A Phase II, Randomized, Open Label Trial of Pre-operative (Neoadjuvant)Letrozole (Femara) vs. Letrozole in Combination With Avastin in Post Menopausal Women With Newly Diagnosed Operable Breast Cancer
Phase: PHASE2
Conditions: Breast Cancer, Cancer of the Breast, Breast Neoplasm
Sponsor: University of Alabama at Birmingham

 
 
A Phase II, Randomized, Open Label Trial of Pre-Operative 
(Neoadjuvant) Letrozole (Femara®) vs. Letrozole in 
Combination with Avastin® in Post-Menopausal Women with 
Newly Diagnosed Operable Breast Cancer (Breast Cancer 
Research Foundation - Johns Hopkins Consortium) 
 
     Study Protocol & Statistical Analysis Plan  
 
 
 
 
   
 
 
 
 
      NCT00530868 
 
 
 
 
 
 
 
 
 
 
 
    March 14, 2011 
 
 
 
 
Andres Forero, M.D., Principal Investigator 
 
 
  University of Alabama at Birmingham 
 
 
 
 
Birmingham, AL 35294 
 
 
 

 
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COMPREHENSIVE CANCER CENTER 
UNIVERSITY OF ALABAMA AT BIRMINGHAM 
BIRMINGHAM, ALABAMA 
 
UAB 0648 and Affiliates:  A Phase II, Randomized, Open Label Trial of Pre-Operative 
(Neoadjuvant) Letrozole (Femara®) vs. Letrozole in Combination with Avastin® in Post-
Menopausal Women with Newly Diagnosed Operable Breast Cancer (Breast Cancer 
Research Foundation – Johns Hopkins Consortium) 
 
Coordinating Center: 
 
Comprehensive Cancer Center 
 
 
 
 
 
 
 
 
 
University of Alabama at Birmingham (UAB) 
 
Principal Investigator: 
 
Andres Forero, M.D. 
 
 
 North Pavilion 2510 
 
 
 1802 6th Ave South 
 
 
 Birmingham,  Alabama 35294-3300 
 
 
 Phone: (205) 975-2837 
 
 
 Fax: (205) 934-1608 
 
 
 
 
 
Email: Andres.Forero@ccc.uab.edu 
 
Breast Cancer Research Consortium Sites 
 
 
 
 
 
 
University of North Carolina at Chapel Hill 
 
 
 
 
 
 
 
 
 
 
 
Georgetown University 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dana Farber Cancer Institute 
 
 
 
 
 
 
University of Alabama at Birmingham 
 
 
 
 
 
 
University of California – San Francisco 
 
 
 
 
 
 
 
 
 
 
 
Indiana University 
 
 
 
 
 
 
Biostatistician: 
 
 
Wenquan Wang, Ph.D., Gretchen Cloud, Ph.D. 
 
Research Coordinator: 
 
Valerie Caterinicchia, R.N., B.S.N., OCN 
 
Clinical Trials Network Office: 
Pam Dixon, R.N., B.S.N., OCN 
 
Study Pharmacist:  
 
Rebecca Quinn, PharmD 
 
Investigational Agent: 
 
Avastin 
 
Support: 
Breast Cancer Research Foundation – Johns Hopkins 
Consortium  
 
 

 
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Genentech, Inc. 
 
 
 
 
 
Breast Cancer Research Foundation of Alabama 
 
 
 

 
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TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
1.0 BACKGROUND 
1.1 
Hormonal therapy for breast cancer 
1.2 
Vascular endothelial growth factor 
1.3 
Anti-VEGF monoclonal antibody 
1.4 
Hypothesis for this study 
1.5 
Study rationale 
2.0 OBJECTIVES 
2.1 
Primary 
2.2 
Secondary 
3.0 STUDY DESIGN 
3.1 
Description of the Study 
3.2 
Rationale for Study Design 
3.3 
Outcome Measures 
3.3.1 Primary Outcome Measures 
3.3.2 Secondary Outcome Measures 
4.0 SAFETY PLAN 
4.1 
Protocol Management and Oversight of Participating Affiliate Sites 
5.0 STUDY SUBJECTS 
5.1 
Subject Selection and registration 
5.2 
Inclusion Criteria 
5.3 
Exclusion Criteria 
6.0 STUDY DESIGN 
6.1 
Treatment Plan 
6.2 
Biopsy specimens 
7.0 STUDY MEDICATION 
7.1 
Avastin  Dosage and Formulation  
7.1.1 Avastin  Administration 
7.1.2 Avastin Storage 
7.1.3 Avastin Dose Modification and Toxicity Management 
7.2 
Letrozole 
7.3 
Other concomitant medications 
8.0 CLINICAL AND LABORATORY EVALUATIONS 
 
Pretreatment Evaluations 
 
Evaluations During Treatment 
 
 

 
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Post-Treatment Evaluations 
9.0 SUBJECT DISCONTINUATION 
10.0 STUDY DISCONTINUATION 
11.0 STATISTICAL METHODS 
11.1 
Determination of Sample Size 
11.2 
Statistical Analysis 
12.0 ADMINISTRATIVE RULES OF THIS PROTCOL 
12.1 
Compliance with the Protocol and Protocol Reviews 
12.2 
Informed Consent 
12.3 
Records and Reports 
12.4 
Study Management  
13.0 ASSESSMENT OF SAFETY 
13.1 
Adverse Event Reporting and Definitions 
13.2 
 Reporting of Serious Treatment Emergent Adverse Events 
14.0 
REFERENCES 
 
APPENDICES 
 
Appendix A: Study Flowchart 
 
Appendix B: NCI Common Toxicity Criteria Version 3.0 
Appendix C: FDA MedWatch 3500 Form 
Appendix D: Evaluation of Proteinuria 
Appendix E: Performance Status Scale 
Appendix F: NYHA Guidelines 
Appendix G: RECIST Criteria  
Appendix H: Blood and Biopsy Procedures 
 
 
Appendix I: 
Implementation Plan for Off-Site Conduct of UAB Comprehensive 
Cancer Center Investigator-Initiated Phase I and II Studies 
 
 

 
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Study Schema 
 
 
 
 
 
 
Post-Menopausal Patients with estrogen receptor and/or progesterone receptor positive,           
Her-2-neu negative, operable breast cancer (T2, T3 or T4a-c / N0-2 / M0). 
Inflammatory breast cancers are excluded (T4d)                        
Informed Consent 
Baseline H & P, CBC, CMP, INR, UA, urinary protein/creatinine ratio (24 hour urine collection if 
indicated), PET/CT scan or [CT of the chest, abdomen, pelvis, with Bone Scan], core needle 
biopsies 
Therapy 
Arm 1: Avastin 15 mg/kg IV every 3 weeks and Letrozole 2.5 mg PO day.  Prior to each Avastin 
administration: CBC, CMP, toxicity assessment, PS. Physical evaluation every 6 weeks and 
urinary protein/creatinine ratio or urine dipstick (and 24 hour urine collection if indicated), every 
6 weeks. Arm 2: Letrozole 2.5 mg PO day. Physical evaluation, CBC, CMP, toxicity assessment, 
PS every 6 weeks. 
Monitor for toxicities 
 
At 6 weeks evaluate for response    
Core needle biopsy will be obtained as well.
Patients with objective response or stable disease at week 6 will continue treatment per protocol 
with restaging at 12 weeks. Patients continuing with objective response or  stable disease at 12 
weeks will   continue treatment per protocol with restaging every 6 weeks until completion of 
therapy (24 weeks) or evidence of disease progression 
Surgery: At least 4 weeks after the last dose of Avastin. Patients will continue Letrozole until 
surgery. Last response evaluation on week 24.  
 
