 
applies to both the phase I and phase II portions of the trial. 
 
4.3 Stopping Rules for Phase II Portion of the Trial  
For efficacy, we will use the stopping rules based on a Simon’s two stage design, 
where the study will seek to determine whether there is an increase in complete 
pathologic response (pCR, defined by RCB score 0). Since the enrollment of 
patients involves both ER+/HER2+ and ER-/HER2+ patients, with the final 
proportion of  ER+ and ER - status patients unknown until the completion of 
enrollment, we  cannot  determine ahead of time the pCR rates to test. After 23 
patients have been enrolled in stage 1, we will compute the proportion of ER+ and 
ER- subjects and compute the pCR under the null and alternative hypothesis as 
outlined in Section 12.1. Using 𝑝𝑝𝑝𝑝𝑅𝑅1𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏  and 𝑝𝑝𝑝𝑝𝑅𝑅1𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 rates 
determined from the stage 1 enrollment of ER+ and ER- patients, we will 
determine the needed number of pCRs to determine if the study should stop or 
proceed to Stage 2.   
 
For safety, there is an interim safety analysis upon completion of phase I and the 
first stage of phase II. For phase II, after the enrollment of the first 6 patients in 
stage 1 of the phase II study, if at any time more than 50% of the treated patients 
experience a G3-4 toxicity related to IFN-γ which leads to chemotherapy 
treatment delays > 14 days or unplanned cessation of neoadjuvant treatment then 
accrual to the trial will be held until discussions with the sponsor and protocol 
monitoring committee are arranged to determine if accrual can resume. Any 
deaths on study treatment will trigger an immediate safety review and accrual 
hold until the event is fully investigated and a determination is made by the study 
sponsor and protocol monitoring committee. 
 
4.4  
Duration of Follow up  
• After all therapy has been completed, patients will be followed for recurrence and 
survival for up to 2 years after registration. (Every 6 months per either medical records or 
phone calls) 


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• Blood samples will be collected for biomarkers at baseline, 3 month, 6 month and 12 
month follow up visits. 
• Any patients who experience adverse events related to the study drug should be followed 
until one of the following occurs: 
o Resolution of the adverse event to ≤ Grade 1 or baseline severity 
o Determination that the adverse event is stable and is unlikely to resolve 
o Initiation of another anti-cancer treatment 
 
5. 
REPORTED ADVERSE EVENTS/DOSE MODIFICATIONS 
5.1 
Reported Adverse Events 
 
5.1.1 Paclitaxel 
• Hematologic: Myelosuppression (neutropenia, leukopenia, thrombocytopenia, 
anemia). 
• Hypersensitivity: Thought to be caused by the Cremophor vehicle. Minor 
symptoms include hypotension, flushing, chest pain, abdominal or extremity pain, 
skin reactions, pruritus, dyspnea, and tachycardia. More severe reactions include 
hypotension requiring treatment, dyspnea with bronchospasm, generalized 
urticaria, and angioedema. The majority (53%) of the reported reactions occurred 
within 2-3 minutes of initiation of treatment and 78% occurred within the first 10 
minutes. Reactions usually occurred with the first and second doses. 
• Cardiovascular: Atrial arrhythmia (sinus bradycardia [usually transient and 
asymptomatic], sinus tachycardia, and premature beats); significant events include 
syncope, hypotension, other rhythm abnormalities (including ventricular 
tachycardia, bigeminy, and complete heart block requiring pacemaker placement), 
and myocardial infarction. Hypertension (possibly related to concomitant 
medication - dexamethasone) may also occur. 
• Neurologic: Sensory (taste changes); peripheral neuropathy; arthralgia and 
myalgia (dose-related, more common when colony-stimulating factors are also 
administered); 
seizures; 
mood 
alterations; 
neuroencephalopathy; 
hepatic 
encephalopathy; motor neuropathy; and autonomic neuropathy (paralytic ileus 
and symptomatic hypotension).  
• Dermatologic: Alopecia (universal, complete and often sudden, between days 14 
and 21); injection site reactions (erythema, induration, tenderness, skin 
discoloration); infiltration (phlebitis, cellulitis, ulceration, and necrosis, rare); 
radiation recall; and rash. 
• Gastrointestinal: Nausea, vomiting, diarrhea, stomatitis, mucositis, pharyngitis, 
typhlitis (neutropenic enterocolitis), ischemic colitis, and pancreatitis.  


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• Hepatic: Increased AST, ALT, bilirubin, alkaline phosphatase; hepatic failure, 
and hepatic necrosis.  
• Other: Fatigue, headache, light-headedness, myopathy, elevated serum creatinine, 
elevated serum triglycerides, and visual abnormalities (sensation of flashing 
lights, blurred vision). 
          5.1.2    Trastuzumab 
Experience with trastuzumab administration has shown that the drug is relatively 
safe. The most significant safety signal observed during clinical trials was cardiac 
dysfunction (principally clinically significant heart failure [CHF]), particularly 
when trastuzumab was given in combination with an anthracycline-containing 
regimen. Much of the cardiac dysfunction was reversible on discontinuation of 
trastuzumab.  
In addition, during the first infusion with trastuzumab, a symptom complex most 
commonly consisting of fever and/or chills was observed in approximately 40% 
of patients. The symptoms were usually mild to moderate in severity and 
controlled with acetaminophen, diphenhydramine, or meperidine. These 
symptoms were uncommon with subsequent infusions. However, in the post-
approval setting, more severe adverse reactions to trastuzumab have been 
reported. These have been categorized as hypersensitivity reactions (including 
anaphylaxis), infusion reactions, and pulmonary events. Rarely, these severe 
reactions culminated in a fatal outcome.  
Trastuzumab appears to be relatively nonimmunogenic. Only 1 of 903 patients 
evaluated developed neutralizing antibodies to trastuzumab. The development of 
anti-trastuzumab antibodies in this patient was not associated with clinical signs 
or symptoms.  
         5.1.3    Pertuzumab  
Pertuzumab is well-tolerated as monotherapy and that it can be given in 
combination with trastuzumab and a range of other therapeutic agents with 
manageable additional toxicity. No new or unexpected toxicities were 
encountered other than those that are known for agents that target the HER family 
of receptors. Serious or severe infusion-associated symptoms have been rarely 
observed in patients receiving pertuzumab. A low level of cardiac toxicities, 
predominantly asymptomatic declines in left ventricular ejection fraction (LVEF), 
has been reported. In the pivotal Phase III trial WO20698/TOC4129g the rates of 
symptomatic and asymptomatic left ventricular systolic dysfunction (LVSD) were 
not higher in patients receiving pertuzumab, trastuzumab and docetaxel than in 
those receiving placebo, trastuzumab and docetaxel.  
No fetal studies in humans have been performed but pertuzumab caused 
oligohydramnios, delayed renal development and embryo-fetal deaths in pregnant 
cynomolgus monkeys. Moreover, in the post-marketing setting, cases of 
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have been reported in pregnant women receiving trastuzumab (for further details, 
see trastuzumab prescribing information). Therefore, pertuzumab should not be 
used in pregnant women. Protocols for ongoing pertuzumab studies indicate that 
highly effective contraceptive measures must be used; continuous pregnancy 
monitoring must be performed during the trials and for six months after the last 
dose of study drug is administered. Because of the long half-life of pertuzumab 
women should be warned not to become pregnant for at least six months after 
completion of treatment. 
5.1.4 IFN-γ   
Interferon gamma -1b (IFN-g 1b, IFN-γ) is a biologic response modifier with 
diverse immunomodulatory properties. As with other interferons, the main risk of 
IFN-g 1b therapy is the development of “flu-like” or constitutional symptoms 
including fever, headache, chills, myalgia, and fatigue.  These events may 
decrease in severity as treatment continues and may be minimized by bedtime 
administration of IFN-g 1b. Additionally, the severity of flu-like symptoms may 
be lessened with initial dose titration. Acetaminophen administration may be 
useful in the prevention/alleviation of fever and headache.  
In addition to constitutional symptoms, patients may experience injection site 
reactions, which are usually mild and transient.  While IFN-g 1b therapy is 
generally well tolerated, reversible neutropenia and thrombocytopenia, as well as, 
elevations in liver function tests (aspartate aminotransferase (AST) and/or alanine 
aminotransferase (ALT)) have been observed. Caution should be exercised when 
administering IFN-g 1b to patients with myelosuppression or in combination with 
other potentially myelosuppressive agents.  IFN-g 1b does not appear to be 
mutagenic and does not appear to provoke neutralizing antibodies. 
 
