Conditions: Breast Neoplasms
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 7
Healthy volunteers: no

Brief summary:
This phase II trial studies the efficacy of trastuzumab treatment in breast cancer patients with stage II-III human epidermal growth factor receptor 2 (HER2)-negative tumors and HER2-expressing bone marrow disseminated tumor cells (DTCs). Administering targeted trastuzumab therapy to these patients may result in the elimination of HER2 expressing disseminated tumor cells and improved disease free survival.

Detailed description:

Arms:
arm 1: Patients receive definitive surgery and best practice standard chemotherapy according to NCCN guidelines. The 5 chemo backbone options are:

* doxorubicin or epirubicin plus cyclophosphamide followed by paclitaxel or docetaxel
* docetaxel plus cyclophosphamide
* single-agent paclitaxel
* docetaxel plus carboplatin
* fluorouracil plus epirubicin plus cyclophosphamide followed by paclitaxel or docetaxel arm 2: Patients receive definitive surgery and best practice standard chemotherapy according to NCCN guidelines. The 5 chemo backbone options are:

* doxorubicin or epirubicin plus cyclophosphamide followed by paclitaxel or docetaxel
* docetaxel plus cyclophosphamide
* single-agent paclitaxel
* docetaxel plus carboplatin
* fluorouracil plus epirubicin plus cyclophosphamide followed by paclitaxel or docetaxel

Patients will also receive trastuzumab IV over 30-90 minutes for 52 weeks starting such that there is a minimum of 8 weeks of overlap with the standard of care chemotherapy. Trastuzumab may be given weekly, every 2 weeks, or every 3 weeks while overlapping with standard of care chemotherapy. Trastuzumab will be given every 3 weeks after all standard of care chemotherapy has concluded. Treatment continues in the absence of disease progression or unacceptable toxicity.

Interventions: intervention 1: Doxorubicin intervention 2: Trastuzumab intervention 3: Cyclophosphamide intervention 4: Paclitaxel intervention 5: Epirubicin intervention 6: Docetaxel intervention 7: Carboplatin intervention 8: Fluorouracil
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None intervention 4: None intervention 5: None intervention 6: None intervention 7: None intervention 8: None

Protocol document:
Prot_SAP_000.pdf: 
Version 11/01/16 
 
page 1 of 94 
 
 
A Phase II Randomized Trial Evaluating the Effect of Trastuzumab on Disease Free 
Survival in Early Stage HER2-Negative Breast Cancer Patients with ERBB2 
Expressing Bone Marrow Disseminated Tumor Cells  
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201309084 
Protocol Version Date:  11/01/16 
 
 
 
Principal Investigator: 
Rebecca Aft, M.D., Ph.D. 
Washington University School of Medicine 
Department of Surgery 
Telephone: 314-747-0063 
Fax: 314-454-5509 
Email: aftr@wudosis.wustl.edu 
 
Co-Principal Investigator: Foluso Ademuyiwa, M.D., MPH 
Washington University School of Medicine 
Department of Medicine 
Telephone: 314-362-7201 
Fax: 314-362-7086 
Email: fademuyi@dom.wustl.edu 
 
Sub-Investigators 
 
 
 
Modality 
Mark A. Watson, M.D., Ph.D. 
 
 
Genetics 
Ron Bose, M.D., Ph.D.  
 
 
Medical Oncology 
Cynthia X. Ma, M.D., Ph.D. 
 
 
Medical Oncology 
Michael J. Naughton, M.D. 
 
 
Medical Oncology 
Rama Suresh, M.D. 
 
 
 
Medical Oncology 
Timothy Eberlein, M.D.  
 
 
Surgery 
William Gillanders, M.D.  
 
 
Surgery 
Julie Margenthaler, M.D. 
 
 
Surgery 
Amy Cyr, M.D.  
 
 
 
Surgery 
Deb Novack, M.D., Ph.D. 
 
 
Pathology 
Kathryn Trinkaus, Ph.D.  
 
 
Biostatistics 
 
Study Drug:  
 
trastuzumab 
IND# 
119483 
ClinicalTrials.gov #:  
NCT01779050 
 


Version 11/01/16 
 
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A Phase II Randomized Trial Evaluating the Effect of Trastuzumab on Disease Free 
Survival in Early Stage HER2-Negative Breast Cancer Patients with ERBB2 
Expressing Bone Marrow Disseminated Tumor Cells  
 
 
SCHEMA 
 
 
 
 
 
 
 
 
Patients with Stage II-III HER2-negative breast cancer who are candidates for adjuvant or 
neoadjuvant chemotherapy 
Bone Marrow (BM) Aspirate 
No ERBB2 expression in 
bone marrow 
ERBB2 overexpression in BM 
Not eligible for the study 
Pre-registration 
Trastuzumab for 12 months with 
standard chemotherapy 
(n= 10-12 patients) 
Randomize 3:1 
At 6-18 months post-1st BM aspirate 
• Bone marrow aspirate for ERBB2 
expression 
• Follow for recurrent disease development 
 
Standard chemotherapy 
alone 
(n= ~7 patients) 


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List of Amendments 
 
Initial Approval 
 
09/20/13 
Amendment #1 
 
11/18/13 
Amendment #2 
 
04/22/14 
Amendment #3 
 
09/12/14 
Amendment #4 
 
01/21/15 
Amendment #5 
 
07/30/15 
Amendment #6 
 
12/21/15 
Amendment #7 
 
08/10/16 
Amendment #8 
 
11/01/16 
 
 
 


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Glossary of Abbreviations 
 
AE 
Adverse event 
ALT (SGPT) 
Alanine transaminase (serum glutamate pyruvic transaminase) 
ANC 
Absolute neutrophil count 
ASCO 
American Society of Clinical Oncology 
AST (SGOT) 
Aspartate transaminase (serum glutamic oxaloacetic transaminase) 
B-HCG 
Beta human chorionic gonadotropin  
BM 
Bone marrow 
BWFI 
Bacteriostatic water for injection 
CAP 
College of American Pathologists 
CBC 
Complete blood count 
CFR 
Code of Federal Regulations 
CI 
Confidence interval 
CK 
Cytokeratins 
CK-ICC 
CK-immunocytochemical 
CNS 
Central nervous system 
CR 
Complete response 
CRF 
Case report form 
CSC 
Cancer stem cell 
CST 
Central standard time 
CT 
Computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DFS 
Disease-free survival 
DNA 
Deoxyribonucleic acid 
DOB 
Date of birth 
DSM 
Data and Safety Monitoring  
DTC 
Disseminated tumor cell 
ECG (or EKG) 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EDTA 
ethylenediaminetetraacetic acid 
EGFR 
Epidermal growth factor receptor 
EMT 
Epithelial mesenchymal transition 
ER 
Estrogen receptor 
FDA 
Food and Drug Administration 
FFPE 
Formalin-fixed paraffin-embedded 
FISH 
fluorescent in situ hybridization 
FWA 
Federal wide assurance 
GCP 
Good Clinical Practice 
G-CSF 
Granulocyte colony stimulating factor, filgrastim (Neupogen) 
HHS 
Department of Health and Human Services’ 


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HIV 
Human Immunodeficiency Virus 
HRPO 
Human Research Protection Office (IRB) 
IHC 
Immunohistochemical 
IRB 
Institutional Review Board 
IV 
Intravenous (i.v.) 
LVEF 
Left ventricular ejection fraction 
MRI 
Magnetic resonance imaging 
MUGA 
Multiple gated acquisition scan 
NC 
Nanostring nCounter 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
OHRP 
Office of Human Research Protections  
ORR 
Overall response rate 
OS 
Overall survival 
PCR 
Polymerase chain reaction 
PD 
Progressive disease 
PI 
Principal investigator 
PR 
Partial response 
PR 
Progesterone receptor 
QASMC 
Quality Assurance and Safety Monitoring Committee 
QOL 
Quality of life 
qRT-PCR 
Quantitative reverse transcriptase polymerase chain reaction 
RNA 
Ribonucleic acid 
RR 
Response rate 
SAE 
Serious adverse event 
SCC 
Siteman Cancer Center 
SD 
Stable disease 
SWFI 
Sterile water for injection 
TN 
Triple negative 
TTP 
Time to progression 
UPN 
Unique patient number 
WBC 
White blood cell (count) 
WHO 
World Health Organization 
ZA 
Zoledronic acid 
 
 


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TABLE OF CONTENTS 
 
SCHEMA ......................................................................................................................................... 2 
1.0 
BACKGROUND AND RATIONALE ..................................................................................... 8 
1.1 
Overview of Clinical Trial ................................................................................................. 8 
1.2 
Her2 Status of the Primary Tumor and Benefit from Trastuzumab Therapy ................... 9 
1.3 
Molecular Classification of Primary Tumors – PAM50 .................................................... 9 
1.4 
Testing for Her2 Positivity of DTCs by PCR .................................................................. 10 
1.5 
HER2 Negative Breast Cancers: Luminal B and Basal-Like ......................................... 10 
1.6 
Disseminated Tumor Cells (Bone Marrow Micrometastasis or Minimal Residual 
Disease) ..................................................................................................................................... 10 
1.7 
Rationale for Testing Trastuzumab for ERBB2-overexpressing DTCs ......................... 19 
1.8 
Correlative Studies ........................................................................................................ 20 
1.9 
Study Rationale ............................................................................................................. 22 
2.0 
OBJECTIVES .................................................................................................................... 22 
2.1 
Primary Objective .......................................................................................................... 22 
2.2 
Secondary Objective...................................................................................................... 22 
2.3 
Exploratory Objectives ................................................................................................... 23 
3.0 
ELIGIBILITY CRITERIA ..................................................................................................... 23 
3.1 
Inclusion Criteria ............................................................................................................ 23 
3.2 
Exclusion Criteria ........................................................................................................... 24 
3.3 
Inclusion of Women and Minorities ................................................................................ 26 
4.0 
REGISTRATION PROCEDURES ..................................................................................... 26 
4.1 
Confirmation of Patient Eligibility ................................................................................... 26 
4.2 
Patient Registration in the Siteman Cancer Center Database ...................................... 27 
4.3 
Assignment of UPN ....................................................................................................... 27 
5.0 
TREATMENT PLAN .......................................................................................................... 27 
5.1 
Pre-Intervention Phase .................................................................................................. 27 
5.2 
Randomization ............................................................................................................... 27 
5.3 
Agent Administration...................................................................................................... 28 
5.4 
General Concomitant Medication and Supportive Care Guidelines .............................. 29 
5.5 
Women of Childbearing Potential .................................................................................. 29 
5.6 
Duration of Therapy ....................................................................................................... 30 
5.7 
Duration of Follow Up .................................................................................................... 30 
6.0 
DOSING DELAYS/DOSE MODIFICATIONS .................................................................... 31 
6.1 
Trastuzumab .................................................................................................................. 31 
6.2 
Standard of Care Chemotherapy ................................................................................... 34 
7.0 
REGULATORY AND REPORTING REQUIREMENTS ..................................................... 35 
7.1 
Adverse Events (AEs).................................................................................................... 35 
7.2 
Unanticipated Problems ................................................................................................ 35 
7.3 
Noncompliance .............................................................................................................. 36 
7.4 
Serious Noncompliance ................................................................................................. 36 
7.5 
Protocol Exceptions ....................................................................................................... 37 
7.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality Assurance 
and Safety Monitoring Committee (QASMC) at Washington University ................................... 37 
7.7 
Reporting to the FDA ..................................................................................................... 37 
7.8 
Reporting to Genentech ................................................................................................ 38 
7.9 
Timeframe for Reporting Required Events .................................................................... 41 
8.0 
PHARMACEUTICAL INFORMATION ............................................................................... 42 
8.1 
Trastuzumab .................................................................................................................. 42 
9.0 
BONE MARROW ANALYSIS FOR STUDY ENTRY AND PRIMARY ENDPOINT ........... 44 
9.1 
Collection of Specimens ................................................................................................ 44 
9.2 
Handling of Specimens .................................................................................................. 46 
9.3 
Bone Marrow Processing .............................................................................................. 46 
10.0 
CORRELATIVE STUDIES ................................................................................................. 48 
10.1 
Existing FFPE or Frozen Tumor Blocks ........................................................................ 48 
10.2 
Optional Fresh Pre-Treatment Tumor ........................................................................... 48 


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10.3 
Multiplex Gene Detection of Bone Marrow .................................................................... 48 
10.4 
Serial Research Blood Collection (required) ................................................................. 49 
10.5 
Quality of Life Assessments .......................................................................................... 50 
11.0 
STUDY CALENDAR .......................................................................................................... 51 
11.1 
Arm 1 Study Calendar ................................................................................................... 51 
11.2 
Arm 2 Study Calendar ................................................................................................... 52 
12.0 
DATA SUBMISSION SCHEDULE ..................................................................................... 53 
13.0 
MEASUREMENT OF EFFECT .......................................................................................... 53 
14.0 
DATA AND SAFETY MONITORING ................................................................................. 54 
15.0 
STATISTICAL CONSIDERATIONS .................................................................................. 55 
15.1 
Study Endpoints ............................................................................................................. 55 
15.2 
Justification of Sample Size ........................................................................................... 55 
15.3 
Analysis Plan ................................................................................................................. 56 
15.4 
Timeline and Feasibility ................................................................................................. 57 
15.5 
Stopping Rules .............................................................................................................. 57 
16.0 
REFERENCES .................................................................................................................. 60 
APPENDIX A: ECOG Performance Status Scale ......................................................................... 70 
APPENDIX B: HFSA Guidelines ................................................................................................... 71 
 
 
 


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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
Overview of Clinical Trial 
 
Metastasis is the most significant contributor to mortality in breast cancer patients.  
Decades of pre-clinical research has revealed a complex cascade of key events 
involving cell motility, intravasation, transit in the blood or lymphatics, arrest at a 
secondary site, extravasation, colonization and growth at a new site. More recent 
data suggests that only a small, unique subset of cells within a primary tumor 
possess metastatic potential1. Thus, therapies that simply reduce primary tumor 
mass often fail to cure patients. Furthermore, molecular profiles of cells released 
from primary, heterogeneous tumors may evolve as the cells transition and 
progress to metastatic foci2.  To develop new therapeutic interventions to monitor 
and prevent overt distant disease development, it is essential to identify and target 
the intermediary cells in the metastatic process since these cells likely have 
biological behavior and therapeutic vulnerabilities which differ from the primary 
tumor. 
  
In breast cancer patients, bone marrow (BM) is thought to serve as a reservoir for 
disseminated tumor cells (DTCs) which are the hypothesized intermediaries in the 
metastatic process.  DTCs are phenotypically heterogeneous and molecularly 
distinct from the primary tumor2. Currently, DTCs are identified by 
immunocytochemical (CK-ICC) detection of cytokeratins (CK) or molecular 
techniques that detect expression of single genes associated with DTCs3-4. Using 
CK-ICC, DTCs have been detected in the BM of up to 40% of stage I-III breast 
cancer patients5.  Several large multi-institutional clinical studies have documented 
the independent prognostic significance of DTCs6. However, not all DTCs have 
equal metastatic potential7. Using an optimized gene detection platform 
(Nanostring nCounter) and a 38-gene expression profile, data from a pilot study 
provided evidence that patients with molecular subsets of DTCs overexpressing 
ERBB2 are at very high risk of developing recurrent disease. Seventy-five percent 
of patients with Her2-negative primary tumors who harbored ERBB2-positive 
DTCs in their BM developed distant disease within 48 months of diagnosis. 
Discordance between the primary tumor and DTCs/ circulating tumor cells 
(CTCs)/metastatic foci have been reported by other investigators 8-10 11. ERBB2 
amplification seems to become more frequent in systemic progression 9-10, 12. 
Targeting and eliminating ERBB2 overexpressing DTCs may result in an improved 
disease-free survival (DFS) and provide a selective therapeutic intervention for 
these high risk patients who are not candidates for ERBB2-targeted therapy based 
on their primary tumor biomarkers. This could also provide proof of principle that a 
strategy of targeting DTCs based on their biomarker profile may lead to an 
interruption of the metastatic process by eliminating the intermediary cells of 
metastases formation. In addition, characterization of DTC-specific expression 
profiles could lead to improved prediction of the metastatic potential of DTCs and 
reveal their vulnerabilities to targeted therapeutics based on the expression / 
activation of specific signaling pathways involving key regulatory genes such as 
ERBB213-14.  
 
 


Version 11/01/16 
 
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1.2 
Her2 Status of the Primary Tumor and Benefit from Trastuzumab 
Therapy 
 
The Her2 protein and/or ERBB2 gene are over expressed or amplified in 
approximately 25% of breast cancers15-16. ASCO/CAP defines Her2 positivity as 
immunohistochemistry (IHC) score of 3+ or gene amplification measure by 
fluorescent in situ hybridization (FISH)17. Her2 positivity of the primary tumor is 
associated with significantly decreased recurrence-free survival and overall 
survival18-20.  Trastuzumab, a monoclonal antibody targeting Her2, is approved by 
for the treatment of Her2 positive cancer in the adjuvant and metastatic setting21. 
Results from multiple clinical trials demonstrate that the administration of adjuvant 
trastuzumab for one year concurrently with chemotherapy significantly improves 
DFS compared with chemotherapy alone in women with Her2 positive breast 
cancers leading to an approximately 50% reduction in disease events22-23 with only 
minor side effects24-26. Treatment of women with Her2 positive early stage breast 
cancer with chemotherapy/trastuzumab has become the standard of care. 
However, it has been reported that patients whose tumors do not meet the criteria 
for Her2-positivity and in fact are Her2-negative by FISH and IHC may still benefit 
from trastuzumab treatment27.  Paik et al examined tissue blocks from NSABP B-
31, which compared standard chemotherapy of doxorubicin/cyclophosphamide 
followed by paclitaxel (ACT) with ACT plus trastuzumab (ACTH) in the adjuvant 
setting.  They found that some patients with normal gene copy numbers appeared 
to benefit from trastuzumab treatment (relative risk for DFS, 0.40; 95% confidence 
interval [CI], 0.18 to 0.89; P=0.026).  Possible explanations for this observation are 
that tumor cells exist which are dependent on the Her2 pathway for growth and 
survival which are not identified with conventional Her2 testing or Her2 expression 
is acquired as tumor cells progress along the metastatic pathway. These results 
have formed the basis for the NSABP B-47 trial which compares adjuvant 
chemotherapy with or without adjuvant trastuzumab in 3260 early stage breast 
cancer with Her2 expression that does not meet ASCO/CAP definition of Her2 
positivity. 
 
1.3 
Molecular Classification of Primary Tumors – PAM50 
 
Gene expression profiling has defined "intrinsic" subtypes of breast cancer which 
have been shown to be superior in predicting long term outcomes of breast 
cancer28-31 and the likelihood of responding to therapy31 than IHC staining for ER, 
PR, Her-2.  The PAM50 assay is a 50-gene, second-generation breast cancer 
molecular profiling test based on the intrinsic gene signatures which can assign 
the breast cancer intrinsic subtypes to individual patient tumors. Molecular profiling 
of primary tumors with PAM50 will be used for the exploratory objectives in this 
study. Recently, Cheang et al compared response of Her2-positive tumors 
identified by conventional IHC/FISH versus applying the PAM50 to identify the 
Her2-enriched subtype31-32. They found that those tumors which were Her2-
positive by both IHC/FISH and by PAM50 were 6-34x more likely to achieve a pCR 
with chemo/trastuzumab therapy than tumors which were Her2-positive by 
IHC/FISH alone.  Within the PAM 50, there are four genes which are over-
represented in the Her2-enriched subtype. 3 of the 4 genes (ERBB2, GRB7, 
FGFR4) are included in the 38-gene profile that we have generated for 
classification of DTCs33 (see below). 
 


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1.4 
Testing for Her2 Positivity of DTCs by PCR 
 
As discussed above, presently Her2 status is defined by positivity by either IHC or 
FISH assays. Multiple studies have compared IHC/FISH with quantitative reverse 
transcriptase polymerase chain reaction (qRT-PCR) using both fresh and paraffin 
fixed tissue. These have demonstrated overall concordance between the two tests 
of 82-100% and Her2 status by qRT-PCR was found to correlate with DFS34-43.  In 
one large prospective study of 466 patients reported by Lehman-Che43, 
concordance between qRT-PCR and IHC was 97%. Lack of concordance was 
generally due to an underestimation of Her2 positivity by qRT-PCR likely due to 
degradation of RNA and rare cases of intratumoral heterogeneity43.  Thus though 
IHC/FISH remains the standard for assessing Her2 positivity in tumors17; studies 
indicate that Her2 testing by qRT-PCR is accurate, though it may underestimate 
the number of positive patients. 
1.5 
HER2 Negative Breast Cancers: Luminal B and Basal-Like 
 
Gene expression profiling has defined "intrinsic" subtypes of breast cancer which 
have been shown to be superior in predicting long term outcomes of breast 
cancer28-31 and the likelihood of responding to therapy31 than IHC staining for ER, 
PR, Her-2.   Based on this methodology, breast cancers have been divided into 
the basal-like, Her2 –positive, luminal A and Luminal B subtypes. The luminal A 
subtype most closely corresponds to ER+/PR+ breast cancers which are very 
responsive to hormonal treatment. Approximately 20% of invasive breast cancers 
are basal like and most closely corresponding to tumor with are negative for the 
expression of estrogen receptor (ER) and HER2 gene amplification (triple 
negative, TN) by IHC29-30, 44.  An increased prevalence of ER-/HER2- tumors has 
been observed in premenopausal African American women, BCRA1 mutation 
carriers, and Hispanics.  Luminal B tumors generally correspond to tumors which 
are ER+ and PR-/Her2- by IHC.  Luminal B and TN are generally high grade and 
the lack of established targeted agents coincides with their particularly high risk of 
early relapse when compared to other breast cancer subtypes. Adjuvant and/or 
neoadjuvant chemotherapy has been shown to improve the outcome for a small 
subset of patients with chemotherapy-sensitive disease but is ineffective in 
preventing relapse in those that have residual cancer despite neoadjuvant 
chemotherapy.  For example, a recurrence rate of 40-50% in the first 5 years is 
observed in patients with triple negative breast cancer who did not achieve a 
complete pathological response to neoadjuvant chemotherapy45-47.  
 