 

 
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1.0 
BACKGROUND 
1.1 
Hormonal Therapy for Breast Cancer 
Breast Cancer is the most common cancer in the United States and the second most deadly 
cancer in woman with an estimated 212,930 invasive breast cancer diagnoses and 40,870 breast 
cancer deaths in 2005; although the incidence has increased for several decades, the mortality 
rate has decreased (1). The introduction of effective therapies, including chemotherapy, 
hormonal therapies and targeted therapies, has been a major factor contributing to improved 
outcomes (2).  
The link between the endocrine system and breast cancer was first recognized more than 100 
years ago with the use of bilateral oophorectomy in patients with breast cancer (3).  It is now 
established that estrogen stimulates the growth of some breast tumors; thus, hormone-sensitive 
breast cancer can be effectively treated with agents that reduce estrogen stimulation of the 
estrogen receptor.  Since the late 1970s, numerous clinical trials have shown that the selective 
estrogen receptor (SERM) antagonist tamoxifen is as effective for the initial treatment of 
metastatic breast cancer as other hormonal therapies or chemotherapy but with fewer secondary 
effects in women with hormone receptor positive tumors (4,5).  As a result, tamoxifen became 
the gold standard for first-line treatment for hormone-sensitive metastatic breast cancer. In 
contrast to tamoxifen, which inhibits the activity of estrogen by competitively binding to the 
estrogen receptor, aromatase inhibitors (AIs) block the conversion of androgens to estrogens and 
reduce estrogen levels in tissue and plasma. Third-generation AIs include the nonsteroidal 
inhibitors letrozole and anastrozole and the steroidal inhibitor exemestane. With daily oral 
administration, anastrozole and exemestane inhibit aromatase activity in vivo by 97 to 98 percent 
and letrozole inhibits aromatase by more than 99 percent.  Over the last years, the aromatase 
inhibitors (AIs) have been developed and evaluated in clinical trials. As first-line treatment for 
metastatic breast cancer, third-generation AIs are equivalent or superior to tamoxifen. Women 
with  metastatic breast cancer who were given letrozole as first-line treatment had a significantly 
higher response rate, a significantly longer time to progression, and a significant improvement in 
one and two-year survival rates, as compared with women given tamoxifen. Thus, these agents 
have been demonstrated to be more effective than tamoxifen in the metastatic setting with less 
secondary effects and they are now considered the first line therapy in postmenopausal patients 
with hormone receptor positive metastatic breast cancer.   
 
- Adjuvant Hormonal therapy for early breast cancer in postmenopausal women 
The proven efficacy of hormonal therapy in metastatic disease also made it the ideal test agent 
for adjuvant hormonal therapy in early stage operable breast cancer (6). In the 1980’s the use of 
tamoxifen expanded as adjuvant therapy for early-stage hormone receptor positive breast cancer 
reducing reoccurrences by 47% and the risk of death by 26% among patients with hormone-
receptor-positive breast cancer.   
Historically, breast cancer was thought to be a local disease with predictable progression through 
the mammary ducts, lymphatics, and draining lymph nodes before more widespread 
dissemination.  A more recent divergent view of breast cancer is the recognition of the potential 
early micrometastases of cancer cells from the primary tumor; in this view, breast cancer is seen 
 
 

 
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as a systemic disease very early in its development.  This later model of breast cancer is 
increasingly felt to be the correct one based on consistent data which demonstrate a reduction in 
the risk of recurrence of breast cancer in women who received adjuvant therapy.  An overview of 
55 trials of adjuvant tamoxifen for 1, 2, or 5 years versus no therapy for patients with early breast 
cancer showed that the treatment produced highly significant benefits in terms of both recurrence 
and survival; reductions in recurrence were 18%, 25%, and 41%, and reductions in death rate 
were 10%, 15%, and 22% for 1, 2, and 5 years of tamoxifen treatment, respectively (7).  These 
benefits are durable as well after discontinuation of tamoxifen with a 47 % reduction in 
recurrence and 27% improvement in overall survival after 10 years of follow up.  These benefits 
were found to occur almost exclusively in the hormone receptor positive population.  The 
optimal duration of tamoxifen is unresolved, however 5 years of adjuvant therapy has 
demonstrated superior recurrence rates, and overall survival compared to shorter durations of 
therapy.  Three additional trials, including the NSABP-14, studying extending the duration of  
tamoxifen beyond 5 years have not showed any benefit and rather an increase in endometrial 
carcinoma and a decrease in the disease-specific survival.  Two definitive trials, ATLAS and 
aTTom, comparing the effect of 5 years of treatment with tamoxifen vs. the effect of more than 5 
years are currently ongoing.  Although the results have not been conclusive, it appears that 5 
years of tamoxifen is the optimal duration in terms of patient benefit (8-12).  In addition, the 
results of the 1998 Oxford Early Breast Cancer Trialist’s Collaborative Group showed that the 
significant improvements in the risk of recurrence and overall survival, with combination 
chemotherapy and tamoxifen in patients with estrogen receptor positive disease were irrespective 
of age or menopausal status (7). 
Based on the anti-tumor activity of the third-generation AIs (letrozole, anastrozole, exemestane) 
in the metastatic disease setting (13-21), these medications are currently being assessed in the 
adjuvant setting.  Multiple trials are being conducted at this time, and the data from five 
randomized trials is now available.  The first trial, ATAC (Arimidex, Tamoxifen, Alone or in 
Combination) randomized 9,366 postmenopausal women with invasive operable breast cancer 
who completed primary therapy to one of the three arms: tamoxifen alone, anastrozole alone or 
the combination of both.  After the first evaluation, the arm using the combination was 
discontinued due to lack of superiority over tamoxifen alone.  At the updated efficacy analysis 
after 68 months of follow up , DFS remained significantly longer in the anastrozole arm than in 
the tamoxifen arm with a HR 0.87; P = .01 with an absolute difference of 3.8%, demonstrating a 
continued benefit after completion of 5 years of therapy.  Time to progression was significantly 
longer for those patients receiving anastrozole compared with the patients receiving tamoxifen 
(HR = 0.79; P = .0005), time to distant metastases (HR = .86; P = .04) and a larger benefit was 
seen in the hormone receptor positive population.  There was also a 42% decrease in 
contralateral breast cancer (P = .01).  Tolerance was better for anastrozole (22-24).  Thus, 
anastrozole was the first AI approved by the FDA for the adjuvant treatment of early breast 
cancer in postmenopausal women with hormone receptor positive disease.  A second adjuvant 
trial, IES (Intergroup Exemestane Study) randomized 4,742 patients after 2-3 years of adjuvant 
tamoxifen to continued tamoxifen versus exemestane for a total of 5 years. After 30.6 months of 
follow up, exemestane demonstrated an improved DFS with exemestane (HR 0.68; P < .001), 
and an absolute difference of 4.7%. Long term data including survival and toxicity data have not 
been presented.  Several other supportive studies with the use of letrozole in the BIG FEMTA 
and BIG 1-98 trials have confirmed similar benefits demonstrating a class effect of the AI.  
 
