A Phase 2 Clinical Trial Assessing the Correlation of Early Changes in Standardized Uptake Value (SUV) on Positron Emission Tomography (PET) With Pathological Complete Response (pCR) to Pertuzumab and Trastuzumab in Patients With Primary Operable HER2-Positive Breast Cancer
Phase: PHASE2
Conditions: Breast Cancer
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

 
 
  
 
A Phase 2 Clinical Trial Assessing the Correlation of Early Changes in Standardized  
Uptake Value (SUV) on Positron Emission Tomography (PET) with Pathological Complete  
Response (pCR) to Pertuzumab and Trastuzumab in Patients with Primary Operable  
HER2-Positive Breast Cancer  
  
  
Protocol Number  
NCT Number 
 
TBCRC 026   
NCT01937117 
Protocol Chair  
 
Roisin Connolly, M.B., B.Ch.  
 
  
  
 
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 
Bunting-Blaustein Cancer Research Building  
1650 Orleans Street, CRB-I 153  
Baltimore, MD 21287  
Phone: 410-614-9217  
Fax: 410-614-4073  
Email: rconnol2@jhmi.edu  
 
Protocol Co-Chair  
 
Vered Stearns, M.D.  
 
  
  
 
 
Phone: 443-287-6489  
Fax: 410-614-4073  
Email: vstearn1@jhmi.edu  
Breast Pathology  
 
Ashley Cimino-Mathews, M.D.  
 
Radiology  
  
 
 
Nagi Khouri, M.D. 
Lilja Solnes M.D.  
Laboratory Correlates  
 
Ben Park, M.D.  
Edith Perez, M.D.  
Saraswati Sukumar, Ph.D.  
E. Aubrey Thompson, Ph.D  
Biostatistics    
  
 
Zhe Zhang, M.S.  
Supporters  
Genentech (Investigator-initiated)  
 
  
 
Translational Breast Cancer Research Consortium (TBCRC)  
Coordinating Center   
 
 
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 
Protocol Version:  
  
July 30, 2015  
 

  
  
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A Phase 2 Clinical Trial Assessing the Correlation of Early Changes in Standardized  
Uptake Value (SUV) on Positron Emission Tomography (PET) with Pathological Complete  
Response (pCR) to Pertuzumab and Trastuzumab in Patients with Primary Operable  
HER2-Positive Breast Cancer  
  
 
Original Protocol:  
 
 
July 18, 2012  
October 28, 2012 (Revised)  
November 21, 2012 (Revised)  
March 11, 2013 (Revised)  
March 28, 2013 (Revised)  
April 3, 2013 (Revised)  
April 11, 2013 (Revised)  
May 30, 2013 (Revised)  
 
Amendment #1:  
 
 
February 27, 2014  
Sections revised:  Title page, Sections 2, 3, 4.2.3, 4.2.5, 4.3.3, 5.3.4, 5.4.2, 5.4.3 
(new), 6.1.2-6.1.3, 7.3.6, 7.4.4, 7.7, 8, 9.1.5, 9.3, 10.2 (new), 11.1.1, 11.1.2.4, 
11.2.1-11.2.2, 13.2, 16.4, and 16.7.2-16.7.3.  
 
Amendment #2:    
 
 
 
 
 
July 30, 2015  
Sections revised:  Title page, Sections 2, 4.3.3, 6.1.5, 7.3.6, 7.4.1, 8.0, 11.1.1.1, 
11.2.2, 11.5, 13.1, and 16.7.3.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
  
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Table of Contents  
  
1.  SUMMARY ...................................................................................................................................................... 6 
2.  SCHEMA .......................................................................................................................................................... 8 
3. HYPOTHESES.................................................................................................................................................. 8 
4. OBJECTIVES ................................................................................................................................................... 9 
4.1  PRIMARY ...................................................................................................................................................... 9 
4.2 SECONDARY .................................................................................................................................................. 9 
4.3 EXPLORATORY .............................................................................................................................................. 9 
5. BACKGROUND AND RATIONALE ............................................................................................................ 10 
5.1  HER2-POSITIVE BREAST CANCER ............................................................................................................... 10 
5.2  NEOADJUVANT THERAPY ............................................................................................................................ 10 
5.3  PERTUZUMAB ............................................................................................................................................. 12 
5.4  BIOMARKERS OF RESPONSE OR RESISTANCE ................................................................................................ 13 
5.5 OTHER BIOMARKERS OF RESPONSE TO THERAPY .......................................................................................... 22 
5.6 STUDY RATIONALE ...................................................................................................................................... 23 
6. PATIENT POPULATION .............................................................................................................................. 25 
6.1  INCLUSION CRITERIA .................................................................................................................................. 25 
6.2 EXCLUSION CRITERIA .................................................................................................................................. 26 
6.3 INCLUSION OF WOMEN AND MINORITIES ...................................................................................................... 26 
7. STUDY DESIGN AND TREATMENT PLAN ............................................................................................... 27 
7.1  RECRUITMENT ............................................................................................................................................ 27 
7.2 DETERMINATION OF ELIGIBILITY ................................................................................................................. 27 
7.3 PREOPERATIVE PERTUZUMAB AND TRASTUZUMAB ....................................................................................... 27 
7.4  DOSE MODIFICATIONS ................................................................................................................................ 29 
7.5  CONCOMITANT AND SUPPORTIVE THERAPY ................................................................................................. 35 
7.6  SURGICAL EVALUATIONS OF PARTICIPANTS ................................................................................................ 35 
7.7  ADDITIONAL TREATMENT ........................................................................................................................... 36 
7.8  EVALUATION OF LONG TERM OUTCOMES .................................................................................................... 37 
7.9 DISCONTINUATION AND WITHDRAWAL OF SUBJECTS .................................................................................... 37 
7.10 ADDITIONAL INFORMATION ....................................................................................................................... 38 
8. STUDY CALENDAR ...................................................................................................................................... 39 
9. PHARMACEUTICAL INFORMATION ....................................................................................................... 41 
9.1  PERTUZUMAB (PERJETA®) ....................................................................................................................... 41 
9.2  TRASTUZUMAB (HERCEPTIN®) ................................................................................................................ 42 

  
  
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9.3 DOCETAXEL (TAXOTERE®) ......................................................................................................................... 43 
9.4 DRUG ACCOUNTABILITY.............................................................................................................................. 43 
10. MEASUREMENT OF EFFECT ................................................................................................................... 43 
10.1 PATHOLOGIC RESPONSE DETERMINATION .................................................................................................. 43 
10.2 CLINICAL RESPONSE DETERMINATION ....................................................................................................... 43 
11. CORRELATIVE STUDIES .......................................................................................................................... 44 
11.1 TUMOR TISSUE SAMPLES ........................................................................................................................... 44 
11.2 BLOOD SAMPLES ....................................................................................................................................... 46 
11.3  POSITRON EMISSION TOMOGRAPHY (PET) IMAGING .................................................................................. 48 
11.4  LEFTOVER SAMPLES ................................................................................................................................. 49 
11.5  ADDITIONAL INFORMATION ...................................................................................................................... 50 
12.  ADVERSE EVENTS .................................................................................................................................... 50 
12.1 GENERAL .................................................................................................................................................. 50 
12.2 DEFINITIONS ............................................................................................................................................. 50 
12.3  RELATIONSHIP .......................................................................................................................................... 52 
12.4 SPECIFIC INSTRUCTIONS FOR RECORDING ADVERSE EVENTS ....................................................................... 53 
12.5 REPORTING PROCEDURES........................................................................................................................... 54 
13. DATA AND SAFETY MONITORING......................................................................................................... 57 
13.1  DATA MANAGEMENT ................................................................................................................................ 57 
13.2 MEETINGS ................................................................................................................................................. 58 
13.3 MONITORING ............................................................................................................................................. 58 
14. ADMINISTRATIVE PROCEDURES .......................................................................................................... 59 
14.1  PROTOCOL AMENDMENTS ......................................................................................................................... 59 
14.2 INFORMED CONSENT.................................................................................................................................. 59 
14.3 ETHICS AND GOOD CLINICAL PRACTICE ..................................................................................................... 59 
14.4 REGULATORY AUTHORITIES....................................................................................................................... 60 
15. COORDINATING CENTER & SITE RESPONSIBILITIES ...................................................................... 60 
15.1  PROTOCOL CHAIR ..................................................................................................................................... 60 
15.2 COORDINATING CENTER ............................................................................................................................ 60 
15.3 PARTICIPATING SITES ................................................................................................................................ 61 
16. STATISTICAL CONSIDERATIONS .......................................................................................................... 62 
16.1  OVERALL ................................................................................................................................................. 62 
16.2  STUDY DESIGN ......................................................................................................................................... 62 
16.3  SAMPLE SIZE AND ACCRUAL ..................................................................................................................... 63 
16.4  INTERIM ANALYSIS AND EARLY STOPPING GUIDELINES ............................................................................. 63 

  
  
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16.5  DEFINITION OF VARIABLES ....................................................................................................................... 65 
16.6  DATA QUALITY AND NORMALIZATION ...................................................................................................... 65 
16.7 ANALYSIS PLAN ........................................................................................................................................ 65 
16.8 REPORTING AND EXCLUSIONS .................................................................................................................... 68 
REFERENCES.................................................................................................................................................... 68 
APPENDICES ..................................................................................................................................................... 73 
APPENDIX A: AMERICAN JOINT COMMITTEE ON CANCER STAGING .................................................................. 74 
APPENDIX B: ECOG PERFORMANCE STATUS SCALE ....................................................................................... 77 
 
APPENDIX C:  PET/CT QUALITY ASSURANCE MANUAL ...................................................................................... 75 
APPENDIX D:  PET/CT IMAGING GUIDELINES ...................................................................................................... 78  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
  
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1. 
Summary  
A large proportion of women with early breast cancer will be offered adjuvant chemotherapy in an 
effort to minimize the risk of disease recurrence. However, because of a lack of predictive 
biomarkers of sensitivity or resistance, most women receiving adjuvant chemotherapy will either 
not require or will not benefit from this approach. In addition, chemotherapy is associated with 
unwanted short and long term side effects, including serious and potentially life threatening 
complications. The management of women with human epidermal growth factor receptor 2 
(HER2)-positive breast cancer has been revolutionized by the development of anti-HER2 therapies 
such as trastuzumab.1 The addition of trastuzumab, however, to adjuvant regimens can further 
potentiate chemotherapy-induced cardiotoxicity. The personalization of systemic treatment for 
early breast cancer has the potential to both reduce breast cancer recurrences and minimize or 
eradicate toxicity. We propose a series of clinical trials, which aim to utilize early imaging and 
plasma/tumor biomarker changes to determine which women with estrogen receptor (ER)negative, 
HER2-positive breast cancer can be treated with biologic therapy alone and be spared the toxicity 
of systemic chemotherapy, without compromising efficacy.   
  
Recent studies in the metastatic and neoadjuvant settings indicate that dual anti-HER2 therapy, 
such as the combination of trastuzumab and pertuzumab (a novel HER2 dimerization inhibitor), is 
more efficacious than single agent anti-HER2 therapy. The phase 3 CLEOPATRA trial in women 
with previously untreated HER2-positive metastatic breast cancer (MBC) patients, randomized 
patients to trastuzumab/docetaxel (control arm) versus trastuzumab/pertuzumab and docetaxel 
(experimental arm). The median progression-free survival was significantly greater in the 
experimental arm (18.5 months) compared to the control arm (12.4 months).2  
  
NeoSphere was a Phase 2 pre-operative randomized trial comparing trastuzumab/docetaxel 
(control arm), trastuzumab/pertuzumab/docetaxel, trastuzumab/pertuzumab (dual antiHER2 
blockade) and pertuzumab/ docetaxel.3 The pathological complete response (pCR) rates were 29%, 
46%, 17% and 24% respectively. For patients with ER-negative disease, an impressive pCR rate 
was observed for dual antiHER2 therapy alone (27%). That a large proportion of women treated 
with biological therapy alone can obtain a pCR, an acceptable surrogate of excellent outcome in 
neoadjuvant clinical trials, has caused great excitement in the breast oncology community. Clearly, 
dual antiHER2 blockade has been shown to be superior to trastuzumab or pertuzumab alone and 
will become a component of standard of care in the near future such that studies investigating 
biomarkers of response in this setting are imperative. The development of biomarkers able to 
identify a subgroup of patients with HER2-positive disease that may potentially be treated with 
biologic therapy alone and be spared systemic chemotherapy is imperative. Several candidate 
biomarkers can be considered including functional imaging and plasma/tissue biomarkers.   
  
  
Fluorodeoxyglucose (FDG) positron-emission tomography (PET) may allow for prediction of pCR 
as early as 2 weeks post commencement of systemic therapy. The NeoALLTO PET substudy is 
the first study to prospectively evaluate changes in SUV (standardized uptake value) on PET as a 
predictor of response to neoadjuvant anti-HER2 therapy (n=86).4 Patients were randomized to 
receive 6 weeks of antiHER2 therapy followed by the addition of weekly paclitaxel for 12 
additional weeks, until definitive surgery.5 This substudy indicated that metabolic changes in the 

  
  
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primary tumor of patients with invasive operable breast cancer treated with anti-HER2 agents can 
be detected as early as after 2 weeks of treatment. pCR rates were found to be twice as high in 
patients who were FDG-PET responders compared to non-responders.   
  
We propose to evaluate for the first time the correlation between early changes in SUV and pCR 
in men and women with ER-negative, HER2-positive breast cancer receiving trastuzumab and 
pertuzumab (PT) pre-operatively. This has not previously been evaluated in patients receiving 
antiHER2 therapy alone and as such is novel and potentially practice changing. The results from 
this phase 2 biomarker study will be used to plan a randomized study using a predefined cut point 
for SUV decline such that we can further attempt to identify a group of individuals with 
HER2positive early breast cancer who do not require cytotoxic chemotherapy in addition to anti-
HER2 agents. This non-invasive biomarker approach will be of great interest to breast cancer 
oncologists and patients by facilitating a personalized approach to managing patients with HER2-
positive disease that will undoubtedly spare toxicity and reduce the costs associated with anti-
cancer strategies, without compromising efficacy.   
  
Importantly, we will also investigate candidate tissue and plasma biomarkers that may predict 
sensitivity (pCR) or resistance (no pCR) to anti-HER2 therapy, with a focus on the PI3K pathway. 
  
  
 

  
  
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2. 
Schema  
  
  
*Participants with complete clinical response, or without complete clinical response who choose not to  
receive additional preoperative treatment, should proceed directly to surgery; participants with disease  
  
progression or with residual disease on biopsy after completion of study treatment may proceed to  
  additional standard preoperative treatment.  
  
3. Hypotheses  
We hypothesize that early changes in SUV on FDG-PET will correlate with pCR in men and 
women with ER-negative, HER2-positive breast cancer receiving pertuzumab and trastuzumab 
(PT). Our results will be used to develop a randomized study, in which we will use a predefined 
SUV decline to randomize patients to antiHER2 therapy alone versus antiHER2 therapy plus 
chemotherapy. We also hypothesize that aberrations of the PI3K pathway, and an immune function 
gene signature will correlate with pCR in this population. Our long term goal is to identify a group 
of patients with HER2-positive disease who can be spared chemotherapy. The study will also 
provide a much needed tissue dataset for future investigation.  
  

  
  
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4. 
Objectives  
4.1  
Primary  
4.1.1 To correlate baseline and change (day 15) in SUV on FDG PET with pathological complete 
response (pCR) in patients treated with preoperative pertuzumab/trastuzumab  
4.2  
Secondary  
4.2.1 To correlate PIK3CA mutation status and other genomic alterations (mutations/somatic 
rearrangements) qualitatively and quantitatively in plasma tumor DNA (ptDNA) with pCR  
4.2.2 To correlate PI3K pathway activation (e.g. PTEN low and/or PIK3CA mutation, HER 1-4 
expression and/or phosphorylation) in tumor samples and pCR  
4.2.3 To correlate a gene immune function signature in tumor samples and pCR  
4.2.4 To correlate baseline and change (day 15) in Ki67 with pCR  
4.2.5 To determine pCR rates to preoperative pertuzumab/trastuzumab + taxane in the setting of 
histologically 
confirmed 
residual 
cancer 
after 
12 
weeks 
of 
preoperative 
pertuzumab/trastuzumab  
4.3  
Exploratory  
4.3.1 To identify baseline biomarkers that may predict sensitivity (pCR) or resistance (no pCR) to 
anti-HER2 therapy, such as ER, Ki67, serum methylation markers, gene expression 
profiles, and others to be decided.  
4.3.2 To explore changes in biomarkers (e.g., PIK3CA, ER, Ki67, serum methylation markers, 
gene expression profiles) through serial samples collected from baseline, end of therapy, at 
the time of definitive surgery and post operatively in an effort to understand resistance to 
anti-HER2 therapy and obtain preliminary data.  
4.3.3 To correlate baseline and change (day 15) in SUV on FDG PET with pCR among patients 
with 
histologic 
confirmed 
residual 
disease 
after 
12 
weeks 
preoperative 
pertuzumab/trastuzumab and subsequent addition of standard therapy per investigator 
discretion.  
 
 
 
  
  

  
  
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5. 
Background and Rationale  
Breast cancer is the most common malignancy in women in Western societies. More than 200,000 
women were diagnosed with breast cancer in the US in 2010.6 Advances in the early detection and 
optimal adjuvant treatment of breast cancer have led to a significant reduction in disease relapse 
and death. Nevertheless, more than 40,000 women per year continue to die from this disease.  
Traditionally, clinicopathologic factors have been used to guide choice of therapy for women 
newly diagnosed with breast cancer, i.e., endocrine therapy for tumors that express hormone 
receptors and trastuzumab for tumors that overexpress HER2. However, it is still difficult to 
precisely identify who will or will not benefit from (neo) adjuvant chemotherapy. Data from the 
Oxford Overview,7 the identification of biologically distinct breast cancer intrinsic subtypes with 
various prognoses,8 and multivariate prediction models such as Adjuvant! Online9 have helped 
further refine this decision-making process. The recent development of multigene predictive tools 
has helped with improved prognostication for patients with hormone receptor-positive, 
HER2negative breast cancer and, in some cases, with prediction of benefit from specific therapies. 
The molecular tools developed thus far offer little aid in clinical decision-making for women with 
ERnegative or HER2-positive disease, and improved prognostic and predictive tools for these 
patients are needed. Consequently, biomarkers able to identify a subgroup of patients that may 
potentially be spared systemic chemotherapy or who need intensification of standard therapy are 
of great interest and forms the rationale for our study.  
5.1  
HER2-Positive Breast Cancer  
Increased expression of HER2 or amplification of the HER2/neu gene had been associated with a 
worse prognosis when compared to other breast cancer phenotypes.10 The NSABP B-31/NCCTG- 
N9831 and HERA adjuvant clinical trial results indicated that the addition of 1 year of trastuzumab 
to adjuvant chemotherapy regimens (predominantly anthracycline and taxane-containing) in a 
HER2-positive population of women resulted in an approximately 50% reduction in breast cancer 
recurrence and 35% reduction in mortality.11,12 A recent updated joint analysis at 4 years of the 
NSABP B-31/NCCTG-N9831 trial has confirmed that the addition of adjuvant trastuzumab to 
chemotherapy maintains both a significant disease-free and overall survival benefit when 
compared to chemotherapy alone.1 Non-anthracycline based regimens have also been evaluated 
and indicate comparable efficacy to anthracycline-based approaches with a more favorable toxicity 
profile.13 It is possible, therefore, that not all women require an anthracycline as part of their (neo) 
adjuvant treatment strategy. Phase 2 and 3 trials evaluating the use of trastuzumab and 
chemotherapy in the neoadjuvant setting are described below and have reported high pCR rates, a 
potential surrogate endpoint for survival that is commonly used in neoadjuvant studies.  
5.2  
Neoadjuvant Therapy  
The neoadjuvant setting has been an attractive area of research for identifying new effective 
treatment strategies while minimizing treatment-related adverse events, studying drug mechanisms 
of action, and developing clinically applicable prognostic and predictive biomarkers in an attempt 
to individualize therapy. Neoadjuvant therapy, also designated primary systemic or preoperative 

  
  
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therapy, was initially employed to downstage inoperable tumors to allow for definitive surgery. 
Similar survival benefits have subsequently been demonstrated for the administration of 
chemotherapy before or after surgery. An International Expert Panel, convened three times over 
the past decade, has recommended that clinicians should consider neoadjuvant chemotherapy in 
any patient with primary operable disease for whom adjuvant chemotherapy is clearly indicated.14 
The neoadjuvant approach may allow for other benefits such as enhancement of breast 
conservation, may offer prognostic information, and enables assessment of in vivo response to 
therapy. Women who achieve a pCR following neoadjuvant therapy are expected to have improved 
outcome compared to those with extensive residual disease.15  
Response to treatment in an individual woman may predict her long-term outcome. Despite the 
varied definitions of pCR in trials completed to date, it has been consistently demonstrated that 
pCR is associated with improved disease-free and overall survival.16 Women without residual 
invasive and noninvasive tumor cells in the breast and in the axillary nodes have substantially 
improved outcomes compared to women with similar stage and tumor characteristics and extensive 
residual disease. Furthermore, clinical response to therapy can be assessed as early as following 
one to two cycles and may help predict who will achieve a pCR. Current consensus 
recommendations for preoperative chemotherapy include anthracycline- and taxane-based therapy. 
This is based on results obtained from several prospective trials suggest that preoperative 
anthracycline and taxane based therapy is associated with high response rates.17-19   
5.2.1 Neoadjuvant therapy for HER2-positive disease  
Women with HER2-positive tumors who are candidates for neoadjuvant chemotherapy should 
receive a trastuzumab-based regimen.20 Several small phase 2 studies have reported high pCR rates 
when trastuzumab was added to neoadjuvant anthracycline and taxane-based regimens. Concurrent 
paclitaxel and trastuzumab, followed by concurrent FEC (5-FU, epirubicin, cyclophosphamide) 
and trastuzumab yielded a pCR rate of 60%, without an increase in cardiac toxicity compared to 
chemotherapy alone.21 A non–anthracycline-based approach combining carboplatin, weekly 
paclitaxel, and trastuzumab was associated with a pCR rate of 76%.22  
In the phase 3 NeOAdjuvant Herceptin (NOAH) trial, the addition of trastuzumab to three cycles 
of doxorubicin and paclitaxel, followed by four cycles of paclitaxel and three cycles of CMF was 
associated with an improvement in overall response rate compared to chemotherapy alone (81% 
vs 73%, P = .18) and pCR rate (43% vs 23%, P = .002).23 The NeoALLTO trial investigated the 
efficacy of lapatinib (a dual HER2/EGFR tyrosine kinase inhibitor) plus paclitaxel, versus 
trastuzumab plus paclitaxel, versus concomitant lapatinib and trastuzumab plus paclitaxel, when 
given as neoadjuvant therapy in patients with HER2-positive breast cancer (n=455). Patients were 
randomized to initially receive 6 weeks of a "biological window" of either lapatinib, or 
trastuzumab, or lapatinib with trastuzumab. Subsequently the same targeted therapy was continued 
with the addition of weekly paclitaxel for a further 12 weeks, until definitive surgery. After surgery, 
patients received 3 cycles of adjuvant FEC followed by the same targeted therapy as in the 
biological window of the neoadjuvant phase for a further 34 weeks (to complete 52 weeks of anti-
HER2 therapy). pCR was significantly higher in the combination arm (lapatinib plus trastuzumab) 
compared with either trastuzumab or lapatinib alone (51.3% vs. 29.5% vs. 24.7%, respectively; P 