1.6 
Disseminated Tumor Cells (Bone Marrow Micrometastasis or Minimal 
Residual Disease) 
 
1.6.1 Clinical Significance of DTCs  
 
Disseminated tumor cells (DTCs) detected in the bone marrow of breast 
cancer patients have been shown to be associated with reduced disease-
free survival and overall survival in breast cancer patients at both primary 
diagnosis and recurrence-free follow-up after local primary local and 
systemic treatment 
48. DTCs, also described as bone marrow 
micrometastasis or minimal residual disease, can be detected in 12–45% 
of patients with primary operable breast cancer as determined by 
immunocytochemistry 6, 49-54.  The presence of DTCs has clearly been 


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shown to be an independent prognostic factor for patients with stage I-III 
breast cancer in multiple studies6, 55. DTCs can thus be used to select 
patients at increased risk for relapse who are likely to benefit from 
additional treatment intervention. The table below summarizes the major 
studies using immunocytochemical detection of DTCs in BM. 
 
 


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Major clinical studies of the prognostic value of DTC detection in BM by 
immunocytochemistry and prognostic value on disease and overall survival 
(univariate and multivariate analysis) 
Reference 
Sampling 
Marker 
No. 
patients 
Detection 
rate (%) 
Follow-
up 
(month) 
Disease 
free 
survival 
Overall 
survival 
Univ 
Multiv 
Univ 
Multiv 
Redding 198356 
Smear 
MUC 
110 
28 
 
NA 
NA 
NA 
NA 
Manegold 198857 
Biopsy 
Smear 
CK/PKK1 
50 
8 
 
NA 
NA 
NA 
NA 
Landys 199858 
Biopsy 
CK/AE1–AE3, 
KL1, CAM 5-2 
128 
19 
240 
NA 
NA 
Yes 
NA 
Salvadori 199059 
Biopsy 
CK/MBr1 
121 
16.5 
48 
No 
No 
NA 
NA 
Mathieu 199060 
Biopsy 
MUC/EMA, 
HMFG2 
93 
1 
 
No 
No 
No 
No 
CK/KL1, AE1–
AE3, CAM5-2 
Kirk 199061 
Smear 
MUC/anti-milk 
fat 
globulin 
LICR.LON.M8.4 
25 
48 
34 
No 
NA 
NA 
NA 
Singletary 199162 
Smear 
CK/AE1, 
AE3, 
MAK-6 
71 
38 
11 
No 
No 
No 
No 
MUC/113F1, 
260F9, 317G5 
Cote 199163 
Smear 
MUC/C26, T16 
49 
36.7 
30 
Yes 
Yes 
NA 
Na 
CK/AE-1 
Schlimok 199264 
Cytospin 
CK18/CK2 
187 
18 
39 
Yes 
Yes 
NA 
NA 
Harbeck 199465 
Smear 
CK 
100 
38 
34 
Yes 
Yes 
No 
Yes 
MUC/EMA 
Ménard 199466 
Cytospin 
CK/MBr1, 
MBr8, 
CK18/CK2, 
MUC1 
197 
31 
NA 
NA 
NA 
NA 
NA 
Molino 199767 
Cytospin 
CK/MBr1, 
MBr8, 
MOV8, 
MOV16 MluC1 
109 
31 
36 
No 
No 
No 
No 
Funke 199668 
Cytospin 
CK18/CK2 
234 
38 
NA 
NA 
NA 
NA 
NA 
Diel 199669-70 
Smear 
MUC/TAG12 
(2E11) 
727 
43.3 
78 
Yes 
Yes 
Yes 
Yes 
Mansi 199950, 71 
Smear 
EMA 
350 
25.4 
150 
Yes 
No 
Yes 
No 
Lyda 200072 
Biopsy 
CK/AE1–AE3, 
35βH11 CAM 5-
2 
54 
31 
38 
Yes 
NA 
NA 
NA 
Untch 199973 
Cytospin 
CK18/CK2 
581 
28 
 
No 
No 
No 
No 
Braun 20005 
Cytospin 
CK/CK8,18,19 
(A45 B/B3) 
552 
36 
36 
Yes 
Yes 
Yes 
Yes 
Gerber 200152 
Cytospin 
CK/CK8,18,19 
(5D3) 
554 
37 
54 
Yes 
Yes 
Yes 
Yes 
Gebauer200153 
Smear 
CK, MUC/EMA 
396 
42 
75 
Yes 
Yes 
Yes 
Yes 
Kasimir-Bauer 
200174 
Cytospin 
CK/CK8,18,19 
(A45 B/B3) 
128 
34 
24 
NA 
NA 
NA 
NA 
Naume 200449 
Cytospin 
CK/AE1/AE3 
819 
13 
49 
Yes 
Yes 
Yes 
Yes 
Braun 20056 
Various 
Various 
4703 
30.6 
63 
Yes 
Yes 
Yes 
Yes 
Bidard 200775 
Cytospin 
CK/CK8,18,19 
(A45 B/B3) 
621 
15 
50 
Yes 
Yes 
Yes 
Yes 
CK, cytokeratin; Muc, mucin; EMA, epithelial membrane antigen; NA, not available 
Table adapted from 55. 
 


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In a pooled analysis of 9 studies comprising 4703 patients with stage I, II, 
or III breast cancer6, the presence of micrometastases at diagnosis was 
detected in 30.6% of patients and was found to be a significant and 
independent prognostic factor with respect to poor overall survival (OS) and 
breast cancer-specific survival (univariate mortality ratios: 2.15 and 2.44, 
respectively; p<0.001 for both outcomes) and poor disease-free survival 
(DFS) and distant DFS during the 10-year observation period (incidence 
rate ratios: 2.13 and 2.33, respectively; p<0.001 for both outcomes).  
 
In an institutional study (HRPO# 02-0778), bone marrow samples were 
collected from women with locally-advanced breast cancer undergoing 
neoadjuvant chemotherapy with or without zoledronic acid (ZA) to study 
the impact of neoadjuvant therapy on occult micrometastases and bone 
density. Bone marrow data was available for 119 patients prior to therapy 
and 112 patients at the time of surgery (at 3 months). Prior to any therapy, 
46% of the ZA-treated and 41% of the no-ZA treatment group had 
detectable DTCs in their bone marrow (p=0.651).  At surgery, 23% of the 
ZA-treated and 36% of the no-ZA treatment arm had detectable DTCs 
(p=0.054). In patients who did not achieve a pCR, about 50% were found 
to have bone marrow micrometastases. Patients with residual bone marrow 
DTCs after neoadjuvant chemotherapy were associated with a poorer 
prognosis than those patients without DTCs (unpublished data from Dr. 
Aft).   
 
Although the application of using DTCs is still investigational according to 
the American Society of Clinical Oncology (ASCO) 2007 update of 
recommendations for the use of tumor markers in breast cancer, its 
incorporation into clinical management algorithms is currently the focus of 
much research. 
 
1.6.2 DTCs as Potential Metastatic Progenitors and a Marker of 
Body-wide Dissemination of Invasive Cancer Cells 
 
Micrometastases have been shown to retain clonogenic and tumorigenic 
capacities in many biological reports76-78. Clinical studies have indicated a 
link between BM DTCs and the onset of bone metastases6, 75, 79, strongly 
supporting the idea of local growth of DTCs into macrometastases. It has 
also been speculated that the bone marrow may act as a long-term 
reservoir for tumor cells, which can re-circulate to other distant organs, 
leading to recurrence80. The high genetic heterogeneity81 of BM 
micrometastatic cells might be responsible for recirculation of some cancer 
seeds from the bone marrow to different host organs. However, there is 
currently no direct evidence suggesting that bone marrow DTCs are 
responsible for the lung or liver metastases. On the contrary, many 
biological models have reported that most of the target organs harbor 
micrometastatic dissemination of mammary tumors82-84. Current literature 
does not provide strong evidence for a common pool of genes responsible 
for coupled homing to bone marrow (or flat bones) and liver. Paget was the 
first to describe the non-random growth of metastases85, and the sustaining 
molecular determinants of cancer cell homing to different organs have been 
recently characterized86-88. Therefore, in the case of distant non-bone or 
local relapses predicted by BM DTCs75, these cells mostly appear as a 


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marker of a body-wide dissemination of invasive cancer cells rather than 
the body’s only long-term reservoir of disseminated cancer cells. 
 
1.6.3 DTCs and CSCs 
 
The role of cancer stem cells (CSCs) in the establishment of metastases 
remains controversial89-92. Experimentally, CD44+/CD24- cancer cells, a 
phenotype associated with a stem cell pattern, exhibit an invasive 
phenotype which is a prerequisite to metastasis93-94. In a report on 50 
cases, most BM DTCs exhibited a stem cell-like immunohistochemistry 
(IHC) phenotype92. 
 
1.6.4 DTCs and Systemic Therapy 
 
There are few trials on systemic treatments in DTC positive patients. 
Cytostatic treatment has no significant effect95-96 possibly due to the low 
expression of proliferation markers suggesting that these cells are 
dormant97-98. Cell cycle independent agents directed against specific DTC 
characteristics might therefore be more promising.  Bisphosphonates clear 
DTCs from bone marrow95. This finding parallels the recently observed 
prolongation of survival in early breast cancer patients99-101.  Three non-
randomized trials targeting treatment to DTCs showed significant DTC 
elimination in patients receiving edrecolomab directed against EpCAM102-
104.  
 
1.6.5 Potential of DTCs in Monitoring Therapy Efficacy in the 
Adjuvant Setting 
 
An important potential application for DTC detection is the monitoring of 
therapeutic efficacy in the adjuvant setting. The effectiveness of adjuvant 
therapy regimens can currently only be assessed retrospectively in large-
scale clinical trials after an observation period of at least 5 years. Bone 
marrow biopsies performed before and following therapy for the presence 
of DTCs make real-time assessments of therapeutic efficacy possible.  
Persistence of DTCs in bone marrow years after diagnosis and initial 
therapy has been shown to be an indicator of subsequent systemic 
treatment failure79, 105-106. Persistence or disappearance of DTCs after 
systemic treatment could therefore be used as a surrogate marker of 
treatment response107. Beyond prognostic relevance, phenotyping of DTC 
can reveal targets for individualized treatment approaches 
 
1.6.6 Rationale for Determining Expression of ERBB2 by DTCs in 
Patients with Her2-negative Breast Cancers 
 
Discordance in Her2 expression between the primary tumor and 
disseminated cells:  Several studies have shown an antigen shift from the 
primary tumor to distant metastases 108-110. Discordance in Her2 expression 
between primary tumors and metastases has been observed in 10-20% of 
cases. Several studies have reported Her2 overexpression in circulating 
tumor cells (CTCs) from metastatic patients with Her2-negative primary 
tumors8-10, 111-112. Her2-positive DTCs in BM from early stage patients with 
Her2-negative primary tumors have also been reported11, 113-114. Her2 


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amplification seems to become more frequent in systemic progression 9-10, 
12. Two hypotheses have been advanced to explain the discordance of 
Her2 expression between the primary tumor and CTCs/DTCs. It has been 
proposed that Her2 amplification is acquired during dissemination and 
disease  progression115. Alternatively, Her2 positive clones of the primary 
tumor may have a greater tendency to break away and form metastases111-
112, 116.  
 
To determine whether Her2-positive DTCs could be eliminated by targeted 
therapy, Rack et al conducted a small non-randomized phase II trial 
evaluating the efficacy of trastuzumab in eliminating DTCs from BM13 in 10 
women with  stage I-III breast cancer.  Of the ten women with Her-2 positive 
DTCs, 4 had Her2 positive tumors. After 12 months of trastuzumab therapy, 
none of the ten women had detectable Her2 positive DTCs in their BM. 
Interestingly, 3 patients continued to have detectable Her2 negative DTCs. 
Thus, in this pilot study, trastuzumab was found to be effective in 
eliminating Her2 overexpressing DTCs. The persistence of Her2 negative 
DTCs illustrates the heterogeneity of DTCs and highlights the need to 
define multiple predictive markers. 
 
Preliminary Data: Her2 expression in DTCs correlates with early 
recurrence: We have examined bilateral iliac crest BM from 20 women 
with newly diagnosed clinical stage II/II breast cancer collected prior to any 
treatment for the expression of our 38 gene panel (HRPO# 05-0648). The 
primary tumors of 7 patients were Her2 positive and these patients received 
treatment with chemo/trastuzumab. Two of these 7 patients had DTCs that 
were ERBB2 positive and none of the 7 Her2-positive patients developed 
metastatic disease with a mean follow-up of 48 months (Figure 1). In 
contrast, of the 13 patients who had Her2-negative tumors, 4 had DTCs 
that were ERBB2-positive and of these 4 patients, 3 (75%) developed 
metastatic disease within 24 months of diagnosis. ERBB2-positivity in 
these BM specimens was confirmed with qRT-PCR with 100% patient 
correspondence to the nCounter assay. Although these numbers are too 
small to reach statistical significance the data suggest that those patients 
with Her2-negative primary tumors and ERBB2 overexpression in their BM 
at the time of diagnosis are at high risk of early metastatic disease 
development and may benefit from ERBB2-directed therapy. Moreover, we 
have found that ERBB2 levels in blood, which were very low, did not 
correlate with levels in the BM or metastatic disease development.  
 


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1.6.7 Molecular Detection of Disseminated Tumor Cells 
 
Several studies have assessed DTC detection by using molecular biology 
techniques such as real-time quantitative polymerase chain reaction (RT-
PCR) determination with several epithelial-specific or organ specific mRNA 
such as CK19, MUC1120, urokinase-type plasminogen activator receptor 
(uPAR), EpCAM121 and mammaglobin122-127.  Though expression of 
mammaglobin, as well as cytokeratin 19 mRNA in BM has been shown to 
give prognostic information128-130, given the heterogeneity of DTC it is 
unlikely that there will be a single marker suitable for the detection of all 
DTC in all breast cancer patients.   
 
Using single gene PCR to detect DTC highlights the difficulty of this 
approach. Since DTC are known to be phenotypically heterogeneous, 
multiple appropriate sensitive and specific markers which are predictive 
need to be identified.  Until now, there has been technical difficulty in 
assessing multiple genes in the same specimen55.  .   
 
Multi-marker qRT-PCR has the potential to overcome some of the above 
concerns by allowing the detection of down-regulated/poor expression of a 
single genes106, 130, 134-135.  In addition, genes associated specifically with 
clinically- and functionally-significant DTCs have recently been identified 
and validated using a conventional qRT-PCR assay136-137. Several of these 
genes when detected in bone marrow have been shown to identify patients 
at high risk of recurrence137. 
 
Nanostring nCounterTM (NC) is a sensitive, multiplex platform developed 
for the analysis of gene expression138. The NC platform can quantitatively 
detect expression of up to 500 genes in a single reaction with similar 
sensitivity and reproducibility as qRT-PCR.  The NC platform counts single 
RNA molecules,  does not require enzymatic reactions or high quality RNA 
 
Figure 1. Recurrent disease development in patients 
with ERBB2-positive DTCs.  Patients with Her2 positive 
tumors received chemotherapy with trastuzumab. 
Patients with Her2-negative tumors received cytotoxic 
chemotherapy alone. All patients had ERBB2-positive 
DTCs. All recurrences detected within 24 months 
(mean=19) of diagnosis. 
0
10
20
30
40
50
60
70
80
Primary tumor 
Her2+/DTC ERBB2+
Primary tumor 
Her2-/DTC ERBB2+
Percentage of pts with metastatic  
disease
Recurrant disease develop in patients with 
ERBB2-positive DTCs 
Percentage of pts with 
recurrant disease 
development


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and simple to use139.  In this technology, a multiplexed probe library is made 
with two sequence specific probes for each gene of interest138. The capture 
probe contains a 35-50 base sequence complementary to a particular 
target mRNA plus a short common sequence coupled to an affinity tag. The 
second reporter probe contains a second 35-50 bp sequence 
complementary to the target mRNA which is coupled to a color-coded tag 
that provides the detection signal (Figure 2). The limit of detection 
corresponds to 0.2-1 mRNA molecules per target cells with a dynamic 
range of 2.5 logs. This technology will allow the rapid, simultaneous 
detection of previously validated gene expression patterns analysis of 
hundreds of genes across many samples. 
 
 
 
The current standard of DTC detection by IHC is labor-intensive which has limited 
the clinical use of DTC detection. We have validated the NC assay by measuring 
expression of genes which are DTC associated in patient BM. We developed a 
panel of 38 genes which are associated with all subtypes of breast cancer and are 
not expressed in normal bone marrow (Figure 3). These genes include those 
associated with epithelial cells and ER/Her2-negative tumors (EpCAM, 
cytokeratins 5,7,8,17, EGFR)28, genes which have shown to be prognostic in 
DTC128-130, 137, 140 (mammaglobin, K19, Twist1, PITX2) and housekeeping genes 
(Table 2). BM specimens were considered to be 'positive' for gene expression if 
expression levels in BM are at least two standard deviations above the mean 
expression of the 10 BM samples from healthy volunteers. Patients were 
considered ‘positive’ for biomarker gene expression when detected in at least one 
of two bone marrow samples analyzed (left or right side).  For multiple gene testing 
by NC or Fluidigm Biomark HD (FBHD), BM is considered positive if any one of 
the validated genes is detected.  
 
 
Figure 2. Mechanism of NC 
a) A schematic representation of the hybridized 
complex (not to scale). The capture probe and 
reporter probe hybridize to a complementary 
target mRNA in solution via the gene-specific 
sequences (see Methods for details on capture 
and 
reporter 
probe 
construction). 
After 
hybridization, the tripartite molecule is affinity-
purified first by the 3'-repeat sequence and then by 
the 5'-repeat sequence to remove excess reporter 
and capture probes, respectively. (b) Schematic 
representation of binding, electrophoresis, and 
immobilization. (i) The purified complexes are 
attached to a streptavidin-coated slide via 
biotinylated capture probes. (ii) Voltage is applied 
to elongate and align the molecules. Biotinylated 
anti-5' oligonucleotides that hybridize to the 5'-
repeat sequence are added. (iii) The stretched 
reporters are immobilized by the binding of the 
anti-5' oligonucleotides to the slide surface via the 
biotin. Voltage is turned off and the immobilized 
reporters are prepared for imaging and counting. 
(c) False-color image of immobilized reporter 
probes. From Geiss et al 


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We 
have 
performed 
several pilot studies with 
NC 
to 
determine 
the 
sensitivity and to optimize 
sample input. Using breast 
cancer cell lines (SKBR3, 
MDAMB231, ZR75) diluted 
into normal human bone 
marrow 
at 
varying 
concentrations, we found 
that the genes K19, Slug, 
S100A3 and EGFR could 
be detected at a 1:100,000 
dilution using 0.5 ug of 
RNA and that these genes 
were expressed at a wide 
range of levels depending 
on the cell line. Moreover, 
the detection was linear 
over 
a 
20-fold 
range 
(Figure 4).  We next 
determined 
whether 
sensitivity would be increased by using Ficoll gradient enrichment of the DTC or 
by increasing the RNA. We found that Ficoll gradient enrichment resulted in a 2-
fold increase in NS detected counts, while increasing the RNA to 5ug increased 
detection by 10-fold without increasing the background which allows us to detect 
certain genes at a level of 1 DTC per 1 million bone marrow cells.  We next tested 
whether we were able to detect genes associated with DTC by NC in patient 
samples and determine the correlation with detection by qRT-PCR (Table 3).  We 
found that each of the genes could be detected in at least one patient specimen 
and that there was good correlation with expression as detected by qRT-PCR.  Our 
initial results indicate that NC is as sensitive as qRT-PCR for the detection of 
several genes which have been associated with DTCs, that the results are linear 
over a wide range of values, and that RNA isolated from whole bone marrow can 
be used for the assay.  
 
As a preliminary test of technical feasibility, we have performed the 38-gene 
nCounter assay on 42 bone marrow specimens from 21 breast cancer patients and 
a set of 8 bone marrow specimens from healthy volunteer women.  Nine patients 
had disease free survival (DFS) of less than 5 years while twelve patients had no 
evidence of metastatic disease at last follow-up. As demonstrated in Figure 5, all 
but two specimens had detectable levels (defined as two standard deviations 
above the mean of the control population) of at least one gene transcript in the 38-
gene signature.  This corresponds to a 90% positivity rate based on the expression 
of at least one gene transcript in at least one bone marrow sample. 
 
Recently, the WU CLIA-certified GPS lab acquired a Fluidigm Biomark HD (FBHD) 
that provides quantitative analysis of cDNAs by qPCR using integrated fluidic 
circuit nanochips. These chips contain fluidic networks that enable automated 
combining of up to 96 cDNA samples with 96 gene assays to perform 9216 qPCR 
reactions simultaneously using 20ul sample cDNA loading volumes. This 
technology can detect specific targets at a minimum of 500-1,000 copies in the 
Figure 3.  Genes represented in the 38-gene DTC 
panel and their biological pathway associations.  
Transcripts detected in the twenty-patient pilot 
cohort are highlighted in bold underline and 
those associated with early metastatic recurrence 
are denoted by asterisks. 


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original volume. Because some genes exhibit low expression, such as those 
associated with DTCs, resulting in more dilute target concentrations, a multiplexed 
14 cycle pre-amplification of the targets of interest is performed in a primer-limited 
environment such that small amounts of cDNA are amplified equally without 
introducing bias abundances. We have employed this technology to analyze a 46-
gene panel in 74 patient BM specimens. This technology will be developed as a 
clinical grade assay for use in the detection of DTCs in this clinical trial. From our 
preliminary work we have found that this technology provides excellent sensitivity 
and specificity for the detection of specific DTC populations. We expect this type 
of assay platform for clinical samples will facilitate translation of multi-gene 
biomarkers into the clinic, identify women at high risk of breast cancer recurrence 
and provide guidance on tailored therapies based on the molecular profile of micro-
metastatic breast tumor cells. 
 
 
1.7 
Rationale for Testing Trastuzumab for ERBB2-overexpressing DTCs 
 
Evidence from the literature and our preliminary data suggest that 
micrometastases that persist despite chemotherapy are likely enriched with cells 
that have stem cell-like features that are responsible for subsequent disease 
recurrence96, 141-142.  Bone marrow disseminated tumor cells (DTCs) can be used 
as a surrogate for systemic micrometastases Discordant Her2 expression between 
primary breast cancer and CTCs/DTCs have been reported.13, 112-113   Clinical trial 
data has identified a yet undefined subgroup of patients with Her2-negative 
primary tumors who benefit from trastuzumab therapy. 
 
The current trial will assess if 12 months of trastuzumab in patients with expression 
of ERBB2 by BM DTC will lead to the elimination of bone marrow micrometastases 
that persist following the standard primary and adjuvant breast cancer therapies. 
Women with clinical stage II/III Her2 negative primary breast cancer who are 
Figure 4.  Comparison of assay linearity for detecting KRT19 (left) and EPCAM (right) gene 
expression in ZR75 breast tumor cells serially diluted into healthy volunteer bone marrow using 
qRT-PCR and nCounter platforms.  qPCR data (left axes) was calculated using the ∆∆Ct 
method, relative to the average signal detected in healthy control bone marrow.  nCounter data 
(right axes) represent signal counts from each sample, after subtraction of average counts from 
healthy control bone marrow.  Note that despite differing units of measure, both assays show 
roughly concordant linearity and sensitivity. 