 

 
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Additional data has also been published using five years of letrozole following five years of 
adjuvant tamoxifen, the MA-17 trial, in postmenopausal patients with hormone receptor positive 
tumors (randomized study between placebo and letrozole).  At a median follow-up of 2.4 years, 
the estimated 4-year DFS rate was 93% in the letrozole group compared with 87% in the placebo 
group (25).       
While both surgery and radiation therapy treatments affect the local disease, adjuvant therapy is 
employed to reduce the risk of systemic disease.  Thus, the current management of breast cancer 
attempts to eradicate breast cancer both at the primary tumor site and by affecting distant 
metastases.  The timing of these therapies is undergoing re-examination.  Observation of effects 
both on the primary tumor and on systemic metastases has suggested that systemic therapy might 
provide greater efficacy if employed earlier in the treatment of breast cancer (neoadjuvant 
therapy).   
- 
Neoadjuvant therapy for breast cancer 
Preoperative chemotherapy has been employed as part of multimodality therapy for patients with 
locally advanced breast cancer (LABC) or inflammatory breast cancer (IBC).  These patients 
fared poorly with single-modality therapy alone. Historical studies demonstrated 5-year disease-
free survival (DFS) rates for LABC to be 41% with surgery alone and 29% with radiation 
therapy (26).  Patients with IBC did worse, with DFS rates of 3 to 10% with surgery or radiation 
alone (27).  In contrast, the combination of primary chemotherapy, surgical resection, and 
radiotherapy has significantly improved the clinical outcome of these patients (28, 29).  As a 
result of the favorable impact that preoperative chemotherapy has demonstrated on the survival 
rates of patients with locally advanced disease, numerous studies have evaluated its use in 
earlier-stage disease (30-32). The largest of the randomized studies comparing preoperative to 
postoperative chemotherapy was the NSABP B-18 study.  This study randomized 1,523 women 
with operable stage I-IIIA breast cancer to receive four cycles of Adriamycin (doxorubicin) and 
cyclophosphamide (AC) either preoperatively or postoperatively.  Although the disease free and 
overall survival rates at 5 years of follow up were similar between the two cohorts (DFS: 66.7% 
vs. 67.3% and OS: 72.3 vs. 73.2%), it became clear that preoperative chemotherapy improved 
the breast conservation rate.  There was an overall response rate of 79% in the patients after 
preoperative chemotherapy with an increase in the number of conservative surgeries (67% vs. 
60%).  More importantly, the pathologic complete response (CR) rate was directly proportional 
to DFS and overall survival (OS) rates (33, 34).  For patients who experienced a pCR, DFS and 
overall survival were significantly improved, with 84% of patients with pCR disease free at 5 
years compared to 60% in patients who had no clinical response.   
 
A similar association between pathologic CR rate and survival has been demonstrated in 
numerous other studies (35-40).  The pCR is increasingly an accepted surrogate marker in 
neoadjuvant trials for overall survival and outcomes.  The majority of published studies of 
preoperative chemotherapy have used anthracycline-based regimens with pCR rates between 10-
15%.  However, owing to the efficacy of the single agent taxanes (paclitaxel and docetaxel) in 
the metastatic setting, both taxanes have been included in numerous trials over the past decade.  
Based on the theoretical benefit of combining alternating cross-resistant chemotherapy agents, 
the addition of paclitaxel and docetaxel to anthracycline-based regimens have increased pCR 
rates to between 20-25%. This was confirmed in a large randomized neoadjuvant study, NSABP-
 
 

 
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27, comparing the addition of docetaxel to an anthracycline based regimen.  The addition 
increased pCR from 13.7 % to 26.1 % at the cost of increasing G4 toxicity from 10.3 % to 23.4 
%, with an incidence of neutropenic fever 7.3 % vs. 21.2 %.  Ongoing studies with longer 
follow-up will clarify the role of preoperative chemotherapy in earlier stage disease and the most 
effective sequence or combination of chemotherapeutic agents. As it is described below, it has 
become apparent that not all breast cancer tumors have the same response to neoadjuvant 
chemotherapy; as an example, patients with estrogen receptor negative tumors have higher 
pathological complete responses to neoadjuvant chemotherapy than those with estrogen receptor 
positive tumors.  Hence, pathologic CR rate is considered to be the most important study end 
point to assess the efficacy of preoperative chemotherapy regimens. 
 
Neoadjuvant chemotherapy has multiple practical and theoretic advantages, enhancing our 
ability to care for patients with breast cancer.  This treatment approach remains a standard of 
care for patients with IBC or LABC and a safe option for patients with early-stage breast cancer.  
Neoadjuvant chemotherapy can downstage both the primary tumor and metastatic disease in the 
axilla, therefore improving the ability to perform breast-conserving therapy without increasing 
the risk for local recurrence.  This approach also allows the physician to evaluate the in vivo 
response to treatment and potentially to avoid ineffective therapies.  The pathologic CR to a 
particular neoadjuvant therapy regimen can predict survival, allowing this end point to be used 
as a surrogate that can identify treatments that will improve survival much earlier than traditional 
trial designs.  The future of neoadjuvant chemotherapy will be to develop classification or 
predictive markers to tailor therapy to an individual and enhance survival.  Neoadjuvant 
chemotherapy is no longer limited to patients with stage III cancer or IBC but is an approach that 
will continue to develop new uses as research continues to move forward. Preoperative 
chemotherapy in primary breast cancer patients is now accepted as an effective treatment, 
showing that the DFS and overall survival are similar to those of patients in whom systemic 
therapy is used after surgery.   
- Neoadjuvant hormonal therapy for operable breast cancer 
One of the most important factors influencing the achievement of pathological CR with 
neoadjuvant chemotherapy is hormone receptor status. ER positive patients achieve 
pathological CR rates of 8-12% (or lower) while ER negative patients have a pathological 
CR rates of 20-40%. Additionally, these chemotherapy regimens are associated with significant 
toxicity including neutropenic fever, cardiomyopathy and alopecia (94-96). Thus, post-
menopausal women with ER positive or PR positive (Her-2-neu negative) tumors may benefit 
from alternative, less toxic neo-adjuvant therapy. Several phase II and III studies conducted with 
tamoxifen have shown that it is possible to obtain good clinical responses with pre-operative 
endocrine therapy. Initial studies in which endocrine therapy was compared with surgery 
demonstrated initial reduction in tumor size for the majority of the older patients receiving 
endocrine treatment without impact in the overall survival for both groups; however, long-term 
local disease control was poor for those patients receiving endocrine therapy alone (41, 42).  
This might not be the case when endocrine therapy is used not “in substitution of” but “before” 
optimal local control with surgery.     
 