5.2 
Dosing Delays/Dose Modifications 
 
5.2.1 Criteria for Administering Chemotherapy Paclitaxel and IFN-γ  
                       See Section 4.1.1 for criteria for administering therapy 
 
• If treatment is delayed due to neutropenia, delay treatment until satisfactory ANC 
(as per Section 4.1.1) and resume at 100% dose; filgrastim (5 µg/kg rounded to 
300 or 480 µg SC daily on days 2-6 of each cycle) may be used if neutropenia 
prevents administration of weekly paclitaxel on schedule. Filgrastim may also be 
used with weekly paclitaxel if a cycle is complicated by grade 3 or 4 febrile 
neutropenia.  
• If the platelet counts recovery is delayed due to thrombocytopenia, delay 
treatment until satisfactory plate count (as per Section 4.1.1) and resume with one 
dose level reduction in paclitaxel. 
• If treatment is delayed > 3 weeks due to protocol associated toxicity, the patient 
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 5.2.2 Dose Modifications for Toxicity 
Pertuzumab – fixed dose, no modification of dose. If the patient is unable to 
tolerate the standard dose, they will be discontinued from study. 
 
Trastuzumab - After consulting with the Principal Investigator, if grade 2 or 
higher infusion reaction occurs and is attributed to Trastuzumab, it may be given 
on weekly dosing of 2mg/kg. 
 
Paclitaxel – After consulting with the Principal Investigator, if grade 2 or higher 
infusion reaction occurs and is attributed to Paclitaxel, nab-Paclitaxel may be 
given on weekly 80mg/m2 dosing. 
 
Dose modifications will be made using the schema outlined in the Table 3 below.  
 
The initial dose level of paclitaxel given during the first cycle is dose level 1. 
Patients who require a dose reduction to dose level -2 will be removed from study.  
 
The following toxicities will require reduction of paclitaxel by one dose level:  
 
• Grade 3 or 4 febrile neutropenia (fever > 38.5°C and ANC < 1,000/mm3) between 
courses. Filgrastim may also be used with weekly paclitaxel if a cycle is 
complicated by grade 3 or 4 febrile neutropenia (see Section 5.2.1 above for 
dosing guidelines)  
• Platelet nadir < 20,000/mm3.  
• Failure to recover adequate platelet count on the planned day of therapy.  
• Grade 3-4 non-hematologic toxicity (excluding neuropathy)  
• Grade 3-4 neuropathy (hold paclitaxel until < grade 2 neuropathy, reduce 
paclitaxel dose one dose level; keep IFN-γ dose stable) 
Table 3 
Dose Modifications for Toxicity 
 
Dose Level 
Paclitaxel 
1 
80 mg/m2 day 1 
-1 
60 mg/m2 day 1 
-2 
Remove from study 
 
IFN-γ Dose 
Reduced Dose 
15 mcg/m2 
Discontinue 
30 mcg/m2 
15 mg/m2 
50 mcg/m2 
30 mcg/m2 
75 mcg/m2 
50 mcg/m2 


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6. 
AGENT FORMULATION AND PROCUREMENT 
6.1 
Paclitaxel 
Other Names: Taxol, Onxol, Nov-Onxol, Paclitaxel Novaplus, NSC# 125973. 
Classification: Antimicrotubule agent. 
Mode of Action: Promotes microtubule assembly and stabilizes tubulin polymers by 
preventing their depolarization, resulting in the formation of extremely stable and 
nonfunctional microtubules, and consequently inhibition of many cell functions. 
Storage and Stability: The intact vials may be stored under refrigeration or at room 
temperature. Freezing does not adversely affect the product. Solutions diluted to a 
concentration of 0.3 to 1.2 mg/mL in normal saline, 5% dextrose, 5% dextrose and 
normal saline, or 5% dextrose in Ringer’s solution are stable for up to 27 hours when 
stored at room temperature and normal room light. 
Dose Specifics: Refer to Section 4.1.1.  
Preparation: Preparation of standard regimens should follow site standards.  
Administration: Administered as an IV infusion over 1 hour with an in-line 0.22 micron 
filter.  
Incompatibilities: Avoid the use of PVC bags and infusion sets due to leaching of DEHP 
(plasticizer).  
Drug Interactions: Ketoconazole may inhibit paclitaxel metabolism, based on in vitro 
data. 
Availability: A concentrated solution of 6 mg/mL in polyoxyethylated castor oil 
(Cremophor EL) 50% and dehydrated alcohol 50% is commercially available in 5 mL 
vials. 100 mg/16.7 mL and 300 mg/50 mL vials are also available. See package insert for 
further information.  
 