  
  
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< 0.01 for both), indicating that dual blockade of the HER2 pathway is a valid concept. No major 
cardiac dysfunctions or toxic deaths were observed during the neoadjuvant phase. There was 
increased, but manageable, toxicity (mainly diarrhea and liver enzyme alterations) in the lapatinib 
arms. A subset of patients also participated in a PET/CT substudy which is described in a later 
section.5 The NeoSPHERE study investigating various combinations of pertuzumab, trastuzumab, 
and docetaxel in the neoadjuvant setting is described below.  
In addition, a number of studies are ongoing or have been completed evaluating dual anti-HER2 
therapies alone in the neoadjuvant setting. A phase 2 neoadjuvant study of lapatinib and 
trastuzumab in patients with operable HER2-positive breast cancer has been presented 
(TBCRC006). Patients received 12 weeks of anti-HER2 therapy pre-operatively, as well as 
letrozole +/- goserelin if they had ER-positive disease (approximately 60% of study population). 
The overall pCR rate was 27%, with a 21% rate for ER-positive patients and 38% for ER-negative. 
The treatment was found to be well tolerated and clinical responses were observed a few weeks 
after starting therapy.24 Another TBCRC study is evaluating whether longer duration (12 versus  
24 weeks) of anti-HER2 therapy prior to surgery will result in a higher rate of pCR (TBCRC023).  
5.3  
Pertuzumab  
Pertuzumab is a humanized monoclonal antibody and is the first of a novel class of HER2-targeted 
agents known as HER2 dimerization inhibitors. This agent bind to a distinct epitope on the 
extracellular domain of the HER2 receptor (the domain II dimerization arm), blocking the 
interaction between HER2 and other HER family receptors.25 Potent inhibition of HER-mediated 
intracellular signaling results in cancer cell growth inhibition and death.26  
5.3.1 Preclinical Studies  
The antitumor activity of pertuzumab and trastuzumab, a HER2-targeted monoclonal antibody, has 
been evaluated both alone and in combination in HER2-positive breast cancer xenografts. The 
combination of trastuzumab and pertuzumab was found to have a greatly enhanced antitumor 
effect, a result not achieved with monotherapy. The enhanced efficacy of the combination was also 
observed after tumor progression during trastuzumab monotherapy and may relate to the different 
mechanisms of action of trastuzumab and pertuzumab; inhibition of HER2 dimerization and 
prevention of p95HER2 formation.26  
5.3.2 Clinical Studies: Pertuzumab Monotherapy  
Pertuzumab was investigated initially as a single agent in metastatic breast cancer, with 
disappointing results. In a study which evaluated pertuzumab monotherapy in patients with 
progressive HER2-negative metastatic breast cancer (MBC), the response rate (RR) was 4.9% and 
the clinical benefit rate (CBR) was 9.8% (n=79). Pertuzumab was relatively well tolerated, with 
most adverse events being mild to moderate. Decline in left ventricular ejection fraction of ≥ 10% 
and/or to ≤ 50% was observed in eight patients, with one case of congestive heart failure.27 In 
another study in HER2-positive patients with MBC progressing on trastuzumab, the objective RR 
was 7.4% with a CBR of 11.1% (n=29). The most frequent adverse events were diarrhea (48%), 

  
  
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nausea (34%), and vomiting (24%), and two patients developed an asymptomatic decline in LVEF 
of > 10% to < 50%. Interestingly, if the tumor failed to respond to pertuzumab monotherapy or 
responded and then progressed, trastuzumab could be added to pertuzumab. This strategy was 
undertaken in 14 patients, and 2 of these 14, achieved confirmed response when trastuzumab was 
added to the pertuzumab. This was the first clinical report providing some evidence of an enhanced 
effect when the antibodies are combined.28  
5.3.3 Clinical Studies: Dual anti-HER2 Therapy  
The combination of pertuzumab and trastuzumab has yielded promising results in early phase 
clinical trials. A phase 2 trial in patients with HER2-positive MBC whose disease had progressed 
during prior trastuzumab-based therapy assessed the efficacy and safety profile of the combination 
of pertuzumab and trastuzumab (n=66). The objective response rate was 24.2%, and the clinical 
benefit rate was 50%, with median progression-free survival (PFS) of 5.5 months. Cardiac 
dysfunction was minimal, and no patients needed to come off study as a result of cardiac-related 
adverse events.29  
5.3.4 Clinical Studies: Dual anti-HER2 Therapy and Chemotherapy  
This combination of HER2-targeted agents has also been investigated in the neoadjuvant setting. 
NeoSphere was a Phase 2 randomized trial of preoperative systemic therapy comparing 
trastuzumab/docetaxel, 
trastuzumab/pertuzumab/docetaxel, 
trastuzumab/pertuzumab, 
and 
pertuzumab/docetaxel. The pCR rates were 29%, 46%, 17% and 24% respectively. When patients 
were analyzed based on ER status, a 63% pCR rate was observed in those with ER-negative disease 
treated with trastuzumab/pertuzumab/docetaxel and 27% in those with ER-negative disease treated 
with trastuzumab/pertuzumab.30 Based on data from NeoSphere and other preoperative studies 
investigating pertuzumab in combination with chemotherapy, the FDA has granted accelerated 
approval in late 2013 to pertuzumab for use preoperatively in combination with a complete 
chemotherapy regimen such as that investigated in NeoSphere. That a large proportion of women, 
however, treated with biological therapy alone in the NeoSphere study, can obtain a pCR has 
caused great excitement in the breast oncology community. There is a clear need to determine who 
these patients are upfront as they could potentially be spared the added toxicity of chemotherapy.  
  
A phase 3 trial in previously untreated HER2-positive MBC patients (CLEOPATRA) which 
randomizes patients to trastuzumab/docetaxel versus trastuzumab/pertuzumab/docetaxel has 
recently been presented. The median progression-free survival was 12.4 months in the control 
group, as compared with 18.5 months in the pertuzumab group (hazard ratio for progression or 
death, 0.62; 95% confidence interval, 0.51 to 0.75; P<0.001). The interim analysis of overall 
survival showed a strong trend in favor of pertuzumab plus trastuzumab plus docetaxel.2 In 
addition, a phase 1 trial is ongoing investigating the combination of trastuzumab, pertuzumab, and 
paclitaxel in patients with metastatic breast cancer.  
5.4  
Biomarkers of Response or Resistance  
As we enter an era of “personalized” cancer treatment, the preoperative period has been recognized 
as ideal for evaluating surrogate biomarkers for the prediction of response to therapy and clinical 
outcome.31 In a recent meta-analysis which included 3776 patients in 16 studies, pathologic 

  
  
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response was found to be a prognostic indicator for relapse-free survival, disease-free survival and 
overall survival; suggesting that patients achieving pCR after neoadjuvant chemotherapy have 
favorable outcomes.32 Valuable information can be obtained using small numbers of patients in a 
short time frame, by assessing tumor response to therapy in vivo and measuring biochemical or 
radiologic changes in malignant tissue prior to, during, and following neoadjuvant therapy. Ideally, 
these surrogate biomarkers can be used early in the treatment paradigm to predict response to 
neoadjuvant chemotherapy. For example, a marker that can separate sensitive and resistant tumors 
to specific agent(s) early in the course of therapy could be used to determine treatment early in the 
course. This could avoid certain patients receiving toxic and futile therapies, and direct other 
patients towards an aggressive or investigational approach in an effort to maximize their breast 
cancer outcome.  
  
Traditionally, standard clinicopathologic factors such as age, receptor status, grade, and 
proliferation index aid in determining the choice of therapy for those with early breast cancer. In 
addition to single genes or proteins, gene-expression profiling allows for the rapid assessment of 
multiple genes rather than single genes using high-throughput DNA sequencing and may be used 
to predict both response to therapy and clinical outcome. Several trials have evaluated multigene 
assays as predictors of response to therapy in the preoperative setting, and validation efforts are 
ongoing.33,34 The molecular tools developed thus far, however, offer little aid in clinical 
decisionmaking for women with ER-negative or HER2-positive disease, and improved prognostic 
and predictive tools for these patients are needed. Of great interest also to the oncology community 
is the development of biomarkers able to identify a subgroup of patients with HER2-positive 
disease that may potentially be treated with biologic therapy alone and be spared systemic 
chemotherapy.  
5.4.1 Functional Imaging to Predict Response to Neoadjuvant Therapy  
Imaging techniques may also provide early information regarding tumor response. PET may allow 
for early prediction for pathologic response as early as 2 weeks post commencement of systemic 
therapy.35 Although several studies have evaluated the role of FDG PET in predicting response to 
therapy, a clear role in this regard has not been established. It must also be noted that the majority 
of studies performed to date included few if any patients with HER2-positive disease and so the 
role of PET in this population is not as clearly defined.  
  
There are a few advantages to using FDG-PET for our study. We have some preliminary data from 
the NeoALLTO PET substudy described below that it may be predictive of response to therapy in 
a HER2-positive population, it is widely available such that all sites can easily do, the baseline 
study is covered by insurance, and finally response criteria for FDG-PET have been established 
(PERCIST).36 Other imaging modalities may be of interest in imaging biomarker studies, such as 
fluoro-L-thymidine (FLT)-PET. Although FLT-PET is promising and interesting, it is not FDA 
approved for use at this time unlike FDG-PET, it is difficult to make available for multi-site studies. 
In addition, other imaging modalities such as MRI have not been proposed for cost reasons and 
due to challenges in multicenter standardization.  
5.4.1.1 Predicting Response to Chemotherapy  

  
  
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Wahl and colleagues, our collaborator at Johns Hopkins, have conducted serial FDG PET studies 
in 11 patients with primary breast cancer >3 cm in size. They observed a rapid and significant 
decrease in glucose metabolism as early as 8 days after the administration of one cycle of 
combination chemotherapy in women who responded to the regimen.37 Patients with a partial 
response had a reduced FDG uptake later in the course of the treatment, while non-responders had 
no significant change in uptake. In another study, FDG PET studies were obtained before and after 
1, 4, and 8 cycles of doxorubicin-based combination chemotherapy administered every 3 weeks. 
Mean baseline values of FDG uptake were higher in lesions that subsequently showed a partial or 
complete pathological response compared to those which did not.38 Moreover, the breast lesions 
that responded to the chemotherapy (clinical or pathological complete response demonstrated a 
statistically significant reduction in FDG uptake following a single cycle of the chemotherapy. 
These findings were confirmed in another study in which 22 patients with primary breast cancer 
underwent FDG PET imaging before and after 1 and 2 cycles of combination epirubicin and 
cyclophosphamide or epirubicin and paclitaxel. Women who had minimal residual disease had a 
decline in tracer uptake while those who had gross residual disease showed only little change in 
tracer uptake.39 These studies demonstrate that FDG PET response following one cycle of 
chemotherapy is predictive of complete pathological response to the therapy (sensitivity 90-100% 
and specificity 74-91%).  
Investigators at Johns Hopkins have also conducted a prospective study in women with stage 2 and 
3 breast cancer who received neoadjuvant chemotherapy with FDG PET being performed at 
baseline and 7 days after the first cycle of chemotherapy. Six of nineteen eligible women 
underwent radiological imaging of FDG-PET/CT and MRI for at least two cycles of treatment. 
Greater reduction in SUV was observed in responders (38%) compared to the non-responder (22%; 
P = 0.03). MRI volumes decreased after cycle 1 by 42% (responders) and 35% (non-responder; P 
= 0.11). Proliferation index Ki-67 performed on biopsy specimens declined in responders in the 
first cycle (median = 47%, range = 29-20%), but increased (4%) in the non-responder.40 Finally, a 
multi-institutional neoadjuvant clinical trial being led by our group at Johns Hopkins has reached 
full patient accrual (n=60) and indicates feasibility of completing studies which incorporate serial 
biopsies and PET imaging with central review in multiple sites (.35  
  
The results of the studies outlined above suggest that early changes in FDG uptake after the first 
cycle of therapy are predictive of subsequent clinical and pathological response to combination 
chemotherapy (4-8 cycles) for breast cancer. To our knowledge, few studies have evaluated change 
in FDG uptake shortly after the administration of combination chemotherapy (7 days). Because 
FDG PET detects changes in tumor biology, it is a very attractive modality to evaluate in our 
clinical model.  
  
Many other studies have evaluated changes in PET as a biomarker of response to neoadjuvant 
therapy at later time points and have identified varying cut offs felt to best predict response to 
therapy. A study of 52 patients undergoing neoadjuvant chemotherapy for stage 2 and 3 breast 
cancer aimed to evaluate early axillary lymph node response. Fifty per cent of baseline SUV was 
considered the best cutoff value to distinguish responders from non-responders. The sensitivity, 
specificity, negative predictive value and accuracy of FDG PET after one course of chemotherapy 

  
  
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were, respectively, 96, 75, 95, and 84%; suggesting that the pathological status of regional axillary 
lymph nodes could be accurately predicted after one course of neoadjuvant chemotherapy using 
PET.41 In another neoadjuvant study (n=52) in which PET was performed after the 1st and 2nd cycle 
of therapy, patients were classified as non responders (NR) when the decrease of SUVmax in the 
primary tumor was less than 15% at the time of the second PET per EORTC criteria.42 The 3-year 
disease-free survival (DFS) rate was significantly longer for metabolic responders than for 
nonresponders (respectively 94% vs. 69%). This prospective study showed that a decrease in the 
SUV less than 15% after the first chemotherapy course was a very potent predictor of failure to 
respond to neoadjuvant chemotherapy.43  
A recent meta-analysis of 19 studies and 920 patients with pCR aimed to predict histopathological 
response in primary breast lesions by PET. The pooled sensitivity, specificity, positive predictive 
value (PPV), negative predictive value (NPV), and diagnostic odds ratio were 84%, 66%, 50%, 
91%, and 11.90, respectively. Subgroup analysis showed that performing a post-therapy PET early 
(after the 1st or 2nd cycle of chemotherapy) was significantly better than later (accuracy 76% vs. 
65%, P = 0.001). In addition, the best correlation with pathology was yielded by employing a 
reduction rate (RR) cutoff value of standardized uptake value between 55 and 65%. Further 
prospective studies were recommended to adequately position PET in treatment management for 
breast cancer patients.44  
Finally, the impact of ER status on the role of PET as a biomarker in this setting remains to be 
defined. A considerable distinction in metabolic activity has been observed between the different 
subtypes of breast cancer. The mean SUVmax in lymph node metastases of ER-positive, 
triplenegative and HER2-positive tumors was 6.6, 11.6 and 6.6, respectively (n=38).45 In a study 
that evaluated 41 patients receiving neoadjuvant anthracycline/taxane based chemotherapy, PET 
scans were obtained at baseline and post 2 cycles of therapy. Thirty-three patients (80%) presented 
SUV reduction > 50% including all 10 patients with pCR (included minimal residual disease) and 
23 patients with no pCR. On the contrary, none of the 8 patients with SUV reduction ≤ 50% 
achieved a pCR and these 8 patients had ER-positive tumors. Hence, out of a total of 24 patients 
with ERpositive tumors, one third was correctly identified early as not obtaining a pCR.46 These 
results indicate that PET may play a role in early identification of patients with ER-positive tumors 
who are unlikely to obtain a pCR.  
5.4.1.2 Predicting Response to Anti-HER2 Therapy  
The NeoALLTO PET substudy is the first study to prospectively evaluate changes in SUV on PET 
as a predictor of response to neoadjuvant anti-HER2 therapy (n=86, 77 evaluable). As a reminder, 
patients in the NeoALLTO study were randomized to initially receive 6 weeks of a "biological 
window" of either lapatinib, or trastuzumab, or lapatinib with trastuzumab. Subsequently, the same 
targeted therapy was continued with the addition of weekly paclitaxel for a further 12 weeks, until 
definitive surgery. PET scans were performed at baseline, week 2, and week 6 after starting 
therapy. A metabolic response was defined as a >15% reduction in SUV at 2 weeks, or >25% 
reduction at 6 weeks per EORTC criteria.42 At week 2, metabolic responders had a pCR rate of 
42% and non-responders 21%. At week 6, metabolic responders had a pCR rate of 44% and 
nonresponders 19%. A more stringent cut off of 30% reduction in SUV from baseline to 2 weeks 

  
  
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is observed in approximately 50% of the population and is associated with a pCR rate of 40% 
approx. It must be emphasized that the patients in this small PET substudy are from all arms of 
NeoALLTO and received only one biologic therapy for the first 6 weeks of the “biological 
window” while the PET scans were being performed, but subsequently also received paclitaxel. 
Therefore, the pathological response reflects response to the chemotherapy and biologic therapy 
while the response on PET reflects response to the biologic therapy alone.  
The association between early changes in SUV on PET in an advanced HER2-positive breast 
cancer population receiving trastuzumab and lapatinib has also recently been presented 
(TBCRC003 PET substudy).47 Metabolic responses were detected as early as 1 week with 
FDGPET and the PET scan at week 1 correlated with the PET scan at week 8 after initiating 
therapy. In exploratory analyses, there appeared to be a relationship between early metabolic 
response and more traditional measures of clinical outcome, such as clinical benefit rate and PFS. 
For example, 45% (9/20) of patients who achieved a metabolic partial response at week 1 had 
clinical benefit by RECIST and 23% (5/23) of patients who achieved metabolic stable disease at 
week 1 had clinical benefit by RECIST.  
We propose to evaluate for the first time the correlation between early changes in SUV and pCR 
in men and women with ER-negative, HER2-positive breast cancer receiving trastuzumab and 
pertuzumab. Based on the low pCR rate observed in those with ER-positive disease, we propose 
to investigate this question in an ER-negative population alone. For the purposes of this clinical trial 
we have decided to use a cut off of <10% for ER/PR staining. The publication in 2011 by Iwamoto et 
al supports this approach as it indicates that few tumors with ER staining of 1-9% show molecular 
features similar to those of ER-positive, potentially endocrine-sensitive tumors, whereas most 
show ER-negative, basal-like molecular characteristics.48 In addition, as we are focusing on a select 
population of patients with HER2-positive disease (approximately 8-10% of early breast cancers), 
and hope to avoid difficulties with accrual, we felt it would be in the best interest of the trial to 
include patients with ER staining 1-10%. These patients will be recommended adjuvant hormonal 
therapy after completion of adjuvant chemotherapy per standard of care.   
The results from this phase 2 biomarker study will be used to plan a randomized study using a 
predefined cut point for SUV decline such that we can further attempt to identify a group of 
individuals with HER2-positive early breast cancer who do not require cytotoxic chemotherapy in 
addition to anti-HER2 agents. The preliminary data from NeoALLTO described below and our 
ongoing experience in HER2-negative disease suggest that this non-invasive biomarker approach 
will be of great interest to breast cancer oncologists and patients by facilitating a personalized  
approach to managing patients with HER2-positive disease which will undoubtedly spare toxicity 
and reduce the costs associated with anti-cancer strategies, without compromising efficacy.  
The NeoALLTO study defined a metabolic responder as a patient with ≥ 15% decline in SUV at 2 
weeks (EORTC criteria). Our collaborators at Johns Hopkins (Wahl and colleagues) have concern 
that obtaining this 15% reduction in SUV may at times be due to chance and believe that 
randomizing patients with a greater reduction in SUV as defined by PERCIST criteria is more 
appropriate when aiming to preselect a population with a high chance of obtaining a pCR.36  The 
EORTC criteria were proposed initially in 1999 when limited data was available in this regard. 