 

 
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While tamoxifen is well tolerated, its adverse events are of important clinical consequences to 
the patient.  Thus, the AIs became an alternative treatment in the neoadjuvant setting.  In a phase 
I-II trial conducted in Scotland, a clinical response rate of 92% was seen in 24 postmenopausal 
estrogen receptor positive patients whose locally advanced breast cancer was treated with 
letrozole for 3 months prior to surgery.  Of note, all 15 patients not eligible for breast 
conservation surgery had sufficient tumor regression to allow breast conservative surgery (43).  
This pilot study led to the conduct of a randomized, double-blind study designed to compare 
efficacy of preoperative letrozole versus preoperative tamoxifen for postmenopausal women 
with estrogen receptor and/or progesterone receptor positive breast cancer ineligible for breast 
conservative surgery (44).  Overall objective response rate was statistically significantly superior 
in the letrozole group (55%) compared with the tamoxifen group (36%).  In addition, 45% of 
patients in the letrozole group underwent breast conservative surgery compared with 35% in the 
tamoxifen group.  Clinical complete response was 10% for the group receiving letrozole 
compared with 4% for the group receiving tamoxifen.  Neoadjuvant letrozole was very tolerable 
as the most common side effect was hot flashes (20%) and nausea (5%).  Semiglazov et al 
presented to the 2004 ASCO Annual Meeting and the 5th European Breast Cancer Conference a 
study randomizing postmenopausal, ER positive patients to neoadjuvant AI (anastrozole or 
exemestane) vs. an anthracyline/taxane combination chemotherapy regimen.  This trial 
demonstrated similar response rates between the two cohorts with a similar disease free survival 
at 3 years of follow-up.  However there was a trend towards increased breast conservation 
surgery in the AI group.  Neoadjuvant hormonal therapy also has similar response rates in both 
Her2 negative (106/154; 64%) and Her2 positive (11/18; 64%) breast cancers.  
The optimal duration of neoadjuvant AI therapy is unknown, with current published trials using 
AI for 3-4 months prior to surgery.  Renshaw et al (San Antonio Breast Cancer Meeting 2004, 
abstract # 405) evaluated 142 women treated with neoadjuvant AI, and demonstrated in the first 
3 months of therapy, 100/142 pt had sufficient tumor regression to allow BCS with a median 
tumor regression of 52%. The 42 remaining patients continued to received letrozole, and 
between 3 and 6 months of therapy, had an additional 57% median tumor regression (CR = 
12/42; 29%).  After 6 months of therapy, 22/42 patients continued neoadjuvant letrozole for an 
additional 6 months. Between 6 and 12 months of therapy there was an additional 66% median 
tumor regression with only 1 patient having progressive disease (CR = 8/22; 36%).   Extended 
therapy of neoadjuvant AI up to 12 months is safe with continued tumor regression. In addition, 
Paepke et al (2003 ASCO Annual Meeting, abstract #321) conducted a trial in which 33 patients 
received letrozole for a minimum of 4 and a maximum of 8 months prior to surgery. They 
reported that longer treatment resulted in a statistically significant increase in tumor size 
reduction. Moreover, 90% of the patients receiving therapy for longer than 4 months had CR or 
PR compared with 57% of patients with CR or PR receiving therapy for up to 4 months.  Thus, 
these studies strongly suggest that patients whose tumors respond to letrozole after 3 
months can expect a further reduction in tumor volume with continued therapy.    
 
 

 
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- Hormone resistance 
While the hormonal agents have demonstrated adequate anti-tumoral activity, the development 
of resistance is at the core of the problem and must be overcome to make additional progress.  
Consequently, efforts aimed at gaining a better understanding of the mechanisms underlying 
hormone resistance are warranted and could lead to strategies to prevent or circumvent 
resistance. Although the mechanisms responsible for tamoxifen resistance remain unknown, over 
the last 20 years, a number of mechanisms for hormone resistance have been proposed including 
increased levels of intracellular antiestrogen binding sites; alterations in the estrogen receptor; 
increased agonistic properties of tamoxifen and ligand independent activation of the estrogen 
receptor; activation of ERβ by antiestrogens and constitutive growth factor signaling conferring 
ER+ breast cancer cells with estrogen independent and hormone resistant phenotypes (45).  In 
addition, a number of biological factors have demonstrated to be related with a favorable or poor 
response to endocrine therapies and they can be used to understand mechanisms of resistance 
and to develop new therapeutic strategies to overcome resistance to these agents in breast cancer 
patients.   
Angiogenesis represent a fundamental step in tumor progression (46); multiple studies have 
shown that node negative as well as node positive breast cancer patients with high  intra-tumoral 
vascular endothelial growth factor (VEGF, a potent angiogenic factor) concentrations have a 
significantly shorter relapse-free survival (47, 48).  In addition, the expression of tyrosine 
kinases such as epidermal growth factor receptor or its family member HER2, the tumor 
suppressor gene TP53, and VEGF have been associated with a poor response to tamoxifen (49, 
50).  Recent studies have demonstrated that oncogenes and tumor suppressor genes associated 
with neoplastic cell transformation play an integral part in activating the angiogenic switch.  
Alterations of these genes, including RAS, SRC, HER2, and TP53, have been characterized as 
inducers of VEGF expression (as an example, mutant TP53 has demonstrated the ability of up-
regulate VEGF) (51).  
Furthermore, studies in UAB Breast Cancer Spore using the MCF-7 tamoxifen murine 
xenografts model by Dr. Fran Kern and collaborators indicate that paracrine effects of VEGF 
over-expression by ERα+ breast cancer cells can reverse the effectiveness of tamoxifen as a 
cytostatic or cytotoxic agent (52-58).  In this MCF-7 model, they demonstrated that increased 
expression of VEGF causes an acquired Tamoxifen resistance and tumor loss of estrogen 
dependence in vivo.  MCF-7 cells with VEGF co-expression (stably transfected or regulated 
expression) had in vitro tumor cell proliferation and estrogen dependence identical to non-
transfected MCF-7, while MCF-7/VEGF cells in vivo displayed increased tumor growth rates, 
higher rates of metastases, and estrogen independence.  These survival and proliferation signals 
may be an indirect effect of VEGF presumably reflecting the enhanced vascularity of tumors 
with secondary growth factor and signal pathway modulation.  Reversal of VEGF over-
expression in vivo returned tumors to estrogen dependent growth. 
In addition, Berns and collaborators evaluated the predictive value of TP53 gene mutation status 
and VEGF levels for response to tamoxifen therapy in 160 advanced breast cancer patients with 
ER+ tumors (59).  In a univariate analysis, both TP53 gene mutation and VEGF levels above the 
median value were significantly associated with a short progression-free survival, post-relapse 
 
 