6.2    Trastuzumab 
Formulation 
Trastuzumab is a sterile, white to pale yellow, preservative-free lyophilized powder for 
intravenous (IV) administration. Each vial of trastuzumab contains 440 mg of 
trastuzumab, 9.9 mg of L-histidine HCl, 6.4 mg of L-histadine, 400 mg of α,α-trehalose 
dihydrate, and 1.8 mg of polysorbate 20, USP. Reconstitution with 20 mL of the supplied 
Bacteriostatic Water for Injection (BWFI) USP, containing 1.1% benzyl alcohol as a 
preservative, yields 21 mL of a multidose solution containing 21 mg/mL trastuzumab, at 
a pH of ~6.  
Dosage 
The recommended initial loading dose is 8 mg/kg trastuzumab administered as a 90-
minute infusion. The recommended maintenance trastuzumab dose is 6 mg/kg q3wk and 
can be administered as a 30-minute infusion if the initial loading dose was well tolerated. 
Trastuzumab may be administered in an outpatient setting. If trastuzumab 6mg/kg q 3 wk 
is not tolerated, trastuzumab 2mg/kg q week can be administered. 
Preparation 


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Use appropriate aseptic technique. Each vial of trastuzumab should be reconstituted with 
20 mL of BWFI, USP, 1.1% benzyl alcohol preserved, as supplied, to yield a multidose 
solution containing 21 mg/mL trastuzumab. Immediately upon reconstitution with BWFI, 
the vial of trastuzumab must be labeled in the area marked “Do not use after” with the 
future date that is 28 days from the date of reconstitution. If the patient has known 
hypersensitivity to benzyl alcohol, trastuzumab must be reconstituted with sterile water 
for injection. Trastuzumab that has been reconstituted with SWFI must be used 
immediately and any unused portion discarded. Use of other reconstitution diluents 
should be avoided. Determine the dose of trastuzumab needed, based on a loading dose of 
8 mg trastuzumab/kg for q3wk dosing schedules or a maintenance dose of 6 mg/kg 
trastuzumab/kg for q3w dosing schedules. Calculate the correct dose using 21 mg/mL 
trastuzumab solution. Withdraw this amount from the vial and add it to an infusion bag 
containing 250 mL of 0.9% sodium chloride, USP. DEXTROSE (5%) SOLUTION 
SHOULD NOT BE USED. Gently invert the bag to mix the solution. The reconstituted 
preparation results in a colorless to pale yellow transparent solution. Parenteral drug 
products should be inspected visually for particulates and discoloration prior to 
administration.  
No incompatibilities between trastuzumab and polyvinylchloride or polyethylene bags 
have been observed. 
Storage  
Vials of trastuzumab are stable at 2C–8C (36F–46F) prior to reconstitution. Do not use 
beyond the expiration date stamped on the vial. A vial of trastuzumab reconstituted with 
BWFI, as supplied, is stable for 28 days after reconstitution when stored refrigerated at 
2C–8C (36F–46F), and the solution is preserved for multiple use. Discard any remaining 
multi-dose reconstituted solution after 28 days.  
6.3    Pertuzumab  
Formulation 
Pertuzumab drug product is provided as a single use formulation containing 30 mg/mL 
pertuzumab in 20 mM L-histidine acetate (pH 6.0), 120 mM sucrose and 0.02% 
polysorbate 20. Each 20 mL vial contains 420 mg of Pertuzumab (14.0 mL/vial). Upon 
receipt, pertuzumab vials are to be refrigerated at 2°C–8°C (36°F–46°F) until use. 
Pertuzumab vials should not be used beyond the expiration date provided by the 
manufacturer. Because the formulation does not contain a preservative, the vial seal may 
only be punctured once. Any remaining solution should be discarded. Vial contents 
should be protected from light, and should not be frozen. The solution of pertuzumab for 
infusion, diluted in PVC or non-PVC polyolefin bags containing 0.9% Sodium Chloride 
Injection, USP, may be stored for up to 24 hours prior to use. Diluted pertuzumab has 
been shown to be stable for up to 24 hours at a temperature range of 2°C–25°C. 
However, since diluted pertuzumab contains no preservative, the diluted solution should 
be stored refrigerated (2°C–8°C). 
 
Dosage  
The initial dose of pertuzumab is 840 mg administered as a 60 minute intravenous 


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infusion, followed every 3 weeks thereafter by a dose of 420 mg administered over a 
period of 30 - 60 minutes.  
 
Preparation 
Prepare the solution for infusion, using aseptic technique, as follows:  
 
 
 
Parenteral drug products should be inspected visually for particulates and discoloration   
           prior to administration.  
 
Withdraw the appropriate volume of pertuzumab liquid concentrate from the vial(s).  
 
Dilute into the 250 mL 0.9% sodium chloride PVC or non-PVC polyolefin infusion bags.  
 
Mix diluted solution by gentle inversion. Do not shake.  
 
Administer immediately once prepared.  
Dextrose (5%) solution should not be used to dilute pertuzumab.  
 
6.4 nab-Paclitaxel    
Other Names: Abraxane 
 
Classification: Microtubule inhibitor 
 
Mode of Action: Microtubule inhibitor that promotes the assembly of microtubules from 
tubulin dimers and stabilizes microtubules by preventing depolymerization. 
 
Storage: Store the vials in original cartons at 20ºC to 25ºC (68ºF to 77ºF). Retain in the 
original package to protect from bright light. 
 
Dose Specifics: Refer to Section 5.2.2 
 
Preparation: Preparation of standard regimens should follow site standards.  
 
Administration: 80 mg/m2 intravenously over 30 minutes every week. 
 
Drug Interactions: Use caution when concomitantly administering with inhibitors or 
inducers of either CYP2C8 or CYP3A4. 
 
Availability: For injectable suspension: lyophilized powder containing 100 mg of 
paclitaxel formulated as albumin-bound particles in single-use vial for reconstitution. See 
package insert for further information.  
 
6.5 IFN-γ 
 
Product description 


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• IFN-γ is a cytokine or biologic response modifier that exists as a single-chain polypeptide 
containing 140 amino acids. Production of this agent is achieved via fermentation of a 
genetically engineered Escherichia coli bacterium containing the DNA which encodes for 
the human protein. The agent is a sterile, clear, colorless solution pre-filled in a single-
use vial for subcutaneous injection. 
• Each 0.5 mL vial contains 100 mcg of IFN-γ formulated in 20 mg of mannitol, 0.37 mg 
disodium succinate hexahydrate, 0.14 mg succinic acid, 0.05 mg polysorbate 20 and 
sterile water.  
Availability 
• Will be supplied by Horizon Pharma, LLC  
Solution preparation 
• Available as described above and per product label.  Distributed as single use vials and 
designated amount per dose to be administered by patient 
Storage requirements and Stability 
• Vials should be refrigerated at 2-8°C (36-46°F) 
• If exposed to room temperature for greater than 12 hours, vials should be discarded 
• Do not freeze 
• Avoid shaking or excessive vigorous activity 
Route of administration 
• Will be self-administered SQ by patients or representative  
• Note: Patients will be trained on appropriate administration technique and appropriate 
documentation of self-administration in the patient diary 
 
  
7. 
CORRELATIVE/SPECIAL STUDIES 
Blood will be collected at the time of screening, 3 months, 6 months and 12 months from the 
start of therapy. For phase II clinical trial, tissue samples from pre-therapy and post-therapy 
(surgical specimen) will be collected. 
 