  
  
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The PERCIST criteria is based on more modern data which indicates that larger drops in SUV of 
more than 30-35% is associated with a good outcome for cancer patients, and thus they propose a 
30% cut off for SUV decline for tumor response. We anticipate that we will identify an SUV 
reduction from baseline which will accurately predict pCR and plan in our next study to randomize 
patients with this identified decline in SUV from baseline to PT versus PT + chemotherapy.36 We 
chose to evaluate day 15 changes to correlate with findings from ongoing studies of endocrine 
therapies, chemotherapies, and novel agents that are commonly evaluating changes in imaging and 
tissue markers approximately 2 weeks after treatment initiation. We will also be able to evaluate 
concomitant changes in FDG uptake and in molecular markers that may be associated with 
response or resistance to chemotherapy detected in the breast cancer tissue obtained at the time of 
imaging. The results of our study can be used to design new studies with novel agents such as the 
randomized design described.  
5.4.2 The PI3K Pathway as a Biomarker of Response to Therapy  
An important focus of this study will be the correlation of tumor PIK3CA mutation status and PI3K 
pathway activation status with pCR. Similar to the HER2 pathway, the PI3K pathway is involved 
with several cellular systems, regulating proliferation, growth, and survival. Prior studies have 
shown that trastuzumab has antitumor effects only in the presence of a normal PI3K pathway and 
dysregulation of the PI3K pathway through genetic alterations results in trastuzumab resistance.49 
Thus, it can be hypothesized that PI3K pathway dysregulation, causing trastuzumab resistance, 
may be associated with inability to achieve pCR and transitively, poorer clinical outcomes. 
Preclinical data also supports a link between PI3K/Akt pathway and pertuzumab with or without 
trastuzumab.26,50 This question is very important because the use of trastuzumab is now standard 
in the treatment of HER2-positive breast cancer, and assessment of PI3K pathway activation in 
this patient population may provide a biomarker to identify patients less likely to respond to 
trastuzumab-based therapy.51   
  
A recent study was conducted to determine the frequency of PIK3CA mutations and human 
epidermal growth factor receptor-2 (HER2) phosphorylation status (pHER2-Tyr1221/1222) and if 
PIK3CA, phosphatase and tensin homolog (PTEN), or pHER2 had an impact on outcome in HER2-
positive early-stage breast cancer patients treated with adjuvant chemotherapy and trastuzumab. 
Samples from 240 patients receiving adjuvant chemotherapy and subsequent trastuzumab were 
evaluated for PTEN and pHER2 expression by immunohistochemistry as well as PIK3CA 
mutations (exons 9 and 20) by pyrosequencing. PIK3CA mutation were identified in 26%, 24% 
were PTEN low, 45% pHER2 high, and 47% patients had increased PI3K pathway activation 
(PTEN low and/or PIK3CA mutation). No significant correlations were observed between the 
clinicopathological variables and PIK3CA, PTEN, and pHER2 status. In both univariate and 
multivariate analyses, patients with PIK3CA mutations or high PI3K pathway activity had a 
significant worse OS despite adequate chemotherapy and trastuzumab.52 Other studies have 
investigated the expression or activation of HER3 as a predictive or prognostic biomarker in 
patients with HER2-positive disease or those treated with pertuzumab. Chiu et al. reported that 
HER3 overexpression was identified in 10.0% of tumors in patients with breast cancer in a 
randomized study and was a clinically significant marker of reduced survival in such patients.3 In 
a randomized phase 2 study, it was found that low HER3 levels corresponded with response to 

  
  
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pertuzumab in patients with platinum-resistant ovarian cancer.4 In the NEOPSHERE and 
CLEOPATRA studies, the level of HER3 protein and mRNA did not correlate with treatment 
outcome. However, it has recently been reported that HER3 expression and activation is modulated 
in response to HER2, HER3, or PI3K pathway inhibition, which may not be reflected by the 
analysis of primary tumors but perhaps in biopsies obtained subsequent to commencement of 
anticancer therapy.53 We propose to investigate these questions in a prospective fashion using both 
archival and frozen tumor samples before, during, and after therapy.  
  
In addition, we propose to evaluate BEAMing (Beads, Emulsification, Amplification and 
Magnetics) as a technique to identify PIK3CA mutations in an ER-negative, HER2-positive early 
breast cancer population at baseline and during therapy, and assess if a correlation exists between 
mutation status and pCR. Because BEAMing is quantitative, we will also determine the correlation 
between pre and post treatment ptDNA blood levels with pCR. Traditional screening for mutations 
such as the PIK3CA mutation relies upon sequencing of archival tumor tissue. However, 
limitations exist with this method including the need to select specimens with a sufficient number 
of tumor cells, tumor heterogeneity, and variable quality of extracted and stored DNA that can 
interfere with accurate analyses. There is a growing need to develop accurate and less invasive 
methods of screening. Our group designed a prospective study to evaluate the feasibility of 
detecting hotspot PIK3CA mutations in both archival tumor and peripheral blood samples from 
patients with metastatic breast cancer using the BEAMing technique, and compared the results to 
those obtained by traditional sequencing of FFPE tissue. BEAMing was also performed on any 
remaining genomic DNA extracted from archival tissue samples used for sequencing.53,54  
  
BEAMing of blood samples revealed that 28% (17/60) of patients had an identifiable hotspot 
PIK3CA mutation. Standard sequencing indicated that 27% (14/51) of tumors (12 primary, 2 
metastatic) had PIK3CA mutation. Of the 51 samples for which both a tissue and plasma sample 
was available, 39 (76.5%) yielded the same results (either all wild type or same hotspot PIK3CA 
mutation identified) by both sequencing and BEAMing. The concordance between results obtained 
by BEAMing and sequencing of the same archival tissue sample was 100%. The investigators 
concluded that BEAMing was a non-invasive method of identifying these mutations. The 23.5% 
discordance observed between mutational status in archival tissue samples and blood samples 
drawn later in the disease course supports recent reports that PIK3CA mutational status changes 
during the progression of breast cancers. The study overall suggest that patients should optimally 
be selected for trials of PI3K inhibitors based on PIK3CA mutational status at time of enrollment, 
rather than on mutational status of archival tissue.  
  
New unpublished preliminary data from Dr. Park’s laboratory demonstrate that second generation 
digital PCR technologies, specifically droplet digital PCR (ddPCR, Bio-Rad) can also detect 
PIK3CA mutations in plasma of patients with early stage breast cancer. PIK3CA mutations were 
detected in 14 of 15 patients tested, including eight patients with Stage Ia (T1, N0, M0) disease, 
yielding a sensitivity of 93.3% and specificity of 100% PIK3CA mutations can therefore be 
reproducibly detected in pre op plasma samples (~2ml of plasma containing as few as 1000 genome 
equivalents) from early stage breast cancer patients with minimal tumor burden. In addition, newer 

  
  
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technologies using next generation sequencing will enable us to use all mutations/genomic 
rearrangements as “biomarkers” that can be qualitatively and quantitatively measured in plasma as 
a surrogate measure of disease burden and response to therapies. As this technology matures, we 
envision expanding this portion of the biomarker study to include other mutations (e.g. TP53) and 
genomic rearrangements and then coupling this with BEAMing and other digital PCR technologies 
to assess how changes in ptDNA correlate with pCR.  
5.4.3 Immune enrichment signatures as a Biomarker of Response to Therapy  
There has been a surge in interest in the role of the immune system, and agents targeting the 
immune system, in breast cancer. Breast carcinomas are often infiltrated by inflammatory cells, 
particularly macrophages and T lymphocytes. These inflammatory cells may represent a 
cellmediated immune response against the carcinoma. A recent study indicated that tumor-
infiltrating CD8+ T lymphocytes have antitumor activity in breast cancer based on the observed 
favorable effect on patients’ survival, suggesting a potential therapeutic strategy that can be 
harnessed.55 The prognostic and predictive roles of tumor-infiltrating lymphocytes (TILs) were 
also assessed in a phase 3 adjuvant breast cancer trial. In the HER2-positive population, there was 
a significant interaction between increasing stromal lymphocytic infiltration (10% increments) and 
benefit with anthracycline-only chemotherapy.56 The importance of the immune system in HER2-
positive breast cancer was also determined in a meta-analysis (n >2,100) in which gene expression 
modules related to key biological processes in breast cancer were evaluated together with clinical 
factors and several prognostic signatures.57 Multivariate analysis demonstrated that the tumor 
invasion and immune response modules were significantly associated with survival in the HER2-
positive tumors. Finally, the adaptive immune system and immune checkpoints were found to be 
associated with response to chemotherapy and anti-HER2 therapy with pertuzumab and 
trastuzumab in the NeoSPHERE neoadjuvant study.[ref] High PD-L1 expression was consistently 
associated with lower pCR rate in all chemotherapy containing arms, with a similar trend in the 
anti-HER2 therapy alone arm. This further supports our understanding of the key role of the 
immune system in contributing to HER2-targeted antibody therapy on top of signaling inhibition.58   
  
Our collaborators at the Mayo clinic have recently completed transcriptome analysis of 1282 
patient samples from the N9831 adjuvant trastuzumab trial.59 RNA was extracted and DASL 
microarray analysis was carried out on 433 patients who received chemotherapy alone and 849 
who received chemotherapy plus trastuzumab. They identified 87 genes which are linked to 
immune functions and which have significant Cox HRs, using relapse free survival as a continuous 
variable end point. A modified surface mapping tool was used to assort HER2 tumors, based on 
the expression of these immune function genes, into two cohorts: those that are enriched for 
immune function genes and those that are not enriched. Both immune-enriched and immune 
notenriched tumors were equally responsive to chemotherapy alone (AC->T, HR=0.98, p=0.94). 
Among trastuzumab-treated patients, those with immune not-enriched tumors exhibited 
relapsefree similar to that observed in patients treated with chemotherapy alone (HR=01.00, 
p=0.99). However, trastuzumab-treated immune enriched tumors exhibited considerably long 
relapse free survival (HR 0.54, p<0.001), when compared to immune not-enriched tumors. It was 
concluded that there is a subset of HER2 tumors that manifest a highly activated immune status 
prior to onset of therapy, and it appears that most of the benefit from adjuvant trastuzumab resides 

  
  
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within this cohort of tumors. These data are consistent with the concept that immune feature, 
present in the tumor prior to onset of therapy, play an integral, perhaps essential role in the 
mechanism of action of trastuzumab.  
  
Based upon the data described above, we hypothesize that tumors with an enhanced immune 
signature, defined as described above, are more likely to undergo pCR after neoadjuvant 
trastuzumab or pertuzumab. We anticipate that we will be able to use RNA from formalin-fixed, 
paraffin-embedded biopsy samples to measure the expression of the genes that we identified from 
the N9831 analysis. A surface mapping model will be used to assign tumors to immune enriched 
and not-enriched cohorts.   
5.4.4 Change in Apoptosis and Proliferation as a Marker of Response to Therapy  
A change in the proliferation index Ki67 has been evaluated predominantly in women receiving 
hormonal treatments for breast cancer. The IMPACT trialists evaluated short term changes in Ki67 
during neoadjuvant treatment of primary ER-positive breast cancer with anastrozole or tamoxifen 
alone or combined. In the 88 ER-positive women who received tamoxifen for 2 weeks and 
underwent repeat Ki-67 assessment, a geometric mean percentage change in Ki-67 expression from 
baseline to 2 weeks of -59.5% (95% CI -68.5 to -47.9) was reported.60 Importantly the IMPACT 
trialists have subsequently performed an exploratory analysis of their original data and have found 
that a higher Ki67 expression after 2 weeks of endocrine therapy was statistically significantly 
associated with a  lower recurrence-free survival (P=0.004) whereas higher Ki67 expression at 
baseline was not.61  In a study of the tumors from 104 postmenopausal women before and after 2 
weeks treatment with anastrozole, luminal A and B tumors obtained similar benefit from therapy 
as measured by the proportional fall in Ki67 upon treatment. Tumors classified as basal and HER2-
like showed poor reductions in Ki67 upon treatment.62 Validation of these results in this study 
would be useful as future studies are planned.  
  
Apoptosis and proliferation have also been examined in a HER2-overexpressing population. A 
neoadjuvant trial was performed in 35 patients with locally advanced HER2-overexpressing breast 
cancers who received weekly trastuzumab for the first 3 weeks, followed by a combination of 
trastuzumab and docetaxel for 12 weeks before surgery. Sequential core biopsies were assessed by 
immunohistochemistry for cell cycle and proliferation, apoptosis and survival, epidermal growth 
factor receptor, and total and p-HER-2. Apoptosis was significantly induced (median increase from 
3.5% to 4.7) within week 1 consistent with a 35% increase above baseline. Tumors with high 
baseline Ki67 were less likely to respond.63  
  
These results suggest that serial core biopsies can be collected from women with primary breast 
cancer who receive neoadjuvant hormonal therapy or chemotherapy and that early change in the 
apoptotic and proliferative indices in the tumors may predict response or resistance to the therapy. 
To our knowledge, early changes in markers of apoptosis and proliferation have not been evaluated 
in patients receiving dual anti-HER2 blockade with pertuzumab and trastuzumab. We will examine 
how baseline and change (day 15) in cleaved caspase 3 and Ki67 are associated with pCR.  

  
  
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5.5  
Other Biomarkers of Response to Therapy  
Ongoing discussions with our collaborators and Genentech will help us define other promising 
biomarkers for evaluation during this study, and may include correlation of changes in candidate 
gene expression or methylation with pCR.  
5.5.1 Candidate Gene Expression Profile Analysis  
The effectiveness of standard systemic therapies for breast cancer may vary among individuals. 
For example, the presence of the ER predicts response to endocrine therapy and perhaps relative 
resistance to chemotherapy. Overexpression or amplification of the HER2 oncogene may be 
associated with a relative resistance to endocrine manipulations and relative sensitivity to 
chemotherapy.64 Review of the literature suggests that hundreds of individual genes or proteins 
may be associated with relative response or resistance to common chemotherapies in breast cancer. 
It is likely that other genes may confer relative resistance or sensitivity to specific treatments, but 
have not yet been identified. It is also possible that there are clusters of tens or hundreds of genes 
that may predict response or resistance to specific therapies.  
  
Gene expression has been used by several investigators to classify breast neoplasms. It is also 
possible that chemosensitive vs. resistant tumors will have distinct gene profiles.65,66 Baylor 
investigators evaluated molecular profiles of breast cancer responsive or resistant to 4 cycles of 
docetaxel administered every 3 weeks. These portraits were different than expected. 67 Similarly, 
MD Anderson investigators have developed a set of pharmacogenomic predictors to predict pCR 
to paclitaxel and anthracycline basal regimen.68 Finally, the prognostic ability of the 70-gene 
Mammaprint assay was assessed in a HER2-positive population (heterogeneous for ER and node 
status) that did not receive adjuvant chemotherapy or trastuzumab (n=89), and 22% of them were 
classified as having as good prognosis (10-year DDFS, 84%) while the other 78% had a poor 
outcome (10-year DDFS 55%). 69  It remains to be seen whether this assay may identify 
HER2postive patients that might be spared adjuvant chemotherapy and trastuzumab. Our 
hypothesis is that distinct baseline gene expression profiles can be correlated with response or 
resistance to preoperative chemotherapy. It is also possible that following therapy, distinct patterns 
can be observed in responsive or resistant tumors.   
5.5.2 Methylation Profiles  
In breast cancer, multiple genes are methylated, and thus silenced, compared to non-cancerous 
tissue. Gene methylation is likely related to cancer progression. HDAC inhibitors may also 
alleviate gene repression that is mediated through promoter hypermethylation. It is presumed that 
HDAC inhibitors can increase expression of the genes that are not methylated but may not induce 
the expression of hypermethylated genes. Some of the genes that are often methylated in breast 
cancer include growth promoting hormone receptors such as estrogen receptor alpha (ER), an 
important predictive factor of response to endocrine manipulations, and the retinoid receptor 
RARbeta, critical for cell differentiation. Other genes that are often hypermethylated in breast 
cancer include cyclin D, Twist, RASSF1A, APC, and HIN-1. Dr. Sukumar and other Johns 
Hopkins investigators have evaluated hypermethylation of a panel of seven genes using 
methylationspecific polymerase chain reaction (MSP) in a variety of breast tissues.70-73 In invasive 
breast carcinomas, up to 100% of specimens contained at least one hypermethylated gene, 80% 

  
  
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contained two, and 60% contained three or more methylated genes.73 In 44 ductal carcinoma in 
situ (DCIS) specimens, 95% had at least one methylated gene. In contrast, the percentage of women 
with benign breast disease having at least one methylated gene was only 15%. Only one of 8 
reduction mammoplasty specimens contained hypermethylated genes. More recently, Dr. Sukumar 
and colleagues have developed a novel method, quantitative multiplex-methylation specific PCR 
(QMMSP) to accurately assess promoter hypermethylation for many genes simultaneously in small 
samples. QM-MSP is highly sensitive (1 in 104-105 copies of DNA) and linear over 5 orders of 
magnitude.71 Because tumor samples may not be accessible in most patients with metastatic breast 
cancer, assays that can be conducted in blood samples are desirable.71,72,74 Gene hypermethylation 
may be seen in circulating DNA and thus represent an attractive surrogate in the proposed study.75  
5.5.3 Pharmacogenomics  
The variability in response to drugs may be in part due to genetic polymorphism of receptors, 
transporters, or drug metabolizing enzymes. Single nucleotide polymorphisms (SNPs) have been 
identified in CYP450 enzymes and may be associated with a different ability to metabolize drugs. 
Due to the small sample size, we do not anticipate that correlation can be made between the 
presence of specific SNPs and response to therapy. However, if a SNP exists in an individual 
patient, it may help us to assess her individual response and toxicity. These results may also assist 
in design of future studies. To our knowledge no candidate genes are known which may predict 
response to anti-HER2 therapy at this time, with mixed data regarding the relevance of Fc receptor 
polymorphisms. At this time we plan to store samples obtained at baseline for potential future 
analyses in the event that further information becomes available from the literature and patients 
will be appropriately consented for same.  
5.6  
Study Rationale  
The management of HER2-positive breast cancer has been revolutionized by the development of 
anti-HER2 therapies such as trastuzumab. Recent studies indicate that dual anti-HER2 therapy is 
more efficacious than single anti-HER2 therapy. NeoSphere was a Phase 2 randomized trial of 
preoperative 
systemic 
therapy 
comparing 
trastuzumab/docetaxel, 
trastuzumab/pertuzumab/docetaxel, trastuzumab/pertuzumab, and pertuzumab/docetaxel. The 
pCR rates were 29%, 46%, 17% and 24% respectively and all arms had acceptable toxicity. The 
fact that a large proportion of women treated with biological therapy alone can obtain a pCR has 
caused great excitement in the breast oncology community. There is a clear need to determine who 
these patients are upfront as they could potentially be spared the added toxicity of chemotherapy.  
  
The NeoALLTO PET substudy has indicated that metabolic changes in the primary tumor of 
patients with invasive operable breast cancer treated with anti-HER2 therapies can be detected as 
early as 2 weeks of treatment. Pathologic CR rates were found to be twice as high in patients who 
are FDG-PET/CT responders compared to non-responders according to EORTC criteria. We 
expect, based on the NeoALLTO data, that early change in SUV on FDG-PET will be associated 
with response to pertuzumab/trastuzumab in patients with primary operable HER2-positive breast 
cancer. We propose to evaluate for the first time the correlation between early changes in SUV and 
pCR in men and women with ER-negative HER2-positive breast cancer receiving pertuzumab and 
trastuzumab alone. Based on the low pCR rate observed in those with ER-positive disease, we 

  
  
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propose to investigate this question in an ER-negative population alone. We anticipate that our 
findings will mirror the NeoALLTO PET substudy results but we hope to identify an SUV decline 
that more accurately predicts pCR. Data from TBCRC006 and the ongoing TBCRC023 trial 
indicate that the administration of dual anti-HER2 therapy alone is feasible in the pre-operative 
setting and is associated with pCR in a proportion of women. Our study will build on this 
experience by attempting to define early in the treatment paradigm the patients who are likely to 
obtain a pCR using changes in SUV on PET. The results from this phase 2 biomarker study, if 
promising, will be used to then plan a randomized study using a predefined cut point for SUV 
decline such that we can further attempt to identify a group of individuals with HER2-positive 
early breast cancer who do not require cytotoxic chemotherapy in addition to anti-HER2 agents. 
We believe this will be of great interest to breast cancer patients and this has been echoed by the 
TBCRC advocates in recent discussions.   
  
In addition, we aim to identify plasma and tissue biomarkers that may predict response to 
pertuzumab and trastuzumab with a focus on the PI3K pathway. We propose to evaluate BEAMing 
as a technique to identify PIK3CA mutations in plasma in a HER2-positive, ER < 10% early breast 
cancer population at baseline and during therapy, and assess if a correlation exists between 
mutation status and pCR. We anticipate that in the future a combination of early changes in SUV 
on PET and tissue biomarkers will provide the optimal way of identifying those patients who do 
or not need chemotherapy or targeted therapies.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
  
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6. Patient Population  
6.1  Inclusion Criteria  
6.1.1 Female and male patients, 18 years old or older  
6.1.2 Histologically proven infiltrating carcinoma of the breast on core needle biopsy that is:  
• 
ER/PR ≤10% staining by IHC   
• 
HER2 positive – IHC 3+, ISH ≥2.0, or average HER2 copy number ≥6.0 signals 
per cell or per current ASCO-CAP (American Society of Clinical Oncology – 
College of American Pathologists) or NCCN (National Comprehensive Cancer 
Network) guidelines.   
Note:  All histological diagnostic material should be reviewed at enrolling institution as 
required per local standards.  
6.1.3 Unresected, untreated breast cancer that meets one of the following clinical stages (see 
Appendix A):  
• 
T2, T3, or T4a-c lesion, any N, M0  
Note: Patients with inflammatory breast cancer (T4d) are not eligible.  Bilateral cancers 
are permitted with approval of the Protocol Chair.  Participants with clinically evaluable 
disease will be followed for response by clinical examination; measurable disease is not 
required for participation.  
6.1.4 ECOG performance status 0-1 (Appendix B)  
6.1.5 Adequate organ function as follows:  
• 
Absolute neutrophil count (ANC) ≥ 1,500/mm3  
• 
Platelet count ≥ 100,000/mm3  
• 
Hemoglobin ≥ 10 g/dL  
• 
Creatinine ≤ 1.5 times the upper limit of normal with creatinine clearance ≥ 50 
mL/min using the Modified Cockcroft-Gault method   
• 
Bilirubin (total) ≤ 1.5 times upper limit normal (with exception of Gilberts 
syndrome)  
• 
AST(SGOT), ALT(SGPT), and alkaline phosphatase ≤ 2 times the upper limit of 
normal   
Note: Exceptions to lab parameters may be allowed with approval of the Protocol Chair  
6.1.6 Adequate cardiac function as defined by LVEF ≥ 50% on echocardiogram or multi-gated 
acquisition scan (MUGA)  

  
  
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6.1.7 Able and amenable to baseline and follow-up PET/CT imaging and study-specific biopsy 
procedures.   
Note:  If there are any imaging concerns that the patient may not be suitable for 
quantitative PET/CT (e.g., a metallic device directly overlies the breast), discussion with 
the local and central radiologists is required to confirm eligibility for the trial.  Also, it is 
expected that subjects have all PET/CT imaging done on pre-qualified machines for the 
study; if baseline imaging done on another machine, please contact the Protocol 
Chair/designee for guidance prior to confirming eligibility.   
6.1.8 The patient, if of childbearing potential, is willing to use effective, non-hormonal 
contraception while on treatment and for at least 6 months following the last dose of 
therapy.  
6.1.9 Patient understands the study regimen, its requirements, risks, and discomforts, and is able 
and willing to sign an informed consent form.  
6.2  Exclusion Criteria  
6.2.1 Received prior or ongoing local (e.g radiation) or systemic treatment (chemotherapy or 
endocrine therapy) for the current breast cancer. Patients who received tamoxifen or 
raloxifene or another agent for prevention of breast cancer may be included as long as the 
patient has discontinued the treatment at least one month prior to baseline study biopsy.  
6.2.2 Systemic treatment for prior cancer within the last 5 years, with the exception of adequately 
treated cone-biopsied in situ carcinoma of the cervix uteri and basal or squamous cell 
carcinoma of the skin.  
6.2.3 Women who are pregnant or nursing  
6.2.4 Current use of any investigational agents  
6.2.5 Known hypersensitivity to trastuzumab or pertuzumab  
6.2.6 Any medical condition that in the opinion of the investigator puts the patient at risk of 
potentially serious complications while on this therapy. Specifically, uncontrolled 
hypertension (systolic >150 and/or diastolic >100), unstable angina, congestive heart 
failure of any New York Heart Association (NYHA) classification, serious cardiac 
arrhythmia requiring treatment (exception: atrial fibrillation, paroxysmal supraventricular 
tachycardia), history of myocardial infarction within 6 months of enrollment.  
6.3  
Inclusion of Women and Minorities  
Individuals of all races and ethnic groups are eligible for this trial. There is no bias towards age or 
race in the clinical trial outlined. This trial is open to the accrual of men and women.  
  