 
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overall survival and a poor response to Tamoxifen.  In a Cox multivariate regression analysis 
including the traditional predictive factors, the addition of TP53 gene mutation and VEGF status, 
alone or in combination significantly predicted a poor response to tamoxifen.  When the two 
factors were combined, a significantly decreased odds ratio was seen for response (odds ratio, 
0.27).  Similarly, an increased hazard ratio was seen for progression-free survival (2.32) and 
post-relapse overall survival (1.68) in the group with mutant TP53 and high VEGF compared 
with the group with booth risk factors absent.  
Dr. Zhican Qu, Ph.D., a recipient of a career development award of the UAB-Breast SPORE, 
conducted preclinical efficacy studies of tamoxifen and bevacizumab in combination for the 
treatment of breast cancer using a novel mouse model.  In her previous studies she showed that 
elevated levels of VEGF increases tumor growth, and promotes metastasis and tamoxifen 
resistance; thus, she hypothesized that a combination of agents targeting both ER and VEGF 
signaling may be a more effective treatment than the use of either drug alone.  She tested this 
hypothesis using a combination of tamoxifen and Avastin in a xenograft nude mouse model with 
regulated-VEGF expression.  The efficacy of the treatment was tested in two groups of mice, one 
with early and one with late stage ER-positive breast cancer.  All mice received orthotopic 
implantation of 107 tumor cells at mammary fat pat and a 17β-estradiol pellet supplement on Day 
1.  Doxycycline was given to all mice to induce expression and secretion of VEGF from tumor 
cells.  Animals in the early stage group started therapy (tamoxifen or Avastin or placebo or 
tamoxifen-Avastin in combination) on Day 7 when the tumor size reached about 100 mm3.  The 
Avastin-tamoxifen combined treatment demonstrated better control of primary tumor growth 
than therapy with tamoxifen or Avastin alone.  The survival rate was superior in the group of 
mice treated with combination therapy compared with the groups treated with single agents or 
placebo.  Animals in the late stage group started therapy on Day 32 when the tumor size reached 
about 1000 mm3.  The combined treatment showed again a better reduction of tumor growth rate 
and a significant better survival.  83% of mice survived in the combined treatment group, while 
only about 50% of mice survived in the monotherapy groups.  These results demonstrated that 
the Avastin-tamoxifen combination is more effective on inhibiting tumor growth of ER positive 
breast cancer and increasing survival rate than either drug treatment alone (2005 AACR Annual 
Meeting, abstract #1091).   
 
1.2 
Vascular Endothelial Growth Factor 
 
A novel approach to the treatment of solid tumors, including breast cancer, is to inhibit the 
growth of new vessels supplying the tumor.  Folkman and others have provided compelling 
evidence linking tumor growth and metastases with angiogenesis (60).  Human cancer cells 
placed in avascular sites in animals form tumors that growth only up to 1-2 mm in diameter (61).  
The same cells implanted in vascular sites grow rapidly to form large, bulky tumors.  Several 
authors have shown a correlation between microvessel density and clinical outcome.  Weidner 
and coworkers have shown a statistically significant correlation between the number and density 
of microvessels in histologic specimens of human breast cancer and clinical outcome, including 
the incidence of metastases as well as overall survival and relapse-free survival (62, 63).  A 
study of a large cohort of patients (836) with operable, invasive breast cancer demonstrated that 
angiogenesis, as measured by Chalkley count, is an independent prognostic indicator of 
recurrence-free and overall survival (64).  Obermair and co-workers have also demonstrated the 
 
 

 
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microvessels density correlates inversely with disease-free survival in breast cancer (65).  In 
addition, Fox and coworkers have shown an association between tumor angiogenesis and the 
presence of bone marrow micrometastases in breast cancer patients prior to surgery (66). 
 
Recognition that angiogenesis is essential to the growth of solid tumors has led to identification 
of angiogenic factors responsible for stimulation of new blood vessel formation.  Of the 
identified angiogenic factors, vascular endothelial growth factor (VEGF; also known as vascular 
permeability factor) is the most potent and specific and has been identified as a crucial regulator 
of both normal and pathologic angiogenesis (67).  VEGF is a highly conserved, homodimeric, 
secreted, heparin-binding glycoprotein whose dominant isoform has a molecular weight of 
approximately 45,000 (67, 68).  VEGF produces a number of biological effects, including 
endothelial cell mitogenesis and migration, induction of proteinases leading to remodeling of the 
extracellular matrix, increased vascular permeability, and maintenance of survival for newly 
form blood vessels (67).  VEGF expression is regulated by hypoxia via molecular pathways 
similar to those regulating erythropoietin gene expressions (67).  The biologic effects of VEGF 
are mediated through binding and stimulation of two receptors on the surface of endothelial 
cells: Flt-1 (fms-like tyrosine kinase) and KDR (kinase domain region) (67).  Increased 
expression of VEGF has been measured in most human tumors examined to date, including 
tumors of lung, breast, thyroid, gastrointestinal tract, kidney, bladder, ovary, and cervix, as well 
as angiosarcomas and glioblastomas (67).  In breast cancer, VEGF mRNA and protein are highly 
expressed in invasive ductal carcinoma, metastatic ductal carcinoma, and comedo-type DCIS but 
not as highly in lobular carcinoma (69).  Normal breast ductal epithelium expresses VEGF 
mRNA at low levels and VEGF receptor mRNA expression has not been detected. Increased 
VEGF mRNA and protein levels have been shown to correlate with shortened relapsed-free 
survival (70, 71). 
 
1.3 
Anti-VEGF Monoclonal Antibody 
 
Inhibition of VEGF using an anti-VEGF monoclonal antibody blocks the growth of a number of 
human cancer cell lines in nude mice (67).  The human cancers represented by these cell lines 
that are growth-inhibited by anti-VEGF antibody include non-small cell lung cancer (Calu-6), 
colorectal cancers (LS174T), HM-7, LSLiM6), breast cancer (MCF-7), prostate cancer (D-145), 
head and neck cancer (KB), ovarian cancer (SK-OV-3), and others (67).  In addition, the 
combination of anti-VEGF antibody and chemotherapy in nude mice injected with human cancer 
xenografts results in an increased anti-tumor effect compared with antibody or chemotherapy 
treatment alone (72). 
 
To test the hypothesis that inhibition of VEGF in patients with cancer results in clinical benefit, a 
recombinant humanized version of a murine anti-human VEGF monoclonal antibody, named 
rhuMAb VEGF (Bevacizumab or Avastin®), was developed (73).  In cynomolgus monkey 
studies, twice weekly IV treatment with rhuMAb VEGF (doses of 2, 10, and 50 mg/kg) for either 
4, 13, or 26 weeks was well tolerated with no overt signs of acute toxicity (74, 75).  There were 
no effects on body weight, food consumption, blood pressure, EKGs, rectal body temperature, 
respiratory rate, ophthalmologic observations, or clinical pathology parameters.  In all active 
treatment groups, animals with open growth plates showed physeal dysplasia.  Focal to diffuse 
 
 

 
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chondroid necrosis and linear fissuring of the cartilaginous growth plate were also observed.  
Females treated with 10 to 50 mg/kg twice weekly had decreased ovarian and uterine weights, 
which were associated with the absence of corpora lutea.  Minor changes in some organ weights 
were noted in the 4-week study but were not produced in the 13- or 26- week study.  No 
antibodies against rhuMAb VEGF were detected.  To assess the effects on wound healing, 
rabbits were given a partial thickness dermal wound on the ear or on the back and treated with 
rhuMAbVEGF every other day for two weeks at doses from 0.5 to 50 mg/kg per day (76).  Dose-
related inhibition of wound healing was exhibited following treatment with the antibody, with 
maximal inhibition observed at a dose level of 10 mg/kg per day.  No specific tissue cross-
reactivity with human, cynomolgus monkey, or rabbit tissue was observed with 400 μg/ml of the 
antibody.  The results of non-clinical efficacy and toxicology studies supported entering clinical 
development. 
 
- Clinical Trials with Avastin (Bevacizumab) 
 
Avastin has been studied in a multitude of Phase I, II, and III clinical trials in more than 5000 
patients and in multiple tumor types. In addition, data are available from 3,863 patients enrolled 
in two post-marketing studies in metastatic colorectal cancer (CRC).  Approximately 130,000 
patients have been exposed to Avastin as a marketed product or in clinical trials.  The following 
discussion summarizes Avastin’s safety profile and presents some of the efficacy results 
pertinent to this particular trial. Please refer to the Avastin Investigator Brochure for descriptions 
of all completed Phase I, II, and III trials reported to date. 
 