7.1 Determine whether IFN-γ combination therapies restore anti-HER-2 CD4 Th1 response 
 
Immune Monitoring: Systemic anti-HER2 CD4pos T-cell responses will be generated from 
autologous peripheral blood mononuclear cells (PBMC) pulsed with HER2 peptides. PBMC will 
be collected at least three time points about 30 cc blood each draw. IFN-ɣ production will be 
measured by enzyme-linked immunosorbent spot (ELISPOT) assays or by in vitro sensitization 
assays, as previously described (26, 27).  
 


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Briefly, ELISPOT PVDF membrane plates will be coated overnight with anti-IFN-ɣ capture 
antibody (Mabtech Inc).  The following day, after the plates will be washed with PBS 
(Mediatech Inc) and blocked with 10% human serum/DMEM, 2x105 PBMCs or SLN cells will 
be plated in each well either unstimulated, pulsed with HER2-derived Class II peptide (4 μg) 
(42-56, 98-114, 328-345, 776-790, 927-941, 1166-1180), or pulsed with anti-human CD3/CD28 
antibodies (0.5 µg/mL) (positive control, BD Pharmingen), and incubated at 37oC with 5% CO2 
for 24-36 hours.  After the plates will be washed with PBS, 100 µL of detection antibody (1 
mg/mL; 7-B6-1-biotin) will add to each well and the plates will be incubated for 2 hours.  The 
plates will be washed again with PBS, 100 µL of 1:1000 diluted streptavidin-HRP will added to 
each well and the plates will be incubated for another hour.  TMB substrate solution (Mabtech 
Inc) will added to reveal spot formation.  Spot forming cells (SFC) were counted using an 
automated reader (ImmunoSpot CTL). 
 
A positive response to an individual HER2 Class II peptide will be defined as a minimum of 20 
SFC/2x105 cells after subtracting the unstimulated background.  Three metrics will be used to 
quantify the CD4pos Th1 response: (1) responsivity (the proportion of patients responding to ≥ 1 
peptide), (2) response repertoire (the number of peptides to which a patient responds), and (3) 
cumulative response (the sum of the SFCs across all 6 Class II HER2 peptides) (12).  We will 
also assess whether there is a change in anti-HER-2 IL-10, IL-4, granzyme B by multicolor 
imaging of a percentage of the samples. Controls will be utilized of normal response proteins 
such as candida, tetanus and anergy will be assessed by CD3/CD28 antibody stimulation. 
 
We will assess the CD8 T cell response against HER-2 as well as validate ELISPOT assays using 
in vitro sensitization, CD8pos T-cells will be selected from the cryopreserved lymphocyte cell 
fractions via negative selection (StemCell Technologies).  Autologous DC1 will be suspended in 
SFM with GM-CSF (10 ng/ml), pulsed with one of the six Class II HER2 peptides, and co-
cultured with the CD4pos T-cells at a ratio of 10:1.  IL-2 (30 IU/m; ThermoFisher ) will be added 
on day 2.  On day 10, T-cells will be harvested and tested against T2 target cells pulsed with one 
of the six class II HER2 peptide or irrelevant controls (p53 and colon cancer peptide).  After 24 
hours, the supernatant will be harvested and analyzed by enzyme-linked immunosorbent assay 
(ELISA).  A positive response to the HER2 peptide will be defined as a two-fold increase in CD8 
or CD4pos T-cell IFN-ɣ production compared to the irrelevant peptide controls.  Again, 
responsivity and response repertoire metrics will be used to quantify the CD4pos immune 
response. 
 
 
7.2 Determine whether IFN-γ combination therapies change in immune infiltrates following 
therapy  
Rationale: There is data to suggest that pCR to HER-2 targeted regimens requires immune 
response (10); we will therefore assess the quality and quantity of the immune infiltrates prior to 
and following therapy to determine predictors of responses to therapy. It will be important to 
know if IFN-γ is more responsive in patients with significant pre-existing immune infiltrate in 
the tumor or not. Being able to identify patients that are likely to respond to immune therapies is 
critical. In our neoadjuvant DCIS vaccine studies there is a trend toward more complete 


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responses to vaccines in patients with higher pre-existing anti-CD4 Th1 cells (manuscript 
submitted). It will be important to search for clues to determining response.  
Approach: Using immunohistochemistry, we will assess the change in the quality and quantity 
of the immune infiltrate in subjects receiving the neoadjuvant IFN-γ regimen compared to a 
control matched group not receiving IFN-γ. We will analyze sections from regions of residual 
tumor.  Sections of the pre-treatment biopsy and of the residual tumor/tumor bed will be stained 
using CD4, CD8, CD20, CD1c, CD11b, CD14,CD141, pan MHC class I, II, FOXP3, CD56, 
CD16, antibodies to study recruitment of TIL, as well as other populations, including NK, B cell 
macrophage and DC. We will also perform IHC for IFN-γ on the tumor cells, macrophages (M1 
and M2) on surrounding tissues. PD1 and PD1-L expression also will be measured. In those with 
sufficient tissue we will perform exploratory analysis examining immune gene signatures using 
standard Affymetrix.  
7.3 Determine whether IFN-γ combination therapies changes known tumor biomarkers 
that predict response in the HER-2pos IBC   
 
Rationale: IFN-γ combination therapy may be effective in many but not all patients.  We would 
like develop assays to determine whether this therapy can prevent recurrence, sparing patients 
from additional therapies.  A serum HER-2 assay that measures cleaved or circulating HER-2 
protein in the serum will be assessed to correlate with response.  
Rationale for Analysis of Serum HER-2: Recent data suggests that soluble serum HER-2 
levels can predict those at risk to fail therapy (28). Since HER-2 is the target antigen and the 
oncodriver it will be useful to determine whether this can serve as a biomarker of response. We 
will analyze serum HER-2 levels in patients prior to following completion of therapy. We will 
also check serum HER-2 at 3 months, 6 months, and 12 months from therapy. .   
 