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candidates for chemotherapy will undergo right and left iliac crest BM aspiration to 
assess levels of ERBB2  both by qRT-PCR and as a component of the 38 gene 
profile using multiplex gene expression analysis (NC or FBHD).  Those women 
with ERBB2 levels 2 standard deviations higher than control normal BM by qRT- 
PCR will be randomized to standard adjuvant chemotherapy with no additional 
treatment or 1 year of trastuzumab administered with standard chemotherapy.  
Results from this trial will provide critical foundation for future definitive studies 
assessing the effectiveness of trastuzumab in reducing breast cancer recurrence 
in these high-risk patients. 
 
1.8 
Correlative Studies 
 
1.8.1 The Effects of Chemotherapy and Trastuzumab on the 38-Gene 
Profile  
 
This specific aim will: 
 
1. Prospectively identify those genes in the 38-gene signature that are 
associated with DTCs but are not eliminated by chemo/trastuzumab 
in patients with ERBB2-positive DTCs. 
2. Determine which genes in the 38 gene profile are eliminated in 
parallel with the ERBB2-positive DTCs. 
 
 
Figure 5.  Simplified matrix of nCounter gene expression data showing patterns of gene expression, 
represented here as detected (filled box) or undetected (empty box) in paired bone marrow 
specimens from 21 breast cancer patients.  Only 17 of the 38 genes detected in at least one patient 
in this trial set are shown.  ‘Detected’ is defined as expression that was 2 SD above the mean 
expression in a set of 8 healthy control women.  ER and Her2 status of each case is shown, as well 
as the number of months until relapse (NA= no relapse to date).  Total samples with detectable 
gene expression (column sums) and total genes detected in a sample (row sum) are indicated.  . 
 


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Based on the data of Rack13, we expect up to 30% of patients to have 
residual ERBB2-negative DTCs after chemo/trastuzumab treatment. By 
comparing the 38-gene profile before and after chemo/trastuzumab 
treatment, we will be able to determine the molecular profile of those DTCs 
that remain after treatment and correlate expression with disease 
recurrence. We will examine which genes in the 38 gene profile are 
eliminated in parallel with the ERBB2-positive DTCs. For example, we will 
examine the relationship between ERBB2 and the cancer stem 
cell/Hedgehog pathway gene PTCH1. In our preliminary data (Figure 4), 
we observed that all patients with Her2-negative tumors/ERBB2-positive 
DTCs also expressed PTCH1. Thus we should be able to determine 
whether these 2 genes are expressed by the same population of DTCs or 
by separate populations of DTCs and the possible relationship of residual 
PTCH1 expression and DFS. 
 
1.8.2 Comparison of Recurrence Rate in Trastuzumab-treated 
Patients with PAM50-defined Her2 Subtype versus Other 
Subtypes  
 
In this specific aim, we will assign each patient’s primary tumor to a 
corresponding intrinsic subtypes using the PAM50 gene expression 
signature 31.  This will be accomplished using the multiplex gene expression 
assays to analyze the tumor specimens for the expression of the 50 genes 
in the signature. This work is ongoing at Washington University. This will 
allow us to: 
1. Compare recurrence rates in trastuzumab treated patients with 
PAM50 defined Her2 subtype versus other subtypes and 
2. Determine whether a specific tumor subtype is associated with 
ERBB2-positive DTCs.  
 
1.8.3 Expected Results and Alternative Strategies 
 
We expect that ERBB2 expression by DTCs will be a predictive biomarker 
identifying those patients who will benefit from trastuzumab therapy, in a 
population of patients who would not otherwise be candidates for this 
therapy based on their primary tumor.  We expect that treatment of these 
patients with trastuzumab will confer a DFS advantage compared to the 
control population of patients. We expect to observe elimination of ERBB2-
positive DTCs with trastuzumab therapy and that this will correlate with an 
improved DFS. Based on our pilot data, we expect >90% correlation 
between ERBB2 positivity testing between qRT-PCR and multiplex assays.  
By analyzing the 38 gene profile on all patient BM, we expect to identify 
new predictive therapeutic markers for targeting by identifying genes which 
persist through chemotherapy. Finally, we expect to define the relationship 
between tumor subtype and DTC profile. We believe that many of the 
tumors with ERBB2 positive DTCs will have the Her2-enriched molecular 
subtype and that an equal number will likely be luminal B-like tumors or 
basal-like.  Most importantly, if successful, we will have demonstrated 
within the context of a clinical trial that therapeutic targeting of DTC 
predictive biomarkers interrupts the metastatic cascade and results in 
improved survival.  This will provide the foundation for future prospective 
trials based on this paradigm. 


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It is possible that we will have underestimated the risk of recurrence or 
benefit of the proposed treatments and need to adjust the number of 
patients needed to accrue into the trial.  To address this, we will perform 
an interim analysis after one year, review the results with our statistician 
and revise the trial accordingly. It is possible that we will observe 
elimination of DTCs with trastuzumab therapy but no improvement in 
recurrence rate. This may be due to the presence of residual 
subpopulations of DTCs with metastatic potential, such as those 
expressing the cancer stem cell/hedgehog pathway gene PTCH1, which 
are not eliminated by trastuzumab/chemotherapy.  If this is the case, we 
will attempt to identify genes which are associated with these cells for future 
targeting within the context of a clinical trial. Finally, we may observe that 
all patients enrolled into the trial will have a Her2-enriched molecular 
subtype of their primary tumor, if this is the case if will obviate the need to 
focus on DTCs for ERBB2 analysis, but we will have identified a population 
of patients who will benefit from ERBB2-directed therapy. 
 
1.9 
Study Rationale 
 
Data suggest that micrometastases that persist despite chemotherapy are likely 
enriched with cells that have stem cell-like features that are responsible for 
subsequent disease recurrence.  Bone marrow disseminated tumor cells (DTCs) 
can be used as a surrogate for systemic micrometastases.  Discordant Her2 
expression between primary breast cancer and CTCs/DTCs have been reported.  
Clinical trial data has identified a yet undefined subgroup of patients with Her2-
negative primary tumors who benefit from trastuzumab therapy. We hypothesize 
that the subgroup of patients with Her2-negative primary tumors and ERBB2-
positive DTCs will benefit from trastuzumab therapy and that administering 
targeted trastuzumab therapy to these patients will result in the elimination of 
ERBB2 overexpressing DTCs and improved DFS as measured by recurrence rate.  
With the correlative studies, we hope to define the relationship between tumor 
subtype and DTC profile as well as identify new predictive therapeutic markers for 
targeting by identifying genes which persist through chemotherapy.  Results from 
this trial will provide a critical foundation for future definitive studies assessing the 
effectiveness of trastuzumab in reducing breast cancer recurrence in these high-
risk patients. 
 
 
2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
Evaluate 3 year recurrence and death rates in patients treated with trastuzumab 
administered with chemotherapy for 12 months versus standard chemotherapy 
alone 
 
2.2 
Secondary Objective 
 
Evaluate the effect of trastuzumab administered for 12 months on the elimination 
of ERBB2 overexpressing bone marrow DTCs in patients with early stage HER2-


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negative breast cancer who have ERRB2 overexpressing bone marrow DTCs in 
their bone marrow prior to treatment (surgery and chemotherapy). 
 
2.3 
Exploratory Objectives 
 
• 
Examine primary breast cancer specimens molecular subtypes of breast 
cancer by PAM50 and correlate with BM expression of ERBB2 and 
treatment outcome  
 
• 
Examine BM for other DTC specific gene expression using multiplex gene 
technology and correlate with response to trastuzumab and outcome 
 
 
3.0 
ELIGIBILITY CRITERIA 
 
Since bone marrow aspiration for DTCs is not routinely performed as standard clinical 
practice, this study includes a pre-registration phase to enroll patients who meet all 
eligibility criteria listed in Section 3.1. In the pre-registration phase, patients will be 
consented for the bone marrow aspiration to test for the presence or absence of DTC 
overexpressing ERBB2. Analysis of the bone marrow will be performed at the Washington 
University Molecular Diagnostics Laboratory at Barnes-Jewish Hospital and Washington 
University School of Medicine.  Patients who are found to have DTCs overexpressing 
ERBB2 in the bone marrow are eligible for further study registration and treatment. 
Patients who do not have DTC which overexpress ERBB2 will not be eligible for further 
study intervention.  Bone marrow collection and testing will be covered by research funds 
and will not be charged to the patient or her insurance. 
 
Note: All patients who are eligible to enroll into the trial after their bone marrow has been 
screened for ERBB2-overexpressing DTCs will be eligible to participate in other trials if 
the primary and secondary endpoints of this trial will not be compromised nor the 
endpoints of the secondary trial which the patient is offered. The endpoints and treatments 
of the secondary trials will be carefully screened to ensure that there will be no interference 
with the interpretation of the endpoints of the primary trial. All patients who are screened 
for this trial but are ineligible to continue due to the status of their bone marrow may 
participate in other trials. 
 
3.1 
Pre-Registration Eligibility 
 
3.1.1 Pre-Registration Inclusion Criteria 
 
1. Histologically confirmed HER2-negative primary invasive ductal or 
invasive lobular breast carcinoma.  For patients enrolling for 
neoadjuvant treatment, diagnosis must be clinical stage II or III; for 
patients enrolling for adjuvant treatment, diagnosis must be pathologic 
stage IIA to IIIC. 
 
Standard HER2 testing will be performed in the surgical specimen at 
Washington University according to the standard of care in the 
Department of Pathology.  A HER2-negative primary breast cancer 
sample from a patient eligible for randomization should have a HER2 
IHC score of 0 or  1+ Those patients with IHC score of 2+ should be 
HER2 FISH-negative in standard testing. 


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Patient will have undergone staging studies including a CT of the 
chest/abdomen/pelvis and bone scan and/or PET scan either prior to 
the initiation of treatment or prior to entry into the trial. 
 
In addition, patients with non-metastatic, HER2-negative, recurrent 
tumors who need chemotherapy are eligible. 
  
2. Planning 
to 
receive 
best 
practice 
adjuvant 
or 
neoadjuvant 
chemotherapy according to institutional guidelines. Adjuvant tamoxifen 
or aromatase inhibitors treatment will be allowed for hormone receptor-
positive patients.  Patients who have failed neoadjuvant endocrine 
therapy will also be eligible. 
 
3. At least 18 years old. 
 
4. ECOG performance status < 1. 
 
5. Patient (or legally authorized representative) must be able to 
understand and willing to sign a written informed consent document. 
 
3.1.2 Pre-Registration Exclusion Criteria 
 
1. Prior chemotherapy for this cancer (excluding initiation of best practice 
chemotherapy to be given as standard of care described in Section 
5.3.1, which may be initiated after the pre-registration bone marrow 
collection but before final confirmation of eligibility and randomization). 
 
2. Previous treatment with trastuzumab or any other Her2 targeted 
therapy. 
 
3. Presence of an uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, symptomatic congestive heart 
failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situations that would limit compliance with study 
requirements. 
 
 


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3.2 
Registration Eligibility 
 
3.2.1 Registration Inclusion Criteria 
 
1. Presence of bone marrow ERBB2 overexpressing DTCs at the time of 
diagnosis; bone marrow aspiration will be performed in consented 
patients to evaluate DTCs following pre-registration provided patients 
meet all eligibility criteria as described in this section. 
 
2. ECOG performance status < 1. 
 
3. Adequate cardiac function as demonstrated by LVEF of >55% 
performed no more than 4 weeks prior to randomization. 
 
4. Normal organ and marrow function as defined below: 
 
• 
leukocytes >3,000/mcL 
• 
absolute neutrophil count >1,500/mcL 
• 
platelets >100,000/mcL 
• 
hemoglobin > 10 g/dL 
• 
total bilirubin within institutional upper limits of normal unless related 
to primary disease 
• 
AST(SGOT)/ALT(SGPT) <2.0 X institutional upper limit of normal 
• 
Creatinine < 1.5  institutional upper limits of normal OR creatinine 
clearance >60 mL/min/1.73 m2 for patients with creatinine levels 
above institutional normal 
 
5. If a woman of childbearing potential, patient must use two forms of 
effective contraception for a minimum of 6 months following 
trastuzumab. Effective methods of birth control include use of 
established oral, injected, or implanted hormonal methods of birth 
control, IUD, IUS, and condoms.   
 
3.2.2 Registration Exclusion Criteria 
 
1. Evidence of distant metastasis present by CT scan, bone scan, or 
physical exam. 
 
2. History of allergic reactions attributed to compounds of similar chemical 
or biologic composition to trastuzumab. 
 
3. Prior chemotherapy for this cancer (excluding initiation of best practice 
chemotherapy to be given as standard of care described in Section 
5.3.1, which may be initiated after the pre-registration bone marrow 
collection but before final confirmation of eligibility and randomization). 
 
4. History of other malignancy < 5 years previous with the exception of 
basal cell or squamous cell carcinoma of the skin which were treated 
with local resection only or carcinoma in situ of the cervix. 
 


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5. Pregnant or breastfeeding.  Patient must have a negative serum 
pregnancy test < 7 days from date of registration (if a woman of 
childbearing potential). 
 
Women of childbearing potential are defined as follows: 
• 
Women with regular menses 
• 
Women with amenorrhea, irregular cycles, or using a contraceptive 
method that precludes withdrawal bleeding 
• 
Women who have had a tubal ligation. 
 
Women are considered not to be of childbearing potential for the 
following reasons: 
• 
The patient has undergone hysterectomy and/or bilateral 
oophorectomy. 
• 
The patient is post-menopausal defined by amenorrhea for at least 
1 year in a woman > 45 years old.  
 
6. Clinically important history of active liver disease, including viral or 
other hepatitis or cirrhosis. 
 
7. Uncontrolled hypocalcemia, hypomagnesemia, hyponatremia, or 
hypokalemia defined as less than the lower limit of normal for the 
institution despite adequate electrolyte supplementation. 
 
8. Symptomatic intrinsic lung disease or extensive tumor involvement of 
the lungs resulting in dyspnea at rest. 
 
3.3 
Inclusion of Women and Minorities  
 
Breast cancer is rare in men and children. Therefore, this trial is only open to 
women of all races and ethnic groups. 
 
  
4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the 
Siteman Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirmation of patient eligibility includes the information listed below: 
 
1. Registering coordinator’s name and contact information 
2. Registering MD’s name 
3. Patient’s race, sex, and DOB 
4. Copy of signed consent form 


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5. Completed eligibility checklist, signed and dated by a member of the study 
team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
Patients must be registered through the Siteman Cancer Center database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has 
no middle initial, a dash will be used on the case report forms (CRFs).  All data will 
be recorded with this identification number on the appropriate CRFs. 
 
 
5.0 
TREATMENT PLAN  
 
5.1 
Pre-Intervention Phase 
 
Patients who meet all eligibility criteria listed in Section 3.1 with the exception of 
bone marrow status for DTCs (inclusion criterion #2) can be pre-registered.  At that 
point, consent will be obtained, and bone marrow will be collected and analyzed 
for ERBB2 expression.  The patient will be informed whether she is eligible to 
continue in the study based on the results of the bone marrow testing. The patient 
is also to be informed that the clinical significance for the presence or absence of 
bone marrow DTCs is not yet clear and is used only as a marker in this trial. 
 
Only patients with ERBB2-positive bone marrow DTCs will be eligible for 
randomization.  The descriptions for the bone marrow collection procedure, sample 
processing, and analysis are detailed in Section 9.0.  Bone marrow collection and 
analysis for DTCs will be covered by protocol funds and will not be charged to the 
patient or her insurance.  Patients without ERBB2-positive bone marrow DTCs will 
be approached for enrollment to a companion trial, HRPO# 201310088. 
 
5.2 
Randomization  
 
5.2.1 Original Randomization Plan 
 
Upon the receipt of the bone marrow DTC status, eligibility will be 
confirmed and signed informed consent verified. Still-eligible and 
consenting patients will be randomized to receive either standard of care 
treatment (Arm 1) or trastuzumab (Arm 2) as recommended below. 
Randomization may take place before or after the start of standard of care 
chemotherapy as long as there is time for at least 8 weeks of overlap of 
SOC chemo and trastuzumab if the patient is randomized to Arm 2.  
Approximately the same number of patients will be assigned to each 
treatment group.  A stratified, permuted block randomization will be used 
to balance as closely as possible the number of patients in each arm by 
lymph node status (positive vs. negative), tumor size (< 3cm vs. ≥ 3cm), 
estrogen receptor status of the primary tumor (positive vs. negative), and 
time of surgery (pre- vs. post-chemotherapy).  Randomization will be in 


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blocks of random size. The randomization table will be uploaded in our 
REDCap system.  Randomization will occur via an online form with entry 
of the patient ID number and stratum information.  Once all information is 
entered, randomization is carried out via a submit button through REDCap.  
The randomization scheme will be created using a formal probability model 
implemented in SAS (version 9.3 or higher). 
 
5.2.2 Current Randomization Plan (Amendment #6) 
 
Five patients have been randomized as described above, 4 to Arm 1 (SOC 
treatment) and 1 to Arm 2 (trastuzumab). Following study reopening to 
accrual, all patients meeting screening requirements will be randomized in 
a 3:1 allocation ratio (trastuzumab (Arm 2) : no trastuzumab (Arm 1)). Ten 
to 12 patients are expected to be randomized. Randomization will be 
unstratified and in blocks of 4. The randomization scheme will be created 
using a formal probability model implemented in SAS v9.4/STAT13.1. The 
randomization table will be uploaded into our REDCap system.  Once the 
randomization table is uploaded is will be locked and unalterable until the 
study is closed to further accrual. Randomization will be carried out by the 
clinical research assistant using an online form. 
 
5.3 
Agent Administration 
 
5.3.1 Standard Chemotherapy 
 
Patients in both arms will receive best practice standard chemotherapy 
according to NCCN guidelines.  The 5 chemo backbone options are: 
• 
Doxorubicin (or epirubicin) plus cyclophosphamide followed by 
paclitaxel (or docetaxel) 
• 
Docetaxel plus cyclophosphamide 
• 
Single agent paclitaxel 
• 
Docetaxel plus carboplatin 
• 
Fluorouracil plus epirubicin plus cyclophosphamide followed by 
paclitaxel (or docetaxel) 
 
Patients are allowed to start chemotherapy after the pre-registration bone 
marrow collection but before confirmation of eligibility and before 
randomization.  Patients randomized to Arm 2 must have a minimum of 8 
weeks of overlap with standard of care chemotherapy and trastuzumab.   
 
5.3.2 Trastuzumab 
 
Patients randomized to the trastuzumab treatment arm (Arm 2) will also 
receive IV trastuzumab for a total of 52 weeks.  Treatment with trastuzumab 
must be initiated such that there is a minimum of 8 weeks of overlap with 
the standard of care chemotherapy.  The dosing of trastuzumab when 
given concurrently with standard of care chemo depends on the treatment 
cycles for the standard of care chemo.  Trastuzumab may be given weekly, 
every 2 weeks, or every 3 weeks.  If given weekly, the loading dose will be 
4 mg/kg IV over 90 minutes and the subsequent doses that overlap with 
the standard of care chemo will be 2 mg/kg IV over 30 minutes.  If given 


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every 2 weeks, the loading dose will be 6 mg/kg IV over 90 minutes and 
the subsequent doses that overlap with the standard of care chemo will be 
4 mg/kg IV over 30 minutes.  If given every 3 weeks, the loading dose will 
be 8mg/kg IV over 90 minutes and the subsequent doses that overlap with 
the standard of care chemo will be 6 mg/kg IV over 30-90 minutes.  Please 
note that trastuzumab shall not be given concurrently with any 
anthracyclines. 
 
After standard of care chemo has concluded, all remaining doses of 
trastuzumab will be given at 6 mg/kg IV over 30-90 minutes every 3 weeks. 
Total length of trastuzumab administration is 52 weeks, including the time 
period during which trastuzumab is being administered concurrently with 
standard of care chemo. 
 
Definitive surgery may be performed prior to the initiation of chemotherapy, 
after the completion of part of the standard of care chemo, or after the 
completion of all of the standard of care chemo at the discretion of the 
treating surgeon. 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
Medications required to treat adverse events and manage cancer symptoms, 
concurrent stable disease (e.g., controlled hypertension), and pain medications 
are allowed. 
 
The patient must notify a member of the research team about any new medications 
she takes after the start of the study medication. 
 
5.5 
Women of Childbearing Potential 
 
Women of childbearing potential (defined as women with regular menses, women 
with amenorrhea, women with irregular cycles, women using a contraceptive 
method that precludes withdrawal bleeding, and women who have had a tubal 
ligation) are required to have a negative urine pregnancy test within 7 days prior 
to the date of registration and again within 7 days prior to the first dose of 
trastuzumab (if randomized to Arm 2).   
 
Patients are required to use two forms of acceptable contraception, including one 
barrier method, during participation in the study and for 7 months following the last 
dose of trastuzumab.  
 
If a patient is suspected to be pregnant, all study drugs should be immediately 
discontinued.  In addition, a positive urine test must be confirmed by a serum 
pregnancy test.  If it is confirmed that the patient is not pregnant, the patient may 
resume dosing. 
 
If a patient becomes pregnant during therapy or within 7 months after the last dose 
of trastuzumab, the investigator must be notified in order to facilitate outcome 
follow-up. 
 
 
 


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5.6 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to 
the patient’s health and/or the patient no longer wishes to continue protocol 
therapy, the protocol therapy should be discontinued and the reason(s) for 
discontinuation documented in the case report forms.  
 
In the absence of treatment delays due to AEs, treatment may continue until the 
end of standard of care chemo (for patients randomized to Arm 1) or approximately 
14 months (time of treatment with standard of care chemo plus one full year of 
trastuzumab) (for patients randomized to Arm 2) or until one of the following criteria 
applies: 
 
• 
Documented and confirmed disease progression 
• 
Death 
• 
Adverse event(s) that, in the judgment of the Investigator, may cause 
severe or permanent harm or which rule out continuation of study drug 
• 
General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
• 
Suspected pregnancy 
• 
Major violation of the study protocol 
• 
Lost to follow-up 
• 
Patient withdrawal 
• 
The PI decides to remove the patient from study 
• 
The Siteman Cancer Center decides to close the study 
 
Subjects who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar, Section 11.0. 
 