Phase I studies:  Two phase I trials were conducted.  The first study was a dose 
escalation, single agent trial in patients with advanced malignancies to evaluate safety and 
pharmacokinetics (77).  Five dose levels were evaluated (0.1 to 10 mg/kg) and subjects received 
a total of four doses over a 42-day treatment period.  The second study evaluated the 
combination with different chemotherapeutic agents in patients with advanced solid 
malignancies (78).  The antibody was administered as 8 weekly doses of 3 mg/kg.  In general, 
Avastin was well tolerated in both studies (see Avastin toxicity profile below).  No antibodies to 
Avastin were detected in either study.  The pharmacokinetics of Avastin appeared to be linear for 
doses greater than 1 mg/kg, with a half-life of 15 days.  The combination with chemotherapy 
agents did not modify the pharmacokinetic behavior of the antibody.  Thus, these phase I studies 
demonstrated that Avastin was well tolerated and had predictable pharmacokinetics when used 
either as a single agent or in combination with cytotoxic agents.  
 
Phase II studies:  Four studies were conducted in NSCLC in combination with 
carboplatin and paclitaxel, hormone-refractory prostate cancer as a single agent, relapsed 
metastatic breast cancer as a single agent, and metastatic colorectal cancer in combination with 
5-FU and Leucovorin (79-81).  Doses varied from 5 to 20 mg/kg every other week.  Antitumor 
activity has been demonstrated in multiple tumor types and with both single agent Avastin and 
combination chemotherapy.  In the prostate study, no patient had an objective response; however 
there were signs of modest activity.  The NSCLC trial reported increased response rates and 
prolongation of time to disease progression compared with chemotherapy alone.  In the 
colorectal trial the hazard of experiencing disease progression was reduced by 57% in patients 
receiving the combination of Avastin and 5-FU/Leucovorin compared with those receiving 
 
 

 
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chemotherapy alone.  Improvements in response rate and median survival were also seen in these 
colorectal patients.  
 
Phase III studies; pivotal trials:  Two trials were conducted in metastatic colorectal 
cancer.  The first one was a phase III randomized study of irinotecan/5-FU/Leucovorin (IFL) 
with or without Avastin in patients with newly diagnosed metastatic colorectal cancer (82).  
Patients received up to 48 doses of Avastin on an every 2-week infusion schedule.  Nine hundred 
twenty five patients were enrolled in the trial.  The results for this study confirmed that the 
addition of Avastin to chemotherapy resulted in an increase in overall survival, response rate, 
and duration of response compared with patients who received chemotherapy alone.  The 
combination resulted in a statistically significant prolongation of survival (p=0.00004) with a 
corresponding increase in median duration of survival from 15.6 months for patients treated with 
chemotherapy alone to 20.3 months for patients treated with the combination of Avastin and 
chemotherapy (hazard ratio of death of 0.660).  The addition of Avastin to chemotherapy also 
resulted in a significant improvement in progression-free survival; median of 6.24 months for 
patients treated with chemotherapy alone and 10.5 months for patients treated with the 
combination.  Similar increases were seen in overall response rate (35% vs. 45%), and duration 
of response (7.1 vs. 10.4 months).  The addition of Avastin to the chemotherapy regimen resulted 
in a small increase in the incidence of grade 3 and 4 adverse events (from 74% to 85%).  No 
increase was seen in adverse events leading to death or study discontinuation or in 60-day 
mortality.  Grade 3 and 4 adverse events that were increased in the combination arm compared 
with the chemotherapy arm included deep thrombophlebitis, hypertension, diarrhea, and 
leucopenia.  No significant differences were observed in the incidence of infusion-related events, 
thromboembolic events, grade 3 and 4 bleeding, or proteinuria of all grades.  The addition of 
Avastin to chemotherapy appeared to increase the risk of the uncommon event of gastrointestinal 
perforation.  
 
The second trial was a double-blind, randomized trial of 5-FU/Leucovorin with or without 
Avastin in patients with metastatic colorectal cancer who were not candidates for treatment with 
irinotecan as a first line (83).  The trial enrolled 209 patients.  Patients received up to 48 doses of 
Avastin (5 mg/kg) on an every-2-week infusion schedule.  The addition of Avastin resulted in a 
statistically significant prolongation of progression-free survival and trends towards improved 
survival, response rate, and duration of response compared with chemotherapy alone.  The 
addition of the antibody was well tolerated.  Grade 3 hypertension and gastro-intestinal 
perforation were increased by the addition of the antibody.  Proteinuria and arterial 
thromboembolic events were also observed. 
 
The results of these trials led to the approval of Avastin in combination with intravenous 5-
FU-based chemotherapy by the FDA in 2004 for first-line treatment of patients with newly 
diagnosed colorectal cancer. 
 
Additional data from Phase III trials in metastatic CRC (E3200), non−small cell lung cancer 
(NSCLC; E4599), and metastatic breast cancer (E2100) have also demonstrated clinical benefit 
from Avastin when added to chemotherapy.  In Study E3200, the addition of Avastin to 
FOLFOX chemotherapy resulted in improved overall survival compared with FOLFOX alone 
 
 

 
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(13.0 vs. 10.8 months, respectively, HR = 0.75; p < 0.01) in a population of previously treated 
CRC patients. 
 
There was also improved overall survival in first-line NSCLC patients (E4599) treated with 
carboplatin/paclitaxel + Avastin compared with chemotherapy alone (12.3 vs. 10.3 months, 
respectively; HR = 0.80; p = 0.003).  The results from this trial were the basis for FDA approval 
of Avastin for use in combination with carboplatin + paclitaxel as first-line treatment of patients 
with unresectable, locally advanced, recurrent or metastatic, non-squamous NSCLC in October 
2006.  Finally, patients with untreated metastatic breast cancer (E2100) who received Avastin in 
combination with weekly paclitaxel had a marked improvement in PFS compared with 
chemotherapy alone (13.3 vs. 6.7 months, respectively; HR = 0.48; p < 0.0001) (see the Avastin 
Investigator Brochure for additional details). 
 
- Avastin in the Treatment of Breast Cancer 
 
The monoclonal antibody has been evaluated in two clinical trials (80, 83).  The first trial was a 
phase II, dose-escalation study evaluating the safety, efficacy, and pharmacokinetics of Avastin 
in patients with metastatic breast cancer that relapsed after at least one conventional 
chemotherapy agent. 18 patients were enrolled at 3 mg/kg every other week, 41 patients were 
enrolled at 10 mg/kg every other week, and 16 patients were enrolled at 20 mg/kg every other 
week.  The overall response rate was 9.3% (7 out of 75 patients).  A PR in a cervical lymph node 
was observed in 1 out of 18 patients treated at the 3 mg/kg dose cohort.  One CR and 4 PRs were 
observed among the 41 patients enrolled in the 10 mg/kg dose cohort.  One PR was observed in 
the group of patients treated in the 20 mg/kg dose cohort.  Toxicity observed in this trial was not 
different from the toxicity in all other trials.  Pharmacokinetic analysis suggested that the profile 
was similar to that in the colorectal and NSCLC phase II trials.  Thus, this trial showed evidence 
of single-agent activity of Avastin in metastatic breast cancer and provided the rationale for a 
phase III trial using Avastin in combination with chemotherapy in this setting. 
 