  
  
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7. Study Design and Treatment Plan  
NOTE:  If at any time the Protocol Chair is not available, the Protocol Co-Chair may provide 
oversight on study questions.  The Study Liaison may also be contacted for guidance.  
7.1  
Recruitment  
Patients will be recruited through the breast cancer clinics at each of the participating centers.  
Local institutions will assign a participant subject number at the time of consent which includes a 
site designation number and sequential subject number (e.g., 1-001, 1-002, etc.)  
7.2  
Determination of Eligibility  
After eligibility is established at the participating institution, the study staff will register 
participants with the Coordinating Center at Johns Hopkins. The following is required to be 
submitted for successful registration:  
  
• 
Registration forms  
• 
Copy of subject consent  
• 
Copies of the following source documents:    
- 
Diagnostic pathology report(s), including receptor status  
- 
Laboratory reports as per the eligibility criteria and pregnancy test, if applicable 
-  
Other documents, if requested.  
  
Upon review of the registration documents, the Coordinating Center at Johns Hopkins will confirm 
successful registration by return email and/or fax to the local study team/designee.  
  
Study treatment cannot begin until the patient is successfully registered with the Coordinating 
Center. In the event a subject’s day 1 is delayed for unforeseeable reasons and a baseline 
assessment(s) fall outside the intended window, it is expected that these be repeated and reviewed 
by the Coordinating Center prior to treatment unless explicit approval is given by the Protocol 
Chair or her designee.  
  
Subjects who sign a consent form, but do not initiate protocol treatment for any reason (e.g., 
subjects who are screen failures), will be replaced and will not count towards our accrual goal.  
7.3  
Preoperative Pertuzumab and Trastuzumab  
7.3.1 Overview  
A total of 4 cycles of combination pertuzumab and trastuzumab will be administered prior to 
definitive breast surgery.   
  
A cycle where treatment is held may be made up if treatment is being tolerated. Treatment will be 
administered on an outpatient basis.  
  

  
  
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The doses of pertuzumab and trastuzumab do not need to be recalculated unless the body weight 
has changed by more than ±10% from baseline.  
  
Note:  Changes in the infusion times for pertuzumab and trastuzumab noted below may be made 
in the event of toxicity (e.g., extension of the infusion timing) or per institutional standard – without 
contacting the Protocol Chair.  
7.3.2 Pertuzumab  
Pertuzumab will be administered before trastuzumab every 3 weeks (840 mg as a loading dose, 
then 420 mg every 3 weeks) intravenously (IV) for 4 doses. The initial dose should be administered 
over about 60 minutes and subsequent administration may be infused over about 30-60 minutes.  
  
Due to possibility of infusion-associated reactions (IARs) with pertuzumab, observation of the 
patient during and for 60 minutes after the first infusion and during and for 30 minutes following 
subsequent infusions is recommended (see Section 7.4.3.1).  
7.3.3 Trastuzumab  
Trastuzumab will be administered after pertuzumab every 3 weeks (8 mg/kg loading dose, then 6 
mg/kg every 3 weeks) IV for 4 doses. The initial dose should be administered over 90 minutes. If 
this is well tolerated, subsequent administration may be infused over about 30 minutes. DO NOT 
ADMINISTER AS AN IV PUSH OR BOLUS.   
7.3.4 Premedications  
Premedication regimens standard to each institution are suggested. This may include 
acetaminophen and diphenhydramine, for example. Each institution may employ the 
premedication regimens considered routine in their practices. The regimens used should be clearly 
noted in the medical record and research file.  
7.3.5 Randomization and Blinding  
This is an open-label, non-randomized phase 2 study.  
7.3.6 Additional Preoperative Treatment  
It is preferred that participants proceed to definitive surgery at the end of study therapy. The 
primary endpoint of this trial is correlation of SUVmax change with pCR. The option to administer 
additional treatment prior to surgery should be reserved for (a) disease progression on study 
treatment, or (b) cases where the investigator and/or patient is concerned about a possible 
incomplete clinical response AND residual disease is histologically confirmed as outlined in the 
study schema.  All cases should be discussed with the Protocol Chair prior to obtaining the 
post-study treatment biopsy (Section 8.0) and commencing additional therapy to ensure 
compliance with the protocol.  
  
In subjects who consider additional treatment, a tumor biopsy and blood sample will be required 
prior to initiating the treatment. In scenario b), the biopsy must be reviewed both locally and 
by the Study Chair or designee to confirm presence of residual disease prior to initiation of 

  
  
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additional treatment; it is requested that additional cores be collected at the same time for 
research purposes (Section 8.12).  Documentation of residual disease by imaging to confirm 
response may be considered at the discretion of the treating team (standard of care procedure) to 
document need for additional treatment (Section 8.9).  
It is recommended that additional preoperative treatment comprise a standard preoperative regimen 
of chemotherapy and HER2-directed therapy per NCCN guidelines. No other treatment options 
should be considered after study therapy and before surgery.  
  
Any further monitoring and tests after completion of the study treatment and during the additional 
preoperative treatment are at the discretion of the treating team. A final toxicity check 30 days after 
the last dose of study treatment will be collected; however, only adverse events believed to be 
related to the study treatment or interventions are collected during this time period.    
  
Upon completion of additional treatment, all post-treatment/surgical research tissue and blood 
samples are still required to be collected. Please see the Study Calendar (Section 8) for additional 
information.  
7.4  
Dose Modifications  
Dose modification of pertuzumab and trastuzumab is not permitted.  
  
If possible, symptoms should be managed symptomatically. In case of toxicity, appropriate medical 
treatment should be used (including anti-emetics for nausea/vomiting, anti-diarrheals for diarrhea, 
etc.). No dose escalation is planned for this study.  
  
Toxicity assessments will be done using NCI Common Terminology Criteria for Adverse Events 
(CTCAE v 4.0) which is available at http://ctep.cancer.gov/reporting/ctc.html).  
7.4.1 Re-Treatment Criteria  
  
A new cycle of treatment may begin when the following are met:  
• 
All non-hematologic toxicity improved to Grade ≤2 (or to baseline)  
• 
ANC ≥ 1,000/mm3 and platelets ≥ 75,000/mm3  
  
For any event, which is apparent at baseline, the dose delays will apply according to the 
corresponding shift in toxicity grade, if the investigator feels it is appropriate. (e.g. if a patient has 
grade 1 asthenia at baseline which increases to grade 2 during treatment, this will be considered as 
a shift of 1 grade and treated as a grade 1 toxicity for dose delay purposes).  
  
A cycle may be delayed as noted below. A total of 4 cycles of treatment should be administered 
whenever possible.   
  
For delayed or missed doses, if the time between 2 sequential infusions is less than 6 weeks, the 
420 mg IV dose of pertuzumab should be administered. Do not wait until the next planned dose. 

  
  
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If the time between 2 sequential infusions is 6 weeks or more, the initial dose of 840 mg 
pertuzumab should be re-administered as a 60 minute IV infusion followed every 3 weeks 
thereafter by a dose of 420 mg IV administered over 30-60minutes.  
  
Pertuzumab should be withheld or discontinued if trastuzumab treatment is withheld or 
discontinued.   
For delayed or missed doses, if the time between 2 sequential infusions is less than or equal to 4 
weeks, the 6 mg/kg maintenance dose of trastuzumab will be administered over about 30 minutes.  
If the time between 2 sequential infusions is 4 weeks or more, the 8 mg/kg loading dose of 
trastuzumab will be administered over 90 minutes, followed every 3 weeks thereafter by a dose of 
6mg/kg administered over 30 minutes.  
7.4.2 Toxicity Management  
NOTE:  Dose delays and recovery windows refer to date of planned dose, not since last dose. For 
example:  if cycle 2 was to start on January 1st, but is delayed due to grade 3 ANC; you may hold 
treatment up to additional 3 weeks from the originally planned cycle 2 day 1.  
  
Figure 1: Actions to be Taken in Case of Pertuzumab and Trastuzumab Hematologic Toxicity  
  
Hematologic toxicity 
related to study treatment  Action  
ANC  
Grade 1 or 2:  Continue with study treatment  
  
Grade 3 or 4:  Hold study treatment  
  
Toxicity resolves to Grade ≤2 within 3 weeks, resume study treatment  
  
Toxicity does NOT resolve to Grade ≤2 within 3 weeks, discontinue 
treatment, or discuss with Protocol Chair  
Platelets  
Grade 1:  Continue with study treatment  
  
Grade 2:  Consider holding treatment per grade 3 or 4 guidelines, or 
continue at treating physician discretion  
  
Grade 3 or 4:  Hold study treatment  
  
Toxicity resolves to Grade ≤2 within 3 weeks, resume study treatment  
  
Toxicity does NOT resolve to Grade ≤2 within 3 weeks, discontinue 
treatment, or discuss with Protocol Chair  

  
  
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Hemoglobin  
Grade 1 or 2:  Continue with study treatment  
  
Grade 3 or 4:  Hold treatment until resolution per below or consider 
transfusion  
  
Toxicity resolves to Grade ≤2 within 3 weeks, resume study treatment  
  
Toxicity does NOT resolve to Grade ≤2 within 3 weeks, discontinue 
treatment, or discuss with Protocol Chair.  
  
Figure 2: Actions to be Taken in Case of Pertuzumab and Trastuzumab Non-Hematologic Toxicity  
  
Non-hematologic toxicity related to study 
treatment, excluding cardiac toxicity  
Action  
1. Grade 1 or 2  
Continue with study treatment  
2. Grade 3 or 4  
Hold study treatment  
 
  
Toxicity resolves to Grade ≤2 within 3 weeks, resume 
study treatment  
  
Toxicity does NOT resolve to Grade ≤2 within 3 weeks, 
discontinue treatment, or discuss with Protocol Chair  
3.  Recurrence of Grade 3 or 4 toxicity 
upon re-challenge  
Discontinue treatment  
  
Figure 3: Actions to be Taken in Case of Pertuzumab and Trastuzumab Cardiac Toxicity  
  
Cardiac toxicity related to study treatment  Action  
1. Asymptomatic drop in LVEF or 
symptomatic congestive heart failure  
Hold study treatment.  
Monitor and continue per Figure 4  
2. Other cardiac toxicities not covered by 
this figure.  
Follow rules 1 and 2 above for non-hematologic toxicity 
7.4.3 Important Toxicities  
7.4.3.1 Infusion-Associated Reactions (IAR)  
Like other monoclonal antibodies, pertuzumab has been associated with infusion associated 
reaction (such as chills, diarrhea, fatigue, headache, nausea, and pyrexia), and with hypersensitivity 
reactions. Close observation of the patient during and for 60 minutes after the first infusion and 
during and for 30 minutes following subsequent infusions is recommended following the 
administration of pertuzumab. If a significant IAR occurs, the infusion should be slowed down or 
interrupted and appropriate medical therapies should be administered. Patients should be evaluated 
and carefully monitored until complete resolution of signs and symptoms. Permanent 
discontinuation should be considered in patients with severe infusion reactions. This clinical 

  
  
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assessment should be based on the severity of the preceding reaction and response to administered 
treatment for the adverse reaction.  
7.4.3.2 Serious Infusion-Associated Events  
Serious adverse reactions to trastuzumab infusion including dyspnea, hypotension, wheezing, 
bronchospasm, tachycardia, reduced oxygen saturation, and respiratory distress have been reported 
infrequently. In rare cases (4 per 10,000), these events were associated with a clinical course 
culminating in a fatal outcome. Serious reactions have been treated with supportive therapy such 
as oxygen, beta-agonists, corticosteroids and withdrawal of trastuzumab as indicated.  
7.4.3.3 Respiratory Symptoms  
A low rate of respiratory events that are compatible with an IAR or hypersensitivity 
reaction/anaphylaxis has been reported. Although pertuzumab targets the HER2 receptor it inhibits 
heterodimerization with other members of the HER family (eg, EGFR [HER1]). Accordingly, it 
may cause toxicities associated with the use of EGFR inhibitors, such as interstitial lung disease 
(ILD). The few reports of ILD occurring in pertuzumab-treated patients received so far also had 
evidence of alternative causes, e.g., concomitant medication, preceding/concurrent neutropenia 
with potential infection or relevant medical history.  
7.4.3.4 Left Ventricular Dysfunction  
Decreases in LVEF have been reported with drugs that block HER2 activity. Trastuzumab and 
pertuzumab both target HER2, thus there is a risk of cardiac dysfunction with these agents. In the 
CLEOPATRA pivotal trial, pertuzumab in combination with trastuzumab and docetaxel was not 
associated with increases in the incidence of symptomatic LVSD or decreases in LVEF compared 
with placebo in combination with trastuzumab and docetaxel.2  Pertuzumab combined with 
trastuzumab and chemotherapy did not result in any significantly greater incidence of symptomatic 
LVSD or decreases in LVEF than trastuzumab and chemotherapy in patients with early breast 
cancer.3 However, in the pivotal MBC trial a greater proportion of patients who developed 
symptomatic LVSD had received prior anthracyclines and/or radiotherapy compared to the 
proportion of patients receiving prior anthracyclines and/or radiotherapy in the overall 
pertuzumab-treated population.2 Therefore, patients who have received prior anthracyclines or 
prior radiotherapy to the chest area may be at higher risk of decreased LVEF.    
  
Pertuzumab has not been studied in patients with: a pretreatment LVEF value of ≤ 50%; a prior 
history of CHF; decreases in LVEF to <50% during prior trastuzumab adjuvant therapy; conditions 
that could impair left ventricular function such as uncontrolled hypertension, recent myocardial 
infarction, serious cardiac arrhythmia requiring treatment or a cumulative prior anthracycline 
exposure to > 360mg/m2 of doxorubicin or its equivalent.  
7.4.3.5 Management of Cardiac Safety  
All patients must have a baseline evaluation of cardiac function including a measurement of LVEF 
by either MUGA or ECHO prior to entry into the study. Only patients with an LVEF ≥ 50% will 
be entered into this study.    
  

  
  
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It is recommended that patients should have cardiac monitoring at regular intervals (e.g., every 
three months) during treatment with pertuzumab and trastuzumab. As part of this study, cardiac 
monitoring will occur at baseline and at conclusion of study treatment (i.e., after about 3 months); 
or additionally if required to monitor toxicity.  ECHO or MUGA scans should be scheduled at the 
same radiology facility where the patient’s baseline ECHO or MUGA was conducted. When a 
cardiac event occurs, the Cardiac Report Form must be submitted within 14 days of learning of the 
event.    
  
During the course of trastuzumab and pertuzumab therapy, patients should be monitored for signs 
and symptoms of CHF (i.e., dyspnea, tachycardia, new unexplained cough, neck vein distention, 
cardiomegaly, hepatomegaly, paroxysmal nocturnal dyspnea, orthopnea, peripheral edema, and 
rapid unexplained weight gain). The confirmation of the CHF diagnosis should include the same 
method used to measure LVEF at baseline (either ECHO or MUGA).      
  
Pertuzumab and trastuzumab should be discontinued in any patient who develops clinical signs 
and symptoms suggesting CHF. CHF should be treated and monitored according to standard 
medical practice.  At present, there are inadequate data available to assess the prognostic 
significance of asymptomatic drops in LVEF.  
  
Figure 4: Algorithm for Continuation/Discontinuation of Study Therapy in Asymptomatic Patients 
Based on LVEF Assessment  
  
  
Note:  The LVEF ≥50% and <50% refers to the current LVEF.  For example:  if the current LVEF is 
<50%, but did not drop at least 10 points from baseline, continue treatment and repeat LVEF in 3 
weeks.  If repeat LVEF still has not dropped at least 10 points, continue treatment and repeat LVEF at 
next scheduled time point.  If LVEF has dropped ≥10 points on repeat, continue treatment and repeat 

  
  
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LVEF in another 3 weeks.  If LVEF at that time is confirmed as having dropped ≥10 points AND LVEF 
is <50%, stop treatment.    
  
The Protocol Chair must be notified if an assessment of LVEF is performed prior to the planned 
assessment after 12 weeks of therapy so guidance can be given if necessary.  
7.4.3.6 EGFR-Associated Toxicities  
Although pertuzumab targets the HER2 receptor, it inhibits heterodimerization with other members 
of the HER family (e.g., EGFR [HER1]). Accordingly, it may cause toxicities associated with the 
use of EGFR inhibitors such as diarrhea, rash and other dermatologic toxicities (eg, dry skin, 
pruritus, nail disorders, mucositis).  
7.4.3.7 Diarrhea  
In the 7-week IV and 26-week toxicity studies in cynomolgus monkeys, there was a 
treatmentrelated increase in the incidence of diarrhea. Diarrhea has been observed in 
approximately 60% of patients (treatment-related diarrhea in 50% of patients) being treated with 
pertuzumab in phase 2 single-agent studies, and up to approximately 70% of patients in 
combination therapy studies. Diarrhea was CTCAE Grade 1 or 2 in the majority of cases. To 
prevent dehydration, early treatment of diarrhea with anti-diarrheal medication should be 
considered and patients treated with fluids and electrolyte replacement, as clinically indicated.   
7.4.3.8 Rash  
Rash has also been observed with EGFR inhibitors, mostly of mild to moderate intensity. Rash has 
been observed in approximately 17% of patients receiving pertuzumab in Phase 2 single-agent 
studies and up to 73% of patients in combination studies. The rash was generally of CTCAE Grade 
1 or 2 in severity. Treatment recommendations for EGFR-associated rash include topical or oral 
antibiotics, topical pimecrolimus, topical or (for severe reactions) systemic steroids. These agents 
may be used in patients experiencing pertuzumab-related rash, as clinically indicated, although 
they have not been studied in this context.  
7.4.4 Additional Preoperative Treatment  
Standard institutional guidelines should be used for dose modifications for additional standard 
treatment received prior to surgery.  It should be noted that for patients in whom taxane is added 
to pertuzumab/trastuzumab, a clinical ECHO or MUGA should be performed every 6 weeks while 
receiving that combination as was done in other preoperative studies using the combination.  
7.4.5 Special Considerations  
• 
For any grade 4 toxicity that recurs despite prophylaxis or dose reduction, study 
treatment should be discontinued.  
• 
Any consideration to modification of the above dose delay guidelines should be 
discussed with the Protocol Chair for approval in advance.  

  
  
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7.5  
Concomitant and Supportive Therapy  
7.5.1 General  
In general, concomitant medications and therapies deemed necessary for the supportive care and 
safety of the subject are allowed, provided their use is documented in the medical records. The 
administration of any other therapies intended to treat the primary condition including 
chemotherapy and biologic agents is NOT permitted. Similarly, the use of other concurrent 
investigational drugs is not allowed.  
7.5.2 Other  
No other targeted therapy, chemotherapy, anti-estrogen therapy, radiation therapy, or 
investigational systemic therapy is allowed concomitant with study treatment. Subjects may 
continue on bisphosphonates. Initiation of bisphosphonate therapy while on study treatment must 
be authorized by the Protocol Chair.   
7.6  
Surgical Evaluations of Participants  
7.6.1 Marker Placement  
A titanium marker or “clip” should be placed at the tumor site(s) prior to starting treatment in 
patients who may be a candidates for breast conservation surgery. This will identify the tumor 
location(s) for the surgical procedure in the event that there is a complete response to preoperative 
treatment.  
7.6.2 Evaluation of Axillary Lymph Nodes  
7.6.2.1 Pre-Treatment  
The standard of care for the institution should be followed (e.g., pre-neoadjuvant  therapy sentinel 
lymph node mapping/lymph node biopsy for clinically node negative patients is generally 
recommended at Johns Hopkins and should be performed on all patients; such evaluations are not 
required for subjects at other sites).  
  
• 
Clinical N0 Disease  
For patients with a clinical N0 disease, it is recommended that sentinel node mapping be 
done at baseline, but this is optional. Patients with negative lymph nodes should not require 
further evaluation at the time of definitive surgery. However, patients with a positive fine 
needle aspiration or a sentinel node should undergo an axillary node dissection at the time 
of definitive surgical procedure.  
  
At the time of sentinel lymph node mapping, the surgeon may obtain a biopsy of the 
sentinel node which will be snap frozen and be evaluated immediately by the pathologist 
for presence or absence of micrometastases. If the frozen biopsy specimen does not show 
micrometastases, the node will be removed and no further evaluation will be required 
unless the permanent section reveals micrometastases. If the frozen biopsy specimen 
reveals micrometastases, the surgeon will either 1) perform an axillary node dissection, or 

  
  
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2) will mark the node and no further evaluation will be done until the definitive surgical 
procedure when a lymph node dissection will be done.  
• 
Clinical N1, N2 or N3 Disease  
For patients with a clinical N1, N2, or N3 disease, it is recommended that a fine needle 
aspiration (FNA) or a core biopsy is obtained prior to starting therapy to document the 
status of the node. Following the biopsy, decisions regarding sentinel node mapping for 
patients with N1 disease will be made at the discretion of the treating surgeon.   
7.6.2.2 Post-Treatment  
Patients with a positive sentinel lymph node or a positive sentinel lymph node biopsy prior to 
starting treatment, patients who underwent an unsuccessful sentinel node mapping, or those who 
did not undergo sentinel lymph node mapping at baseline should generally undergo axillary lymph 
node dissection at the time of the definitive surgical procedure.  
  