5.7 
Duration of Follow Up 
 
Patients will be followed every 3 months for 2 years then every 6 months for 3 
years or until death, whichever occurs first.  Patients removed from study for 
unacceptable adverse events will be followed until resolution or stabilization of the 
adverse event.  Follow-up for all patients begins at the end of standard of care 
chemo or at time of surgery (depending on which is last). 
 
 


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6.0 
DOSING DELAYS/DOSE MODIFICATIONS  
 
6.1 
Trastuzumab 
 
6.1.1 Management of Trastuzumab Side Effects 
 
For the first (loading) dose of trastuzumab, premedication with 
acetaminophen 650 mg PO will be given. 
 
Patients should not miss more than one dose of trastuzumab 
consecutively.  Patients do not have to make up missed doses. 
 
6.1.2 Dose Modifications for Trastuzumab 
 
Dose modification of trastuzumab is not permitted except as described in 
Sections 5.3.2 and 6.1.4. 
 
6.1.3 Infusion-associated Symptoms with Trastuzumab 
 
Infusion reactions consist of a symptom complex characterized by fever 
and chills, and on occasion nausea, vomiting, pain (in some cases at tumor 
sites), headache, dizziness, hypotension, rash, and asthenia.  In 
postmarketing reports, serious and fatal infusion reactions have been 
reported.  Severe reactions which include bronchospasm, anaphylaxis, 
angioedema, hypoxia, and severe hypotension were usually reported 
during or immediately following the initial infusion.  However, the onset and 
clinical course were variable including progressive worsening, initial 
improvement followed by clinical deterioration, or delayed post-infusion 
events with rapid clinical deterioration.  For fatal events, death occurred 
within hours to days following a serious infusion reaction. 
 
Interrupt trastuzumab infusion in all patients experiencing dyspnea, 
clinically significant hypotension, and intervention of medical therapy 
administered, 
which 
may 
include: 
epinephrine, 
corticosteroids, 
diphenhydramine, bronchodilators, and oxygen.  Patients should be 
evaluated and carefully monitored until complete resolution of signs and 
symptoms.  The rate of infusion should be decreased for mild or moderate 
(CTCAE 4.0 grade 1 or 2) infusion reactions.  Permanent discontinuation 
should be strongly considered in all patients with severe (grade 3 or 4) 
infusion reactions. 
 
6.1.4 Cardiac Dysfunction 
 
Signs and symptoms of cardiac dysfunction were observed in a number of 
women who received trastuzumab alone or in combination with 
chemotherapy, most often anthracycline-based treatment.  Cardiac 
dysfunction was observed most frequently among patients who received 
trastuzumab plus AC chemotherapy (28%), compared with those who 
received AC alone (7%), trastuzumab plus paclitaxel (11%), paclitaxel 
alone (1%), or trastuzumab alone (7%).  Severe disability or fatal outcome 
due to cardiac dysfunction was observed in ~1% of all patients. 
 


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The nature of the observed cardiac dysfunction was similar to the 
syndrome of anthracycline-induced cardiomyopathy.  The signs and 
symptoms of cardiac dysfunction usually responded to treatment.  
Complete and partial responses were observed among patients with 
cardiac dysfunction.  The risk appears to be independent of tumor response 
to therapy.  Analysis of the clinical database for predictors of cardiac 
dysfunction revealed only advanced age and exposure to an anthracycline 
as possible risk factors.  In the clinical trials, most patients with cardiac 
dysfunction responded to appropriate medical therapy, often including 
discontinuation of trastuzumab.  In many cases, patients were able to 
resume treatment with trastuzumab.  In a subsequent study using weekly 
paclitaxel and trastuzumab as first-line treatment for metastatic breast 
cancer, the observed incidence of serious cardiac dysfunction was 3% 
(N=95) (Seidmen et al. 2001).  Since the occurrence of cardiac dysfunction 
in the trastuzumab plus chemotherapy trial was an unexpected 
observation, no information is available regarding the most appropriate 
method for monitoring cardiac function in patients receiving trastuzumab.  
Significant advances in the understanding and treatment of CHF have been 
made in the past several years, with many of the new drugs demonstrating 
the ability to normalize cardiac function. Patients who develop symptoms 
of congestive heart failure while on trastuzumab should be treated 
according to the HFSA guidelines (Appendix B).  
 
All patients randomized to Arm 2 must have a MUGA scan at baseline, and 
on a regular schedule throughout the course of the study.  Investigators are 
strongly urged to schedule MUGA scans at the same radiology facility 
where the patient’s baseline MUGA scan was done whenever possible.  
MUGA scans are required at protocol-specified time points and after any 
patient has any of the following: discontinuation of protocol therapy, 
congestive heart failure, breast cancer recurrence, or a second primary 
cancer.  
 
Post-surgical radiation therapy may be required in patients at risk for 
recurrence.  Whenever possible, irradiation to the internal mammary nodes 
should be avoided because of the concern for possible additional 
cardiotoxicity from the combination of trastuzumab and radiation therapy.  
Efforts should be taken to ensure that the volume of the heart irradiated is 
minimal.  Investigators are encouraged to discuss cardiac toxicity concerns 
with their radiation oncologists to ensure careful planning of the ports of 
left-sided lesions.   
 
Recommended Cardiac Monitoring 
Conduct thorough cardiac assessment, including history, physical 
examination, and determination of LVEF by MUGA scan. The following 
schedule is recommended: 
• 
Baseline LVEF measurement immediately prior to initiation of 
trastuzumab 
• 
LVEF measurements every 3 months during and upon completion 
of trastuzumab using the same modality  and same facility as used 
for baseline 
• 
Repeat LVEF measurement at 4 week intervals if trastuzumab is 
withheld for significant left ventricular cardiac dysfunction 


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• 
LVEF measurements every 6 months for at least 2 years following 
completion of trastuzumab as a component of adjuvant therapy. 
 
Asymptomatic Patients 
If a patient does not have significant symptoms related to LV dysfunction, 
administration of trastuzumab will depend on the absolute change in LVEF 
between baseline and follow-up assessments. 
 
Trastuzumab should be initiated in an asymptomatic patient if: 
• 
The LVEF increased or stayed the same; 
• 
The LVEF decreased by < 15 percentage points but is still at or 
above the lower limit of normal for the radiology facility. 
 
Trastuzumab is PROHIBITED in an asymptomatic patient if: 
• 
The LVEF decreased < 15 percentage points and is below the limit 
of normal for the radiology facility: 
• 
The LVEF decreased by 16 percentage points or more (regardless 
of lower limits of normal for the radiology facility)  
 
Withhold trastuzumab dosing for at least 4 weeks for either of the following: 
• 
> 16% absolute decrease in LVEF from pre-treatment values 
• 
LVEF below institutional limits of normal and > 10% absolute 
decrease in LVEF from pretreatment values. 
• 
Trastuzumab may be resumed if, within 4-8 weeks, the LVEF 
returns to normal limits and the absolute decrease from baseline is 
< 15%. 
• 
Permanently discontinue trastuzumab for a persistent (> 8 weeks) 
LVEF decline or for suspension of trastuzumab dosing on more 
than 3 occasions for cardiomyopathy. 
 
If a patient has significant symptoms related to left ventricular (LV) 
dysfunction, cardiac ischemia, or arrhythmia, initiation of trastuzumab is 
prohibited. 
 
6.1.5 Pulmonary Events 
 
Severe pulmonary events leading to death have been reported rarely with 
the use of trastuzumab in the postmarketing setting. Signs, symptoms and 
clinical findings include dyspnea, pulmonary infiltrates, pleural effusions, 
non-cardiogenic pulmonary edema, pulmonary insufficiency and hypoxia, 
and acute respiratory distress syndrome. These events may or may not 
occur as sequelae of infusion reactions. Patients with symptomatic intrinsic 
lung disease or with extensive tumor involvement of the lungs, resulting in 
dyspnea at rest, may be at greater risk of severe reactions. Other severe 
events reported rarely in the postmarketing setting include pneumonitis and 
pulmonary fibrosis. 
 
6.1.6 Hematologic Toxicity 
 
Hematologic toxicity is infrequent following the administration of 
trastuzumab as a single agent, with an incidence of Grade III toxicities for 


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WBC, platelets, hemoglobin all < 1%. No Grade IV toxicities were 
observed. 
 
6.1.7 Diarrhea 
 
Of patients treated with trastuzumab as a single agent, 25% experienced 
diarrhea. An increased incidence of diarrhea, primarily mild to moderate in 
severity, was observed in patients receiving trastuzumab in combination 
with chemotherapy. 
 
6.1.8 Infection 
 
An increased incidence of infections, primarily mild upper respiratory 
infections of minor clinical significance or catheter infections, was observed 
in patients receiving trastuzumab in combination with chemotherapy. 
 
6.1.9 Other Serious Adverse Events 
 
The following other serious adverse events occurred in at least one of the 
958 patients treated with trastuzumab in clinical studies: 
 
Body as a Whole: cellulitis, anaphylactoid reaction, ascites, hydrocephalus, 
radiation injury, deafness, amblyopia 
Cardiovascular: vascular thrombosis, pericardial effusion, heart arrest, 
hypotension, syncope, hemorrhage, shock, arrhythmia 
Digestive: hepatic failure, gastroenteritis, hematemesis, ileus, intestinal 
obstruction, colitis, esophageal ulcer, stomatitis, pancreatitis, hepatitis 
Endocrine: hypothyroidism 
Hematological: pancytopenia, acute leukemia, coagulation disorder, 
lymphangitis 
Metabolic: 
hypercalcemia, 
hypomagnesemia, 
hyponatremia, 
hypoglycemia, growth retardation, weight loss 
Musculoskeletal: pathological fractures, bone necrosis, myopathy 
Nervous: convulsion, ataxia, confusion, manic reaction 
Respiratory: apnea, pneumothorax, asthma, hypoxia, laryngitis 
Skin: herpes zoster, skin ulceration 
Urogenital: hydronephrosis, kidney failure, cervical cancer, hematuria, 
hemorrhagic cystitis, pyelonephritis 
 
6.2 
Standard of Care Chemotherapy 
 
All dose modifications for the standard of care chemotherapy regimens should be 
performed as per routine during standard of care administration. 
 


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7.0 
REGULATORY AND REPORTING REQUIREMENTS  
 
The entities providing oversight of safety and compliance with the protocol require 
reporting as outline below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all 
events meeting the definition of unanticipated problem or serious noncompliance be 
reported as outlined in Section 7.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined 
in Section 7.4.  In addition, any fatal or life-threatening adverse experiences where there 
is a reasonable possibility of relationship to study intervention must be reported. 
 
Genetech requires that all events be reports as outlined in Section 7.5. 
 
7.1 
Definitions 
 
7.1.1 Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject 
including any abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will 
be utilized for all toxicity reporting.  A copy of the CTCAE version 4.0 can 
be downloaded from the CTEP website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the 
definitions for the terms listed that should be used are those provided by 
the Department of Health and Human Services’ Office for Human Research 
Protections (OHRP).  A copy of this guidance can be found on OHRP’s 
website:  http://www.hhs.gov/ohrp/policy/advevntguid.html.   
 
7.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results 
in any of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical 
judgement, may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
 


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7.1.3 Unexpected Adverse Experience 
 
Definition:  any adverse drug experience, the specificity or severity of 
which is not consistent with the current investigator brochure (or risk 
information, if an IB is not required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA. 
 
7.1.4 Life-Threatening Adverse Experience 
 
Definition:  any adverse drug experience that places the subject (in the 
view of the investigator) at immediate risk of death from the reaction as it 
occurred, i.e., it does not include a reaction that, had it occurred in a more 
severe form, might have caused death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
7.1.5 Unanticipated Problems 
 
Definition: 
• 
unexpected (in terms of nature, severity, or frequency) given (a) the 
research procedures that are described in the protocol-related 
documents, such as the IRB-approved research protocol and informed 
consent document; and (b) the characteristics of the subject population 
being studied; 
 
• 
related or possibly related to participation in the research (in this 
guidance document, possibly related means there is a reasonable 
possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and 
 
• 
suggests that the research places subjects or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized. 
 
7.1.6 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies 
that govern human subjects research or failure to follow the determinations 
of the IRB.  Noncompliance may occur due to lack of knowledge or due to 
deliberate choice to ignore regulations, institutional policies, or 
determinations of the IRB. 
 
7.1.7 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in 
substantial harm to subjects or others, or that materially compromises the 
rights or welfare of participants. 
 
 


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7.1.8 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under 
the research team’s control. Exceptions apply only to a single participant 
or a singular situation. 
 
Local IRB pre-approval of all protocol exceptions must be obtained prior to 
the event. 
 
7.2 
Reporting to the Human Research Protection Office (HRPO) at 
Washington University 
 
The PI is required to promptly notify the IRB of the following events: 
 
• 
Any unanticipated problems involving risks to participants or others which 
occur at WU, any BJH or SLCH institution, or that impacts participants or 
the conduct of the study. 
 
• 
Noncompliance with federal regulations or the requirements or 
determinations of the IRB. 
 
• 
Receipt of new information that may impact the willingness of participants 
to participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence 
of the event or notification to the PI of the event.  The death of a research 
participant that qualifies as a reportable event should be reported within 1 working 
day of the occurrence of the event or notification to the PI of the event. 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgement via 
email to a QASMC auditor. 
 
7.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem at WU or any 
BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the 
review process need not be reported to QASMC.) 
 
7.4 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the Washington University principal investigator to report any 
unanticipated problem to the FDA as follows: 
 
• 
Report any unexpected fatal or life-threatening adverse experiences 
(Section 7.1.4) associated with use of the drug  by telephone or fax no later 
than 7 calendar days after initial receipt of the information.   


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Report any serious, unexpected adverse experiences (Section 7.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.  All MedWatch forms will be sent by the 
investigator or investigator’s team to the FDA at the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
7.5 
Reporting to Genentech 
 
Safety assessments will consist of monitoring and reporting AEs and SAEs that 
are considered related to trastuzumab, all events of death, and any study-specific 
issue of concern. 
 
For purposes of reporting to Genentech, an AE is any unfavorable and unintended 
sign, symptom, or disease temporally associated with the use of an investigational 
medicinal product or other protocol-imposed intervention, regardless of attribution.  
This includes the following: 
 
• 
AEs not previously observed in the subject that emerge during the protocol-
specified AE reporting period, including signs or symptoms associated with 
breast cancer 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). 
• 
If applicable, AEs that occur prior to assignment of study treatment 
associated with medication wash-out, no treatment run-in, or other protocol 
mandated intervention. 
• 
Pre-existing medical conditions (other than breast cancer) judged by the 
investigator to have worsened in severity or frequency or changed in 
character during the protocol-specified AE reporting period. 
 
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 investigational medicinal product. 
• 
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.) 
 


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All AEs and SAEs whether volunteered by the subject, discovered by study 
personnel during questioning, or detected through physical examination, 
laboratory test, or other means will be 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} (see 
following guidance), and actions taken. 
 
To ensure consistency of AE and SAE causality assessments, investigators should 
apply the following general guideline: 
 
Yes 
 
There is a plausible temporal relationship between the onset of the AE and 
administration of trastuzumab 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 trastuzumab; and/or the AE abates or 
resolves upon discontinuation of trastuzumab or dose reduction and, if applicable, 
reappears upon re-challenge. 
 
No 
 
Evidence exists that the AE has an etiology other than trastuzumab (e.g., 
preexisting medical condition, underlying disease, intercurrent illness, or 
concomitant medication); and/or the AE has no plausible temporal relationship to 
trastuzumab administration (e.g., cancer diagnosed 2 days after first dose of 
trastuzumab). 
 
Expected adverse events are those adverse events that are listed or characterized 
in the Package Insert or current Investigator Brochure.  
 
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. 
 
7.5.1 Specific Instructions for Recording Adverse Events 
 
Investigators should use correct medical terminology/concepts when 
reporting AEs or SAEs. Avoid colloquialisms and abbreviations. 
 
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. 


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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”. 
 
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 re-assessed 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”). 
 
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. 
 
Pregnancy 
If a female subject becomes pregnant while receiving investigational 
therapy or within 90 days after the last dose of study drug, a report should 
be completed and expeditiously submitted to the 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 exposed 
to the {study drug} should be reported as an SAE. 
 
Post-Study Adverse Events 
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 


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conceived offspring of a female subject who participated in the study, this 
should be reported as an SAE.  
 
Reconciliation 
The Sponsor agrees to conduct reconciliation for the product. 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. 
 
7.5.2 Reporting Instructions 
 
Investigators must 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 the trastuzumab (regardless 
of causality) will be transmitted to Genentech within fifteen (15) 
calendar days of the Awareness Date. 
• 
Serious AE reports that are unrelated to the trastuzumab will be 
transmitted to Genentech within thirty (30) calendar days of the 
Awareness Date. 
• 
Additional Reporting Requirements to Genentech include the 
following: 
o Any reports of pregnancy following the start of administration 
with the trastuzumab will be transmitted to Genentech within 
thirty (30) calendar days of the Awareness Date.  
o All Non-serious Adverse Events originating from the Study will 
be forwarded in a semi-annual report to Genentech. 
 
All written IND safety report submitted to the FDA by the investigator must 
also be faxed to Genentech Drug Safety at one of the numbers above.  
Additionally, all IND annual reports submitted to the FDA by the Sponsor-
Investigator should be copied to Genentech.  Copies of such reports should 
be faxed to Genentech Drug Safety at one of the numbers above.  Any 
study report submitted to the FDA by the Sponsor-Investigator 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.  Copies of such 
reports should be mailed to the assigned Clinical Operations Contact for 
the study. 
 
7.6 
Timeframe for Reporting Required Events  
 


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The study period during which all AEs and SAEs must be reported begins after 
informed consent is obtained and initiation of study procedures and ends 30 days 
following the last day of study treatment or study discontinuation/termination, 
whichever is earlier.  After this period, investigators should only report SAEs that 
are attributed to prior study treatment. 
 
 
8.0 
PHARMACEUTICAL INFORMATION 
 
8.1 
Trastuzumab 
 
8.1.1 Description  
 
Trastuzumab (Herceptin) is a recombinant DNA-derived humanized 
monoclonal antibody that selectively binds with high affinity to the 
extracellular domain of HER2 (Kd = 5 nM)143-144.  The antibody is an IgG1 
kappa that contains human framework regions with the complementarity-
determining regions of a murine antibody (4D5) that binds to HER2.  
 
8.1.2 Pharmacokinetics and Drug Metabolism 
 
Trastuzumab administered once weekly demonstrated dose-dependent 
pharmacokinetics. Mean half-life increased and clearance decreased with 
increasing dose level. The half-life averaged 1.7 and 12 days at the 10 and 
500 mg dose levels, respectively. Trastuzumab’s volume of distribution was 
approximately that of serum volume (44 mL/kg). At the highest weekly dose 
studied (500 mg), mean peak serum concentrations were 377 mcg/mL.  
 
In studies using a loading dose of 4 mg/kg followed by a weekly 
maintenance dose of 2 mg/kg, a mean half-life of 5.8 days (range = 1 to 32 
days) was observed. Between Weeks 16 and 32, trastuzumab serum 
concentrations reached a steady state with a mean trough and peak 
concentrations of approximately 79 microgram/mL and 123 microgram/mL, 
respectively.  
 
Data suggest that the disposition of trastuzumab is not altered based on 
age or serum creatinine (up to 2.0 mg/dL). No formal interaction studies 
have been performed.  
 
8.1.3 Supplier(s) 
 
Trastuzumab will be purchased from Genentech.   
 
 
8.1.4 Dosage Form and Preparation 
 
Trastuzumab is a sterile, white to pale yellow, preservative-free lyophilized 
powder for intravenous (IV) administration.  Each vial of trastuzumab 
contains 400 mg of trastuzumab, 9.9 mg of L-histidine HCl, 6.4 mg of 
L-histadine, 400 mg of α,α-trehalose dihydrate, and 1.8 mg of polysorbate 
20, USP.  Reconstitution with 20 mL of the supplied Bacteriostatic Water 
for Injection (BWFI) USP, containing 1.1% benzyl alcohol as a preservative, 


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yields 21 mL of a multidose solution containing 21 mg/mL trastuzumab, at 
a pH of ~6. 
 
Use appropriate aseptic technique.  Each vial of trastuzumab should be 
reconstituted with 20 mL of BWFI, USP, 1.1% benzyl alcohol preserved, as 
supplied, to yield a multidose solution containing 21 mg/mL trastuzumab.  
Immediately upon reconstitution with BWFI, the vial of trastuzumab must 
be labeled in the area marked “Do not use after” with the future date that is 
28 days from the date of reconstitution. 
 
If the patient has known hypersensitivity to benzyl alcohol, trastuzumab 
must 
be 
reconstituted 
with 
Sterile 
Water 
for 
Injection 
(see 
PRECAUTIONS).  Trastuzumab which has been reconstituted with SWFI 
must be used immediately and any unused portion discarded.  Use of other 
reconstitution diluents should be avoided. 
 
Determine the dose of trastuzumab needed. Calculate the correct dose 
using 21 mg/mL trastuzumab solution.  Withdraw this amount from the vial 
and add it to an infusion bag containing 250 mL of 0.9% sodium chloride, 
USP.  DEXTROSE (5%) SOLUTION SHOULD NOT BE USED.  Gently 
invert the bag to mix the solution.  The reconstituted preparation results in 
a colorless to pale yellow transparent solution.  Parenteral drug products 
should be inspected visually for particulates and discoloration prior to 
administration. 
 
No incompatibilities between trastuzumab and polyvinylchloride or 
polyethylene bags have been observed. 
 
Refer to Section 5.3.2 for dosing information. 
 
 


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8.1.5 Storage and Stability 
 
Vials of trastuzumab are stable at 2-8°C (36-46°F) prior to reconstitution. 
Do not use beyond the expiration date stamped on the vial. A vial of 
trastuzumab reconstituted with BWFI, as supplied, is stable for 28 days 
after reconstitution when stored refrigerated at 2-8°C (36-46°F), and the 
solution is preserved for multiple use. Discard any remaining multi-dose 
reconstituted solution after 28 days. If unpreserved SWFI (not supplied) is 
used, the reconstituted trastuzumab solution should be used immediately 
and any unused portion must be discarded. DO NOT FREEZE 
TRASTUZUMAB THAT HAS BEEN RECONSTITUTED. 
  
The solution of trastuzumab for infusion diluted in polyvinylchloride or 
polyethylene bags containing 0.9% Sodium Chloride Injection, USP, may 
be stored at 2-8°C (36-46°F) for up to 24 hours prior to use. Diluted 
trastuzumab has been shown to be stable for up to 24 hours at room 
temperature (2-25°C). However, since diluted trastuzumab contains no 
effective preservative, the reconstituted and diluted solution should be 
stored refrigerated (2-8°C).  
 