The first Phase III trial in breast cancer was a randomized, open-label study that evaluated safety 
and efficacy of Avastin in combination with capecitabine in patients with metastatic disease 
previously treated.  Patients received up to 35 doses of Avastin on an every-3-week infusion 
schedule (15 mg/kg per dose).  The trial enrolled 462 patients.  The results of the primary 
efficacy analysis showed that the treatment with the combination of Avastin and capecitabine did 
not result in a statistically significant prolongation in progression-free survival compared with 
capecitabine alone.  There was a statistically significant increase in the secondary endpoint of 
objective response rate; however, this did not result in an improvement in progression-free 
survival.  The adverse event profile was similar to that for the completed phase III trials. 
 
More recently Dr. K Miller presented the results of an ECOG phase III trial (E2100) in which 
patients with untreated metastatic  breast cancer were randomized to paclitaxel versus paclitaxel 
and Avastin were presented (Proc ASCO 2005 and Breast Cancer 94(1): Abstract #3, 2005). A 
total of 722 women with recurrent or metastatic breast cancer who had not previously received 
systemic chemotherapy for their recurrent or metastatic disease were enrolled in this study 
between December 2001 and May 2004. Patients whose tumors overexpressed HER-2 were not 
included in the study unless they had previously received trastuzumab (HerceptinTM) or were 
 
 

 
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unable to receive trastuzumab. Also excluded were patients who had received adjuvant 
chemotherapy treatment with paclitaxel within the previous 12 months, as well as patients with a 
prior history of blood clots or who were anticoagulated.   In this trial the combination showed 
statistically significant prolongation of disease free survival (10.9 months vs. 6.1 months) and 
response rate (28.2% versus 14.2%).  The tolerance of the combination was excellent.  Avastin 
combination was well tolerated and now is being evaluated in the adjuvant setting.  Thus, 
efficacy of Avastin has been demonstrated in breast cancer. 
 
A pilot trial using the combination of Letrozole and Avastin (15 mg/kg every 3 weeks) in the 
neoadjuvant setting for post-menopausal, ER positive patients with locally advanced breast 
cancer was initiated in our institution at the end of 2005.  Although it is early to evaluate efficacy 
at this time (August 2006), the combination has showed to be safe in the 18 patients enrolled 
(same toxicity profile of Letrozole and Avastin).  Of the 18 patients, 8 have completed therapy 
per protocol and of those 2 had a pCR, 3 had a very good partial response, 2 had stable disease 
and 1 progressed within the first 12 weeks of therapy and was taken off the study at that time.  
Of the other 10 patients, 2 came out of the study due to refractory hypertension and 8 are in 
active therapy now.  Of the 8 patients that had surgery, none has had a problem with healing.  
The Avastin dose selected for this trial was based on the pharmacokinetic data provided by 
Genentech (similar to 10 mg/kg every 2 weeks) as well as the experience in previous clinical 
trials.  It is anticipated that preliminary results will be presented at the 2007 ASCO annual 
meeting.   
 
The safety of the combination between Letrozole and Avastin has also been demonstrated by 
Traina et al (2006 proceedings of the ASCO annual meeting, abstract #3050). Metastatic breast 
cancer patients were eligible for the trial. Therapy consisted of letrozole (2.5 mg daily) and 
Avastin (15 mg/kg IV q3 weeks). The primary endpoint was frequency of Grade (Gr) 4 toxicity. 
Thirty two patients were enrolled and at the time of publication 28 were evaluable. After a 
median of 8 cycles, therapy-related toxicities observed were: Gr 2: hypertension 4, headache 4, 
proteinuria 3, fatigue 6, joint pain 5, hot flashes 1, epistaxis 1; Gr 3: hypertension 5, headache 1, 
proteinuria 1. There was no therapy-related Gr 4/5 toxicity. They concluded that the combination 
was safe and well tolerated.    
 
- Avastin Safety Profile 
 
In the initial Phase I and II clinical trials, four potential Avastin-associated safety signals were 
identified: hypertension, proteinuria, thromboembolic events, and hemorrhage.  Additional 
completed Phase II and Phase III studies of Avastin as well as spontaneous reports have further 
defined the safety profile of this agent. Avastin-associated adverse events identified in phase III 
trials include congestive heart failure (CHF), gastrointestinal perforations, wound healing 
complications, and arterial thromboembolic events (ATE). These and other safety signals are 
described in further detail as follows and in the Avastin Investigator Brochure. 
 
Hypertension: Hypertension has been commonly seen in Avastin clinical trials to date 
and oral medications have been used to manage the hypertension when indicated. Grade 4 and 5 
hypertensive events are rare. Clinical sequelae of hypertension are rare but have included 
hypertensive crisis, hypertensive encephalopathy, and reversible posterior leukoencephalopathy 
 
 

 
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syndrome (RPLS) (Ozcan et al., 2006; Glusker et al., 2006). RPLS may include signs and 
symptoms of headache, altered mental function, seizures, and visual disturbances / cortical 
blindness and requires treatment, which should include control of hypertension, management of 
specific symptoms, and discontinuation of Avastin. 
 
 
There is no information on the effect of Avastin in patients with uncontrolled 
hypertension at the time of initiating Avastin therapy.  Therefore, caution should be exercised 
before initiating Avastin therapy in these patients.  Monitoring of blood pressure is 
recommended during Avastin therapy.  Optimal control of blood pressure according to standard 
public health guidelines is recommended for patients on treatment with or without Avastin. 
 
 
Temporary interruption of Avastin therapy is recommended in patients with hypertension 
requiring medical therapy until adequate control is achieved.  If hypertension cannot be 
controlled with medical therapy, Avastin therapy should be permanently discontinued.  Avastin 
should be permanently discontinued in patients who develop hypertensive crisis or hypertensive 
encephalopathy. 
 
 
Proteinuria: An increased incidence of proteinuria has been observed in patients treated 
with Avastin compared with control arm patients.  In the Avastin-containing treatment arms of 
clinical trials (across all indications), the incidence of proteinuria (reported as an adverse event) 
was up to 38% (metastatic CRC Study AVF2192g). The severity of proteinuria has ranged from 
asymptomatic and transient events detected on routine dipstick urinalysis to nephrotic syndrome; 
the majority of proteinuria events have been grade 1. NCI-CTC Grade 3 proteinuria was reported 
in up to 3% of Avastin-treated patients, and Grade 4 in up to 1.4% of Avastin-treated patients.  
The proteinuria seen in Avastin clinical trials was not associated with renal impairment and 
rarely required permanent discontinuation of Avastin therapy.  Avastin should be discontinued in 
patients who develop Grade 4 proteinuria (nephrotic syndrome). 
 
 
Patients with a history of hypertension may be at increased risk for the development of 
proteinuria when treated with Avastin.  There is evidence from the dose-finding, Phase II trials 
(AVF0780g, AVF0809s, and AVF0757g) suggesting that Grade 1 proteinuria may be related to 
Avastin dose. 
 
 
Proteinuria will be monitored by urine protein:creatinine (UPC) ratio at least every 6 
weeks.  If the UPC ratio is not available, a dipstick urinalysis may be used to allow treatment to 
proceed. 
 