Patients who had a positive FNA prior to treatment may undergo a sentinel node mapping at the 
time of breast surgery. Patients whose sentinel node is negative may or may not undergo node 
dissection at discretion of the surgeon. However, it is highly recommended that such patients will 
undergo node dissection. Patients whose sentinel node is positive should be recommended axillary 
lymph node dissection. If a patient with an FNA-positive node at baseline does not go axillary 
node dissection documentations should be made regarding the reason.   
7.6.3 Breast Surgery  
Following study therapy, patients will undergo breast conserving surgery or a mastectomy at the 
discretion of the treating surgeon. It is recommended that the definitive surgery take place 2-4 
weeks after the last dose of study medication.  
7.7  
Additional Treatment  
7.7.1 Chemotherapy, Pertuzumab and Trastuzumab  
Additional standard preoperative treatment with pertuzumab, trastuzumab, and taxane per standard 
of care may be considered in cases of disease progression or incomplete clinical response to study 
treatment.    
   
Postoperative chemotherapy and trastuzumab per standard of care (NCCN guidelines) is 
recommended for those who are medically fit for same and may be administered at the discretion 
of the treating team. It is preferred that an anthracycline-taxane based adjuvant chemotherapy be 
administered such as AC (adriamycin/cyclophosphamide) followed by paclitaxel plus trastuzumab 
per institutional preferences and per physician discretion; if not administered after the study 
treatment and prior to definitive surgery. An alternative for those with pre-existing cardiac risk 
factors is the docetaxel, carboplatin, trastuzumab regimen per the BCIRG006 study.13 Trastuzumab 
should be administered to complete one year of therapy per standard practice.    
7.7.2 Radiation Therapy  

  
  
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Radiation therapy will be administered at the conclusion of all protocol treatment, per institutional 
standards.   
7.7.3 Hormonal Therapy   
Adjuvant hormonal therapy will be administered to patients with ER 1-9% after completion of 
adjuvant chemotherapy and local therapy, per standard of care and physician discretion.   
7.8  
Evaluation of Long Term Outcomes  
The subject’s medical record will be reviewed approximately every 6 months from the start of 
therapy for information regarding recurrence of the breast cancer (local or systemic), development 
of a new cancer, or death. (Note: Only those subjects who initiate protocol treatment will be 
followed.)   
  
In the event that a subject does not continue her post-study care at the institution, every attempt 
will be made to collect this information either by direct contact or through communication with 
her outside physician(s).  
7.9  
Discontinuation and Withdrawal of Subjects  
All patients who initiate protocol treatment will be included in the overall evaluation of response 
(intent-to-treat analysis). All reasons for discontinuation of therapy should be documented clearly 
in the medical record.   
  
If a subject discontinues or withdraws from the study, every attempt will be made to get a 
tissue/tumor biopsy and study bloods if the subject is able and willing to do so.  
7.9.1 Discontinuation of Treatment  
The reasons for discontinuation or protocol treatment include:  
  
• 
Evidence of disease progression during study therapy at the discretion of the treating 
investigator and after discussion with the Protocol Chair (see Section 7.3.6).  
• 
Non-compliance with the study protocol; including, but not limited to not attending the 
majority of scheduled visits. The Protocol Chair will determine when non-compliance 
should lead to removal from study.   
• 
Unacceptable major toxicity.   
• 
Intercurrent illness or condition that would, in the judgment of the treating investigator, 
affect assessment of clinical status to a significant degree or require discontinuation of 
study treatment.  
• 
At subject’s own request. Note: The reason for discontinuation from the study must be 
documented. The patients will be included in the overall evaluation of response (intent-
totreat analysis) if any protocol therapy was administered prior to withdrawal.  
• 
Study is closed for any reason (e.g. new information shows that the patient’s welfare would 
be at risk if she continued study treatment).  

  
  
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7.9.2 Withdrawal from Study  
The reasons for withdrawal from the study include:  
  
• 
Subject withdraws consent for follow-up.  
• 
Subject is lost to follow-up.  
• 
Study is terminated for any reason.  
7.10 Additional Information  
Subjects may be given parking vouchers (if applicable) to cover parking costs during the study, 
depending on the preferences of each participating institution. No other subject remuneration is 
planned.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
  
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8. Study Calendar  
Procedure  
Baseline1  Cycle 1 
Day 1  
Cycle 1  
Day 15  
(-0/+3 days) 
Every 3 
weeks&  
(Cycles 2-4)  
Post-  
Study Tx2  
Post-  
Surgery  
CLINICAL ASSESSMENTS/TESTS:  
  
  
  
  
  
  
Physical Exam  
X  
  
  
X  
X  
X  
Clinical Tumor Measurements3  
X  
  
  
X  
X  
  
ECOG PS, Vital Signs (Height, baseline)4  
X  
  
  
X  
  
  
CBC with diff, platelets  
X#  
X#  
  
X  
X  
  
Chemistries5  
X#  
X#  
  
X  
X  
  
Electrocardiogram (ECG)6  
X  
  
  
  
  
  
MUGA/Echocardiogram  
X  
  
  
  
X  
  
Pregnancy Test (Blood or Urine)7  
X*  
X*  
  
  
  
  
Axillary Nodal Evaluation8  
X  
  
  
  
  
  
Breast Imaging9  
X  
  
  
  
X  
  
TREATMENT:  
  
  
  
  
  
  
Trastuzumab10  
  
X  
  
X  
  
  
Pertuzumab10  
  
X  
  
X  
  
  
Additional preoperative treatment11  
  
  
  
  
X  
  
CORRELATIVE BLOOD/TISSUE STUDIES:12  
 
 
 
 
Pharmacogenomics (whole blood)  
X  
  
  
  
  
  
Serum, plasma  
X  
  
X  
  
X  
X  
PET/CT Scan  
X  
  
X  
  
  
  
Image guided breast tumor biopsy  
X  
  
X  
  
X  
  
Tumor sample- diagnostic/archival  
X  
  
  
  
  
  
Tumor sample- from surgery  
  
  
  
  
X  
  
OTHER ASSESSMENTS:  
  
  
  
  
  
  
Concomitant Medication Review  
X  
  
  
X  
X  
  
Symptom/Toxicity Assessment13  
X  
  
  
X  
X  
X  
Definitive Surgery14  
  
  
  
  
X  
  
Follow-up/Survival (q6 months)15  
  
  
  
  
  
X  
 #   Required ≤ 14 days prior to first treatment (i.e., day 1); baseline labs may be used if within window. *  
Required ≤ 7 days prior to first treatment (i.e., day 1) unless otherwise noted. &   All assessments for cycles 
2-4 required ≤ 3 days prior to each specified treatment; the same window applies to the start of each cycle 
due to scheduling delays outside of those related to adverse events (exceptions may be made to extend this 
window, with approval by the Protocol Chair/designee).  

  
  
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Note: The schedule should be followed as closely as is realistically possible; however, the schedule may be 
modified due to problems such as scheduling delays or conflicts (e.g., clinic closure, poor weather 
conditions, vacations, etc.) with the guidance of the Protocol Chair/designee, as appropriate, and will not 
be reportable as a deviation unless the endpoints of the study are affected.   
8.0 Study Calendar (continued)  
  
1. Baseline assessments are required within 28 days of first treatment with exception of axillary nodal 
evaluation and breast imaging, if performed, where no window applies, and unless otherwise noted.  
2. Post-study treatment assessments may be done as soon as 2 weeks, and preferably within 4 weeks, after the 
last dose of study treatment (pertuzumab/pertuzumab); these must take place prior to additional standard 
preoperative treatment (if applicable) or definitive surgery.  
3. Any patient with clinically evaluable disease should have tumor measurements completed at baseline, prior 
to each cycle of treatment, and post-study treatment (including after additional standard preoperative 
treatment, if applicable).    
4. Height at baseline only.  
5. Chemistries to include measurement of sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose, 
total bilirubin, calcium, total protein, albumin, AST, ALT, and alkaline phosphatase.  
6. ECG may be repeated at the discretion of the treating team at any time during or following treatment.  
7. Pregnancy test (serum or urine) is required for women of childbearing potential; ≤ 7 days before initiating 
study therapy and must be negative to commence the study.  
8. Axillary nodal evaluation is recommended prior to initiation of preoperative treatment and can be performed 
as per standard practice at each institution (no baseline window applies to this assessment).  
9. Standard of care imaging may be performed at baseline and also to confirm response prior to additional 
preoperative treatment, and may include mammogram, ultrasound, magnetic resonance imaging (MRI) 
and/or other, at the discretion of the treating team.  
10. Treatment will consist of Pertuzumab and Trastuzumab (both administered every 3 weeks, IV).  
11. For cases of progression or residual clinical disease additional standard preoperative treatment should 
comprise of a standard preoperative regimen of chemotherapy and HER2-directed therapy per NCCN 
guidelines In these cases, all items under the “Post-Study Tx” must be done prior to start of additional 
treatment, including biopsy.    
12. Correlative blood/tissue studies:  
• 
Blood for serum, plasma, and pharmacogenomics (baseline only) samples will be collected at baseline, 
on day 15 (window days 15-18), post-completion of study treatment (and prior to surgery/additional 
standard preoperative treatment), and about 1-4 weeks following the surgical procedure prior to 
initiation of any postoperative treatment.  In patients who receive additional preoperative treatment, a 
sample is also requested at the completion of the additional treatment and prior to the surgical procedure.  
• 
PET scan should be performed prior to tumor biopsy at baseline and on day 15 (window days 15-18); 
additional window (>28 days) may be permitted for baseline PET with advance approval from Protocol 
Chair.  
• 
Tumor biopsy at baseline and on day 15 (window days 15-18).  In patients who receive additional 
preoperative treatment, a biopsy is also required at the completion of study treatment and prior to the 
initiation of additional treatment.  Note:  A separate tumor biopsy is not needed on the day of surgery; 
tissue will be taken from the surgical specimen.  Tissue from the original diagnostic and surgical 
procedures will also be collected for study tests.   
• 
All correlative blood/tissue and PET studies should be performed on the same day.  
• 
The diagnostic/archival tumor samples should only be submitted upon request from coordinating center.  

  
  
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13. All subjects must have a final toxicity assessment at least 30 days following the last dose of study drug. In 
subjects who initiate additional preoperative treatment and/or have surgery during the 30 day time period, 
only toxicities believe to be related to the study treatment/interventions will be captured.  
14. Definitive surgery consists of lumpectomy or mastectomy, with or without nodal evaluation (i.e., sentinel 
node and/or axillary node dissection) and will occur after completion of study treatment with or without 
additional preoperative treatment.   
15. Subjects will be followed approximately every 6 months after completion of study treatment for disease 
status and survival.  
 
9. Pharmaceutical Information  
9.1  
Pertuzumab (PERJETA®)  
9.1.1 Classification  
Pertuzumab is a recombinant, humanized monoclonal antibody based on the human IgG1 (κ) 
framework sequences and consists of two heavy chains (449 residues) and two light chains (214 
residues). Like trastuzumab, pertuzumab is directed against the extracellular domain of HER2. 
However, it differs from trastuzumab in the epitope-binding regions of the light chain (12 amino 
acid differences) and heavy chain (29 amino acid differences). As a result, pertuzumab binds to an 
epitope within what is known as subdomain 2 of HER2 while the epitope for trastuzumab is 
localized to subdomain 4.  
9.1.2 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).   
9.1.3 Preparation and Administration  
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.   
(Note: Dextrose (5%) solution should not be used to dilute 
pertuzumab.)  Mix diluted solution by gentle inversion. Do not shake.  
Administer immediately once prepared.  
9.1.4 Storage and Stability  
Upon receipt, pertuzumab vials are to be refrigerated at 2°C–8°C (36°F–46°F) until use. 
Pertuzumab vials may 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 

  
  
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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).  
9.1.5 Supply  
Pertuzumab will be supplied by Genentech/F Hoffman La Roche Inc., free of charge to subjects 
for the duration of participation in the study regimen.  Sites will order supplies of pertuzumab 
directly from Genentech or a central distribution group, as directed by the Coordinating Center, 
during the study.  NOTE:  Pertuzumab supply will be commercial in participants who receive 
additional standard preoperative treatment.  
9.1.6 Adverse Effects  
The package insert should be consulted for the most current and complete information.  
9.2  
Trastuzumab (HERCEPTIN®)  
9.2.1 Classification  
Trastuzumab is a recombinant humanized monoclonal antibody, produced by Chinese hamster 
ovary cells in culture.    
9.2.2 Formulation  
Trastuzumab is a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous 
(IV) administration.  Each vial of trastuzumab contains 400 mg of tratuzumab, 9.9 mg of L-
histidine HCl, 6.4 mg of L-histadine, 400 mg of , -trehalose dihydrate, and 1.8 mg of polysorbate 
20, USP.  
9.2.3 Preparation and Administration  
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..  
9.2.4 Storage and Stability  
Vials of trastuzumab are stable at 2 C–8 C (36 F–46 F) prior to reconstitution.  Do not use 
beyond the expiration date stamped on the vial.    
9.2.5 Supply  
Trastuzumab will be obtained from commercial supply and the patient or patient’s insurance 
company will be responsible for the costs.   
9.2.6 Adverse effects  
Please see the trastuzumab Package Insert for more details on the known precautions, warnings, 
and adverse reactions of trastuzumab.  
9.2.7 Other  

  
  
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The package insert should be consulted for the most current and complete information.  
9.3  
Docetaxel (Taxotere®)  
Additional preoperative treatment with pertuzumab, trastuzumab, and docetaxel (or other taxane 
per institutional standard after discussion with Study Chair) will follow current package and 
institutional guidelines.  The treatment and dose modifications guidelines will not be mandated by 
this protocol.   
9.4  
Drug Accountability  
The research pharmacy will keep records for medication receipts, dispensation, and destruction as 
per standard practice at each institution for supplied agents.  
   
10. Measurement of Effect  
10.1 Pathologic Response Determination  
We will evaluate pathological response in the breast in the axilla.   
10.1.1 Endpoint Definitions for Pathologic Response  
• 
Pathologic Complete Response (pCR): No viable invasive cancer in pathologic 
specimen (breast and axillary nodes), as determined by histological examination. We 
will report whether in situ disease only was present in the specimen versus no disease. 
We will also report for each woman whether she had a pCR in the breast only, in the 
lymph nodes only, or in both. We will also report the size of residual disease within the 
breast.  
• 
Nodal Disease: We will report the number of subjects who had negative lymph node 
prior to starting treatment, or at the time of surgery: 0, 1-3, 4-10, or >10 lymph nodes.  
10.2 Clinical Response Determination  
We will evaluate clinical response in the breast, and when appropriate, in the axilla, infraclavicular 
and supraclavicular regions, and skin. At the time of study enrollment, patients will have a breast 
examination. Physical examination findings will be documented including clinical tumor size, 
character, mobility, and location of the breast mass. In addition, T4 features must be described. 
The patient will be examined by one of the treating investigators or his/her designee at each 
specified time point and after the final dose of treatment.  Whenever possible, the same method 
(e.g., calipers or ruler) and investigator should be used to confirm consistency in the measurements.  
10.2.1 Endpoint Definitions for Clinical Response (UICC criteria)  
• 
Complete response (cCR) in the breast on physical exam will be defined as the absence 
of any palpable abnormality: i.e., no skin or breast thickening, mass, or associated skin 
or nipple changes. CR will be recorded separately for the breast and axilla.  
• 
Partial response (cPR) in the breast will be defined as a 50% or greater decrease in the 
product of biperpendicular diameters as measured with a ruler, compared with the 
prechemotherapy measurement.  

  
  
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• 
Stable disease (cSD) in the breast will be defined as palpable disease which does not 
fit the definition of PR or PD.  
• 
Progressive disease (cPD) will be defined as an increase in the product of 
biperpendicular diameters of 25% or greater compared to the original measurement.  
• 
Inevaluable disease (cID) is defined as breast cancer that is not palpable in two 
dimensions.  
11. Correlative Studies  
11.1 Tumor Tissue Samples  
In addition to the fresh tumor tissue samples below, sites may be asked to provide samples of 
routinely collected and processed tissues (e.g., formalin-fixed paraffin-embedded tissues) – 
including those from the original diagnostic and surgical procedures for the study tests.  The 
samples will be shipped to Johns Hopkins for study as below, when requested.  In the event of 
paraffin-embedded tissues where the blocks will not be released, 10-15, 5-10 micron, slides should 
be submitted for each applicable sample.  
11.1.1 Collection  
Up to 6 core-biopsies will be taken at each designated time point. The biopsies will be obtained at 
the surgical clinic, mammography suite, or the Oncology Center, by a skilled practitioner. These 
research biopsies may be obtained with mammography or ultrasound guidance as necessary. These 
core biopsies will be obtained in addition to diagnostic core needle biopsy material; however, 
whenever possible, and after a consent form has been signed, attempts will be made to coordinate 
diagnostic and study biopsies. Alternatively, patients who undergo sentinel node mapping or a 
lymph node biopsy may have study biopsy at the same time.   
  
Biopsies will be obtained at the following time points:  
  
• 
Prior to starting treatment:  Attempts will be made to coordinate the baseline biopsy with 
the diagnostic biopsy or with the sentinel node mapping or lymph node biopsy. (Note: A 
titanium marker or “clip” may be placed at the discretion of the investigator performing the 
biopsy.)  
• 
Day 15 (-0/+3 days):  All day 15 blood/tissue and PET studies should be performed on the 
same day, with the PET scan taking place before the biopsy.  
• 
Post-study treatment/Prior to additional preoperative treatment (14-28 days after last dose 
of study medication):  In patients who receive additional preoperative treatment, a biopsy 
after completion of study medications and prior to initiation of additional treatment is 
required; samples at the time of surgery are still requested if tumor is present.  The biopsy 
should be reviewed and reported to confirm presence of residual disease prior to initiation 
of treatment at the treating institution.  
• 
Definitive surgery (i.e., mastectomy or lumpectomy):  A separate tumor biopsy is not 
needed on the day of surgery; tissue will be taken from the surgical specimen.    

  
  
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Note:  In any instance where a subject refuses a study biopsy after initiation of study treatment, it 
will not be considered a protocol deviation.  
11.1.1.1 Specimen Handling, Transportation, Storage, and Processing  
The study staff will be notified when a biopsy is taking place. The following procedures 
documented on the study tissues worksheet will be followed:  
  
Up to three core-biopsy specimens will be rapidly frozen for future RNA extraction. Up to 3 core 
biopsy specimens will be suspended in 10% buffered formalin and after approximately 24 hours 
of suspension in formalin, the cores will be embedded in paraffin; a single slide should be cut and 
H&E stained for use by the study pathologist to evaluate presence of tumor.  The block and slide 
will be shipped to Johns Hopkins for the appropriate studies listed below.  Note:  Cores to be placed 
in formalin specifically for the study at the time of definitive surgery are not required; a block or 
sections from the routine pathologic formalin-fixed paraffin-embedded (FFPE) specimens will be 
requested at the time the case if finalized, if available.  
11.1.2 Methods  
11.1.2.1 Immunohistochemistry   
Serial biopsies will be stained using commercially available monoclonal antibodies including 
cleaved caspase 3 antibody and ki67. Other proteins related to the apoptotic process such as 
activated caspase 3, PARP, may be determined based on promising literature. Quantification will 
be performed by the study pathologist.  
11.1.2.2 PIK3CA mutation status, PTEN expression, HER1-4 expression/activation  
The study pathologist will retrieve specimens containing sufficient tissue for analysis; 
formalinfixed paraffin embedded (FFPE) and frozen tissues, if available, may be used.  The 
specimens will then be processed in pathology and serial sections prepared and delivered to the 
Park Laboratory for PIK3CA analysis including hot spot mutation analysis and PTEN expression. 
DNA will be extracted from tissues and used for sequencing genes known to be altered in the 
PI3Kinase pathway including, but not limited to HER1, 2, 3 and 4, PIK3CA, PTEN, PIK3R and 
AKT1.  If frozen tissue is available, additional proteomics analyses will be performed including, 
but not limited to, reverse phase protein array (RPPA) to examine the amount and phosphorylation 
status of key proteins in the HER2/PI3Kinase pathways including HER12,3 and 4.  FFPE samples 
will also be used for in situ hybridization analyses of genes known to be amplified in the PI3Kinase 
pathway as well as immunohistochemical staining for genes that are altered in breast cancers that 
could also influence response to therapies (e.g. TP53).  
11.1.2.3 RNA Extraction for Gene Expression Analysis  
Tissues will be homogenized in Trizol (Gibco BRL) using a polytron homogenizer (Brinkman 
Instruments) and total RNA isolated according to the standard Trizol protocol. The total RNA 
obtained will be further subjected to an additional round of phenol chloroform extraction, 
precipitated and resuspended in RNAse free water. We will determine RNA concentrations by 
comparing the optical density ratios (OD260/OD280) obtained spectrophotometrically. Since 

  
  
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standard gel electrophoresis to assess RNA quality requires almost the entire RNA sample, we will 
use an Agilent 2100 analyzer and RNA 6000 LabChip kits (RNA microelectroseparation and 
analysis; Agilent Technologies, New Castle, DE).   
  
RNA will be preserved for future studies of gene expression arrays. Gene expression analysis may 
be conducted for sets of genes currently suspected to be involved in the response of cells to 
pertuzumab and trastuzumab using quantitative reverse transcription PCR (RT-PCR).  
  