8.1.6 Administration 
 
Treatment may be administered in an outpatient setting by intravenous (IV) 
infusion over 90 minutes. DO NOT ADMINISTER AS AN IV PUSH OR 
BOLUS. Patients should be observed for fever and chills or other infusion-
associated symptoms. 
 
 
If trastuzumab is being administered concomitantly with chemotherapy, 
trastuzumab administration should precede chemotherapy administration.  
Patients 
should 
be 
observed 
for 
fever 
and 
chills 
or 
other 
infusion-associated symptoms.  If prior infusions are well tolerated, 
subsequent doses may be administered over 30 minutes. 
 
8.1.7 Special Handling Instructions 
 
None. 
 
 
9.0 
BONE MARROW ANALYSIS FOR STUDY ENTRY AND PRIMARY ENDPOINT  
 
9.1 
Collection of Bone Marrow Specimens 
 
Bone marrow aspirations for DTCs will be obtained at two time points: 
• 
Baseline (in order to determine eligibility for randomization)  
• 
6-18 months after 1st bone marrow aspiration  
 
Bone marrow collection and analysis will be paid for by research funds and will not 
be charged to the patient or her insurance. 
 


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The baseline bone marrow aspiration will be performed in the operating room at 
the time of portacath placement (for patients receiving neoadjuvant chemotherapy) 
or at the time of surgery (for patients receiving adjuvant chemotherapy).  The 6-18 
month bone marrow aspiration will be performed in the operating room at the time 
of portacath removal (for patients randomized to Arm 2) or at a scheduled 
appointment in the Center for Advanced Medicine (for patients randomized to Arm 
1 who do not keep the portacath for the full year).  
 
 
 
Sixteen cc of bone marrow will be collected from the right and/or left anterior iliac 
crest. After the collection of 8 cc the needle will be redirected to minimize the 
collection of blood.  If the patient tolerates the collection of bone marrow from one 
side, then bone marrow will be collected from the contralateral side. 
 
Subjects will be in the supine position, the right or left posterior superior iliac crest 
region or the anterior iliac crest will be identified, and the area will be sterilely 
prepped with an alcohol based solution.  
 
The region to be entered will be anesthetized with lidocaine and/or 
lidocaine/bupivacaine mixture and, following adequate anesthesia, an Illinois 
needle will be inserted into the iliac crest region.  
collection of bone marrow in the operating room
1 tube provided by Biocept or other vendor, 1-3 
Pax tubes per side, remainder in EDTA tubes
16 ml total from each iliac crest
1-3 Pax tubes/side to Cytogenomics 
and Molecular Pathology Lab
EDTA tubes to SCC TPC for storage
Pax tubes -
RNA preparation 
Perform  ERBB2 gene expression analysis on 
Fluidigm Biomark HD
multiplex gene detection system
or direct detectionof Her2-postive DTCss 
through commercial vendor
review  data for  presence of 
ERBB2 positive DTCs
extra RNA to tumor bank
Biocept (or other 
commercial lab)
total of 2mL (1mL from 
each iliac crest)


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The bone marrow cavity will be penetrated and approximately 16cc of bone 
marrow will be obtained from each iliac crest.  If BM is collected from both iliac 
crests, then 1-4 cc’s of BM from each side will be combined and placed in a tube 
provided by a commercial vendor for DTC enumeration/Her2+ determination.  The 
remainder of the BM will be divided between 1-3 PAXgene bone marrow RNA 
tubes (2 ml each) (Qiagen) and EDTA tubes.  
 
The procedure will be repeated on the contralateral side.  After aspiration, the 
needle will be removed.  
 
The puncture sites will be cleaned and bandaged. Each participant will receive 
printed wound care instructions containing contact information for medical 
assistance if the participant experiences any adverse symptoms or problems 
following the procedure.  
 
9.2 
Handling of Bone Marrow Specimens 
 
All tubes containing bone marrow will be labeled with an assigned code number 
and the site of collection.  
 
All PAXgene tubes from each side (1-3 per side, or 2-6 total) will be transported to 
the Molecular Diagnostics Laboratory (MDL) at Washington University and stored 
until analysis is requested.  If analyzed, two of the tubes will be used for RT-PCR 
analysis, and one tube will be stored.  The Cee Sure tubes or tubes from other 
commercial vendors will be shipped in the kits provided by the vendor to Biocept 
or other commercial vendor according to the vendor’s instructions. The EDTA 
tubes will be transported to the Siteman Cancer Center Tissue Procurement Core 
for processing and storage.  (Please refer to sample flow diagram.)   
 
9.3 
Bone Marrow Processing 
 
RT-PCR detection of bone marrow DTCs will be performed at the CLIA-certified 
Genomic and Pathology Service/Molecular diagnostics Laboratory (MDL)  at 
Washington University (GPS@WU) (CLIA# 26D0698685).  Direct detection of 
Her2+ DTCs will be performed by Biocept or other commercial vendor. 
 
ERBB2 status of bone marrow will be determined by a clinical grade qRT-PCR 
assay for ERBB2 expression in BM developed in the CLIA certified Molecular 
Diagnostic Laboratory using guidelines for the development of biomarkers145.   
RNA will be processed and purified from 2 PAXgene tubes per iliac crest side (right 
and left) using a commercially available protocol/kit designed for PAX tubes 
(Qaigen). Her2-expressing cell lines diluted into normal bone marrow will serve as 
the positive control. Commercially available primers/probe for ERBB2 and ERBB2 
related genes will be used (Applied Biosystems). PCR reactions will be run on a 
Fluidigm Biomark HD. Levels of expression relative to normal BM will be calculated 
as previously described using an established gene as an endogenous control137. 
Samples will be considered positive/over expressed if ERBB2 overexpression 
levels are 2 standard deviations above a pool of control bone marrows.  Validation 
and interassay variation coefficients will be determined using the breast cancer cell 
line SKBr3 cells (Her2 expressing) diluted into normal BM. Only patients ERRB2 
expression in their BM will be eligible to continue on the study and receive 


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trastuzumab for 12 months. A second BM aspirate will be performed after the 
completion of drug treatment. The unused portion of the BM specimens will be 
banked. 
 
9.3.1 Analysis of Her2+ DTCs by Commercial Vendors 
 
DTCs will be analyzed by Biocept or other commercial vendor for Her2+ 
expression by FISH.  Briefly, after Ficoll gradient enrichment, DTCs are 
immunomagnetically isolated using a cocktail of 10 antibodies.  The 
retrieved cells are stained for standard epithelial markers and for Her2+ 
amplification by FISH.  Any BM specimen that contains a Her2+ 
overexpressing DTC will be considered positive and the patient enrolled 
into the trial. 
 
9.3.2 RNA Quantification and Quality Control 
 
Aglient Bioanalyzer with Agilent Nano 6000 kits will be used to assess the 
quality of each RNA sample.  The Nanodrop 1000 spectrophotometer will 
be used to assess the quantity of each sample.  All RNAs to be used for 
qRT-PCR or NC analysis will have demonstrated a RIN score of > 7.0.  
 
cDNA will be prepared from RNA according to standard procedures. 
 
9.3.3 Analysis of Gene Expression 
 
(If funding is available only.) 
 
Samples of 300 ng will be loaded onto a Fluidigm Biomark HD Each sample 
will be run in duplicate, for a total of 8 samples per patient (2 from each iliac 
crest analyzed in duplicate). 
 
For each sample, each gene duplicate will be averaged and the ddCT will 
be calculated using reference genes. Fold overexpression from normal 
bone marrow will be calculated. Any gene 2 STD over the expression of 
the normal bone marrow pool from healthy volunteers will be considered 
overexpressed in that bone marrow. If a gene is overexpressed in both 
bone marrow specimens from the same side, the patient’s bone marrow 
will be considered positive for expression.  Bone marrows will be 
considered negative if  the all of the bone marrow samples are less than 2 
STD above normal bone marrow pool or if only one specimen on each side 
is 2STD above the normal bone marrow pool. 
 
 
 


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10.0 
CORRELATIVE STUDIES 
 
10.1 
Existing FFPE or Frozen Tumor Blocks 
 
Existing FFPE or frozen tumor blocks from a prior diagnostic biopsy or breast 
surgery will be requested and logged onto the Siteman Cancer Center Tissue 
Procurement Core. 
 
Tumor blocks will then be processed in the Tissue Procurement Core for tumor 
DNA/RNA extraction and tissue sectioning for IHC analysis.  H&E staining will be 
performed before the tissue processing to ensure high tumor content for future 
analysis.  If needed, micro or macro dissection will be performed to isolate tumor 
cells from the surrounding stroma. 
 
The following analyses will be performed for their potential predictive value in 
treatment response to trastuzumab: 
• 
Targeted DNA sequencing will be performed on tumor DNA for somatic 
mutations which could affect treatment response 
• 
To define the breast cancer molecular subtype; tumor RNA will be analyzed 
by at the Washington University Human Genome Sequencing Center for 
PAM50 classification of the tumor. 
 
10.2 
Optional Fresh Pre-Treatment Tumor 
 
A tumor biopsy using a 14-gauge needle may be performed prior to initiation of 
treatment if the tumor is easily accessible and the patient consents to this optional 
biopsy.  The tissue specimen will be taken to the Tissue Procurement Core for 
storage and shared with other investigators who have appropriate IRB approvals. 
 
10.3 
Multiplex Gene Detection of Bone Marrow  
 
(If funding is available only.) 
 
All bone marrow specimens will be analyzed by a multiplex gene detection system 
for expression of 38-53 genes associated with the presence of DTCs.  Gene 
expression analysis will be correlated with trastuzumab treatment either before or 
after microfilter enrichment for DTCs. 
 
Analysis to be performed based on genes expression and analysis of tumor 
specimens include: 
 
1. To correlate time to recurrence  with reduction in ERBB2 gene expression 
in bone marrow 
2. Correlate time to recurrence with the elimination of DTCs 
3. Assess the effect of trastuzumab on alterations in expression of other DTCs 
genes/pathways using multiplex gene detection   
 
 


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10.3.1 Analysis of DTC Expression Using Multiplex Gene Expression 
Assays 
 
RNA will be prepared from BM nucleated cells and examined for 
expression of a panel of genes using NC or FBHD. The assay will be 
performed using the manufacturer’s methods. Expression data will be 
normalized and analyzed using the manufacturer’s standard procedures139, 
146. All analyses will be performed in duplicate. Positive and negative 
controls will be included in each assay to normalize for assay based 
variation (differences in hybridization, purification and binding efficiency).  
Additionally, the data will be normalized against internal reference genes 
(housekeeping genes) to correct for differences in number of cells, absolute 
mRNA content and sample preparation. In replicate measurements, the 
average, the standard deviation and coefficient of variation percentage 
(%CV) is calculated using the normalized data. BM specimens will be 
considered to be 'positive' for gene expression if expression levels in BM 
are at least two standard deviations above the mean expression of the 20 
BM samples from healthy volunteers. Patients will be considered ‘positive’ 
for biomarker gene expression when detected in at least one of two bone 
marrow samples analyzed (left or right side).  For multiple gene testing by 
NC, BM will be considered positive if any one of the panel of genes is 
detected. 
 
10.4 
Serial Research Blood Collection (required) 
 
Thirty mL of blood (10 mL in a red top silica clot activator serum tube for serum, 
10 mL in a lavender top EDTA tube for plasma, and 10 mL in the cell-free DNA 
BCT tube for plasma circulating DNA) will be collected for future analysis of 
circulating markers at the following time points: 
 
• 
Baseline 
Note: an additional 10 mL of blood in a lavender top EDTA tube will be 
collected at this time point for germline DNA 
• 
Pre-treatment 
• 
Every 6 months (+/- 1 month) post-initiation of treatment for 5 years (or until 
disease relapse or end of study participation, whichever comes first)  
 
These samples will be transported to the Siteman Cancer Center Tissue 
Procurement Core for further processing and storage as described below.  
 
10.4.1 Serum Processing Instructions 
 
Ten mL blood in red top silica clot activator serum tube will be centrifuged 
at 1200G for 10 minutes at 4°C.  The serum should then be stored as 3 to 
5 vials of 1 mL aliquots at -70°C. 
 
10.4.2 Plasma Processing Instructions 
 
Ten mL blood in lavender top EDTA tube is mixed several times to ensure 
adequate anticoagulation and placed on ice.  Deliver tube to laboratory 
within 30 minutes of draw and spin at 1000G for 10 minutes at 4°C.  The 


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plasma will then be aspirated off in 3 to 5 vials of 1 mL aliquots at -70°C. 
 
10.4.3 Germline DNA Processing Instructions 
 
Ten mL blood in lavender top EDTA tube should be transported at room 
temperature and processed to cell pellet. 
 
10.4.4 Cell-free Plasma DNA Processing Instructions 
 
Ten mL blood in the cell-free DNA BCT (from Streck Clinical Laboratory 
Products) is immediately mixed by gentle inversion 8 to 10 times and 
transported at room temperature and processed to cell-free plasma for 
storage at -70°C.  Spin twice to ensure the plasma layer has no cellular 
contamination.  
 
10.5 
Quality of Life Assessments 
 
The EORTC QLQ-C30 and BR23 will be administered by a research coordinator 
to assess quality of life at the following time points: 
 
• 
Before initiation of chemotherapy 
• 
At one year (at the same time as the bone marrow aspiration) 


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page 51 of 94 
11.0 
STUDY CALENDAR 
 
11.1 
Arm 1 Study Calendar 
 
If the patient's condition is deteriorating, laboratory evaluations should be repeated 
no more than 48 hours before the next cycle of therapy. 
 
Pre- 
Reg 
Baseline 
Day 1 of 
each cycle 
Follow-
upa 
Off 
Study 
Informed consent 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical history  
X 
 
 
X 
X 
Con meds 
X 
 
X 
X 
X 
Adverse events 
X 
 
X 
X 
 
Physical exam, VS, ECOG PS 
X 
 
X 
X 
X 
Height 
X 
 
 
 
 
Weight 
X 
 
X 
 
X 
CBC w/diff, plts 
 
Xm 
X 
X 
X 
CMP  
 
Xm 
X 
X 
X 
β-HCGd 
 
Xm 
 
 
X 
ECHO (preferred) or MUGA 
Xk 
Xk 
 
 
 
CT/bone scan or PET scan 
Xj 
Xj 
 
 
 
Bone marrow collection 
Xe 
 
 
Xc 
 
Research blood draw 
X 
X 
Xn 
 
Tumor tissue 
 
Xh 
 
 
 
Definitive surgeryf 
 
Xf 
 
Xf 
 
Portacath placement 
 
X 
 
 
 
Chemotherapyb 
 
 
 
 
 
QOL assessments 
 
Xm 
 
Xc 
 
Mammogramg 
X 
 
 
X 
 
a. Q 3 mo for yrs 1-2, q 6 mo for yrs 3-5 starting at the end of standard of care chemo or surgery 
(depending on which is last)  
b. Best practice standard of care chemo to be given according to NCCN guidelines.  May start any 
time after the baseline bone marrow collection.  See Section 5.3.1 for details. 
c. 6-18 months after 1st bone marrow aspiration and, for research blood, one year following that 6-
18-month time point. 
d. Women of childbearing potential only. 
e. After eligibility is confirmed. 
f. Surgery may take place prior to initiation of chemotherapy, at any point during standard of care 
chemo, or at the end of standard of care chemo. 
g. Annual mammograms for patients with remaining breast tissue 
h. Archival tissue required, fresh tissue optional. 
j. This imaging need only occur once at either the pre-registration or baseline time point. 


Version 11/01/16 
 
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k. Only one ECHO or MUGA need take place; this must occur no more than 4 weeks prior to 
randomization. 
m. Must occur no more than 4 weeks prior to initiation of chemotherapy 
n. Q 6 mo after start of treatment for 5 yrs. 
 
11.2 
Arm 2 Study Calendar 
 
In the event that the patient's condition is deteriorating, laboratory evaluations should be 
repeated within 48 hours prior to initiation of the next cycle of therapy. 
 
 
Pre- 
Reg 
Baseline 
Active Treatment 
Follow-
upa 
1 year from 
the date of 
trastuzumab 
completion 
 
Off 
Study 
Day 1 of 
each cycle 
12 mo after 
start of 
trastuzumab 
Informed consent 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Medical history  
X 
 
 
 
X 
X 
X 
Con meds 
X 
 
X 
 
X 
X 
X 
Adverse events 
X 
 
X 
X 
X 
 
 
Physical exam, VS, ECOG PS 
X 
 
Xq 
 
X 
X 
X 
Height 
X 
 
 
 
 
 
 
Weight 
X 
 
X 
 
 
 
X 
CBC w/diff, plts 
 
Xo 
Xr 
X 
X 
X 
X 
CMP  
 
Xo 
Xr 
X 
X 
X 
X 
β-HCGe 
 
Xo 
Xe 
 
 
X 
X 
ECHO (preferred) or MUGAb 
Xn 
Xn 
Xb 
 
Xb 
 
 
CT/bone scan or PET scan 
Xm 
Xm 
 
 
 
 
 
Bone marrow collection 
Xf 
 
 
Xg 
 
 
 
Research blood draw 
X 
X 
Xp 
 
Tumor tissue  
 
Xk 
 
 
 
 
 
Definitive surgeryh 
 
 
 
 
 
 
 
Portacath placement and removal 
 
X 
 
X 
 
 
 
Chemotherapyc 
 
 
 
 
 
 
 
Trastuzumabd 
 
 
 
 
 
 
 
QOL assessments 
 
Xo 
 
Xg 
 
 
 
Mammogramj 
X 
 
 
 
X 
 
 
a. Q 3 mo for yrs 1-2, q 6 mo for yrs 3-5 starting at the end of standard of care chemo or surgery (depending on 
which is last).  
b. Repeat LVEF using the same methodology as at baseline will be obtained every 3 months following initiation of 
trastuzumab, or every 4 weeks if trastuzumab is held for significant left ventricular cardiac dysfunction.  LVEF 
measurements should be taken after completion of trastuzumab as clinically indicated. 
c. Best practice standard of care chemo to be given according to NCCN guidelines.  May start any time after the 
baseline bone marrow collection.  See Section 5.3.1 for details. 
d. Patients randomized to receive trastuzumab must have a minimum of 8 weeks of overlap with standard of care 


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chemo and trastuzumab.  Trastuzumab is to be administered first on days when it is given with standard of care 
chemo.  See Section 5.3.2 for details. 
e. Women of childbearing potential only – at screening and within 7 days of the first dose of trastuzumab. 
f. After eligibility is confirmed. 
g. 6-18 months after 1st bone marrow aspiration. 
h. Surgery may take place prior to initiation of chemotherapy, at any point during standard of care chemo, or at the 
end of standard of care chemo. 
j. Annual mammograms for patients with remaining breast tissue 
k. Archival tissue required, fresh tissue optional. 
m. This imaging need only occur once at either the pre-registration or baseline time point. 
n. Only one ECHO or MUGA need take place; this must occur no more than 4 weeks prior to randomization. 
o. Must occur no more than 4 weeks prior to initiation of chemotherapy. 
p. Q 6 mo after start of treatment for 5 yrs. 
q. Patient should be seen on Day 1 of each cycle while receiving SOC chemo, but may be seen every 3rd cycle 
once SOC chemo has concluded and patient is receiving trastuzumab only. 
r. CBC and CMP should be drawn on Day 1 of each cycle while receiving SOC chemo, and then every 3rd cycle 
once SOC chemo has concluded and patient is receiving trastuzumab only. 
 
 
12.0 
DATA SUBMISSION SCHEDULE 
 
Form 
Submission schedule 
Original Consent Form 
Bone Marrow Form 
Prior to registration 
Eligibility Checklist 
On Study Form 
Prior Therapy Form 
Research Blood Form 
EORTC QLQ-C30 Form 
EORTC BR23 Form  
At baseline  
Randomization Form 
Time of randomization 
On Study Standard Chemo Summary 
Form 
End of standard chemotherapy 
Trastuzumab Treatment Record 
Within 4 weeks of each cycle during 
trastuzumab therapy 
Bone Marrow Form 
EORTC QLQ-C30 Form 
EORTC BR23 Form 
6-18 months after first BM aspiration 
1 year after second BM aspiration (blood 
only) 
Research Blood Form 
Screening 
Baseline 
Every 6 months for 5 years after start of 
treatment 
Treatment Summary Form 
End of treatment 
Follow-up Form 
Every 3 months for the first 2 years, then 
every 6 months for the next 3 years 
Adverse Events Form 
ongoing 
SAE Reporting Form 
At time of any SAE 
 
 
13.0 
MEASUREMENT OF EFFECT 
 
The primary endpoints are 3 year recurrence rate and 3 year death rate.  Disease 
recurrence is defined as the documented appearance of local (breast, chest wall, axillary, 
supraclavicular nodes) or distant disease.  Assessment for recurrence will begin at the 
time at which the patient is considered to be disease-free; for the purposes of this protocol, 
this time will be the time point at which the patient has completed both standard 
chemotherapy and definitive surgery.  Surgery may take place either before, during, or 


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after standard chemotherapy.  Patients will be followed for 3 years after the completion of 
standard chemotherapy + surgery for the detection of any recurrent disease.  For patients 
randomized to Arm 2, some of this assessment for recurrent disease will take place during 
the time when they are receiving trastuzumab. 
 
The secondary endpoints of this study are the effectiveness of trastuzumab in clearing 
ERBB2-positive bone marrow DTCs.  Bone marrow DTCs are evaluated by RT-PCR 
performed on specimens collected 6-18 months apart (one before and one after therapy).  
The proportion of samples turned negative after therapy will be calculated.   Samples will 
be considered negative for ERBB2-expression if expression from bone marrow collected 
from each iliac crest is less than 2 standard deviations above the ERBB2-level in pooled 
normal bone marrow specimens.   
 
The exploratory endpoints are the primary tumor molecular subtype by PAM50 assay, 
DTC-specific gene expression by multiplex gene expression analyses and response to 
trastuzumab. Their association with BM expression of ERBB2, response to trastuzumab 
and treatment outcome (disease progression and death) will be examined. 
 