Approach: Dr. Frank Pass and Martell Co have developed several high-sensitivity HER-2 serum 
assays that will be utilized to detect decreases in serum HER-2 as another potential biomarker of 
response; we will evaluate these biomarkers in the context of the proposed therapy regimens. 
These assays will be run on pre and post plasma specimens from patients on clinical trials. 
Samples of serum or plasma will be sent to Martell for analysis. The assays are based off ELISA 
technology that has been modified to improve sensitivity. 
7.4 Anti-drug (IFN-γ) neutralizing antibody assay will be done at baseline and at the end of 
treatment (3 months) to evaluate whether IFN-γ stimulates a detectable host antibody 
response 
 
 
 
 
 
 
 


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8. 
STUDY CALENDAR 
a) Pre-study assessments obtained during standard of care is acceptable for use in this study. Pre-study 
complete blood count (CBC, with differential and platelet count), serum creatinine, and liver function tests 
must be obtained within 14 days prior to registration. 
b) CT scan of the chest/abdomen/pelvis and bone scan (or PET/CT) should be performed within 30 days of 
registration. 
c) MUGA scan or echocardiogram to determine left ventricular ejection fraction must be obtained within 30 
days of registration. 
d) A negative serum or urine beta HCG is required for women with childbearing potential within 7 days of 
registration. 
e)  All the study procedures including labs, visits, scans and treatment (during study therapy) have windows 
+/- 3 days.  
 
 
 
Pre- 
Study 
Week 
   1 
 
   2 
  
 3 
 
 4 
  
 5 
  
 6 
  
 7 
  
 8 
  
 9 
  
 10 
  
 11 
  
 12 
After 
therapy 
completed 
(a) 
Informed consent  
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility checklist 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
History and Physical 
examination 
x 
x 
x 
 
x 
 
 
x 
 
 
x 
 
 
X (a) 
Concurrent meds 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event 
evaluation  (b) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Vital signs  
x 
x 
x 
 
x 
 
 
x 
 
 
x 
 
 
x 
Height 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
x 
 
 
 
x 
 
 
x 
 
 
x 
 
 
x 
CBC w/diff, plts  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Serum chemistry (c) 
x 
x 
x 
x 
x 
 
 
x 
 
 
x 
 
 
x 
Serum b-HCG 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
ECHO or MUGA(d) 
x 
 
 
 
 
 
x 
 
 
 
 
 
 
x 
EKG 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
x 
Tumor 
measurements by 
clinical exam (phase 
II only) 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 
CT of chest/abdomen 
/pelvis  
(bone scan if 
clinically indicated) 
(e) 
x (k) 
 
 
 
 
 
 
 
 
 
 
 
 
x (for phase 
I only) 
IFN-γ (f) 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Paclitaxel (g) 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Trastuzumab (h) 
 
x 
 
 
x 
 
 
x 
 
 
x 
 
 
 
Pertuzumab(h) 
 
x 
 
 
x 
 
 
x 
 
 
x 
 
 
 
Pathology 
submission (i) 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 


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(a)    Off-study treatment evaluation should be completed within 30 days after last chemotherapy treatment.  
After all therapy has been completed, patients will be followed for recurrence and survival for up to 2 years after 
registration. (every 6 months per either medical records or phone calls) 
(b)  Adverse event evaluation (by study physician and/or research nurse, physician assistant) should be performed per study 
calendar. All adverse events must be documented and graded using CTCAE version 4.03. 
(c)   Serum chemistry- Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose,         
potassium, total protein, SGOT [AST], SGPT [ALT], sodium. 
(d)   MUGA or ECHO should be performed at baseline and after completion of therapy in all patients in the phase I and phase II   
portions of the trial. The same modality should be used at each assessment.  
(e)    Repeat CT chest/abdomen /pelvis after completion of all study therapy in patients with metastatic breast cancer. Bone scan 
may be repeated if performed at baseline and if clinically indicated.  
(f)    IFN-γ will be administered by patient or representative under supervision for first dose on cycle 1 day 1.  Subsequent dosing 
will be self-administered by patient or representative at home on three times weekly basis through end of combination phase 
unless schedule change coordinated by clinician and study coordinator, except on C1D1 when self-administered at MCC. 
The clinician or designee will train patient or representative on appropriate administration technique and appropriate 
documentation of self-administration in the diary. 
(g)   Paclitaxel 80mg/m2 IV weekly x 12  
(h)   Trastuzumab and Pertuzumab per the standard of care every 3 weeks IV x 4  
(i)    Pretreatment paraffin specimens should be submitted within one month of registration (phase II) if available; representative 
paraffin specimens from the mastectomy/lumpectomy specimen should be submitted within one month following surgery 
(Phase II) if available. 
(j)   Blood sample will be collected for biomarker at baseline (either pre-study or pre-treatment on week 1), 3 months, 6 months, 
and 12 months (window +/- 4 weeks). Eight, 10mL green top heparin tubes and 3, 10mL red top tubes will be collected at 
baseline, 3 months, 6 months and 12 months 
(k)  Baseline CT scan is required for phase I subjects only. For phase II subjects, CT scan is only if clinically indicated. 
 
9. 
MEASUREMENT OF EFFECT 
9.1  
Definitions 
9.1.1 Pathological Response 
For patients with early breast cancer (phase II), the primary endpoint of this study is 
pathologic complete response in the breast at definitive surgery after completion of 
protocol therapy. The pathologic response to treatment will be assessed by the 
institutional pathologist (preferably a single pathologist for all patients treated at that 
institution). The institutional pathologist (Laila Khazai, MD; H. Lee Moffitt Cancer 
Center) will evaluate response by the “Residual Cancer Burden”(RCB) for each patient as 
described in the online calculator 
(http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3).  
9.1.2 Clinical Response 
For patients with early breast cancer (phase II), a secondary endpoint will be clinical 
complete response and partial response based upon tumor measurements obtained on 
physical examination at baseline, after completion of 4 cycles of study therapy. Tumor 
measurements obtained by physical exam should be performed using a caliper, using 
metric notation, preferably by the same examiner with the patient lying in the supine 
position with the ipsilateral arm placed behind the head (or in the same position on each 
exam), and will be recorded in study specific case report forms. Factors that will be 
evaluated include: 
Blood (biomarkers) 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
X (j) 


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• Breast mass(es) - size (longest dimension) 
• Axillary lymph node(s) - size (longest dimension) 
• Skin edema of the breast - present worse, present unchanged, present 
improved, or absent 
• Skin erythema of the breast - present worse, present unchanged, present 
improved, or absent  
 9.1.3   Radiologic Response  
For patients with metastatic breast cancer (phase I), response and progression will be 
evaluated in this study using the Response Evaluation Criteria in Solid Tumors (RECIST) 
criteria, version 1.1. RECIST criteria will be used for assessing response in patients with 
metastatic disease using the baseline and post treatment imaging studies, plus physical 
examination when appropriate. For patients with locally advanced disease, physician 
examination will be used and measurements recorded at baseline, after completion of 
study therapy. 
 