The overall goal of these studies is to identify candidate markers for response and molecular 
profiles that might be relevant to an understanding of drug mechanisms. Although statistical 
methods will provide some indication of significance of particular molecular changes, validation 
will ultimately require additional testing in independent samples.   
11.1.2.4 RNA Extraction for Immune Signature Endpoint  
Tissue punches will be obtained from demarcated areas of invasive tumor from FFPE tissue using 
a 1 mm Biopsy Punch with Plunger (Fisher Scientific). Total RNA will be extracted from at least 
1 mm tissue punch. In brief, a 1 mm tissue punch will be deparaffinized in Citrisolv ; Fisher 
Scientific) at room temperature for 30 minutes. The Citrisolv will be aspirated and the tissue was 
washed with 100% ethanol, vortexed, and centrifuged twice. Ethanol will be removed and the 
tissue dried at 37oC for 10 minutes. The sample will then be incubated in Proteinase K Digestion 
(PKD) buffer and proteinase K (1 μg/μl) for overnight (at least 8 hours) at 56oC. The digested 
tissue was incubated for 15 minutes at 80oC and centrifuged (14000 rpm) for 2 min at room 
temperature. The supernatant will be collected and the RNA extraction, including DNase I 
treatment, will be completed using the RNeasy FFPE kit on an automated QIAcube platform 
according to the manufacturer’s instructions (QIAGEN, Valencia, CA). The concentration of the 
purified RNA will be determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop 
Technologies; Wilmington, DE). Purified total RNA was stored at -80oC. Samples will be analyzed 
using a Nanostring custom codeset that includes immune function genes associated with RFS in 
N9831.59  
11.2 Blood Samples  
11.2.1 Collection  
Blood samples will be drawn as per the time points identified on study calendar and include:  
  
• 
Prior to starting treatment.  
• 
Day 15 (-0/+3 days):  All day 15 blood/tissue and PET studies should be performed on 
the same day, whenever possible.  
• 
Post-study treatment/Prior to additional preoperative treatment (14-28 days after last dose 
of study medication):  In the event that a subject receives additional preoperative 
treatment, every attempt should be made to get a blood sample after completion of the 
study treatment and prior to additional treatment, as well as after additional treatment and 
prior to surgery.  

  
  
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• 
Definitive surgery (i.e., mastectomy or lumpectomy):  This sample should be collected 
after completion of all treatment and prior to surgery (collection on the day of surgery 
preferred, if feasible).    
• 
About 1-4 weeks after the definitive surgery:  This blood sample should be drawn prior to 
initiation of any planned postoperative treatment (e.g., additional chemotherapy, radiation 
therapy).  
11.2.2 Specimen Handling, Transportation, Storage, and Processing  
The following samples will be collected and studied as outlined in the methods:  
  
• 
Serum samples  
About 20-30 milliliters of blood will be collected at each time point.   
  
• 
Plasma samples  
About 30 milliliters of blood will be collected at each time point.   
  
• 
Pharmacogenomic samples  
About 10 milliliters (mL) of blood will be collected at baseline only.   
  
The samples will be processed and stored at -70°C or below until transfer for analysis; some 
samples may be batched/shipped ambient for processing by Johns Hopkins as outlined in the 
separate study bloods worksheet.     
11.2.3 Methods  
11.2.3.1 BEAMing and digital PCR for ptDNA  
There is a great need to devise improved methods for screening patients for PIK3CA mutations and 
following responses to therapies. Based upon preliminary data, Dr. Ben Park and his research team 
has already demonstrated that BEAMing (Beads, Emulsification, Amplification and Magnetics) 
can reliably and accurately detect PIK3CA mutations from cell-free, plasma-derived tumor DNA 
(ptDNA) in metastatic breast cancer patients.53 Similar to studies by others, they have also shown 
that PIK3CA mutational status can change or convert with recurrent metastatic disease. Pre-
surgical plasma will be obtained from patients to assess both the presence and amount of mutant 
PIK3CA ptDNA by BEAMing. In addition, blood samples post-surgery and during followup will 
also be studied from patients identified with PIK3CA mutations. ptDNA will be isolated from these 
samples and also screened for the presence and amount of PIK3CA mutations by BEAMing. 
Mutant PIK3CA levels before and after surgery will be compared in order to assess whether 
changes in disease burden correlate with changes in ptDNA. Preliminary sample processing will 
be performed in Dr. Park’s laboratory as per Inostics Standard Operating Procedures. These 
samples will then be batch-shipped to Inostics laboratory, Germany where ctDNA and BEAMing 
techniques will be performed on each sample per Inostics standard operating procedures. Every 
sample will be tested for the two PIK3CA hotspot mutations on Exon 9 (E542K, E545K) and a 
further hotspot mutation on Exon 20 (H1047R).  

  
  
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In addition to PIK3CA mutational status, newer next generation sequencing technologies will 
enable the research team to discover somatic mutations (e.g.TP53 mutations) and genomic 
rearrangements which are unique for the patient’s cancer cells relative to their non-cancerous cells. 
Although such cancer specific alterations may or may not play a role in drug sensitivity, they do 
serve as cancer specific “biomarkers” for each individual. As this technology matures, we will be 
able to identify and measure these additional genomic alterations for every patient, not just those 
with PIK3CA mutations, thus broadening the scope and applicability of using ptDNA as 
biomarkers for disease burden and response to therapies.  We therefore propose the following: 
before and after therapy, a tube of blood is drawn from the patient and genomic DNA from plasma 
is extracted and then banked. A portion of the patient’s tumor is obtained to extract genomic DNA. 
The genomic DNA is then subjected to next gen sequencing to identify at least 10 to 20 genomic 
rearrangements/mutations that are present only in cancer cells. Having several unique 
rearrangements ensures that we can follow the tumor over time as genetic instability could, in 
theory, lead to the loss of one or more of these personalized markers. Once markers have been 
identified, we will then query the pre- and post-therapy blood specimens for these markers and use 
BEAMing/digital PCR to quantitate the amount of ptDNA at these two time points.  
11.2.3.2 Methylation  
We expect that over 70% of patients will have circulating DNA at baseline. DNA methylation 
studies will be performed at the Breast Cancer Research Laboratories at Johns Hopkins under the 
direction of Dr. Sara Sukumar. QM-MSP studies will be done on DNA extracted from serum or 
plasma obtained from each subject. Primers and probes for Q-MSP analysis have been designed to 
specifically amplify hypermethylated promoter sequences of Cyclin D2, RAR-beta, APC-1, Twist, 
RASSF1A, HIN-1, and ER-alpha. PCR will be performed in separate wells for each primer/probe 
set. Fluorogenic PCR amplification will be carried out using a 7900 Sequence detector (Perkin-
Elmer Applied Biosystems) as previously described.  
11.2.3.3 Pharmacogenomics  
Genomic DNA isolated from whole blood at baseline will be used to genotype candidate genes that 
may affect pharmacodynamic effects of response and toxicity. We will store the sample obtained at 
baseline for potential future analyses in the event that further information becomes available from the 
literature about promising candidate genes that may predict response to anti-HER2 therapy, and 
patients will be appropriately consented for same.  
11.3 Positron Emission Tomography (PET) Imaging  
11.3.1 Collection  
FDG PET/CT will be performed at baseline and on day 15 (window days 15-18); it is preferred 
that imaging be performed prior to biopsy.   
  
Sites are asked to administer oral contrast per local standards, if applicable; unless otherwise 
contraindicated in the subject. The preferred oral contrast is READI-CAT 2 (barium sulphate 
suspension, 2.1% w/v); though site specific preferences should prevail with the caveat that the 

  
  
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contrast to be used does not contain any additives (e.g., flavoring) or glucose. The dose of oral 
contrast to be administered should be administered with enough water to provide for a total liquid 
volume of 500 milliliters at each administration – half 15 minutes prior to injection and the 
remaining half 15 minutes prior to scanning.    
  
The FDG dose will be calculated based on the weight (kg) of the patient as follows: Dose (mCi) = 
Weight (kg) * 0.22(mCi/kg), where the maximum dose is 25mCi with weights greater than 114kg 
or 250lbs. (NOTE:  A range of FDG dosing may be used of 0.1-0.25 mCi/kg after approval from 
the nuclear medicine collaborators at Johns Hopkins.)  
  
A full description of timing of FDG administration and scan image acquisition, copies of which 
will be provided electronically (e.g., CD) to Johns Hopkins for central analysis, is provided in 
Appendix D.  Note:  As stated in the manual, the guidelines are recommendations only; sites should 
not contravene from site-specific procedures and should contact the Protocol Chair/her designee 
with any questions or concerns.  
11.3.2 Methods  
A PET scan produces a three-dimensional image of the sugar's distribution throughout the body. 
Since malignant cells use more sugar than normal cells, the radioactive sugar tends to find its way 
to and concentrate in cancerous tissue. Tumor standardized uptake values (SUV), defined as the 
ration of tumor FDG concentration (mCi/kg) to whole body concentration (injected dose divided 
by weight of the patient in kg) will be used to assess treatment response.   
  
The presence and location of the primary tumor and any metastases will be qualitatively assessed. 
There will be no qualitative scoring in this study. The normal liver SUV corrected for lean body 
mass (SUL) will be measured as outlined in the PERCIST criteria.36 For the purposes of the 
primary endpoint, maximal SUV (SUVmax) will be correlated with pCR, however, we may also 
explore other criteria per PERCIST in an exploratory fashion.  
  
Prior to imaging of the first study participant, all sites are required to comply with the Quality 
Assurance Manual process outlined in Appendix C.  This will allow the best and most comparable 
images for central analysis.  
11.4 Leftover Samples  
Any leftover study blood and tissue samples will be stored in the Breast Cancer Laboratory at Johns 
Hopkins for future research studies. These samples may be released for use in future studies after 
approval by the Protocol Chair and other regulatory bodies, as appropriate.   
  
In addition, the study PI and collaborators have approval by the TBCRC to use all research 
biospecimens collected during the conduct of this trial to address the research questions described 
in the protocol document.  All future use of residual or repository specimens collected in this trial 
for purposes not prospectively defined will require review and approval by the TBCRC according 
to its established policies, whether the specimens are stored in a central site or at a local institution 
in a virtual repository.  

  
  
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Subjects will be asked to consent to the future use of samples in the consent document.  
11.5 Additional Information  
The study coordinator will keep a log (separate logs will be kept for the blood and tissue samples, 
and radiology/imaging exams) that includes the study number and identifying participant 
information. The technicians will keep a log with the collection conditions, processing and storage 
information.  
  
The laboratory and radiology investigators will be blinded to the subject identifiers and clinical 
data while generating the research data; additionally, the reported results will not disclose any 
unique patient identifiers.  
  
A separate study tissues and study bloods worksheet will be provided to all participating sites 
outlining specific procedures for collection and processing of samples.  
12. Adverse Events  
12.1 General  
This study will use the descriptions and grading scales found in the revised National Cancer 
Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0 for adverse 
event reporting that can be found at http://ctep.cancer.gov/reporting/ctc.html.  
  
Information about all adverse events, including those volunteered by the subject, discovered by 
investigator/study personnel questioning, or detected through physical examination, laboratory test 
or other means, will be collected, followed, and reported appropriately.  Each reported AE or SAE 
will be described by its duration (i.e., start and end dates), regulatory seriousness criteria if 
applicable, suspected relationship to the study drug(s)/intervention(s), and actions taken.   
  
All adverse events experienced by subjects will be collected from the time of first dose of study 
medication, throughout the study and until the final assessment as outlined in the Study Calendar 
(Section 8). Subjects continuing to experience toxicity after discontinuation of the study drug may 
be contacted for additional assessments until the toxicity has resolved or is deemed irreversible. 
Any adverse event experienced during additional preoperative treatment or after the surgical 
procedure that the investigator feels is related to study treatment will be captured.  
12.2 Definitions  
12.2.1 Adverse event (AE)  
An AE is any unfavorable and unintended sign, symptom, or disease temporally associated with 
the use of an investigational medicinal product (IMP) or other protocol-imposed intervention, 
regardless of attribution.  
  
This includes the following:  

  
  
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• 
AEs not previously observed in the subject that emerge during the protocol-specified AE 
reporting period, including signs or symptoms associated with that were not present prior 
to the AE reporting period.  
• 
Complications that occur as a result of protocol-mandated interventions (e.g., invasive 
procedures such as cardiac catheterizations).  
  
Medical conditions/diseases present before starting study treatment are only considered adverse 
events if they worsen after starting study treatment (any procedures specified in the protocol).   
  
Abnormal laboratory values or test results constitute adverse events only if they induce clinical 
signs or symptoms or require therapy.  
12.2.2 Serious adverse event or reaction  
An AE should be classified as an SAE if the following criteria are met:  
• 
It results in death (i.e., the AE actually causes or leads to death).  
• 
It is life threatening (i.e., the AE, in the view of the investigator, places the subject at 
immediate risk of death. It does not include an AE that, had it occurred in a more severe 
form, might have caused death.).  
• 
It requires or prolongs inpatient hospitalization.  
• 
It results in persistent or significant disability/incapacity (i.e., the AE results in substantial 
disruption of the subject’s ability to conduct normal life functions).  
• 
It results in a congenital anomaly/birth defect in a neonate/infant born to a mother exposed 
to the IMP.  
• 
It is considered a significant medical event by the investigator based on medical judgment 
(e.g., may jeopardize the subject or may require medical/surgical intervention to prevent 
one of the outcomes listed above).  
  
Note: Any occurrence of pregnancy must also be reported in the same time frame as a serious 
adverse event.   
12.2.3 Other information:  
• 
Unexpected adverse event: Unexpected adverse events are those not listed in the Package 
Insert (P.I.) or current Investigator Brochure (I.B.) or not identified. This includes adverse 
events for which the specificity or severity is not consistent with the description in the P.I. 
or I.B. For example, under this definition, hepatic necrosis would be unexpected if the P.I. 
or I.B. only referred to elevated hepatic enzymes or hepatitis.    
  
In addition per IRB regulations of the Coordinating Center, any adverse event that is not 
included in the informed consent, but is believed to be considered related to the study 
intervention, is also considered “unexpected”.  
  
• 
Expected (known) adverse event: An adverse event, which has been listed or characterized 
in the I.B. or P.I.    

  
  
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In addition per IRB regulations of the Coordinating Center, an adverse event is considered 
“expected” only if it is included in the informed consent document as a risk.  
12.3 Relationship  
To ensure consistency of AE and SAE causality assessments, the causality/relationship of all 
events to study medication(s) will be assessed by an investigator and assigned as follows:  
12.3.1 “Yes” to the question of causality:  
There is a plausible temporal relationship between the onset of the AE and administration of the 
study drug(s) and the AE cannot be readily explained by the subject’s clinical state, intercurrent 
illness, or concomitant therapies; and/or the AE follows a known pattern of response to the study 
drug(s); and/or the AE abates or resolves upon discontinuation of the study drug(s) or dose 
reduction and, if applicable, reappears upon re-challenge.  
  
The causality in this case may also be classified as follows:  
  
• 
Definitely: An adverse event which has a timely relationship to the administration of the 
investigational drug/agent, follows a known pattern of response, for which no alternative 
cause is present.   
• 
Probably: An adverse event, which has a timely relationship to the administration of the 
investigational drug/agent, follows a known pattern of response, but for which a potential 
alternative cause may be present.   
• 
Possibly: An adverse event, which has a timely relationship to the administration of the 
investigational drug/agent, follows no known pattern of response, but a potential alternative 
cause does not exist.  
12.3.2 “No” to the question of causality:  
Evidence exists that the AE has an etiology other than the study drug(s) (e.g., preexisting medical 
condition, underlying disease, intercurrent illness, or concomitant medication); and/or the AE has 
no plausible temporal relationship to study drug(s) administration (e.g., cancer diagnosed 2 days 
after first dose of study drug).  
  
The causality in this case may also be classified as follows:  
  
• 
Unlikely: An adverse event which does not have a timely relationship to the administration 
of the investigational drug/agent, follows no known pattern of response, does not reappear 
or worsen after re-administration of the investigational drug/agent (if applicable), and for 
which there is evidence that it is related to a cause other than the investigational drug/agent.  
• 
Unrelated: An adverse event, for which there is evidence that it is definitely related to a 
cause other than the investigational drug/agent. In general, there is no timely relationship 
to the administration of the investigational drug/agent, or if there is a timely relationship, 
the event does not follow a known pattern of response, and there is an alternative cause.   

  
  
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12.4 Specific Instructions for Recording Adverse Events  
Investigators should use correct medical terminology/concepts when reporting AEs or SAEs, while 
avoiding colloquialisms and abbreviations.  
12.4.1 Diagnosis vs. Signs and Symptoms  
If known at the time of reporting, a diagnosis should be reported rather than individual signs and 
symptoms (e.g., record only liver failure or hepatitis rather than jaundice, asterixis, and elevated 
transaminases).  However, if a constellation of signs and/or symptoms cannot be medically 
characterized as a single diagnosis or syndrome at the time of reporting, it is ok to report the 
information that is currently available.  If a diagnosis is subsequently established, it should be 
reported as follow-up information.  
12.4.2 Deaths  
All deaths that occur during the protocol-specified AE reporting period, regardless of attribution, 
will be reported to the appropriate parties.  When recording a death, the event or condition that 
caused or contributed to the fatal outcome should be reported as the single medical concept.  If the 
cause of death is unknown and cannot be ascertained at the time of reporting, report “Unexplained 
Death.”  
12.4.3 Preexisting Medical Conditions  
A preexisting medical condition is one that is present at the start of the study. Such conditions 
should be reported as medical and surgical history. A preexisting medical condition should be 
reassessed throughout the trial and reported as an AE or SAE only if the frequency, severity, or 
character of the condition worsens during the study.  When reporting such events, it is important 
to convey the concept that the preexisting condition has changed by including applicable 
descriptors (e.g., “more frequent headaches”) or including the new grade of the event with a 
comment.  
12.4.4 Hospitalizations for Medical or Surgical Procedures  
Any AE that results in hospitalization or prolonged hospitalization should be documented and 
reported as an SAE.  If a subject is hospitalized to undergo a medical or surgical procedure as a 
result of an AE, the event responsible for the procedure, not the procedure itself, should be reported 
as the SAE.  For example, if a subject is hospitalized to undergo coronary bypass surgery, record 
the heart condition that necessitated the bypass as the SAE.  
  
Hospitalizations for the following reasons do not require reporting:  
• 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for preexisting conditions;  
• 
Hospitalization or prolonged hospitalization required to allow efficacy measurement for 
the study; or,   
• 
Hospitalization or prolonged hospitalization for scheduled therapy of the target disease of 
the study.  
12.4.5 Post-Study Adverse Events  

  
  
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The investigator should expeditiously report any SAE occurring after a subject has completed or 
discontinued study participation if attributed to prior study drug exposure.  If the investigator 
should become aware of the development of cancer or a congenital anomaly in a subsequently 
conceived offspring of a female subject who participated in the study, this should be reported as 
an SAE.  
12.5 Reporting Procedures  
12.5.1 General  
All adverse events will be captured on the appropriate study-specific case report forms (CRFs), 
sponsor-provided, or in a designated database.   
  
All serious adverse events, regardless of causality to study drug, will be reported to the 
Coordinating Center.  
12.5.2 Adverse Events of Special Interest  
AEs of Special Interest are defined as a potential safety problem, identified as a result of safety 
monitoring of the Product.    
  
The following adverse events are of special interest for pertuzumab and should be reported in an 
expedited manner:  
  
• 
Left Ventricular Dysfunction  Infusion-associated reactions  
• 
Hypersensitivity reactions/anaphylaxis  
• 
Embryo-fetal toxicity or birth defects  
• 
Pregnancy:  If a female subject becomes pregnant while receiving investigational therapy 
or within 6 months after the last dose of study drug, a report should be completed and 
expeditiously submitted to the Coordinating Center and to Genentech, Inc.  Follow-up to 
obtain the outcome of the pregnancy should also occur. Abortion, whether accidental, 
therapeutic, or spontaneous, should always be classified as serious, and expeditiously 
reported as an SAE.  Similarly, any congenital anomaly/birth defect in a child born to a 
female subject should be reported as an SAE.  
  
All serious adverse events must be reported to the Coordinating Center within 1 business day after 
the investigator becomes aware of the event. Events should be reported using a MedWatch form 
(3500) as available on the FDA website (see link below).   
  
Follow-up information must also be reported within 1 business day of receipt of the information 
by the investigator.  
  
The Coordinating Center will disseminate information regarding serious adverse events to the 
participating sites within 5 days of review of the information by the Protocol Chair (or her designee 
in the event of extended absence) only in the case that the event(s) is believed to be related (i.e., 

  
  
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possibly, probably, or definitely) related to the study medication. The Coordinating Center will be 
responsible for reporting of events to the FDA and supporters, as appropriate (outlined below).  
12.5.3 Institutional Review Board  
All adverse events and serious adverse events will be reported to the IRB per current institutional 
standards. If an adverse event requires modification of the informed consent, these modifications 
will be provided to the IRB with the report of the adverse event. If an adverse event requires 
modification to the study protocol, these modifications will be provided to the IRB as soon as is 
possible.  
12.5.4 Genentech  
12.5.4.1 Reconciliation  
Genentech and the Sponsor will agree to the reconciliation periodicity and format, but agree at 
minimum to exchange monthly line listings of cases received by the other party. If discrepancies 
are identified, the Sponsor and Genentech will cooperate in resolving the discrepancies. The 
responsible individuals for each party shall handle the matter on a case-by-case basis until 
satisfactory resolution.  
12.5.4.2 SAE Reporting  
The Coordinating Center will report all SAEs to Genentech within the timelines described below. 
The completed Medwatch/case report should be faxed immediately upon completion to Genentech 
Drug Safety at:  
  
(650) 225-4682   OR   (650) 225-5288  
  
• 
Relevant follow-up information should be submitted to Genentech Drug Safety as soon as 
it becomes available.  
• 
Serious AE reports that are related to pertuzumab and AEs of Special Interest (regardless 
of causality) will be transmitted to Genentech within fifteen (15) calendar days of the 
Awareness Date.  
• 
Serious AE reports that are unrelated to pertuzumab will be transmitted to Genentech 
within thirty (30) calendar days of the Awareness Date.  
• 
Additional Reporting Requirements to Genentech include the following:  
• 
Any reports of pregnancy following the start of administration with pertuzumab will be 
transmitted to Genentech within thirty (30) calendar days of the Awareness Date.   
• 
All Non-serious Adverse Events originating from the Study will be forwarded in a quarterly 
report to Genentech.  
• 
The Genentech-provided “SAFETY REPORTING FAX COVER SHEET” for 
Genentechsupported research should be used for all correspondence.  
  