 
14.0 
DATA AND SAFETY MONITORING 
  
In compliance with the Washington University Institutional Data and Safety Monitoring 
Plan, the Principal Investigator will provide a Data and Safety Monitoring (DSM) report to 
the Washington University Quality Assurance and Safety Monitoring Committee (QASMC) 
semi-annually beginning six months after accrual has opened (if at least five patients have 
been enrolled) or one year after accrual has opened (if fewer than five patients have been 
enrolled at the six-month mark).  This report will include: 
 
• 
HRPO protocol number, protocol title, Principal Investigator name, data 
coordinator name, regulatory coordinator name, and statistician 
• 
Date of initial HRPO approval, date of most recent consent HRPO 
approval/revision, date of HRPO expiration, date of most recent QA audit, 
study status, and phase of study 
• 
History of study including summary of substantive amendments; summary of 
accrual suspensions including start/stop dates and reason; and summary of 
protocol exceptions, error, or breach of confidentiality including start/stop dates 
and reason 
• 
Study-wide target accrual and study-wide actual accrual  
• 
Protocol activation date 
• 
Average rate of accrual observed in year 1, year 2, and subsequent years 
• 
Expected accrual end date and accrual by arm 
• 
Objectives of protocol with supporting data and list the number of participants 
who have met each objective 
• 
Measures of efficacy 
• 
Early stopping rules with supporting data and list the number of participants 
who have met the early stopping rules 
• 
Summary of toxicities separated by arm 
• 
Abstract submissions/publications 
• 
Summary of any recent literature that may affect the safety or ethics of the 
study  
 


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The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient 
Coordinator becomes aware of an adverse event, the AE will be reported to the HRPO 
and QASMC according to institutional guidelines. 
 
 
15.0 
STATISTICAL CONSIDERATIONS 
 
15.1 
Study Endpoints 
 
The primary objective is to determine the 3-year recurrence and death rates in 
patients treated with trastuzumab administered with chemotherapy for 12 months 
versus standard chemotherapy alone.   The primary outcome measures for each 
patient are: 1) disease free survival, defined as the time from completion of 
standard chemotherapy + definitive surgery to the detection of local or distant 
recurrence, death, or last follow-up; and 2) time to death, defined as time from 
diagnosis to death from any cause or last follow-up. The secondary endpoint is 
ERBB2 expression (positive vs. negative) in bone marrow samples from right and 
left iliac crests after one year of therapy. All eligible patients will have detectable 
ERBB2 overexpression in the bone marrow at time of enrollment, so baseline 
ERBB2 status is not an endpoint. Exploratory endpoints include molecular breast 
cancer subtype measured by PAM50 assay, determination of DTC genes 
associated with recurrent disease development and response to trastuzumab and 
treatment outcome.  
 
15.2 
Justification of Sample Size 
 
Based on our preliminary data, we estimated that 25% of stage II/IIIHer2-negative 
breast cancer patients harbor ERBB2-positive DTCs in their bone marrow. 
Approximately 200 patients were expected to have their bone marrow screened 
for ERBB2-positive DTC to enroll 50 patients into the trial. Less than 10% of 
patients were expected to be lost to follow-up before primary and secondary 
endpoints were measured, leaving 46 patients for analysis of primary and 
secondary endpoints.  Based on the literature and preliminary data, the expected 
3-year recurrence rate is 75% in the standard chemotherapy arm and 40% in the 
trastuzumab arm. 
 
15.2.1 Revised Sample Size Justification without Interim Analysis 
 
All patients who meet screening requirements during the next 12 months 
will be enrolled in addition to the 5 patients already on study. No more than 
one patient is expected to be lost to follow-up, providing 10-12 patients for 
analysis of primary and secondary endpoints. The primary endpoint is 3 
year progression free survival, estimated to be 25% without trastuzumab 
and 60% with trastuzumab. The power to detect this difference will be < 
.50, given a two-sided log rank test at a .05 significance level (power ~ .30 
with 13 patients, .25 with 10 patients and .20 with 8 patients).  
 
The detectable difference with 12 patients, assuming balanced or nearly 
balanced numbers in the two arms, is a hazard ratio ~ .076, corresponding 
to 25% 3 year PFS in the no-trastuzumab arm vs. 90% in the trastuzumab 
arm. 


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The principal secondary endpoint is the proportion of patients who have no 
evidence of disseminated tumor cells in their bone marrow. The expected 
proportion to eliminate DTCs is <10% in the no-trastuzumab arm and >80% 
in the trastuzumab arm. Given 12 patients, 6 in each arm, a one-sided 
Fisher’s Exact test with significance level .05 will have power = .73 to detect 
this difference and power = .82 to detect a 5% greater difference (5% vs. 
80% or 10% vs. 85%). 
 
15.3 
Analysis Plan 
 
15.3.1 Original Analysis Plan 
 
Cox proportional hazards models were to be used to estimate 3-year 
recurrence and death rates adjusted for the 4 stratification factors. Median 
time to recurrence and time to death was also to be calculated by these 
models, as were hazard ratios by study arm.  Based on our data and the 
published DFS advantage observed with trastuzumab treatment, we 
estimated the expected 3-year recurrence rate will be 75% in the placebo 
arm and 40% in the trastuzumab arm.  One interim analysis was planned 
after the first 15 patients (~ 1/3 of the total) had completed 3 years of follow-
up after the end of standard chemotherapy + definitive surgery and 3 year 
recurrence rates had been determined.  Conditional study power was to be 
used to evaluate the estimates on which study power has been calculated, 
and the sample size was to be adjusted, if necessary.  Analysis of the 
primary endpoints was to be carried out at a significance level of 0.0052 for 
the interim analysis and 0.048 for the final analysis in order to preserve an 
overall significance level of 0.05. 
 
Logistic regression was to be used to compare the proportion of patients 
who eliminate ERBB2-positive DTCs from BM in the two study arms 
adjusted for the four stratification factors.  Heatmaps, cluster diagrams and 
histograms were to be used to display expression changes by study arm 
and ERBB2 status.  Change in the 38-gene signature was to be analyzed.  
Based on Fisher’s Exact test (because the smaller sample size may 
provide sparse or empty cells), the minimum detectable difference was 
estimated to be 60% (if ~10% are expression positive), 43% (if 30% 
expression positive) or 40% (if 50% expression positive).  
 
A linear model or nonparametric alternative was to be used to compare 
mean or median expression of ERBB2 expression in BM, by molecular 
subtype. Stratification factor-adjusted Cox proportional hazards models 
were to be used to compare the hazard of recurrent disease and death of 
any cause by molecular subtype.  The frequency of patients with ERBB2 
positive DTCs in each primary tumor PAM50 Her2 subtype was to be 
tabulated, plotted using histograms and described with a relative risk of 
remaining ERBB2 positive and 95% confidence interval. Difference of 
proportions was to be tested with Fisher’s Exact test and with stratification-
adjusted logistic regression models.   
 
 
 


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15.3.2 Current Analysis Plan 
 
Unstratified Cox proportional hazards regression will be used to estimate 
the primary endpoint, 3-year progression-free survival, with 95% 
confidence intervals in the two study arms. If median times are reached, 
medians time to recurrence and time to death will be calculated along with 
hazard ratios and 95% confidence by study arm. The principal secondary 
endpoint is the proportion of patients who eliminate ERBB2-positive DTCs 
from BM.  Fisher’s Exact test will be used to compare the proportion of 
patients who eliminate ERBB2-positive DTCs from BM in the two study 
arms. Heatmaps, cluster diagrams and histograms may be used to display 
expression changes by study arm and ERBB2 status.  Change in the 38-
gene signature will be examined in an exploratory fashion gene by gene.   
 
15.4 
Timeline and Feasibility 
 
15.4.1 Original Timeline and Feasibility 
 
We evaluate and treat approximately new 500 patients with early stage 
breast cancer each year at Siteman Cancer Center (data from year 2008).  
Among these patients, approximately 350 patients each year will have 
HER2- disease (data from year 2008) and can be offered the trial. 
Conservatively, if 25% of these patients (87 patients) are pre-registered for 
the bone marrow testing, we can enroll approximately 21 patients (25% of 
patients) with ERBB2 positive bone marrow DTCs each year.  In addition, 
since the interest to this trial for this high risk population will likely be high, 
we anticipate that a significant number of patients will be referred from 
community oncologists for consideration of this trial. Therefore, the accrual 
goal of 50 patients with positive DTCs to this trial will be easily reached in 
2.5 years. 
 
15.4.2 Current Timeline and Feasibility 
 
Once the study reopens for accrual all patients who meet screening 
requirements during the 12 months will be enrolled. We evaluate and treat 
approximately 500 patients with early stage breast cancer each year at the 
Siteman Cancer Center (data from 2008). Among these patients 
approximately 350 patients each year are expected to have HER2 negative 
disease. Conservatively, if 25% of these patients (87 patients) are pre-
registered for bone marrow testing and 10%-15% have confirmed presence 
of DTCs at diagnosis, the study can enroll 8-13 new patients during the 
next year.  
 
 
15.5 
Stopping Rules 
 
15.5.1 Original Stopping Rule 
 
A 2012 meta-analysis of five major trials plus three others enrolling 11,991 
women with HER2-positive early breast cancer confirmed a significantly 
increased risk for severe heart failure (2.5 versus 0.4 percent, relative risk 
[RR] 5.11, 90% CI 3.00-8.72) and reduction in left ventricular ejection 


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fraction (RR 1.83, 90% CI 1.36-2.47) in patients treated with trastuzumab 
versus non-trastuzumab-based adjuvant or neoadjuvant chemotherapy. 
There was no difference in the cardiotoxicity profile in trials with concurrent 
as compared to sequential administration of chemotherapy and 
trastuzumab. 
 
Based on these data, the incidence of severe cardiac dysfunction would be 
0.1 in the 25 patients with no trastuzumab; that is, 1/10th of 1 event. It 
would be 0.625 in the 25 patients with trastuzumab (or about 6/10ths of 1 
event). Based on these estimates of risk, the probability of 0 events is 0.53 
(slightly greater than ½) in the trastuzumab arm and 0.97 (about 97 in 100) 
in the control arm. The probability of >2 events in the trastuzumab arm is 
.024 (or 2-3 chances in 100) and .0001 (about 1 in 10,000) in the control 
arm.  
 
If the relative risk is higher than expected, for example, as high as 9 (the 
upper limit of the 95% confidence interval for the relative risk), the risk in 
the trastuzumab group would be about 3.6, instead of 2.5, and the 
probability of > 2 events is .059 (about 6 in 100). The study would expect 
to see 0.9 events (9/10th of 1 event). That is, at the maximum estimated 
risk, there might be 1 event in the trastuzumab group, whereas it’s unlikely 
that any will be observed in the no-trastuzumab group. Based on this 
analysis, if more than 2 patients in the treatment arm develop severe 
cardiac dysfunction the study will be halted. 
 
15.5.2 Current Stopping Rule 
 
A 2012 meta-analysis of five major trials plus three others enrolling 11,991 
women with HER2-positive early breast cancer confirmed a significantly 
increased risk for severe heart failure (2.5 versus 0.4 percent, relative risk 
[RR] 5.11, 90% CI 3.00-8.72) and reduction in left ventricular ejection 
fraction (RR 1.83, 90% CI 1.36-2.47) in patients treated with trastuzumab 
versus non-trastuzumab-based adjuvant or neoadjuvant chemotherapy. 
There was no difference in the cardiotoxicity profile in trials with concurrent 
as compared to sequential administration of chemotherapy and 
trastuzumab. 
 
Based on these data, the incidence of severe cardiac dysfunction would be 
.024-.028 in the 6-7 patients with no trastuzumab; that is, 2/100ths-
3/100ths of 1 event. It would be .15-.175 in the 6-7 patients with 
trastuzumab (or about 1/10th-2/10ths of 1 event). Based on these estimates 
of risk, the probability of 0 events is .84-.86 in the trastuzumab arm and 
.97-.98 in the control arm. The probability of >2 events in the trastuzumab 
arm is .009-.012 (or 1 chance in 100) and .0002-.0003 (about 2-3 in 10,000) 
in the control arm.  
 
If the relative risk is higher than expected, for example, as high as 9 (the 
upper limit of the 95% confidence interval for the relative risk), the risk in 
the trastuzumab group would be about 3.6, instead of 2.5, and the 
probability of > 2 events is .018-.024 (about 2-3 in 100). The study would 
expect to see .2 events (2/10th of 1 event). That is, at the maximum 
estimated risk, there might be 1 event in the trastuzumab group, whereas 


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it’s unlikely that any will be observed in the no-trastuzumab group. Based 
on this analysis, if more than 2 patients in the treatment arm develop severe 
cardiac dysfunction the study will be halted. 
 
 


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APPENDIX A: ECOG Performance Status Scale 
 
 
Grade 
 
 
Description 
0 
Normal activity.  Fully active, able to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory.  Restricted in physically strenuous activity, 
but ambulatory and able to carry out work of a light or sedentary nature 
(e.g., light housework, office work). 
2 
In bed <50% of the time.  Ambulatory and capable of all self-care, but 
unable to carry out any work activities.  Up and about more than 50% 
of waking hours. 
3 
 
In bed >50% of the time.  Capable of only limited self-care, confined to 
bed or chair more than 50% of waking hours. 
 
4 
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  
Totally confined to bed or chair. 
5 
 
Dead. 
 
 
 
 


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APPENDIX B: HFSA Guidelines 
Recommendations for Pharmacological Therapy: 
Left Ventricular Systolic Dysfunction 
 
ß-Adrenergic Receptor Blockers 
 
Background for Recommendations 
 
The single most significant addition to the pharmacological management of heart failure 
since the publication of previous guidelines involves the use of ß-receptor antagonists. This 
represents a noteworthy departure from traditional doctrine in which ß-blocking agents 
were classified as contraindicated in the setting of left ventricular systolic dysfunction. A 
solid foundation of both clinical and experimental evidence now firmly supports their use 
in heart failure with the aim of reducing both morbidity and mortality (16,22,23). 
 
ß-Blocker therapy for heart failure has been advocated by some investigators since the 
1970s (24). During the subsequent 2 decades, many small- to medium-sized placebo-
controlled trials, which used a variety of agents, showed several common findings: 1) the 
use of ß-blockers in mild to moderate heart failure was generally safe when initiated at low 
doses and gradually uptitrated under close observation; 2) improvement in left ventricular 
ejection fraction was observed in all trials that lasted at least 3 months; and 3) there was 
wide variability in the effects of ßblockade on exercise tolerance but improvement in 
outcome and symptomatic benefits was noted in many studies. These generally positive 
findings stimulated additional, large-scale clinical trials that have provided an impressive 
body of evidence that supports the use of ß-blockers in patients with heart failure caused 
by left ventricular systolic dysfunction. The recommendations that follow are derived from 
nearly 2 decades of research that include basic science data, animal models, and clinical 
trial experience in over 10,000 patients (25,26). 
 
Although this is a major advance in efficacy, identification of appropriate candidates for 
ßblocker therapy is essential to ensure safe and effective treatment. Prescribing physicians 
should understand the potential risks of ß-blocker therapy, as well as the benefits. The 
interested practitioner who is unfamiliar with ß-blocker initiation and titration may first 
seek further education and counsel from sources such as the Heart Failure Society of 
America or local and regional heart failure specialty centers. 
 
Recommendation 1. ß-blocker therapy should be routinely administered to 
clinically stable patients with left ventricular systolic dysfunction (left 
ventricular ejection fraction less than or equal to 40%) and mild to 
moderate heart failure symptoms (ie, NYHA class II-III, Appendix A) 
who are on standard therapy, which typically includes ACE inhibitors, 
diuretics as needed to control fluid retention, and digoxin (Strength of 
Evidence = A). 
 
The most persuasive outcome in heart failure management remains all-cause mortality. 
Combined endpoints, including mortality or hospitalization and mortality or hospitalization 
for heart failure, have also emerged as key outcomes. These latter endpoints reflect a more 
comprehensive assessment of the influence of therapy on quality of life and disease 


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progression and are assuming more importance as mortality rates decline with treatment 
advances. The substantial beneficial effect of ß-blocker therapy on these endpoints has 
been well shown in clinical trials of symptomatic patients (NYHA class II - III) treated 
with carvedilol, bisoprolol, or metoprolol controlled release/extended release (CR/XL) 
(27-29). Trials with these agents encompass the combined, worldwide experience with ß-
blocker therapy in patients with chronic 
heart failure who were stable on background therapy, including ACE inhibitors (over 90%) 
and diuretics (over 90%). Digoxin was common as background therapy, particularly in 
studies conducted in the United States. Trial results indicate that both selective and 
nonselective ßblockers, with and without ancillary properties, have significant efficacy in 
heart failure. ß-Blocking agents with intrinsic sympathomimetic activity appear to have a 
negative impact on survival and should not be used in heart failure patients. 
 
Metoprolol. The MDC Study was an early trial that included 383 patients with heart 
failure caused by nonischemic causes, NYHA class II-III symptoms, and a left ventricular 
ejection fraction of less than or equal to 40% (30). Patients with coronary artery disease 
were excluded. Study results showed a 34% reduction in risk in patients treated with 
metoprolol, although this strong trend toward benefit (P = .058) was entirely attributable 
to a reduction in the frequency of cardiac transplantation listing in the treatment group. In 
fact, the absolute number of deaths in the metoprolol group was higher than in the placebo 
group (23 v 19, P = .69). 
 
    The MERIT-HF Trial evaluated the effect of metoprolol CR/XL with all-cause mortality 
as the primary endpoint. The trial included 3,991 patients with NYHA class II-IV heart 
failure, although 96% of the study patients were functional class II or III (31). In this study, 
investigators were allowed to select the starting dose of metoprolol CR/XL. Seventy-nine 
percent chose 25 mg as the starting dose for class II patients, and 77% chose 12.5 mg for 
class III-IV patients. The target dose was 200 mg and doses were up-titrated over a period 
of 8 weeks. Premature discontinuation of blinded therapy occurred in 13.9% of those 
treated with metoprolol CR/XL and 15.3% of those in the placebo group (P = .90). The 
study results revealed a 34% reduction in mortality in the metoprolol group (relative risk 
of .66; 95% confidence interval [CI], .53 to .81; p=.0062 after adjustment for interim 
analyses), with annual mortality rates of 11% in the placebo 
and 7.2% in the metoprolol CR/XL group (29). 
 
Bisoprolol. The CIBIS Study evaluated the effects of bisoprolol in 641 patients with left 
ventricular systolic dysfunction caused by ischemic or nonischemic causes and NYHA 
class III-IV heart failure (32). The primary endpoint was all-cause mortality, and 
hospitalization for worsening heart failure was one of the secondary outcomes of interest. 
The initial bisoprolol dose was 1.25 mg/day, which was increased to a maximum dose of 
5 mg/day. The trial found no significant reduction in all-cause mortality in patients treated 
with bisoprolol (20% reduction bisoprolol v placebo, P = .22) (32). The risk of 
hospitalization was significantly reduced by 34% (28% placebo group v 19% bisoprolol 
group, P < .01). 
    The favorable trends seen in CIBIS led to the larger CIBIS II Study, which ultimately 
was prematurely terminated as a result of a significant reduction in mortality in the 
bisoprolol arm (28). These results were obtained in 2,647 patients who were followed for 


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an average of 1.3 years. Over 80% of the patients were judged to be NYHA class III at 
enrollment. Background therapy included ACE inhibitors in 96% and diuretic in 99% of 
the study patients, whereas 52% were taking digoxin. In contrast to the original CIBIS 
study, CIBIS II had a similar starting dose of 1.25 mg but had a greater target dose of 10 
mg daily of bisoprolol. More stringent criteria for defining ischemic cardiomyopathy were 
used. Treatment with bisoprolol reduced the annual mortality rate by 34% (13.2% placebo 
v 8.8% bisoprolol; hazard ratio .66; 95% CI, .54 to .81; P< .0001). Hospitalizations for 
worsening heart failure were also decreased by 32% (18% placebo v 12% bisoprolol, 
hazard ratio .64; 95% CI, .53 to .79; P< .0001). Although a post hoc 
analysis of the CIBIS Study had suggested benefit might be consigned to patients without 
coronary disease, the survival benefit, with significant reductions apparent in both ischemic 
or nonischemic patients, was not influenced by disease origins. 
 
Carvedilol. Carvedilol, a nonselective ß-blocker and α-blocker, has been extensively 
investigated for treatment of heart failure caused by left ventricular systolic dysfunction. 
In the United States carvedilol trials, 4 separate study populations were examined and the 
data from 1,094 patients were combined to evaluate the effect of carvedilol therapy on the 
clinical progression of heart failure (27). Clinical progression was defined as worsening 
heart failure leading to death, hospitalization, or, in one study, a sustained increase in 
background medications. Patients with a left ventricular ejection fraction of 35% or less 
and NYHA class II-IV were eligible if they tolerated 6.25 mg of carvedilol twice per day 
for a 2-week, open-label, run-in period. Although this run-in phase biased the ultimately 
randomized patient population, less than 8% of eligible patients failed the open-label 
challenge. Target dosages for the studies were 50 to 100 mg/day of carvedilol that were 
administered in divided doses twice daily. Patients completing the run-in period were 
randomized based on results from their 6- minute walk test into mild, moderate, or severe 
trials. These studies were prematurely terminated (median follow-up 6.5 months) by the 
Trial Data and Safety Monitoring Board because of reduced mortality across the 4 
combined trials of patients treated with carvedilol. 
    Data from these combined trials indicated a substantial benefit from carvedilol 
treatment. The risk of mortality was 65% lower (7.8% placebo v 3.2% carvedilol; 95% CI, 
39% to 80%; P< .001) and the combined risk of hospitalization or death was reduced by 
38% (20% on placebo v 14% on carvedilol; 95% CI, 18% to 53%; P< .001). A significant 
mortality reduction was also noted when deaths that occurred in the run-in period were 
included in the analysis. The statistical validity of the survival analysis across the trials has 
been questioned because mortality was not the primary endpoint, and only 1 of the 4 trials 
achieved a significant result when analyzed based on the primary endpoint. Nevertheless, 
the magnitude of the survival benefit and the reduction in hospitalization were impressive. 
The survival benefit was not influenced by the cause of disease, age, gender, or baseline 
ejection fraction. Overall, 7.8% of the placebo group and 5.7% of the carvedilol group 
discontinued study medication. Data from the individual trials, PRECISE and MOCHA, 
which evaluated patients with moderate to severe heart failure, found that carvedilol 
reduced the risk of the combined endpoint of mortality or heart failure hospitalization by 
39% to 49% (33,34). The MOCHA Study provided strong evidence for increased benefit 
from higher dosages (25 mg twice per day) versus lower dosages (6.25 mg twice per day) 
of carvedilol, so uptitration of carvedilol dosages to 25 mg twice per day is generally 
recommended. However, favorable effects were noted at 6.25 mg twice per day, so 
intolerance of high doses should not be a reason for discontinuation of therapy. 