 
10. REGULATORY AND REPORTING REQUIREMENTS 
10.1 
Adverse Event 
An adverse event (AE) is defined as the appearance of (or worsening of any pre-existing) 
undesirable sign(s), symptom(s), or medical condition(s) after starting study drug (or therapy) 
even if the event is not considered to be related to study drug (or therapy). An AE will be graded 
using the National Cancer Institute CTCAE version 4.03. AEs will be followed until the safety 
follow up visit (30 days after last dose). A laboratory test abnormality considered clinically 
relevant, e.g., causing the subject to withdraw from the study, requiring treatment or causing 
apparent clinical manifestations, or judged relevant by the investigator, should be reported as an 
AE. 
All AEs will be recorded on the appropriate case report forms (CRF and in the subject’s medical 
records starting from the first treatment. The Investigator will also identify the date of onset, date 
of resolution, seriousness, outcome, and the relationship to study drug. Every effort should be 
made to determine the cause of each AE and whether or not it is related to the study drug. The 
relationship of the AE to the study drug must be rated and recorded following the guidelines 
outlined in the CTCAE v4.03. All the AEs will be reviewed by the Principal Investigator of the 
study. The 3 categories for AE grading are: 
Unrelated 
The lack of a temporal relationship of the event to study treatment makes a
causal relationship not reasonably possible, or by any other drugs, therapeutic 
interventions or underlying conditions that provide a sufficient explanation 


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Possible 
The temporal relationship of the event to study treatment makes a causal 
relationship reasonably possible, and the event is more likely explained by 
exposure to the study treatment than by any other drugs, therapeutic 
interventions or underlying conditions 
Definite 
The temporal relationship of the event to study treatment makes a definitive 
relationship, and the event is more likely explained by exposure to the 
study treatment than by any other drugs, therapeutic interventions or 
underlying conditions 
 
10.2 
Serious Adverse Event 
AEs are classified as serious or non-serious. A serious adverse event (SAE) is any AE that is:  
1. 
is fatal or life-threatening 
2. 
requires in-patient hospitalization or prolonged hospitalization 
3. 
results in persistent or significant disability/incapacity  
4. 
constitutes a congenital anomaly or a birth defect 
5. 
is medically significant, may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
Pregnancy, although not itself a serious adverse event, should also be reported on a serious 
adverse event form or pregnancy form and be followed up to determine outcome, including 
spontaneous or voluntary termination, details of birth, and the presence or absence of any birth 
defects or congenital abnormalities. 
Events not considered to be serious adverse events are hospitalizations for the:  
1.      routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition. 
2.      treatment, which was elective or pre-planned (e.g. surgery as per Phase II of this 
study) for a pre-existing condition that did not worsen 
3.      treatment on an emergency, outpatient basis for an event not fulfilling any of the 
definitions of serious given above and not resulting in hospital admission.  
The sponsor is responsible for submitting reports of AEs that are associated with the use of the 
drug that are both serious and unexpected to the FDA. Any serious adverse event occurring after 
the patient started taking the study medication, and until 30 days after the patient has stopped 
study participation must be reported. Information about all serious adverse events will be 
collected and recorded on the FDA MedWatch 3500a form within 48 hours of the notification. 
SAEs and pregnancy reports will be reported to Horizon Pharma as well. 


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In addition, all SAEs should be reported to H. Lee Moffitt Cancer Center & Research Institute 
through Oncore for review by our Protocol Review & Monitoring Committee. They will be 
submitted to IRB per institution policy.   
SAE reporting begins after the patient has signed the ICF and has received study treatment. 
However, if an SAE occurs after signing the ICF, but prior to receiving study treatment, it needs 
to be reported ONLY if it is considered reasonably possibly related to study procedure. SAE 
reporting forms will be completed within 24 hours of the event and e-mailed to: 
Email AdverseEvents@horizonpharma.com 
FAX for SAEs: 1-800-860-7836  
The drug provider will medically review all SAEs.  
The following detailed information must be recorded for each serious adverse event in the SAE 
report form: 
• 
A description of the AE in medical terms  
• 
The severity grade as assessed by the investigator according to the definitions in NCI-
CTC Version 4.03 
• 
The date of becoming serious and the date of becoming known (if different) 
• 
The reason for seriousness 
• 
The outcome of the SAE at the time of the report 
• 
Information on administration of the study drug and chemotherapy and any action taken 
• 
Information on any treatment procedures necessary for the SAE, concomitant 
medications, relevant lab tests and relevant medical history 
If in any one subject the same SAE occurs on several occasions, then the SAE in question must 
be documented and assessed anew each time. 
The investigator is required to submit SAE Follow-up reports until the SAE has resolved or 
stabilized and all queries have been answered. 
10.3 
Laboratory Test Abnormalities 
 
Laboratory test results will be recorded on the laboratory results pages of the CRF. In the event 
of unexplained abnormal laboratory test values, the tests should be repeated immediately and 
followed up until they have returned to the normal range and/or an adequate explanation of the 
abnormality is found.  


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Laboratory test value abnormalities as such should not be reported on the AE page of the CRF as 
adverse events unless they are treatment-emergent and they satisfy one or more of the following 
conditions for clinical significance: 
1. 
Accompanied by clinical symptoms 
2. 
Leading to a change in study medication (e.g. dose modification, interruption or 
permanent discontinuation) 
3. 
Requiring a change in concomitant therapy (e.g. addition of, interruption of, 
discontinuation of, or any other change in a concomitant medication, therapy or 
treatment) 
Please note: Any laboratory result abnormality fulfilling the criteria for a serious adverse event 
(SAE) should be reported as such, in addition to being recorded as an adverse event in the CRF. 
10.4 
Pregnancy 
Female patients must be instructed to immediately inform the investigator if they become 
pregnant during the study. The study treatment must immediately be stopped and the patient 
must be withdrawn from the study. Pregnancies occurring up to 3 months after the completion of 
the last treatment cycle must also be reported to the investigator. The investigator must report all 
pregnancies within 24 hours to the sponsor and drug provider within 24 hours. The investigator 
should counsel the patient; discuss the risks of continuing the pregnancy and the possible effects 
on the fetus. The patient should be monitored until the conclusion of the pregnancy. 
Pregnancy occurring in the partner of a patient participating in the study should also be reported 
to the investigator, the sponsor and the drug provider. The partner should be counseled and 
followed as described above. 
10.5 
Adverse Drug Reactions with Concomitant Medication 
The investigators must be aware that for all concomitant medications the regulations of post-
marketing reporting for suspected adverse drug reactions apply, i.e. reporting to the marketing 
authorization holder or the local regulatory bodies. 
10.6 
Adverse Events Updates / IND Safety Reports 
Horizon Pharma shall notify the Investigator via an IND Safety Report of the following 
information:  
• 
Any AE associated with the use of study drug in other studies that is both serious and 
unexpected.  
• 
Any finding from tests in laboratory animals that suggests a significant risk for human 
subjects including reports of mutagenicity, teratogenicity, or carcinogenicity.  
The Investigator shall notify his/her IRB/EC promptly of these new serious and unexpected 
AE(s) or significant risks to subjects.  