Initial report:  The report should include the following information within the Event 
Description (section 5) of the MedWatch 3500A form:  
• 
Protocol description (and number, if assigned)  

  
  
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• 
Description of event, severity, treatment, and outcome if known  
• 
Supportive laboratory results and diagnostics  
• 
Investigator’s assessment of the relationship of the adverse event to each investigational 
product and suspect medication  
  
Follow-up report:  Additional information may be added to a previously submitted report by 
any of the following methods:  
• 
Adding to the original MedWatch 3500A report and submitting it as follow-up  
• 
Adding supplemental summary information and submitting it as follow-up with the original 
MedWatch 3500A form   
• 
Summarizing new information and faxing it with a cover letter including patient identifiers 
(i.e. D.O.B. initial, patient number), protocol description and number, if assigned, brief 
adverse event description, and notation that additional or follow-up information is being 
submitted (The patient identifiers are important so that the new information is added to the 
correct initial report)  
  
Occasionally Genentech may contact the reporter for additional information, clarification, or 
current status of the patient for whom and adverse event was reported. For questions regarding 
SAE reporting, you may contact the Genentech Drug Safety representative noted above or the MSL 
assigned to the study. Relevant follow-up information should be submitted to Genentech Drug 
Safety as soon as it becomes available and/or upon request.  
  
MedWatch 3500A (Mandatory Reporting) form is available at:  
http://www.fda.gov/AboutFDA/ReportsManualsForms/Forms/default.htm  
  
For questions related to safety reporting, please contact Genentech Drug Safety at (888) 835-2555 
(phone) or fax (see above).    
12.5.5 Food and Drug Administration (FDA)  
The Coordinating Center will be responsible for reporting to the FDA as follows:  
  
• 
For Investigator-Sponsored IND Studies, some additional reporting requirements for the 
FDA apply in accordance with the guidance set forth in 21 CFR § 600.80.  
• 
Events meeting the following criteria need to be submitted to the Food and Drug 
Administration (FDA) as expedited IND Safety Reports according to the following 
guidance and timelines:  
  
7 Calendar Day Telephone/Fax Report:  The Investigator is required to notify the FDA of 
any fatal or life-threatening adverse event that is unexpected and assessed by the investigator 
to be possibly related to the use of trastuzumab and pertuzumab. An unexpected adverse event 
is one that is not already described in the trastuzumab and pertuzumab Investigator Brochure. 
Such reports are to be telephoned or faxed to the FDA and Genentech within 7 calendar days 
of first learning of the event.  

  
  
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15 Calendar Day Written Report:  The Investigator is also required to notify the FDA and 
all participating investigators, in a written IND Safety Report, of any serious, unexpected AE 
that is considered reasonably or possibly related to the use trastuzumab and pertuzumab. An 
unexpected adverse event is one that is not already described in the trastuzumab and 
pertuzumab investigator brochure.  
  
Written IND Safety reports should include an Analysis of Similar Events in accordance with 
regulation 21 CFR § 312.32. All safety reports previously filed by the investigator with the IND 
concerning similar events should be analyzed and the significance of the new report in light of the 
previous, similar reports commented on.  
  
Written IND safety reports with Analysis of Similar Events are to be submitted to the FDA, 
Genentech, and all participating investigators within 15 calendar days of first learning of the event. 
The FDA prefers these reports on a Medwatch 3500 form, but alternative formats are acceptable 
(e.g., summary letter).  
  
NOTE:  Any study report(s) submitted to the FDA should be copied to Genentech.  This includes 
all IND annual reports and the Clinical Study Report (final study report).  Additionally, any 
literature articles that are a result of the study should be sent to Genentech. All FDA/IND 
correspondence should be faxed to Genentech Drug Safety at (650) 225-4682 or (650) 225-5288; 
whereas copies of literature articles should be mailed to the assigned Clinical Operations contact 
for the study.  
13. Data and Safety Monitoring  
13.1 Data Management  
All information will be collected on study-specific case report forms by the study staff at each 
institution. The necessary forms will be provided to each site by the Coordinating Center.   
  
The completed forms will be forwarded to the Coordinating Center for central review and inclusion 
in the study dataset with relevant source documentation as outlined in the case report forms. The 
data submission schedule is as follows:  
  
At the time of registration:   
  
- 
Registration Form  
- 
Informed Consent Form (signed by the subject)  
- 
Eligibility Checklist  
- 
Source documents related to eligibility   
  
Within 2 weeks after registration:  
  
- 
Baseline study case report forms  

  
  
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- 
Pertinent source documents  
  
Within 2 weeks after each cycle is completed:  
  
- 
Per-cycle case report forms  
- 
Pertinent source documents  
  
Every 6 months during follow-up:  
  
- 
Follow-up case report forms  
- 
Pertinent source documents (if necessary)  
  
All study data will be reviewed for completeness and accuracy by the Protocol Chair. The Principal 
Investigator (or his/her designee) at each respective institution is responsible for review, and 
ensuring the completeness and accuracy, of the data generated by his/her institution. The study 
data will also be periodically reviewed by the Sidney Kimmel Comprehensive Cancer Center 
Clinical Research Office.  
13.2 Meetings  
The Coordinating Center will schedule teleconferences to take place as needed depending on the 
rate of accrual, and will include the Protocol Chair and Principal Investigators from each site. The 
following study team members involved with the conduct of the trial will be included as 
appropriate: study coordinators, data managers, research nurses, sub-investigators, collaborators 
(if applicable), and statistician.  
  
During these meetings matters related to the following will be discussed: enrollment rate relative 
to expectation, characteristics of participants, retention of participants, adherence to protocol 
(potential or real protocol violations), validity and integrity of the data, safety data, analysis of 
samples by Drs. Park, Perez, Thompson and Sukumar’s labs, and progress of data for objectives.  
  
At these meetings, the data pertaining to clinical response and toxicity will also be reviewed, 
dependent upon rate of accrual, as outlined in Section 16.4.  
  
A summary of the items discussed at each teleconference will be prepared by the Coordinating 
Center and forwarded to each participating site. In addition, study investigators meet 2-3 times 
each year in regularly scheduled TBCRC meetings.  
13.3 Monitoring   
As an IND is required for this study, it will be conducted in accordance with the data and safety 
monitoring program established by the Clinical Research Office of the Sidney Kimmel 
Comprehensive Cancer Center at Johns Hopkins (SKCCC CRO).  As required, a monitoring plan 
will be established for the study with monitoring to be conducted by the SKCCC CRO Quality 

  
  
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Assurance staff or other group, if agreed upon to be appropriate.  A medical expert committee or 
formal data safety monitoring board are not planned or required at this time.    
  
As above, all data will be sent to the Coordinating Center for central collation and review.  Subject 
cases from sites will be chosen for a thorough review; it is unclear how often this may occur; 
however, in the event that a case is chosen for central review by the Coordinating Center, each site 
will be asked to provide copies of all source documents and to ensure that all data is current.  There 
are no formal on-site evaluations planned by the Coordinating Center; however, these may occur 
depending on site accrual rate, identified problems or concerns, or other reasons, as appropriate.  
  
In addition, a Data Monitoring Committee (DMC) comprising of breast cancer experts and the 
study statistician will be constituted to review all cases where progressive disease is confirmed or 
suspected during the neoadjuvant setting.  Please see Section 16 for additional information 
regarding this review.  
  
  
  
  
14. Administrative Procedures  
14.1 Protocol Amendments  
Any changes to the protocol will be made in the form of an amendment and must be approved by 
the IRB before implementation. The Protocol Chair (or her designee) is responsible for the 
coordination and development of all protocol amendments, and will disseminate this information 
to the participating centers.  
14.2 Informed Consent  
An investigator will explain to each subject the nature of the study, its purpose, procedures 
involved, expected duration, potential risks and benefits. Each subject will be informed that 
participation in the study is voluntary and that she may withdraw from the study at any time, and 
that withdrawal of consent will not affect her subsequent medical treatment. This informed consent 
will be given by means of a standard written statement and will be submitted for IRB approval 
prior to use. No patient will enter the study before her informed consent has been obtained. In 
accordance with the Health Insurance Portability and Accountability Act (HIPAA), the written 
informed consent document (or a separate document to be given in conjunction with the consent 
document) will include a subject authorization to release medical information to the study sponsor 
and supporting agencies and/or allow these bodies, a regulatory authority, or Institutional Review 
Board access to subjects’ medical information that includes all hospital records relevant to the 
study, including subjects’ medical history.  
14.3 Ethics and Good Clinical Practice  
This study must be carried out in compliance with the protocol and Good Clinical Practice, as 
described in:  

  
  
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1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996.  
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 
and 56 concerning informed consent and IRB regulations).  
3. Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996).  
  
The investigator agrees to adhere to the instructions and procedures described in it and thereby to 
adhere to the principles of Good Clinical Practice that it conforms to.  
14.4 Regulatory Authorities  
14.4.1 Institutional Review Board  
Information regarding study conduct and progress will be reported to the Institutional Review 
Board (IRB) per the current institutional standards of each participating center.  
14.4.2 Food and Drug Administration (FDA)  
This trial falls under Investigational New Drug (IND) application #117913 and is held by the 
Protocol Chair.  All reporting required with regards to the clinical trial outlined will comply with 
FDA regulations and be handled by the Protocol Chair and Coordinating Center.   
15. Coordinating Center & Site Responsibilities  
15.1 Protocol Chair  
The Protocol Chair is responsible for performing the following tasks:  
  
• 
Coordinating, developing, submitting, and obtaining approval for the protocol as well as 
its subsequent amendments.  
• 
Assuring that all participating institutions are using the correct version of the protocol.  
• 
Taking responsibility for the overall conduct of the study at all participating institutions 
and for monitoring the progress of the study.  
• 
Reviewing and ensuring reporting of Serious Adverse Events (SAE).  
• 
Reviewing data from all sites.  
15.2 Coordinating Center  
The Coordinating Center is responsible for performing the following tasks:  
  
• 
Ensuring that IRB approval has been obtained at each participating site prior to the first 
patient registration at that site, and maintaining copies of IRB approvals from each site.  
• 
Managing central patient registration.  
• 
Collecting and compiling data from each site.  

  
  
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• 
Establishing procedures for documentation, reporting, and submitting of AE’s and SAE’s 
to the Protocol Chair, and all applicable parties.  
• 
Facilitating audits by securing selected source documents and research records from 
participating sites for audit, or by auditing at participating sites.  
15.3 Participating Sites  
Participating sites are responsible for performing the following tasks:  
  
• 
Following the protocol as written, and the guidelines of Good Clinical Practice (GCP).  
• 
Submitting data to the Coordinating Center.  
• 
Registering all patients with the Coordinating Center by submitting patient registration 
form, and signed informed consent promptly.  
• 
Providing sufficient experienced clinical and administrative staff and adequate facilities 
and equipment to conduct a collaborative trial according to the protocol.  
• 
Maintaining regulatory binders on site and providing copies of all required documents to 
the Coordinating Center.  
• 
Collecting and submitting data according to the schedule specified by the protocol.  
  
Additional information for participating sites:  
15.3.1 Staffing  
The participating sites will provide experienced staff, and adequate equipment and facilities to 
support this clinical trial. The participating sites will also be responsible for research staff training 
in computer applications, human subjects research, and HIPAA compliance, as well as the 
continuing education in these areas as required by local institutional standards.  
15.3.2 Documentation  
Each participating site is responsible for submitting copies of all relevant regulatory documentation 
to the Coordinating Center. The required documents include, but are not limited to the following: 
local IRB approvals (i.e., protocol, consent form, amendments, patient brochures and recruitment 
material, etc.), IRB membership rosters, summary of unanticipated problems or protocol 
deviations, and documentation of expertise of the investigators. The Coordinating Center will 
provide each participating site with a comprehensive list of the necessary documents. It is the 
responsibility of the participating sites to maintain copies of all documentation submitted to the 
Coordinating Center.  
  
The requirements for data management, submissions, and monitoring are outlined below.  
15.3.3 Confidentiality  
All unpublished information that the Coordinating Center gives to the investigator shall be kept 
confidential and shall not be published or disclosed to a third party without the prior written consent 
of the Protocol Chair (or her designee).  

  
  
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15.3.4  Record Retention  
Following closure of the study, each participating center will maintain a copy of all site study 
records in a safe and secure location. The Coordinating Center will inform the investigator at each 
site at such time that the records may be destroyed.  In general, the FDA guidelines will be followed 
and communicated to site by the Coordinating Center; guidance may be found here:   
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=312.62.  
15.3.5 Publication  
It is understood that any manuscript or releases resulting from the collaborative research will be 
circulated to all participating sites prior to submission for publication or presentation. The Protocol 
Chair will be the final arbiter of the manuscript content.  
15.3.6 Additional Information  
Each participating site is responsible for submitting additional information as requested by the 
Protocol Chair (or her designee). The Coordinating Center may terminate the study at a 
participating site in the event that these conditions are not followed.  
16. Statistical Considerations  
16.1 Overall  
This is a single arm phase 2 study in the neoadjuvant setting aimed to correlate early change (day 
15) in maximal standardized uptake values (SUVmax) on FDG-PET with pathological response to 
pertuzumab/trastuzumab (PT) in patients with primary operable HER2-positive and ERnegative 
breast cancer. We hypothesized that PT will have an impact on SUVmax and changes in SUVmax 
on FDG-PET as early as 15 days after treatment are associated with response to PT. Our ultimate 
goal would be to determine the predictive ability of early changes in SUVmax and identify an 
optimal cut point in SUVmax % reduction that accurately predicts pathological complete response 
(pCR) to PT such that this information can be used in future studies to guide therapy for a similar 
patient population.  
16.2 Study Design  
Eligible patients will receive 12 weeks of PT in the pre-operative setting. An FDG-PET scan will 
be performed at baseline and 15 days (window days 15-18) after commencement of therapy. Tumor 
biopsies will also be performed at baseline, after 2 weeks of therapy and at surgery to assess 
biomarkers with a particular focus on the PI3K pathway and novel predictive gene-expression 
profiles, and response or resistance to therapy. We choose to undertake this study in ER-negative 
disease alone in that 1) a considerable distinction in metabolic activity has been observed between 
the different subtypes of breast cancer, 2) the NeoALLTO PET study also indicated that there was 
not a strong association between change in SUV and response in the ER-positive group, and 3) the 
pCR rate for ER-positive group was low based on the data from the NeoALLTO PET substudy, 
which requires a much larger sample size to reach a reasonable number of responders. We 
anticipate that our findings will mirror the NeoALLTO PET substudy results and meanwhile we 
hope to identify a cut point in SUVmax decline through a Receiver operating characteristic (ROC) 
analysis that more accurately predicts pCR rather than using pre-specified thresholds as in previous 
studies.  

  
  
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16.3 Sample Size and Accrual  
Eligible patients will need to have both baseline and day 15 FDG-PET scan performed and 
SUVmax data collected as well as surgery specimen successfully evaluated to be considered 
evaluable for the primary analysis. A total of 80 evaluable patients will be accrued onto this study.   
  
It is expected that early change in SUVmax has good discriminating potential for predicting pCR 
to PT. We chose to power the study to detect a significant difference from a null value based on 
AUC, a measure used to quantify the overall predictive power of the biomarker. Specifically, we 
are interested in finding that percent SUVmax reduction predicts response to PT with an AUC 
significantly greater than 0.65, a predictive power that is deemed minimally acceptable under the 
scope of this study. For a test of the hypothesis H0: AUC ≤0.65 vs. H1: AUC > 0.65, a total of 80 
evaluable patients with a projected pCR rate of 25% (i.e., 20 responders and 60 non responders) 
will have 81% or greater power to detect a true AUC of 0.80 at one-sided type I error rate of 0.10. 
The power calculation is based on the asymptotic z-test and standard error of AUC estimated using 
the Hanley-McNeil method.76 Given 20 responders and 60 non-responders, precision of the 
sensitivity and specificity estimates will be no wider than ±20% and ±12%, respectively. The table 
below presents, for several observed levels of sensitivity or specificity, the 90% confidence 
intervals and their half widths, which were constructed using exact binomial procedures.  For 
observed sensitivity and specificity of 80%, the 90% confidence intervals will be 60% to 93% (± 
16%) and 70% to 88% (± 9%), respectively. We will be fairly confident that the decline in SUVmax 
has sensitivity of at least 70% if the observed estimate reaches 90%. Similarly, we will be fairly 
confident that the test has specificity of at least 80% if the observed value is 90%.  
  
Table: Precision (indicated by half width of exact 90% CI) for estimating sensitivity and specificity 
for a sample size of 80 patients and a pCR rate of 25% (i.e., 20 pathological responders and 60 
non-responders).  
  
Observed  
sensitivity  
Exact 90% CI  
Half width 
of 90% CI  
Observed  
specificity 
Exact 90% CI  Half width 
of 90% CI  
0.60  
0.39 – 0.78  
0.19  
0.60  
0.49 – 0.71  
0.11  
0.70  
0.49 – 0.86  
0.18  
0.70  
0.59 – 0.80  
0.10  
0.80  
0.60 – 0.93  
0.16  
0.80  
0.70 – 0.88  
0.09  
0.90  
0.72 – 0.98  
0.13  
0.90  
0.81 – 0.96  
0.07  
  
The anticipated accrual rate for this group of patients is approximately 3-5 per month and the 
accrual period is expected to be 24 months. We anticipate accruing additional 10% (i.e., 8 patients) 
to account for major protocol violation as well as potential missing data due to incomplete 
information on decline in SUVmax and pCR. Therefore, the maximum overall accrual of this study 
will be 88 patients.  
16.4 Interim Analysis and Early Stopping Guidelines  
There are no plans to stop the study early for futility with respect to the primary objective (i.e., 
association of change in SUVmax with pCR) considering the small number of pathological 

  
  
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responders (~20). Meanwhile, preliminary data of change in SUVmax with PT are not available 
and it would be difficult to presume a priori what % reduction in SUVmax would be a meaningful 
decline before we correlate it with treatment response. However, there is concern that patients may 
not derive sufficient benefit (i.e., the lesion shows clear signs of progression) from receiving 
antiHER2 agents (PT) without chemotherapy during the neoadjuvent setting, and thus we will carry 
out interim futility analyses based on proportion of clinical progression by cycle 1. Clinical 
response will be assessed at every cycle by clinical breast examination during neoadjuvant 
treatment (Cycles 1-4). Chemotherapy may be administered pre-operatively if evidence of disease 
progression during PT, at the discretion of the treating investigator and after discussion with the 
Protocol Chair (see Section 7.3.6). A Data Monitoring Committee (DMC) comprising of breast 
cancer experts and the study statistician will be constituted to review all cases where progressive 
disease is confirmed or suspected during the neoadjuvant setting, or incomplete response after 12 
weeks PT,. All available data pertaining to these scenarios will be transmitted to the DMC, to aid 
with assessment of disease progression. If it becomes evident that the proportion of progression 
convincingly exceeds 10%, the accrual will be suspended and the study may be terminated. A fully 
amended protocol will be submitted with treatment modifications (e.g., adding chemotherapy to 
PT for all patients).   
  
The following early stopping rules will serve as guidelines for the DMC to hold enrollment if the 
posterior probability of progression by cycle 1 during the neoadjuvant setting being greater than 
10% is 75% or higher. The prior for this monitoring rule is beta (0.5, 7.5), assuming the proportion 
of progression is 6.3%3  with 90% probability that this proportion is between 0.3% and 23%.  
  
Stop if # clinical 
progressions  
2  
3  
4  
5  
6  
7  
8  
9  
10  
11  
Out of N  
2-6  7-14  15-22 23-31 32-39 40-48 49-57 58-66  67-75 
76- 
80  
  
The following table shows the operating characteristics of the stopping rules based on 5000 
simulations.  
  
Underlying risk of clinical progression  
0.05  
0.10  
0.15  
0.20  
Probability of early stopping  
7.3%  
43.5%  83.0%  97.6%  
Average sample size  
75.2  
56.0  
33.6  
19.4  
  
While there is not a formal independent data safety monitoring board, toxicity will be monitored, 
and information will be reviewed with investigators and appropriate staff from all participating 
sites at routine study teleconferences (Section 13.2). If any safety concerns arise with the 
presentation of further results from the NeoSPHERE or CLEOPATRA trials we may amend the 
protocol to incorporate formal safety monitoring plans.  