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    The Australia-New Zealand Carvedilol Trial enrolled 415 patients with ischemic 
cardiomyopathy and a left ventricular ejection fraction of less than 45% (35). Although 
patients with NYHA functional classes I-III were eligible, the majority enrolled were 
NYHA functional class I (30%) or II (54%). ACE inhibitors were used in 86% of the 
participants, whereas 76% were on diuretic therapy, and 38% were on digoxin. This trial 
also had a run-in phase during which 6% of the patients discontinued ß-blocker therapy. 
During an average follow-up of 19 months, carvedilol decreased the combined risk of all-
cause mortality or any hospitalization by 26% (relative risk .74; 95% CI, .57 to .95; P= 
.02). Overall mortality was 12.5% in the placebo group and 9.6% in the carvedilol group 
which was not statistically significant (relative risk .76; 95% CI, .42 to 1.36; P > .10). 
 
Unreported or Ongoing Trials. Studies that are underway will provide additional data 
concerning specific aspects of the efficacy of ß-blocker therapy in heart failure. The effect 
of bucindolol on mortality and morbidity in patients with moderate to severe heart failure 
has been evaluated in the BEST Study. This study enrolled a substantial number of women 
so the potential influence of gender on the efficacy of ß-blocker therapy can be 
investigated. The trial has been stopped, and no results are available for analysis. 
     
The COPERNICUS Trial is designed to assess the effect of carvedilol treatment on disease 
progression and survival in patients with advanced heart failure with symptoms at rest or 
on minimal exertion. The COMET protocol is a 3,000 patient study that directly compares 
the survival benefit of carvedilol versus metoprolol. This trial will provide important data 
concerning the relative efficacy of a selective ß-blocker versus a nonselective ß-blocker 
with ancillary properties. 
 
Recommendation 2. ß-blocker therapy should be considered for 
patients with left ventricular systolic dysfunction (left ventricular 
ejection fraction less than or equal to 40%) who are asymptomatic 
(ie, NYHA class I) and standard therapy, including ACE inhibitors 
(Strength of Evidence = C). 
 
    Data from the SOLVD Prevention Trial prospectively illustrated the efficacy of ACE 
inhibitors in delaying the onset of heart failure symptoms and the need for treatment or 
hospitalization for heart failure in asymptomatic patients with a left ventricular ejection 
fraction less than or equal to 35% (36). Similar controlled, clinical trial data that support 
the use of a ß-blocker in this clinical circumstance are not available. However, significant 
support for the use of ßblocker therapy in patients with asymptomatic left ventricular 
dysfunction can be derived from clinical trials in coronary artery disease and hypertension. 
Previous data indicate that ß-blocker therapy should be used in patients after myocardial 
infarction (MI) and in patients with myocardial revascularization who have good 
symptomatic and functional recovery but residual ventricular systolic dysfunction. Trials 
in hypertension indicate that ß-blocker therapy decreases the risk of developing heart 
failure. Given the potential of ß-blockers to retard disease progression and improve 
ventricular function, the risk to benefit ratio seems sufficiently low to support ß-blocker 
use in asymptomatic patients with left ventricular dysfunction, especially when the 
dysfunction is marked, and coronary artery disease is present. 


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Recommendation 3. To maximize patient safety, a period of clinical 
stability on standard therapy should occur before ß-blocker therapy 
is instituted. Initiation of ß-blocker therapy in patients with heart 
failure requires a careful baseline evaluation of clinical status 
(Strength of Evidence = B). 
 
    Initiation of ß-blocker therapy has the potential to worsen heart failure signs and 
symptoms. This risk increases with the underlying severity of the heart failure that is 
present. To minimize the likelihood of worsening failure, a period of treatment with 
standard therapy and evidence of clinical stability without acute decompensation or fluid 
overload is recommended before initiation of ß-blocker therapy. The majority of the large-
scale, ß-blocker heart failure trials required that chronic heart failure be present 3 months 
or more before initiation of ß-blocker therapy. Patients enrolled in these trials were 
typically treated with ACE inhibitors (if tolerated), diuretic, and digoxin for at least 2 
months and were observed to be clinically stable for 2 to 3 weeks before beginning ß-
blocker therapy. Thus, many heart failure clinicians favor a minimum of 2 to 4 weeks of 
clinical stability on standard therapy before ß-blocker therapy is instituted. Likewise, most 
clinicians discourage the initiation of ß-blocker therapy in the hospital setting after 
treatment for new or decompensated heart failure (with or without associated inotrope 
administration). Some experienced clinicians initiate ß-blocker therapy in the hospital in 
selected patients who have responded well to inpatient treatment and who can be followed 
closely after discharge. 
 
Recommendation 4. There is insufficient evidence to recommend the use of 
ß-blocker therapy for inpatients or outpatients with symptoms of heart 
failure at rest (ie, NYHA class IV) (Strength of Evidence = C). 
 
    ß-Blocker therapy cannot be routinely recommended for NYHA class IV patients 
because there are currently no clinical trial data to indicate favorable long-term efficacy 
and safety of ßblocker therapy in this patient population. A substantial body of 
observational data indicates that successful institution of ß-blocker therapy in patients with 
this degree of heart failure is problematic. If used, these agents may precipitate 
deterioration, and patients so treated should be monitored by a physician who has expertise 
in heart failure. 
The number of patients with class IV heart failure at the time of ß-blocker initiation in 
controlled clinical trials is small. Available trials, which report data on patients with severe 
heart failure mostly labeled as NYHA class III, show the potential problems of ß-blocker 
therapy in this part of the heart failure spectrum. This experience is reflected in a 14-week 
study that evaluated the effects of ß-blocker therapy in 56 patients (51 NYHA class III and 
5 NYHA class IV at randomization) with severe left ventricular dysfunction (average left 
ventricular ejection fraction of 16% ± 1% and left ventricular filling pressure of 24 mm Hg 
± 1 mm Hg) (37). These patients had significant impairment of exercise capacity (mean 
VO2 max of 13.6 mL/kg/min ± 0.6 mL/kg/min) despite ACE-inhibitor, digoxin and 
diuretic therapy. Patients were believed to be clinically stable (requiring no medication 
adjustments) for a 2-week period before an open-label challenge was conducted. Seven 
patients (12%) failed to complete the open-label, run-in period, during which 5 died and 2 
had nonfatal adverse reactions. Clinical parameters did not distinguish these patients from 


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those who were able to continue in the trial. Eighteen of the 49 patients (37%) completing 
the run-in period experienced worsened dyspnea or fluid retention during this phase. Also, 
22% experienced dizziness and required medication adjustment, which delayed up-titration 
during the run-in. Subsequently, an additional 12% of the patients randomized to carvedilol 
withdrew from the blinded arm of the study. One of the United States carvedilol trials 
studied patients with severe left ventricular dysfunction who had markedly reduced 
exercise capacity as assessed by the 6-minute walk test (38). In this trial, 131 patients with 
a mean left ventricular ejection fraction of 22% and severe impairment in quality of life 
underwent a 2-week, open-label challenge phase of 6.25 mg of carvedilol twice per day. 
Ten of these 131 patients (8%) were unable to complete this run-in phase, most because of 
worsening heart failure, dyspnea, or dizziness. Subsequently, 11% of the patients 
randomized to carvedilol withdrew, as did a similar number of patients (11%) in the 
placebo group. In the recently completed large-scale BEST Trial, the mortality trend in 
NYHA class III-IV patients favored the ß-blocker bucindolol, but the difference from 
placebo was not significant. Further analysis of these preliminary findings is necessary, but 
the data suggest that the striking benefit of ß-blockers in mild-to-moderate heart failure 
may not be extrapolated to those with severe symptoms. 
 
Recommendation 5. ß-Blocker therapy should be initiated at low doses 
and up-titrated slowly, generally no sooner than at 2-week intervals. 
Clinicalreevaluation should occur at each titration point and with worsening 
of patient symptoms. Patients who develop worsening heart failure or 
other side effects after drug initiation or during titration require adjustment 
of concomitant medications. These patients may also require a reduction in 
ß-blocker dose and, in some cases, temporary or permanent withdrawal of 
this therapy (Strength of Evidence = B). 
 
    ß-Blocker therapy should be initiated at doses substantially less than target doses. 
Clinical trials required patient reassessment at up-titration of each dose. This careful 
evaluation by trained nurses and/or heart failure specialists likely contributed to the 
relatively low withdrawal rates and safety profiles observed in the clinical trials. 
    Treatment for symptomatic deterioration may be required during ß-blocker titration, but 
with appropriate adjustments in therapy, most patients can be maintained and generally 
achieve target doses. There is a risk of worsening heart failure, and vasodilatory side effects 
may occur with certain agents. Worsening heart failure is typically reflected by increasing 
fatigue, lower exercise tolerance, and weight gain. Increased diuretic doses may be required 
for signs and symptoms of worsened fluid retention. Treatment options also include 
temporary down-titration of the ß-blocker to the last tolerated dose. Abrupt withdrawal 
should be avoided. A minimum period of stability of 2 weeks should occur before further 
up-titration is attempted. Hypotensive side effects may often resolve with reduction in 
diuretic dose. Temporary reductions in ACE inhibitor dose may be helpful for symptomatic 
hypotension not obviated by staggering the schedule of vasoactive medications. 
Administration of carvedilol with food may alleviate vasodilatory side effects as well. 
    If ß-blocker treatment is interrupted for a period exceeding 72 hours and the patient is 
still judged a candidate for this therapy, drug treatment should be reinitiated at 50% of the 
previous dose. Subsequent up-titration should be conducted as previously described. 
 


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Recommendation 6. In general, patients who experience a deterioration in clinical 
status or symptomatic exacerbation of heart failure during chronic maintenance 
treatment should be continued on ß-blocker therapy (Strength of Evidence = C). 
 
Clinical decompensation that occurs during stable maintenance therapy is less likely caused 
by chronic ß-blocker therapy than other factors (diet or medication noncompliance, 
ischemia, arrhythmia, comorbid disease, infection, or disease progression). In these 
situations, maintaining the current ß-blocker dose while relieving or compensating for the 
precipitating factor(s) is most often the best course. Data from patients randomized to 
continue or discontinue ß-blocker therapy in this setting are not currently available. 
However, studies of the withdrawal of ß-blocker therapy in patients with persistent left 
ventricular systolic dysfunction but improved and stable clinical heart 
failure have revealed a substantial risk of worsening heart failure and early death after 
discontinuation of ß-blocker therapy (39,40). 
 
Recommendation 7. Patient education regarding early recognition of 
symptom exacerbation and side effects is considered important. If clinical 
uncertainty exists, consultation with clinicians who have expertise in heart 
failure and/or specialized programs with experience in ß-blocker use in 
patients with heart failure is recommended (Strength of Evidence = B). 
 
In certain patients, frequent return visits for dose-titration may be difficult to accommodate 
in a busy clinical practice. Trained personnel, including nurse practitioners, physicians’ 
assistants, and pharmacists with physician supervision, may more efficiently perform 
patient education and reevaluation during up-titration. Heart failure specialty programs are 
more likely to have the resources to provide this follow-up and education (41). 
Consultation or referral may be particularly beneficial when the clinical heart failure status 
of the patient is uncertain or problems arise during initiation of therapy or dose-titration 
that may cause unwarranted discontinuation of therapy. Ideal patients for ß-blocker therapy 
should be compliant and have a good understanding of their disease and their overall 
treatment plan. Patients should be aware that symptomatic deterioration is possible early 
in therapy and that symptomatic improvement may be delayed for weeks to months. 
 
Unresolved Therapeutic Issues 
 
Combining ß-Blocking Agents With Amiodarone Therapy. Concomitant use of 
amiodarone was generally precluded in the trials evaluating carvedilol and most other ß-
blockers. However, the use of this agent for rate control of atrial arrhythmia or for 
maintenance of sinus rhythm is common in heart failure patients. Drug interactions 
between ß-blockers and amiodarone are possible, including symptomatic bradycardia, and 
may limit the maximum tolerated dose of the ß-blocker. When the combination is used, the 
smallest effective dose of amiodarone should be employed. Given the lack of a clear 
survival benefit, amiodarone is not a substitute for ß-blocker therapy in heart failure 
patients who are candidates for this therapy. 
 
Implantation of Cardiac Pacemakers. Given the strength of evidence that supports ß-
blocker therapy in patients with symptomatic heart failure, some physicians would consider 


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pacemaker implantation when symptomatic bradycardia or heart block occur during the 
initiation of this therapy, although no data are available to support such use. Consideration 
should be given, after weighing risks and benefits, to the withdrawal of other drugs that 
may have bradycardia effects. 
Duration of Therapy. Whether patients experiencing marked improvement in left 
ventricular systolic dysfunction and heart failure symptoms during therapy can be 
successfully withdrawn from ß-blocker therapy remains to be established. Concern 
continues that such patients would experience worsening after ß-blocker withdrawal, either 
in systolic function or symptoms, over a time period that is undefined. Until clinical trial 
data indicate otherwise, the duration of ßblocker therapy must be considered indefinite. 
 
Digoxin 
Background for Recommendations 
Although little controversy exists as to the benefit of digoxin in patients with symptomatic 
left ventricular systolic dysfunction and concomitant atrial fibrillation, the debate continues 
over its current role in similar patients with normal sinus rhythm. Recent information 
regarding digoxin’s mechanism of action and new analyses of clinical data from the DIG 
Trial and the combined PROVED and RADIANCE Trial databases provide additional 
evidence of favorable efficacy that was unavailable to previous guideline committees (42-
47). In fact, this information has recently formed the basis of Food and Drug 
Administration (FDA) approval of digoxin for the treatment of mild to moderate heart 
failure (48). Digoxin, a drug that is inexpensive and can be given once daily, represents the 
only orally effective drug with positive inotropic effects approved for the management of 
heart failure. The committee’s consensus is that digoxin, when used in combination with 
other standard therapy, will continue to play an important role in the symptomatic 
management of the majority of patients with heart failure. 
    The efficacy of digoxin for the treatment of heart failure caused by systolic dysfunction 
has traditionally been attributed to its relatively weak positive inotropic action that comes 
from inhibition of sodium-potassium adenosine triphosphatase (ATPase) that results in an 
increase in cardiac myocyte intracellular calcium. However, in addition to positive 
inotropy, digitalis has important, neurohormonal-modulating effects in patients with 
chronic heart failure, including a sympathoinhibitory effect that cannot be ascribed to its 
inotropic action (49,50). Digoxin also ameliorates autonomic dysfunction as evidenced by 
studies of heart rate variability, which indicates increased parasympathetic and 
baroreceptor sensitivity during therapy (51). 
 
Recommendation 1. Digoxin should be considered for patients who have 
symptoms of heart failure (NYHA class II-III, Strength of Evidence = A 
and NYHA class IV, Strength of Evidence = C) caused by left ventricular 
systolic dysfunction while receiving standard therapy. 
 
    Digoxin increases left ventricular ejection fraction and alleviates symptomatic heart 
failure as evidenced by drug-related improvement in exercise capacity and reductions in 
heart-failureassociated hospitalization and emergency room visits. Digoxin should be used 
in conjunction with other forms of standard heart failure therapy including ACE inhibitors, 
diuretics and ß-blockers. 


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    The DIG Trial, a randomized, double-blind, placebo-controlled trial in over 7,000 
patients with heart failure, showed a neutral effect on the primary study endpoint and 
mortality from any cause during an average follow-up of approximately 3 years (42). In 
the main trial, 6,800 patients with left ventricular ejection fraction less than or equal to 
45% were randomized to digoxin or placebo, in addition to diuretics and ACE inhibitors. 
A total of 1,181 deaths occurred on digoxin (34.8%) and 1,194 on placebo (35.1%) for a 
risk ratio of .99 (95% CI, .91 to 1.07; P = .80). These results differ from other oral agents 
with inotropic properties that have been associated with an adverse effect on mortality. In 
addition, the need for hospitalization and cointervention (defined as increasing the dose of 
diuretics and ACE inhibitors or adding new therapies for worsening heart failure) was 
significantly lower in the digoxin group, even in those patients who were not previously 
taking digoxin. Fewer patients on digoxin compared with placebo were hospitalized for 
worsening heart failure (26.8%v 34.7%; risk ratio .72; 95% CI, .66 to .79; P < .001). These 
long-term data are consistent with recent results obtained from an analysis of the combined 
PROVED and RADIANCE databases (45). In this analysis, patients who continued 
digoxin as part of triple therapy with diuretics and an ACE inhibitor were much less likely 
to develop worsening heart failure (4.7%) than those treated with a diuretic alone (39%, P 
< .001), diuretic plus digoxin (19%, P = .009) or diuretic plus an ACE inhibitor (25%, P = 
.001). 
    Although there are no clinical trial data (level A evidence) for the efficacy of digoxin in 
patients with NYHA Class IV heart failure, there is evidence that digoxin works across the 
spectrum of left ventricular systolic dysfunction. A prespecified subgroup analysis of 
patients enrolled in the DIG Trial with evidence of severe heart failure (as manifested by 
left ventricular ejection fraction less than 25%, or cardiothoracic ratio [CTR] greater than 
.55) showed the benefit of digoxin (48). The following reductions in the combined endpoint 
of all-cause mortality or hospitalization were seen on digoxin compared with placebo: 16% 
reduction (95% CI, 7% to 24%) in patients with a left ventricular ejection fraction of less 
than 25%, and a 15% reduction (95% CI, 6% to 23%) in patients with a CTR of greater 
than .55 (43). Reductions in the risk of the combined endpoint of heart-failure related 
mortality or hospitalization were even more striking: 39% (95% CI, 29% to 47%) for 
patients with left ventricular ejection fraction less than 25%, and 35% (95% CI, 25% to 
43%) for patients with a CTR greater than .55 (48).  
    Evidence for the efficacy of digoxin in patients with mild symptoms of heart failure has 
been provided by a recent retrospective, cohort analysis of the combined PROVED and 
RADIANCE data (52). The outcome of patients in these trials who were randomized to 
digoxin withdrawal or continuation was categorized by using a prospectively obtained 
heart failure score based on clinical signs and symptoms. Patients in the mild heart failure 
group (heart failure score of 2 or less) who were randomized to have digoxin withdrawn 
were at increased risk of treatment failure and had deterioration of exercise capacity and 
left ventricular ejection fraction compared with patients who continued digoxin (all P < 
.01). Patients in the moderate heart failure  group who had digoxin withdrawn were 
significantly more likely to experience treatment failure than either patients in the mild 
heart failure group or patients who continued digoxin (both P < .05). These data suggest 
that patients with left ventricular systolic dysfunction benefit from digoxin despite only 
mild clinical evidence of heart failure. 
In summary, a large body of evidence supports the efficacy of digoxin in patients with 
symptomatic heart failure caused by left ventricular systolic dysfunction. Digoxin has been 
shown to decrease hospitalizations, as well as emergency room visits; decrease the need 


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for cointervention; and improve exercise capacity (42-44,53,54). Taken as a whole, these 
clinical trial data provide support for digoxin’s beneficial effect on morbidity and neutral 
effect on mortality (42). 
 
Recommendation 2. In the majority of patients, the dosage of digoxin 
should be .125 mg to .25 mg daily (Strength of Evidence = C). 
 
    Recent data suggest that the target dose of digoxin therapy should be lower than 
traditionally assumed. Although higher doses may be necessary for maximal hemodynamic 
effects (55), beneficial neurohormonal and functional effects appear to be achieved at 
relatively low serum digoxin concentrations (SDC) typically associated with daily doses 
of .125 mg to .25 mg of digoxin (55-57). The utility of lower SDC is supported by recent 
clinical trial data; the mean SDC achieved in the RADIANCE Trial was 1.2 ng/mL and in 
the DIG Trial was 0.8 ng/mL (42,44). Recent retrospective, cohort analysis of the combined 
PROVED and RADIANCE databases indicates that patients with a low SDC (less than .9 
ng/mL) were no more likely to experience worsening symptoms of heart failure on 
maintenance digoxin than those with a moderate (.9 to 1.2 ng/mL) or high (greater than 1.2 
ng/mL) SDC (41). All SDC groups were significantly less likely to deteriorate during 
follow-up compared with patients withdrawn from digoxin.  
    Therefore, patients with left ventricular systolic dysfunction and normal sinus rhythm 
should be started on a maintenance dosage of digoxin (no loading dose) of .125 or .25 mg 
once daily based on ideal body weight, age, and renal function For patients with normal 
renal function, a dosage of digoxin of .25 mg/day will be typical. Many patients with heart 
failure have reduced renal function and should begin on .125 mg daily. In addition, patients 
with a baseline conduction abnormality, or who are small in stature or elderly, should be 
started at .125 mg/day, which can be up-titrated if necessary. Once dosing has continued 
for a sufficient period for serum concentration to reach steady state (typically in 2 to 3 
weeks), some clinicians consider the measurement of a SDC, especially in elderly patients 
or those with impaired renal function in which the digoxin dose is often not predictive of 
SDC. SDC measurements may be considered when 1) a significant change in renal function 
occurs; 2) a potentially interacting drug (amiodarone, quinidine, or verapamil) is added or 
discontinued; or 3) confirmation of suspected digoxin toxicity is necessary in a patient with 
signs or symptoms and/or electrocardiographic changes consistent with this diagnosis. 
Samples for trough SDC should be drawn more than 6 hours after dosing. Otherwise, the 
result is difficult to interpret because the drug may not be fully distributed into tissues. 
 
Recommendation 3. In patients with heart failure and atrial fibrillation 
with a rapid ventricular response, the administration of high doses of 
digoxin (greater than .25 mg) for the purpose of rate control is not 
recommended. When necessary, additional rate control should be achieved 
by the addition of ß-blocker therapy or amiodarone (Strength of Evidence 
= C). 
    Digoxin continues to be the drug of choice for patients with heart failure and atrial 
fibrillation. However, the traditional practice of arbitrarily increasing the dose (and SDC) 
of digoxin until ventricular response is controlled should be abandoned because the risk of 
digoxin toxicity increases as well. Digoxin alone is often inadequate to control ventricular 
response in patients with atrial fibrillation, and the SDC should not be used to guide dosing 


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to achieve rate control. Therefore, digoxin should be dosed in the same manner as in a 
patient with heart failure and normal sinus rhythm. 
    Digoxin slows ventricular response to atrial fibrillation through enhancement of vagal 
tone. However, with exertion or other increases in sympathetic activity, vagal tone may 
decrease and ventricular rate accelerate. Addition of a ß-blocker or amiodarone 1) 
complements the pharmacological action of digoxin and provides more optimal rate 
control; 2) allows the beneficial clinical effects of digoxin to be maintained; and 3) limits 
the risk of toxicity that may occur if digoxin is dosed to achieve a high SDC (58). For 
patients who have a contraindication to ßblockers, amiodarone is a reasonable alternative. 
If amiodarone is added, the dose of digoxin should be reduced, and the SDC should be 
monitored so that the serum concentration can be maintained in the desired range. Some 
clinicians advocate the short-term, intravenous administration of diltiazem for the acute 
treatment of patients with very rapid ventricular response, especially those with 
hemodynamic compromise. This drug is not indicated for long-term management because 
its negative inotropic effects may worsen heart failure.  
 