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11. DATA MANAGEMENT 
11.1 
Data Collection  
The Clinical Research Coordinators and Investigators of each site will be responsible for the 
recording of the site’s data into the electronic data capture system, ONCORE.  
11.2 
Protocol Monitoring Committee  
The Protocol monitoring committee (PMC) will be composed of medical and statistical 
independent reviewers and will meet to review the efficacy and safety data and determine a 
risk/benefit analysis in this subject population. The purpose of the PMC is to advise on serious 
safety considerations, lack of efficacy and any other considerations within the charge to the 
Committee. The PMC may request additional meetings or safety reports as deemed necessary 
upon discussion with the principal investigator. The PMC may stop the study following review 
of results from each interim analysis. The PMC meets once a month. 
11.3 
Study Monitoring and Auditing  
11.3.1 Investigator Responsibilities  
Investigator responsibilities are set out in the ICH guideline for Good Clinical Practice (GCP) 
and in the US Code of Federal Regulations.  
Investigators, or a designated member, must enter study data onto CRFs or other data collection 
system. The Investigator will permit study-related monitoring visits and audits by Horizon 
Pharma or its representatives, IRB/EC review, and regulatory inspection(s) (e.g., FDA, EMEA, 
TPP), providing direct access to the facilities where the study took place, to source documents, to 
CRFs, and to all other study documents.  
The Investigator, or a designated member of the Investigator’s staff, must be available at some 
time during monitoring visits to review data and resolve any queries and to allow direct access to 
the subject’s records (e.g., medical records, office charts, hospital charts, and study related 
charts) for source data verification. The data collection must be completed prior to each visit and 
be made available to the Horizon Pharma representative so that the accuracy and completeness 
may be checked. 
11.3.2 Site Responsibilities  
A conference call/study meeting will be held monthly to review patient enrollment and accrual, 
safety and toxicity data, and treatment results, as available.  
11.3.3 Monitoring  
Data will be captured in Oncore, Moffitt’s Clinical Trials Database. Regulatory documents and 
case report forms will be monitored internally according to Moffitt Cancer Center Monitoring 
Policies. The monitoring will include source data verification, utilizing research subjects’ 
medical records. 


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The Sponsor maintains a robust pharmacovigilance system comprising a governance framework 
and standard operating procedures supporting a systematic process for review, evaluation, and 
management of accumulating safety data from clinical trials and other sources to identify a 
potential new safety signal and ensure that an investigational product’s risks are adequately 
assessed and communicated to investigators, IRBs/IECs, and regulatory bodies during clinical 
development. 
For this study, safety monitoring activities will include but are not limited to review and 
evaluation of single serious adverse event (SAE) occurrences in real-time as reported through the 
SAE reporting process outlined in this section of the protocol, and review and evaluation, in real-
time, of one or more occurrences of an uncommon SAE that is not commonly associated with 
product exposure. 
Findings and/or safety data obtained during this study will provide information for the overall 
review of safety that is conducted by the Sponsor on a routine basis. The Sponsor will report 
expeditiously any findings from clinical trials (ongoing or completed), epidemiological studies, 
pooled analysis of multiple studies, and findings from animal or in vitro testing that suggest a 
significant risk in humans exposed to the study product.  
Safety data collection for this study begins at the time of the subject’s receiving the first dose of 
study medication.  The investigator is responsible for the detection and documentation of events 
that meet the definition of an unanticipated problem (refer to protocol Section 10.1), AE or SAE. 
11.4    Interim Analyses  
Efficacy interim analysis (Phase II): The first analysis will take place after the first 23 patients 
are accrued and stopping criteria are outlined in the statistical section.  
 
Safety interim analysis: The safety data will be continually assessed as patients are enrolled on 
the study. After the enrollment of the first 6 patients in stage 1 of the phase II study, if at any 
time more than 50% of the treated patients experience a G3-4 toxicity related to IFN-γ which 
leads to chemotherapy treatment delays > 14 days or unplanned cessation of neoadjuvant 
treatment then accrual to the trial will be held until discussions with the sponsor and protocol 
monitoring committee are arranged to determine if accrual can resume. Any deaths on study 
treatment should trigger an immediate safety review and accrual hold until the event is fully 
investigated and a determination is made by the study sponsor and protocol monitoring 
committee. 
12.  STATISTICAL CONSIDERATIONS 
12.1 
Sample Size Determination 
Phase I (metastatic disease): There are 2 dose levels, with 3 to 6 patients treated at each dose 
level. Therefore, a minimum of 6 and maximum of 12 patients will be treated in the phase I 
portion. Adverse effects will be summarized using frequency tables. The proportion of patients 
who completed all 12 cycles of paclitaxel (at full dose or with dose modification) will be 
tabulated. Dose escalation, AEs, overall safety, and efficacy will be monitored by the Moffitt 
Cancer Center Data Safety Monitoring Committee. 


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Phase II (early stage breast cancer): The study will seek to determine whether there is an 
increase in complete pathologic response (pCR, defined by RCB score 0).  
We will use a Simon’s two stage design with 90% power and a type I error rate of 0.10. The 
choice of this power and type I error rate will ensure an appropriate stopping rule for stage 1 and 
to ensure adequate power after stage 2 to determine if the treatment is promising. Since the 
enrollment of patients involves both ER+/HER2+ and ER-/HER2+ patients, with the final 
proportion of  ER+ and ER - status patients unknown until the completion of enrollment, we  
cannot  determine ahead of time the pCR rates to test. Table 4 presents the designs for the two 
extreme cases, the scenarios in which all the patients enrolled are either all ER+ or all ER- 
patients. In both extremes, the number of stage 1 and total number of patients are the same. Thus, 
we will enroll 23 patients into stage 1. After 23 patients have been enrolled, we will compute the 
proportion of ER+ and ER- subjects and compute the pCR under the null and alternative 
hypothesis based on the following formulas. 
• Will assume baseline/standard of care and study drug rates of 50% (𝑝𝑝0.𝐸𝐸𝐸𝐸−)  and 70% 
(𝑝𝑝1.𝐸𝐸𝐸𝐸−) for ER- patients. 
• Will assume baseline/standard of care and study drug rates of 25% (𝑝𝑝0.𝐸𝐸𝐸𝐸+) and 45% 
(𝑝𝑝1.𝐸𝐸𝐸𝐸+) for ER+ patients. 
• Let 
 and  
 represent the number of ER+ and ER- patients in the study at the 
conclusion of stage 1. 
• Stage 1 baseline pCR rate will be computed as 
. 
• Stage 1 treatment pCR rate will be computed as 
. 
• Using 
 and 
 we will determine the needed number of pCRs to 
determine if the study should stop or proceed to Stage 2.   
If the study proceeds to stage 2, following the enrollment of all patients, the final rates 
(
 and 
) to be tested will be based on a weighted average as follows.  
• Let 
 and  
 represent the final number of ER+ and ER- patients in the study 
(stage 1 + stage 2). 
• Final baseline pCR rate will be computed as 
. 
• Final treatment pCR rate will be computed as 
. 
• Using 
 and 
 we will determine the needed number of pCRs to 
determine if the treatment is promising and warrants additional future studies.  
 