  
  
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16.5 Definition of Variables  
Clinical and pathologic responses are dichotomous variables which were defined in section 10.1.1 
and 10.2.1. For each of the variables (e.g., apoptosis, cell proliferation, SUV metrics, gene 
expression levels, gene methylation), we will look at how each change from baseline to the first 
follow-up, and from baseline to surgery. Apoptosis and proliferation will be measured as the 
percentage (%) of cells that stain for the marker. Change will be defined as a percent change from 
baseline. For PET imaging, standardized uptake values (SUV), defined as the ratio of tumor FDG 
concentration (mCi/kg) to whole body concentration (injected dose divided by weight of the patient 
in kg) will be measured per PERCIST and corrected for lean body mass (SUL). The SUVmax, 
defined as the maximum single voxel SUV measured within a defined ROI or VOI, will be used 
for the primary analysis. For gene expression profiles, we will be using cDNA arrays and 
expression levels will be in the form of log ratios comparing samples to a reference sample (see 
below for description of measures for proteomic measures). For gene methylation, we will be using 
the QM-MSP to measure promoter hypermethylation of the genes and the relative amount of 
methylation will be calculated as percent methylation defined by Dr. Sukumar’s lab.   
16.6 Data Quality and Normalization  
Standard approaches will be used to evaluate the quality of the data. For cell proliferation and 
apoptosis as well as for SUV metrics, histograms and boxplots will be generated to look for 
skewness and outliers. Appropriate transformations will be taken to symmetrize the data if needed.  
16.7 Analysis Plan  
16.7.1 Primary Endpoint  
Change in SUVmax at day 15 will be calculated as a % reduction from baseline SUVmax. Results 
will be summarized with descriptive statistics (e.g., mean, standard deviation, median and range), 
classified by pathological responders and non-responders. Distributions of % reduction in 
SUVmax will be compared between the two groups using a t test or nonparametric Wilcoxon rank 
sum test where appropriate. The discriminative and predictive ability of early changes in SUVmax 
will be evaluated by constructing ROC curves. The ROC curve combines the information of the 
true positive rate and true negative rate. In order to identify the most useful criterion of early change 
in SUVmax, sensitivity, specificity, positive predictive value (PPV), negative predictive value 
(NPV), overall accuracy and area under the curve (AUC) along with 90% confidence intervals will 
be estimated for the entire range of threshold values in SUVmax % reduction. As our long term 
goal is to identify a group of individuals with HER2-positive early breast cancer who could 
potentially be spared the added toxicity of chemotherapy in addition to anti-HER2 agents and 
meanwhile to direct other patients towards an aggressive or investigational approach, we aim to 
identify a cut point that has the right balance in sensitivity (1 – false negative rate) and specificity 
(1 – false positive rate). PET scan will be considered a positive test if the resulting % reduction in 
SUVmax >= optimal cut point and a negative test if the resulting % reduction in SUVmax < 
optimal cut point. We are generally more concerned about getting a false positive, meaning a 
woman may end up omitting additional chemotherapy when she does need it. Without prior 
knowledge of predictive ability of change in SUVmax on PET for response to anti-HER2 therapy, 
however, explicit criteria for choosing an optimal cut point are difficult to construct a priori as 
‘optimality’ ultimately depends on practical constraints and clinically relevant trade-offs. We will 

  
  
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evaluate the ROC curves for sensitivity and specificity combinations that would be appropriate for 
clinical decision. As a general rule, an optimal cut point will be defined by maximizing the sum of 
sensitivity and specificity while giving more weight to specificity than sensitivity in the sense that 
we would like to have a lower false positive rate than a false negative rate. A final decision will be 
carefully discussed among the team constituted by the study PI, PET imaging experts and the study 
statistician. A bootstrap re-sampling approach will be used for obtaining a point estimate and a 
confidence interval for the cut point as well as an interval validation. Additionally, logistic 
regression will be used to determine if change of SUVmax predicts response independent of 
standard clinicopathologic factors such as age, tumor size, grade, and proliferation index. A 
potential of baseline SUVmax as a predictor of pCR will be assessed in a similar fashion. 
Imputation for missing data is not planned in this study due to unverifiable assumptions. Patients 
who progress prior to surgery may receive additional preoperative therapy. These patients along 
with death related to disease prior to surgery will be considered as pathological non-responders 
(failures on PT) in the primary analysis. A sensitivity analysis will be performed with the exclusion 
of these patients and results will be reported as well. We will explore SUV metrics using other 
criteria per PERCIST in a similar fashion.  
16.7.2 Secondary Endpoints  
PIK3CA mutation in peripheral tumor DNA obtained by blood sample will be evaluated both 
qualitatively (present/absent) and quantitatively (amount of the abnormal DNA in circulation). 
Results will be summarized using descriptive statistics including mean, standard deviation, 
median, range (for quantitative measure), frequencies and proportions (for qualitative measure).  
Measurements will be performed at baseline, D15, post treatment/pre surgery and post surgery. 
Paired comparisons between baseline and other time points will be made using paired t test for 
quantitative measures and McNemar's test for qualitative measures. The time course of quantitative 
markers will be investigated graphically depicting aggregate and subject-level trends over time. 
Patterns of change in the values over time and how the patterns differ by response status will be 
evaluated using linear mixed effects models. Mutation status and percentage change from baseline 
will be compared between pathological responders and non-responders using Fisher’s exact test 
and t-test and, respectively. We will further use  random-effects models that allows for estimation 
of the subject-specific effects on the repeated observations. Relevant covariates (e.g., standard 
clinicopathologic factors) will be included in the models to control for confounding effects. ROC 
analysis described as above will be performed as appropriate. Concordance of the mutation status 
between ptDNA and tumor specimens will be assessed by kappa statistic with a 95% CI or by Lin’s 
correlation coefficient for continuous data.  Somatic mutations and additional genomic alterations 
will be further sought using next generation sequencing data analysis. Pre and post changes will 
be analyzed in a similar fashion. Markers identified will be coupled with PIK3CA mutation data 
and correlated with pCR using logistic regression approach and ROC analysis.  
  
Key proteins involved in the PI3K pathway activation (e.g., PTEN low and/or PIK3CA mutation, 
HER 1-4 expression and/or phosphorylation) and other secondary outcomes (novel gene 
expression profiles, baseline and change in Ki67) will be correlated with pCR in a similar fashion 
as described above. The expression of the genes that we identified from the N9831 analysis will 
be measured using RNA from formalin-fixed, paraffin-embedded biopsy samples. A modified 

  
  
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surface mapping tool will be applied to assign tumors to immune enriched and not-enriched 
groups.59 We will use Fisher’s exact test to evaluate the association of this immune function 
signature with pCR and estimate the odds ratio along with the 95% confidence interval via logistic 
regression. pCR rate to preoperative PT + taxane will be estimated with the 95% confidence 
interval in the setting of histologically confirmed residual cancer after 12 weeks of preoperative 
PT. Long term outcomes (e.g., recurrence-free survival and overall survival) will be summarized 
using the Kaplan-Meier method. Results from the secondary analyses will be largely exploratory 
and will provide valuable preliminary data for future studies.   
16.7.3 Exploratory Endpoints  
Baseline biomarkers (e.g., KI67, serum methylation, gene expression profiles) will be summarized 
using descriptive statistics. Histograms and plots will be generated to assess the distribution of the 
data for each marker. Suitable transformations will be performed on skewed marker data as 
appropriate to achieve normality. The significance of each molecular marker as predictors of 
response to anti-HER2 therapy will be initially assessed using the t test or Wilcoxon rank sum test 
for continuous variables and Fisher’s exact test for categorical variables. Mantel-Haenszel 
chisquare test will be used for ordered levels of measurements. The significance of multiple 
molecular markers as predictors of response or resistance to anti-HER2 therapy will be evaluated 
using the multiple logistic regression approach. ROC analysis described as above will be 
performed as appropriate, and sensitivity, specificity, PPV, NPV, AUC and overall accuracy will 
be estimated as well. The above analyses of multiple molecular markers will be considered 
exploratory in this small Phase 2 trial and other statistical approaches may be applied during the 
course of the exploration. Furthermore, the multiple testing of individual markers will warrant 
caution in the interpretation of significance tests.  
  
Changes in biomarkers (e.g., PIK3CA, ER, Ki67, serum methylation markers, gene expression 
profiles) through serial samples collected at different time points will be summarized with 
descriptive statistics and described using graphical presentation (e.g., scatterplots, histograms, or 
boxplots). We will create plots that display changes in serial samples over time with tumor tissue 
and blood samples overlaid and blocks of treatment window (e.g., PT, neoadjuvant chemotherapy, 
and definitive surgery) indicated. Standard statistical tests will be used to explore whether or not 
the data shows evidence of changes in patients who are responsive or resistant to anti-HER2 
therapy. The analyses will be exploratory, aimed to provide preliminary data for subsequent 
studies.  
  
Additional exploratory analyses include correlating baseline and change (day 15) in SUV on  
FDG PET with pCR among patients with histologic confirmed residual disease after 12 weeks PT 
and subsequent addition of standard therapy per investigator discretion (Section 7.3.6), using 
Wilcoxon rank sum test and logistic regression model as well.   
16.7.4 Safety  
All subjects will be evaluable for toxicity from the time of their first dose of study medication. The 
assessment of safety will be based mainly on the frequency of adverse events. Adverse events will 
be summarized in each arm based on NCI CTCAE Version 4.0. Frequencies and percentages of 

  
  
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patients having any adverse event will be presented. Safety profiles will contribute to decisions 
whether to move the regimen forward in further studies.   
16.8 Reporting and Exclusions  
16.8.1 Enrollment  
A sample size of up to 88 subjects is planned (accounting for 8 inevaluable patients).  In the event 
that a subject does not initiate study treatment after registration, that subject will not count towards 
the accrual goal.   
16.8.2 Evaluation of Toxicity  
All subjects will be evaluable for toxicity from the time of their first dose of therapy.  
16.8.3 Evaluation of Response  
Analysis will be by intention to treat. All subjects included in the study must be assessed for 
response to treatment even if there are major protocol treatment deviations or if they terminate 
treatment early. All subjects who meet the eligibility criteria and receive a first dose of study 
medication will be included in the main analysis of the response rate.   
REFERENCES  
  
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24. 
Chang JCN, Mayer  IA. , Forero-Torres A, et al. TBCRC 006: A multicenter phase II study 
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31. 
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32. 
Kong X, Moran MS, Zhang N, Haffty B, Yang Q. Meta-analysis confirms achieving 
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33. 
Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core 
biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J 
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34. 
Ayers M, Symmans WF, Stec J, et al. Gene expression profiles predict complete pathologic 
response to neoadjuvant paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide 
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35. 
Connolly RM, Leal JP, Goetz MP, et al. Early  change in 18-fluorodeoxyglucose (FDG) 
uptake on positron emission tomography (PET) predicts response to preoperative systemic therapy 
(PST) in HER2-negative primary operable breast cancer: Translational Breast Cancer Research 
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meeting 2012. J Clin Oncol 30 (suppl; abstr 10509), 2012.  
36. 
Wahl RL, Jacene H, Kasamon Y, Lodge MA. From RECIST to PERCIST: Evolving 
Considerations for PET response criteria in solid tumors. J Nucl Med 2009;50 Suppl 1:122S-50S. 
37. Wahl RL, Zasadny K, Helvie M, Hutchins GD, Weber B, Cody R. Metabolic monitoring of 
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Clin Oncol 1993;11:2101-11.  

  
  
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38. 
Smith IC, Welch AE, Hutcheon AW, et al. Positron emission tomography using 
[(18)F]fluorodeoxy-D-glucose to predict the pathologic response of breast cancer to primary 
chemotherapy. J Clin Oncol 2000;18:1676-88.  
39. 
Schelling M, Avril N, Nahrig J, et al. Positron emission tomography using 
[(18)F]Fluorodeoxyglucose for monitoring primary chemotherapy in breast cancer. J Clin Oncol 
2000;18:1689-95.  
40. 
Jacobs MA, Ouwerkerk R, Wolff AC, et al. Monitoring of neoadjuvant chemotherapy using 
multiparametric, (2)(3)Na sodium MR, and multimodality (PET/CT/MRI) imaging in locally 
advanced breast cancer. Breast Cancer Res Treat 2011;128:119-26.  
41. 
Rousseau C, Devillers A, Campone M, et al. FDG PET evaluation of early axillary lymph 
node response to neoadjuvant chemotherapy in stage II and III breast cancer patients. Eur J Nucl 
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42. 
Young H, Baum R, Cremerius U, et al. Measurement of clinical and subclinical tumour 
response using [18F]-fluorodeoxyglucose and positron emission tomography: review and 1999 
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43. 
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neoadjuvant chemotherapy failure in breast cancer using FDG-PET after the first course. J Clin 
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44. 
Wang Y, Zhang C, Liu J, Huang G. Is 18F-FDG PET accurate to predict neoadjuvant 
therapy response in breast cancer? A meta-analysis. Breast Cancer Res Treat 2011.  
45. 
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response of axillary lymph node metastases to neoadjuvant chemotherapy in breast cancer patients. 
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46. 
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in patients (pts) undergoing preoperative chemotherapy (PCT) for breast cancer (BC). J Clin Oncol 
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47. 
Yap JT, Locascio T, Najita JS, et al. Metabolic Response By FDG-PET in Patients 
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48. 
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expression in breast cancers that are 1% to 10% ER-positive by immunohistochemistry. J Clin 
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49. 
Wang L, Zhang Q, Zhang J, et al. PI3K pathway activation results in low efficacy of both 
trastuzumab and lapatinib. BMC Cancer 11: 248, 2011.  
50. 
Nahta R, Hung MC, Esteva FJ. The HER-2-targeting antibodies trastuzumab and 
pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res 2004;64:2343-6. 
51. Berns K, Horlings HM, Hennessy BT, et al. A functional genetic approach identifies the PI3K 
pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell;12:395-
402, 2007.  
52. 
Jensen JD, Knoop A, Laenkholm AV, et al. PIK3CA mutations, PTEN, and pHER2 
expression and impact on outcome in HER2-positive early-stage breast cancer patients treated with 
adjuvant chemotherapy and trastuzumab. Ann Oncol 2011.  

  
  
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53. 
Higgins MJ, Jelovac D, Barnathan E, et al. Detection of tumor PIK3CA status in Metastatic 
Breast Cancer using Peripheral Blood. Clin Cancer Res 18(12):3462-9, 2012.  
54. 
Higgins MJ, Jelovac D, Barnathan E, et al. Improving detection methods for PIK3CA 
mutations in breast cancer using peripheral blood from patients with metastastic breast cancer 
(MBC). J Clin Oncol 29: 2011 (suppl; abstr 10512)   
55. 
Mahmoud SM, Paish EC, Powe DG, et al. Tumor-infiltrating CD8+ lymphocytes predict 
clinical outcome in breast cancer. J Clin Oncol 2011;29:1949-55.  
56. 
Loi S, Sirtaine N, Piette F, et al. Prognostic and predictive value of tumor-infiltrating 
lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer 
comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 
02-98. J Clin Oncol 2013;31:860-7.  
57. 
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Response to Pertuzumab and Trastuzumab (PT) in the NeoSphere Study (Abs S6-7) Presented at 
the San Antonio Breast Cancer Symposium 2012.  
59. 
Perez EA, Eckel-Passow JE, Ballman KV , et al. Predictive Genomic Markers to 
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60. 
Dowsett M, Ebbs SR, Dixon JM, et al. Biomarker changes during neoadjuvant anastrozole, 
tamoxifen, or the combination: influence of hormonal status and HER-2 in breast cancer--a study 
from the IMPACT trialists. J Clin Oncol 2005;23:2477-92.  
61. 
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presurgical endocrine therapy for primary breast cancer. J Natl Cancer Inst 2007;99:167-70.  
62. 
Dunbier AK, Anderson H, Ghazoui Z, et al. Association between breast cancer subtypes 
and response to neoadjuvant anastrozole. Steroids 2011;76:736-40.  
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Mohsin SK, Weiss HL, Gutierrez MC, et al. Neoadjuvant trastuzumab induces apoptosis 
in primary breast cancers. J Clin Oncol 2005;23:2460-8.  
64. 
Yamauchi H, Stearns V, Hayes DF. When is a tumor marker ready for prime time? A case 
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Lonning PE, Sorlie T, Perou CM, Brown PO, Botstein D, Borresen-Dale AL. Microarrays 
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Staunton JE, Slonim DK, Coller HA, et al. Chemosensitivity prediction by transcriptional 
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67. 
Chang JC, Wooten EC, Tsimelzon A, et al. Gene expression profiling for the prediction of 
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70. 
Evron E, Dooley WC, Umbricht CB, et al. Detection of breast cancer cells in ductal lavage 
fluid by methylation-specific PCR. Lancet 2001;357:1335-6.  
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Fackler MJ, McVeigh M, Mehrotra J, et al. Quantitative multiplex methylation-specific 
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Pu RT, Laitala LE, Alli PM, Fackler MJ, Sukumar S, Clark DP. Methylation profiling of 
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Fackler MJ, McVeigh M, Evron E, et al. DNA methylation of RASSF1A, HIN-1, RARbeta, 
Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma. Int J Cancer 2003;107:970-
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Muller HM, Widschwendter A, Fiegl H, et al. DNA methylation in serum of breast cancer 
patients: an independent prognostic marker. Cancer Res 2003;63:7641-5.  
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Dulaimi E, Hillinck J, Ibanez de Caceres I, Al-Saleem T, Cairns P. Tumor suppressor gene 
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APPENDICES  
  
A 
American Joint Commission on Cancer Staging  
B 
ECOG Performance Status  
C 
PET/CT Quality Assurance Manual  
D 
PET/CT Imaging Guidelines  
 

  
  
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APPENDIX A: American Joint Committee on Cancer Staging   
  
T – Primary Tumor  
TX  
Primary tumor cannot be assessed  
T0  
No evidence of primary tumor  
Tis  
Carcinoma in situ  
Tis (DCIS)  
Ductal carcinoma in situ  
Tis (LCIS)  
Lobular carcinoma in situ  
Tis (Paget)  
Paget’s disease of the nipple with no tumor  
 Note: Paget’s disease associated with a tumor is classified according to the size of the tumor.  
T1  
Tumor ≤ 2 cm in greatest dimension  
T1mic  
Microinvasion ≤ 0.1 cm in greatest dimension  
T1a  
Tumor > 0.1 cm but not > 0.5 cm in greatest dimension  
T1b  
Tumor > 0.5 cm but not > 1 cm in greatest dimension  
T1c  
Tumor > 1 cm but not > 2 cm in greatest dimension  
T2  
Tumor > 2 cm but not > 5 cm in greatest dimension  
T3  
Tumor > 5 cm in greatest dimension  
T4  
Tumor of any size with direct extension to  
(a) chest wall or  
(b) skin, only as described below  
T4a  
Extension to chest wall, not including pectoralis muscle  
T4b  
Edema (including peau d’orange” or ulceration of the skin of the breast, or satellite skin 
nodules confined to the same breast  
T4c  
Both T4a and T4b  
T4d  
Inflammatory carcinoma  
  
N – Regional lymph nodes  
NX  
Regional lymph nodes cannot be assessed (e.g., previously removed)  
N0  
No regional lymph node metastasis  
N1  
Metastasis in movable ipsilateral axillary lymph node(s)  
N2  
Metastases in ipsilateral axillary lymph nodes fixed or matted, or in clinically apparent 
ipsilateral internal mammary nodes in the absence of clinically evident axillary lymph node 
metastasis  
N2a  
Metastasis in ipsilateral axillary lymph nodes fixed to one another (matted) or to other 
structures  
N2b  
Metastasis only in clinically apparent ipsilateral internal mammary nodes and in the absence 
of clinically evident axillary lymph node metastasis  

  
  
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N3  
Metastasis in ipsilateral infraclavicular lymph node(s), or in clinically apparent ipsilateral 
internal mammary lymph node(s) and in the presence of clinically evident axillary lymph 
node metastasis; or metastasis in ipsilateral supraclavicular lymph node(s) with or without 
axillary or internal mammary lymph node involvement  
N3a  
Metastasis in ipsilateral infraclavicular lymph node(s) and axillary lymph node(s)  
N3b  
Metastasis in ipsilateral internal mammary lymph node(s) and axillary lymph node(s)  
N3c  
Metastasis in ipsilateral supraclavicular lymph node(s)  
  
APPENDIX A (CONTINUED): American Joint Committee on Cancer Staging   
  
PN – Regional lymph nodes  
pNX  
Regional lymph nodes cannot be assessed (e.g., previously removed or not removed for 
pathologic study)  
pN0  
No regional lymph node metastasis histologically, no additional examination for isolated 
tumor cells  
pN0(i-)  
No regional lymph node metastasis histologically, negative IHC  
pN0(i+)  
No regional lymph node metastasis histologically, positive IHC, no IHC cluster > 0.2 mm  
pN0(mol-)  
No regional lymph node metastasis histologically, negative molecular findings (RT-PCR)  
pN0(mol+)  
No regional lymph node metastasis histologically, positive molecular findings (RT-PCR)  
pN1mi  
Micrometastasis (> 0.2 mm, none > 2.0 mm)  
pN1  
Metastasis in one to three axillary lymph nodes and/or in internal mammary nodes with 
microscopic disease detected by sentinel lymph node dissection but not clinically apparent  
pN1a  
Metastasis in one to three axillary lymph nodes  
pN1b  
Metastasis in internal mammary nodes with microscopic disease detected by sentinel lymph 
node dissection but not clinically apparent  
pN1c  
Metastasis in one to three axillary lymph nodes and in internal mammary lymph nodes with 
microscopic disease detected by sentinel lymph node dissection but not clinically apparent  
pN2  
Metastasis in four to nine axillary lymph nodes, or in clinically apparent internal mammary 
lymph nodes in the absence of axillary lymph node metastasis  
pN2a  
Metastasis in four to nine axillary lymph nodes (at least one tumor deposit > 2.0 mm)  
pN2b  
Metastasis in clinically apparent internal mammary lymph nodes in the absence of axillary 
lymph node metastasis  
pN3  
Metastasis in 10 or more axillary lymph nodes, or in infraclavicular lymph nodes, or in 
clinically apparent ipsilateral internal mammary lymph nodes in the presence of one or more 
positive axillary lymph nodes; or in more than three axillary lymph nodes with clinically 
negative microscopic metastasis in internal mammary lymph nodes; or in ipsilateral 
supraclavicular lymph nodes  
pN3a  
Metastasis in 10 or more axillary lymph nodes (at least one tumor deposit > 2.0 mm), or 
metastasis to the infraclavicular lymph nodes  

  
  
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pN3b  
Metastasis in clinically apparent ipsilateral internal mammary lymph nodes in the presence of 
one or more positive axillary lymph nodes; or in more than three axillary lymph nodes and in 
internal mammary lymph nodes with microscopic disease detected by sentinel lymph node 
dissection but not clinically apparent  
pN3c  
Metastasis in ipsilateral supraclavicular lymph nodes  
  
  
M – Distant metastasis  
MX  
Distant metastasis cannot be assessed  
M0  
No distant metastasis  
M1  
Distant metastasis  
  
APPENDIX A (CONTINUED): American Joint Committee on Cancer Staging   
  
  
Stage Grouping  
T  
N  
M  
0  
Tis  
N0  
M0  
I  
T1  
N0  
M0  
IIA  
T0  
N1  
M0  
T1  
N1  
M0  
T2  
N0  
M0  
IIB  
T2  
N1  
M0  
T3  
N0  
M0  
IIIA  
T0  
N2  
M0  
T1  
N2  
M0  
T2  
N2  
M0  
T3  
N1  
M0  
T3  
N2  
M0  
IIIB  
T4  
N0  
M0  
T4  
N1  
M0  
T4  
N2  
M0  
IIIC  
Any T  
N3  
M0  
IV  
Any T  
Any N  
M1  
  
  

  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 77  
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APPENDIX B: ECOG Performance Status Scale  
   
Score  
Definition  
0  
Asymptomatic  
1  
Symptomatic, fully ambulatory  
2  
Symptomatic, in bed less than 50% of day  
3  
Symptomatic, in bed more than 50% of day,  but 
not bedridden  
4  
Bedridden  
  
  
  

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
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Page 78  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
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APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 79  
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Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026  
  
 
  

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 80  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026  
 
  

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 81  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026  
 
  

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 82  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026  
 
 

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 83  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026  
 

  
APPENDIX D:  PET/CT Imaging Guidelines  
  
  
Version: July 30, 2015 (Amendment #2)  
  
Page 84  
Preoperative Pertuzumab and Trastuzumab  
Protocol Chair:  Roisin Connolly, M.B, B.Ch  
TBCRC 026