Unresolved Therapeutic Issues 
 
Combination With ß-blockers. ß-Blocker therapy has become pivotal in the management 
of heart failure. However, the majority of patients enrolled in controlled clinical trials that 
study the efficacy of digoxin were not taking ß-blockers. Therefore, it is uncertain whether 
or not digoxin should be routinely included as part of a ß-blocker regimen for symptomatic 
heart failure caused by left ventricular systolic dysfunction. There are attractive features of 
combining digoxin with ß-blocker therapy in the treatment of heart failure. The majority 
of heart failure patients have coronary artery disease and may be at risk for transient 
episodes of myocardial ischemia that could cause catecholamine release and sudden 
cardiac death. Combining digoxin with a ß-blocker may preserve the beneficial effects of 
digoxin on the symptoms of heart failure while minimizing the potential detrimental effects 
of this therapy on catecholamine release in the setting of ischemia (47). 
Combination with Diuretics. Non-potassium-sparing diuretics can produce electrolyte 
abnormalities such as hypokalemia and hypomagnesemia, which increases the risk of 
digoxin toxicity. The combination of digoxin with a potassium- sparing diuretic would be 
a potentially safer alternative. Further study will be necessary to carefully elucidate the 
efficacy and safety of combining digoxin with these agents. 
 
Anticoagulation and Antiplatelet Drugs 
Background for Recommendations 
    Patients with heart failure are recognized to be at increased risk for thromboembolic 
events that can be arterial or venous in origin. In addition to atrial fibrillation and poor 
ventricular function (which promote stasis and increase the risk of thrombus formation), 
patients with heart failure have other manifestations of hypercoagulability. Evidence of 
heightened platelet activation; increased plasma and blood viscosity; and increased plasma 
levels of fibrinopeptide A, ßthromboglobulin, D-dimer, and von Willebrand factor (59-61) 
have been found in many patients. Despite a predisposition, estimates regarding the 
incidence of thromboemboli in patients with heart failure vary substantially between 1.4 
and 42 per 100 patient years (62-65). Although variability in the reported incidence likely 
results from differences in the populations studied and the methods used to identify these 
events, the consensus is that pulmonary and systemic emboli are not common in heart 


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failure patients. Traditionally, the issue of anticoagulation in patients with heart failure 
centered on warfarin. Growing recognition of the importance of ischemic heart disease as 
a cause of heart failure suggests that the role of antiplatelet therapy must be considered in 
patients with this syndrome as well. 
    Previous guidelines have recommended warfarin anticoagulation in patients with heart 
failure complicated by atrial fibrillation and in heart failure patients with prior 
thromboembolic events (18,19). Warfarin anticoagulation specifically was not 
recommended in patients with heart failure in the absence of these indications. There have 
been no randomized, controlled trials of warfarin in patients with heart failure. Therefore, 
recommendations regarding its use, in the absence of atrial fibrillation or clinically overt 
systemic or pulmonary thromboemboli, must be made on the basis of cohort data and expert 
opinion. The likely incidence of thromboembolic events and the possibility of averting 
them with warfarin are important considerations for any guideline recommendation. In 
addition, the potential beneficial effects of warfarin on coronary thrombotic events, 
independent of embolic phenomenon, must be taken into account. The 
substantial clinical trial data that reflect the beneficial effects of antiplatelet therapy in 
patients with ischemic heart disease suggest that new guideline recommendations for heart 
failure should address the role of this form of therapy in patients with left ventricular 
dysfunction. 
 
Anticoagulation 
 
Recommendation 1. All patients with heart failure and atrial fibrillation 
should be treated with warfarin (goal, international normalized ratio (INR) 
2.0 to 3.0) unless contraindicated (Strength of Evidence = A). 
 
The committee agrees with previous guideline recommendations that concern warfarin 
therapy in patients with heart failure complicated by atrial fibrillation. The benefit of 
warfarin anticoagulation in this setting is well established through several randomized trials 
(66). Patients with heart failure commonly have atrial fibrillation. Warfarin anticoagulation 
should be implemented in all of these patients unless clear contraindications exist. 
 
Recommendation 2. Warfarin anticoagulation merits consideration for 
patients with left ventricular ejection fraction of 35% or less. Careful 
assessment of the risks and benefits of anticoagulation should be undertaken 
in individual patients (Strength of Evidence = B). 
 
    Cohort analyses examining the relationship between warfarin use and noncoronary 
thromboembolism in patients with heart failure have not consistently yielded positive 
findings (62,63,65,67-69). It is possible that the lack of consistent benefit was related to 
the low incidence of identifiable embolic events in these populations. However, these 
studies do not make a convincing argument for the use of warfarin to prevent embolic 
events in the absence of atrial fibrillation or a previous thromboembolic episode. 
    In contrast, a recent cohort analysis of the SOLVD population focused on the relation 
between warfarin use and the risk of all-cause mortality rather than risk for embolic events 
(70). After adjustment for baseline differences, patients treated with warfarin at baseline 
had a significantly lower risk of mortality during follow-up (adjusted hazard ratio .76; 95% 


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CI, .65 to .89, P = .0006). In addition to a mortality benefit, warfarin use was also associated 
with a significant reduction in the combined endpoint of death or hospitalization for heart 
failure (adjusted hazard ratio .82; 95% CI, .72 to .93, P = .002). In the SOLVD population, 
the benefit associated with warfarin use was not significantly influenced by 1) presence or 
absence of symptoms (treatment trial v prevention trial), 2) randomization to enalapril or 
placebo, 3) gender, 4) presence or absence of atrial fibrillation; 5) age, 6) ejection fraction, 
7) NYHA class, or 8) origins of disease. 
    The benefit associated with warfarin use in the cohort analysis of the SOLVD population 
was related to a reduction in cardiac mortality. Specifically, there was a significant 
reduction among warfarin users in deaths that were identified as sudden, in deaths 
associated with heart failure, and in fatal MI. In contrast (yet in agreement with previous 
cohort analyses), there was no significant difference in deaths considered cardiovascular 
but noncardiac, including pulmonary embolism and fatal stroke. Some caution is needed 
in consideration of this finding because the number of cardiovascular deaths that were 
noncardiac was far less than the number of cardiac deaths. 
    Reduction in ischemic events is one potential explanation for the apparent benefit from 
warfarin in the SOLVD Study. Warfarin users showed a reduced rate of hospitalization for 
unstable angina or nonfatal MI. Prior investigations of patients after acute MI showed that 
warfarin anticoagulation, when started within 4 weeks, reduces the incidence of fatal and 
nonfatal coronary events, as well as pulmonary embolus and stroke (71). 
    As with other post hoc, cohort analyses, it is possible that the findings from the SOLVD 
Study may result from differences between the treatment groups that were not identified 
and for which statistical correction could not adequately adjust. For this reason, evidence 
from any cohort study must be considered less powerful compared with evidence derived 
from randomized, controlled trials. Nevertheless, in the absence of randomized data, the 
SOLVD cohort analysis represents reasonable evidence to support more aggressive use of 
warfarin anticoagulation than previously recommended in patients with reduced left 
ventricular ejection fraction and sinus rhythm. The data from this analysis provide no 
information regarding the ideal warfarin dose in this patient population. Therefore, the 
dosing recommendation should likely conform to that derived from previous randomized 
trials performed in patients without mechanical prosthetic  valves (INR 2.0 to 3.0). 
 
Antiplatelet Drugs 
 
Recommendation 1. With regard to the concomitant use of ACE 
inhibitors and acetylsalicylic acid (ASA), each medication should be 
considered on its own merit for individual patients. Currently, there is 
insufficient evidence concerning the potential negative therapeutic 
interaction between ASA and ACE inhibitors to warrant withholding either 
of these medications in which an indication exists (Strength of Evidence = 
C). 
 
    Strong evidence supports the clinical benefit of aspirin in ischemic heart disease and 
atherosclerosis (72-75). However, recent post hoc analyses of large randomized trials 
involving ACE inhibitors in heart failure and post-MI suggest the possibility of an adverse 
drug interaction between ASA and ACE inhibitors (76-78). A retrospective cohort analysis 
of the SOLVD Study found that patients on antiplatelet therapy (assumed to be ASA in the 


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great majority of patients) derived no additional survival benefit from the addition of 
enalapril. Data from CONSENSUS II and GUSTO-1 in post-MI patients, suggest not only 
no additive benefit, but the possibility of a negative effect on mortality from the 
combination of ASA and ACE inhibition. In contrast, an unadjusted, retrospective registry 
study in patients with chronic coronary artery disease did not support an adverse interaction 
(79). Interestingly, in an adjusted analysis of the subset of  patients with heart failure in 
this study, the beneficial effects of aspirin seemed less evident in patients taking ACE 
inhibitors. Despite these provocative post hoc findings, no prospective studies have yet 
been reported that concern the possible adverse interaction between ACE inhibitors and 
aspirin. To date, there is no clear evidence of harm from the combination of ASA and ACE 
inhibitors in patients with heart failure (76). 
    There is also some evidence that the potential interaction between ASA and ACE 
inhibitors may be dose related. A recent meta-analysis of all hypertension and heart failure 
patients who have received both ASA and ACE inhibitors suggests that ASA at doses equal 
to or less than 100 mg showed no interaction with ACE inhibitors (80). Any interaction, if 
observed, occurred at higher doses of aspirin. 
    A potential mechanism for the hypothesized adverse interaction between ASA and ACE 
inhibitors in patients with heart failure involves prostaglandin synthesis. ACE inhibition is 
believed to augment bradykinin which, in turn, stimulates the synthesis of various 
prostaglandins that may contribute vasodilatory and other salutary effects. In the presence 
of ASA, the bradykinin-induced increase in prostaglandins should be attenuated or 
blocked, which potentially reduces the benefits of ACE inhibition. Invasive hemodynamic 
monitoring has shown that the acute hemodynamic effect of enalapril is blunted by 
concomitant administration of aspirin (81). Another possibility is that ASA and ACE 
inhibitors act in a similar fashion in heart failure, therefore no added benefit is gained from 
the combination. ACE inhibitors appear to reduce ischemic events in heart failure patients 
possibly through antithrombotic effects, which could mimic those of antiplatelet agents. 
Recent study results that suggest ASA may have independent beneficial action on 
ventricular remodeling support the hypothesis of similar mechanisms of action for ACE 
inhibitors and ASA (82). 
    Development of the adenosine diphosphate (ADP) antagonists, ticlopidine and 
clopidogrel, provides alternative therapy for platelet inhibition that does not appear to 
influence prostaglandin synthesis (83). In direct comparison with aspirin, large-scale 
clinical trial results have established the efficacy of clopidogrel in the prevention of 
vascular events in patients with arteriosclerotic disease (84). Clinical data are limited with 
ADP antagonists in heart failure. However, hemodynamic evaluation found a similar 
reduction in systemic vascular resistance in heart failure patients treated with the 
combination of ACE inhibitors and ticlopidine versus ACE inhibitors alone, which 
suggests no adverse hemodynamic interaction with ACE inhibition with this type of 
antiplatelet compound (85). Definitive resolution of the therapeutic implications of the 
ASA/ACE inhibitor interaction and the appropriate alternative therapy, if any, in heart 
failure awaits the results of additional clinical research studies. 
 
Angiotensin II Receptor Blockers 
 
Background for Recommendations 
    Angiotensin ll (AT) receptor blockers (ARBs) differ in their mechanism of action 
compared with ACE inhibitors. Rather than inhibiting the production of AT by blockade 


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of ACE, ARBs block the cell surface receptor for AT. ARBs that are currently available 
are selective and only effectively inhibit the AT1 subtype of this receptor. Theoretical 
benefits of ARBs include receptor blockade of AT produced by enzymes other than ACE 
and maintenance of ambient AT to maintain or increase stimulation of AT2 receptors. AT1 
receptor antagonism is important because this receptor appears to mediate the classical 
adverse effects associated with AT in heart failure. In contrast, the AT2 receptor subtype 
appears to counterbalance AT1 receptor stimulation by causing vasodilation and inhibiting 
proliferative and hypertrophic responses (86). Thus, the selective receptor blockade of the 
current ARBs may be particularly advantageous. Theoretical concerns about ARB therapy 
include the potential deleterious effects of increased AT levels and AT2 receptor-mediated 
enhancement of apoptosis. Whether ARBs have beneficial effects similar to ACE inhibitors 
on the course of coronary artery disease remains to be determined. ARBs may or may not 
influence bradykinin concentrations, which are anticipated to rise with ACE inhibitor 
therapy and may contribute to their efficacy. 
    The hemodynamic actions of ARBs have, thus far, been similar to ACE inhibitors for 
reduction of blood pressure in hypertension and lowering of systemic vascular resistance 
in heart failure (87). ARBs have a similar mild-to-modest effect on exercise capacity and 
produce a comparable reduction in norepinephrine relative to ACE inhibitors (88). 
 
Recommendation 1. ACE inhibitors rather than ARBs continue to be the 
agents of choice for blockade of the renin-angiotensin system in heart 
failure, and they remain the cornerstone of standard therapy for patients 
with left ventricular systolic dysfunction with or without symptomatic heart 
failure (Strength of Evidence = A). 
 
    At present, it is not possible to predict where ARBs will ultimately reside among 
accepted therapies for heart failure. Although the initial small ELITE Trial suggested a 
greater benefit from a losartan dosage of 50 mg daily than from a captopril dosage of 50 
mg 3 times daily on mortality in elderly patients with heart failure (89), the ELITE II 
Mortality Trial, which included more than 3,000 patients (90), showed no comparative 
benefit from losartan and a trend for a better outcome and fewer sudden deaths with 
captopril (91). This result provides no evidence that the low dose (50 mg ) of losartan that 
was tested is better than an ACE inhibitor for treating heart failure, but it does not exclude 
the efficacy of a higher dose designed to provide continuous inhibition of the AT1 receptor. 
Tolerability of losartan was better than of captopril, primarily because of an ACEinhibitor 
cough. But the well-established efficacy of the ACE inhibitors on outcome in the post-MI 
period, in diabetes, in atherosclerosis, and in heart failure mandates that this drug group 
remains agents of choice for inhibiting the renin-angiotensin system in heart failure. The 
RESOLVD Trial suggested no major differences in efficacy of candesartan and enalapril, 
with a trend favoring enalapril during the study period of 43 weeks (92). The OPTIMAAL 
and VALIANT Studies will provide information specifically about the role of ARBs versus 
ACE inhibitors in the post-MI population. 
    Currently, ACE inhibitors continue to be regarded as the therapy of choice to inhibit the 
renin-angiotensin system in patients with asymptomatic and symptomatic left ventricular 
dysfunction. There is no current rationale to recommend initiating ARBs in patients with 
new onset heart failure or for switching from a tolerated ACE-inhibitor regimen to an ARB 
in patients with chronic heart failure. 


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Recommendation 2. All efforts should be made to achieve ACE 
inhibitor use in patients with heart failure caused by left ventricular 
dysfunction. Patients who are truly intolerant to ACE inhibitors 
should be considered for treatment with the combination of hydralazine and 
isosorbide dinitrate (Hyd-ISDN) (Strength of Evidence = B) or an ARB 
(Strength of Evidence = C). 
 
    Previous large-scale trials do not specifically address the role of ARB and Hyd-ISDN in 
patients who are intolerant to ACE inhibitors. One arm of the CHARM Study has been 
specifically designed to test the effectiveness of candesartan in patients with systolic 
dysfunction who are intolerant to ACE inhibitors. The primary endpoint in this study will 
be a composite of cardiovascular death and time until first hospitalization for heart failure. 
For now, ARBs offer a reasonable alternative in the heart failure or post-MI patient who is 
truly intolerant to ACE inhibition. Intolerance because of cough should always trigger a 
careful reevaluation for congestion. If congestion is present, cough should abate with 
increases in diuretic that should allow ACE-inhibitor use to continue (93). It should be 
emphasized that patients intolerant to ACE inhibitor because of renal dysfunction, 
hyperkalemia, or hypotension are often intolerant to ARBs as well. ACE inhibitor 
intolerance because of persistent symptomatic hypotension in advanced heart failure may 
represent severe dependence on the hemodynamic support of the renin-angiotensin system, 
which generally would predict hypotension with ARB use as well. 
    The combination of Hyd-ISDN has not been studied in the post-MI population, but 
sufficient experience exists to support its use in the ACE-inhibitor-intolerant patient with 
symptomatic heart failure. Hydralazine blocks the development of nitrate tolerance, which 
argues for the use of combination therapy. Although they were not studied alone in a heart 
failure mortality trial, oral nitrates represent another reasonable alternative for patients 
intolerant to both ACE inhibitors and hydralazine. 
 
Unresolved Therapeutic Issues  
 
Combination Therapy With ACE Inhibitors and ARBs. Interest has grown in the 
potential utility of combining ACE inhibitors and ARBs in patients with heart failure. 
Initial data suggest that the combination yields more vasodilation and decreased blood 
pressure than either agent alone. The addition of losartan to an ACE inhibitor has been 
found to improve exercise capacity compared with an ACE inhibitor alone (94). 
Preliminary data from the RESOLVD Trial suggest that ventricular dilation and 
neuroendocrine activation may be best reduced with combination therapy, but other 
endpoints were not clearly affected. Trials are currently underway to determine the safety, 
as well as benefit, of more complete blockade of the renin-angiotensin system. The Val-
HeFT Trial is a large-scale investigation of the effect of valsartan in addition to ACE 
inhibitors on morbidity and mortality in symptomatic patients with heart failure caused by 
systolic dysfunction. One arm of the CHARM Study will also examine the effect of the 
addition of candesartan in patients with symptomatic, systolic dysfunction treated with an 
ACE inhibitor. Preliminary data from the RESOLVD Trial suggest that combination 
therapy may be even more efficacious when used in conjunction with ß-blocker treatment. 


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Results from Val-HeFT and CHARM in the subset of patients treated with ß-blocker 
therapy will provide more information concerning this strategy. 
    Combination therapy represents a rational option when treating severe hypertension or 
other vasoconstriction but cannot, at present, be recommended as routine therapy in the 
absence of a proven superiority to ACE-inhibitor therapy alone. 
 


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HFSA Guidelines 
Criteria for NYHA functional classification for chronic heart failure patients, 
functional capacity (130) 
 
CLASS 1  
No limitation of physical activity. Ordinary physical activity does 
not cause undue fatigue, palpitation, or dyspnea. 
 
CLASS 2  
Slight limitation of physical activity. Comfortable at rest, but 
ordinary physical activity results in fatigue, palpitation or 
dyspnea. 
 
CLASS 3  
Marked limitation of physical activity. Comfortable at rest, but 
less than ordinary activity causes fatigue, palpitation or 
dyspnea. 
 
CLASS 4  
Unable to carry out any physical activity without discomfort. 
Symptoms of cardiac insufficiency at rest. If any physical 
activity is undertaken, discomfort is increased. 


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HFSA Guidelines 
Glossary of Clinical Trials 
 
AVID  
 
Antiarrhythmics Versus Implantable Defibrillators 
BEST  
 
Beta-blocker Evaluation of Survival Trial 
CAMIAT  
 
Canadian Amiodarone Myocardial Infarction Arrhythmia Trial 
CAPRIE  
 
Clopidogrel vs Aspirin in Patients at Risk of Ischemic Events 
CASH  
 
Cardiac Arrest Study Hamburg 
CHF-STAT  
 
Congestive Heart Failure-Survival Trial of Antiarrhythmic Therapy 
CHARM  
Candesartan Cilexetil in Heart Failure Assessment of Reduction in 
Mortality and Morbidity 
CIBIS  
 
Cardiac Insufficiency BIsoprolol Study 
CIBIS II  
 
Cardiac Insufficiency Bisoprolol Study II 
CIDS  
 
Canadian Implantable Defibrillator Study 
COMET  
 
Carvedilol or Metoprolol European Trial 
CONSENSUS  
Cooperative North Scandinavian Enalapril Survival Study 
CONSENSUS II  
Cooperative New Scandinavian Enalapril Survival Study II 
COPERNICUS  
Carvedilol Prospective Randomized Cumulative Survival Trial 
DEFINITE  
 
Defibrillators in Nonischemic Cardiomyopathy Treatment Evaluation 
DIAMOND  
 
Danish Investigation of Arrhythmia and Mortality on Dofetilide 
DIG   
 
Digitalis Investigation Group 
ELITE  
 
Evaluation of Losartan In The Elderly 
ELITE II  
 
Losartan Heart Failure Survival Study - ELITE II 
EMIAT  
 
Infarction Amiodarone Trial 
GESICA  
 
Grupo de Estudio de Sobrevida en Insuficiencia Cardiaca en Argentina 
GUSTO 1  
Global Utilization of Streptokinase and TPA for Occluded coronary arteries 
MADIT  
 
Multicenter Automatic Defibrillator Implantation Trial 
MADITII  
 
Multicenter Automatic Defibrillator Implantation Trial II 
MDC   
 
Metoprolol in Dilated Cardiomyopathy trial 
MERIT-HF  
Metoprolol CR/XL Randomized Intervention Trial in Heart Failure 
MOCHA  
Multicenter Oral Carvedilol in Heart-failure Assessment 
MTT  
Myocarditis Treatment Trial 
OPTIMALL  
Optimal Therapy in Myocardial Infarction with the Angiotensin II Antagonist 
Losartan 
PRECISE  
Prospective Randomized Evaluation of Carvedilol In Symptoms and 
Exercise 
PROVED  
Prospective Randomized study Of Ventricular failure and the Efficacy of 
Digoxin 
RADIANCE  
Randomized Assessment of Digoxin on Inhibitors of the Angiotensin 
Converting Enzyme 
RALES  
Randomized Aldactone Evaluation Study 
RESOLVD  
Randomized Evaluation of Strategies for Left Ventricular Dysfunction 
SAVE  
Survival And Ventricular Enlargement 
SCD-HeFT  
Sudden Cardiac Death in Heart Failure: Trial of prophylactic amiodarone 
versus implantable defibrillator therapy 
SOLVD  
Studies Of Left Ventricular Dysfunction 
SWORD  
Survival With Oral D-sotalol 
ValHeFT  
Valsartan Heart Failure Trial 
VALIANT  
Valsartan in Acute Myocardial Infarction 
 
 


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