Table 4  Simon’s 2-stage designs for the two extreme cases (90% power, 10% type I error rate) 


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Design 
Stage 1 
N1 
Stage 
1 R1 
Stage 
2 N2 
Total 
N 
R  
Ave 
N 
PET 
Constraint 
25% vs 45% (if all were ER+) 
23 
5 
16 
39 
13 
31.5 
0.468 
Mimimax 
50% vs 70% (if all were ER-) 
23 
11 
16 
39 
23 
31 
0.500 
Mimimax 
N1 & N2 are the sample size in the first and second stage. 
R1 is the drug rejection number in the first stage. 
PET is the probability of early termination of the study. 
N is the combined sample size of both stages. 
R is the combined drug rejection number after both stages. 
Ave N is the average sample size if this design is repeated many times. 
Stop rules for the Simon’s two-stage design will be completed using the current version of PASS 
software. The sample sizes the scenarios with all ER+ or all ER- patients, presented in Table 2, 
was computed using PASS version 15.0.1.   
Adverse effects will be summarized using frequency tables. The estimated pCR along with exact 
confidence interval will be presented. The statistical analysis corresponding to the correlative 
study will be descriptive and exploratory in nature. Therefore, results will be summarized using 
descriptive statistics and associations will be evaluated using Fisher exact test. 
12.2 
Reporting and Exclusions 
Evaluation of toxicity: All patients will be evaluable for toxicity from the time of their first 
treatment with protocol therapy. 
Evaluation of clinical and pathologic response (Phase II): All patients included in the study 
must be assessed for clinical and pathologic response to treatment, even if there are major 
protocol treatment deviations or if they are ineligible. The definitions of clinical and pathologic 
responses are provided in Section 9. Each patient will be assigned one of the following clinical 
response categories:  1) complete response, 2) partial response, 3) stable disease, 4) progressive 
disease, 5) early death from malignant disease, 6) early death from toxicity, 7) early death 
because of other cause, or 9) unknown (not assessable, insufficient data).  [Note:  By arbitrary 
convention, category 9 usually designates the “unknown” status of any type of data in a clinical 
database.]  A patient with unknown pathologic response due to refusal of surgery for non-
medical reasons will be replaced with another patient.  
All of the patients who met the eligibility criteria should be included in the main analysis of the 
response rate. Patients in response categories 4 to 9 should be considered as failing to respond to 
treatment (disease progression). Thus, an incorrect treatment schedule or drug administration 
does not result in exclusion from the analysis of the response rate. Precise definitions for 
categories 4 to 9 will be protocol-specific.  
All conclusions should be based on all eligible patients. Subanalyses may then be performed on 
the basis of a subset of patients, excluding those for whom major protocol deviations have been 
identified (e.g., early death due to other reasons, early discontinuation of treatment, major 
protocol violations).  However, these subanalyses may not serve as the basis for drawing 
conclusions concerning treatment efficacy, and the reasons for excluding patients from the 
analysis should be clearly reported.  The 95% confidence intervals should also be provided.  


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3. Slamon, D.J., et al., Human breast cancer: correlation of relapse and survival with 
amplification of the HER-2/neu oncogene. Science, 1987. 235(4785): p. 177-82. 
4. Smith, I., et al., 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-
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5.. Romond, E.H., et al., Trastuzumab plus adjuvant chemotherapy for operable HER2-positive 
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6.. Gianni, L., et al., Neoadjuvant and adjuvant trastuzumab in patients with HER2-positive 
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recurrence in patients with HER2-positive breast cancer treated with adjuvant trastuzumab. Ann 
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8. Datta J, Rosemblit C, Berk E, et al. Progressive Loss of Anti-HER2 CD4+ T-helper Type 1 
Response in Breast Tumorigenesis and the Potential for Immune Restoration. OncoImmunology. 
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9. Datta, J., et al., Anti-HER2 CD4(+) T-helper type 1 response is a novel immune correlate to 
pathologic response following neoadjuvant therapy in HER2-positive breast cancer. Breast 
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11. Datta J, Fracol M, McMillan MT, Berk E, Lowenfeld L, Xu S, Goodman N, Lewis DA,                          
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APPENDIX A - Page 1 of 2 
Correlative Studies: Paraffin Tissue 
 
Formalin fixed, paraffin-embedded specimens must be forwarded to Dr. Brian Czerniecki at the 
Moffitt Cancer Center:  
 
 
 
Patient study ID #: 
 
 
 
 
______________________________ 
 
 
 
 
 
 
 
______________________________ 
Date of shipment of specimens: 
 
 
______________________________ 
Name of study coordinator sending specimens: 
______________________________ 
Contact information of study coordinator: 
 
 
     Telephone:  
 
 
 
 
______________________________ 
     Fax : 
 
 
 
 
 
______________________________ 
     Email: 
 
 
 
 
 
______________________________  
 
Specimen  
Date 
Pathology Specimen No. 
Pretreatment diagnostic biopsy specimen(s).  
 
 
 
Mastectomy or lumpectomy specimens. 
Representative sections should be selected as 
outlined on next page 
 
 
If specimen not available, please indicate N.A. in table above.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
.   
 
 
 
 
 
 


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APPENDIX A - Page 2 of 2 
Pathologic Response Form – Submission of Pathologic Materials for Review 
 
All patients with early stage breast cancer treated per phase II study who undergo 
mastectomy/lumpectomy will be reviewed by a single pathologist. Procedures are indicated 
below. Indicate identification numbers in tables below. Attach copy of pathology report with 
identifiers removed and study sequence number indicated on report. 
 
Patient sequence number: _____________   Pathology specimen number: ________________ 
Dates of specimen: _____________ 
 
Mastectomy/lumpectomy with residual primary tumor (including those with DCIS) with/without 
positive nodes:  
 
Date 
Block identification 
number(s) 
At least one representative tumor block with 
the least necrosis 
 
 
At least one representative block of a positive 
lymph node 
 
 
One representative block of histologically 
normal breast 
 
 
 
Mastectomy/lumpectomy with no residual primary tumor with negative nodes: 
 
Date 
Block identification 
number(s) 
At least one representative block from the 
tumor area 
 
 
One representative block of histologically 
normal breast 
 
 
 
Mastectomy/lumpectomy with no residual primary tumor but with positive nodes: 
 
Date 
Block identification 
number(s) 
At least one representative block from the 
tumor area 
 
 
At least one representative block of a positive 
lymph node 
 
 
One representative block of histologically 
normal breast