Thromboembolic Events: Both venous and arterial thromboembolic (TE) events, 
ranging in severity from catheter-associated phlebitis to fatal, have been reported in patients 
treated with Avastin in the colorectal cancer trials and, to a lesser extent, in patients treated with 
Avastin in NSCLC and breast cancer trials.  
 
 
Venous thromboembolism (including deep venous thrombosis, pulmonary 
embolism, and thrombophlebitis:  In the phase III pivotal trial in metastatic CRC, there was a 
slightly higher rate of venous TE events that was not statistically significant in patients treated 
with Avastin plus chemotherapy compared with chemotherapy alone (19% vs. 16%).  
 
 

 
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In Study AVF2107g, a Phase III, pivotal trial in metastatic CRC, VTE events, including 
deep venous thrombosis, pulmonary embolism, and thrombophlebitis, occurred in 15.2% of 
patients receiving chemotherapy alone and 16.6% of patients receiving chemotherapy + Avastin. 
 
 
The incidence of NCI-CTC Grade ≥ 3 venous VTE events in one NSCLC trial (E4599) 
was higher in the Avastin-containing arm compared to the chemotherapy control arm (5.6% vs. 
3.2%).  One event (0.2%) was fatal in the Avastin-containing arm; not fatal events were reported 
in the carboplatin/paclitaxel arm (see Avastin Investigator Brochure).  In metastatic CRC clinical 
trials, the incidence of VTE events was similar in patients receiving chemotherapy + Avastin and 
those receiving the control chemotherapy alone.  
 
 
In clinical trials across all indications the overall incidence of VTE events was 
2.8%−17.3% in the Avastin-containing arms compared with 3.2%−15.6% in the chemotherapy 
control arms.  The use of Avastin with chemotherapy does not substantially increase the risk of 
VTE event compared with chemotherapy alone.  However, patients with metastatic CRC who 
receive Avastin and experienced a VTE event may be at higher risk for recurrence of VTE event. 
 
 
Arterial Thromboembotic Events:  An increased incidence of ATE events was 
observed in patients treated with Avastin compared with those receiving control treatment.  ATE 
events include cerebrovascular accidents, myocardial infarction, transient ischemic attacks 
(TIAs), and other ATE events.  In a pooled analysis of data from five randomized Phase II and 
III trials (mCRC [AVF2107g, AVF2192g, AVF0780g]; locally advanced or metastatic NSCLC 
[AVF0757g]; metastatic breast cancer [AVF2119g]), the incidence rate of ATE events was 3.8% 
(37 of 963) in patients who received chemotherapy + Avastin compared with 1.7% (13 of 782) in 
patients treated with chemotherapy alone.  ATE events led to a fatal outcome in 0.8% (8 of 963) 
of patients treated with chemotherapy + Avastin and 0.5% (4 of 782) of patients treated with 
chemotherapy alone.  Cerebrovascular accidents (including TIAs) occurred in 2.3% of patients 
treated with chemotherapy + Avastin and 0.5% of patients treated with chemotherapy alone.  
Myocardial infarction occurred in 1.4% of patients treated with chemotherapy + Avastin 
compared with 0.7% of patients treated with chemotherapy alone (see the Avastin Investigator 
Brochure for additional details). 
 
Aspirin is a standard therapy for primary and secondary prophylaxis of arterial thromboembolic 
events in patients at high risk of such events, and the use of aspirin ≤ 325 mg daily was allowed 
in the five randomized studies discussed above. Use of aspirin was assessed routinely as a 
baseline or concomitant medication in these trials, though safety analyses specifically regarding 
aspirin use were not preplanned. Due to the relatively small numbers of aspirin users and arterial 
thromboembolic events, retrospective analyses of the ability of aspirin to affect the risk of such 
events were inconclusive. However, similarly retrospective analyses suggested that the use of up 
to 325 mg of aspirin daily does not increase the risk of grade 1-2 or grade 3-4 bleeding events, 
and similar data with respect to metastatic colorectal cancer patients were presented at ASCO 
2005 (Hambleton et al., 2005). Further analyses of the effects of concomitant use of Avastin and 
aspirin in colorectal and other tumor types are ongoing. 
 
 
 

 
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Gastrointestinal perforation: Patients with metastatic carcinoma may be at increased 
risk for the development of gastrointestinal perforation when treated with Avastin and 
chemotherapy. Avastin should be permanently discontinued in patients who develop 
gastrointestinal perforation. A causal association of intra-abdominal inflammatory process and 
gastrointestinal perforation to Avastin has not been established. Nevertheless, caution should be 
exercised when treating patients with intra-abdominal inflammatory processes with Avastin. 
Gastrointestinal perforation has been reported in other trials in non-colorectal cancer populations 
(e.g., ovarian, renal cell, pancreas, breast, and NSCLC) and may be higher in incidence in some 
tumor types. 
 
 
Fistula:  Avastin use has been associated with serious cases of fistulae including events 
resulting in death.  Fistulae in the GI tract are common (1%–10% incidence) in patients with 
metastatic CRC, but uncommon (0.1%−1%) or rare (0.01%–0.1%) in other indications.  In 
addition, fistulae that involve areas of the body other than the GI tract (e.g., tracheoesophageal, 
bronchopleural, urogenital, biliary) have been reported uncommonly (0.1%–1%) in patients 
receiving Avastin in clinical studies and postmarketing reports.  Events were reported at various 
timepoints during treatment, ranging from 1 week to > 1 year following initiation of Avastin, 
with most events occurring within the first 6 months of therapy. 
 
 
Permanently discontinue Avastin in patients with tracheoesophageal fistulae or any 
Grade 4 fistula.  Limited information is available on the continued use of Avastin in patients 
with other fistulae.  In cases of internal fistula not arising in the GI tract, discontinuation of 
Avastin should be considered. 
 
Wound healing complications: Wound healing complications such as wound 
dehiscence have been reported in patients receiving Avastin. In an analysis of pooled data from 
two trials in metastatic colorectal cancer, patients undergoing surgery 28-60 days before study 
treatment with 5-FU/LV plus Avastin did not appear to have an increased risk of wound healing 
complications compared to those treated with chemotherapy alone (Scappaticci et al., 2005). 
Surgery in patients currently receiving Avastin is not recommended. No definitive data are 
available to define a safe interval after Avastin exposure with respect to wound healing risk in 
patients receiving elective surgery; however, the estimated half life of Avastin is 20 days. 
Avastin should be discontinued in patients with severe wound healing complications. 
 
 
If patients receiving treatment with Avastin require elective major surgery, it is 
recommended that Avastin be held for 4–8 weeks prior to the surgical procedure.  Patients 
undergoing a major surgical procedure should not begin or restart Avastin until 4 weeks after 
that procedure (in the case of high-risk procedures such as liver resection, thoracotomy, or 
neurosurgery, it is recommended that chemotherapy be restarted no earlier than 6 weeks and 
Avastin no earlier than 8 weeks after surgery). 
 
Hemorrhage: Overall, grade 3 and 4 bleeding events were observed in 4.0% of 1132 
patients treated with Avastin in a pooled database from eight phase I, II, and III clinical trials in 
multiple tumor types (Avastin Investigator Brochure, October 2005). The hemorrhagic events 
that have been observed in Avastin clinical studies were predominantly tumor-associated 
hemorrhage (see below) and minor mucocutaneous hemorrhage. 
 
 

 
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Tumor-Associated Hemorrhage:  Major or massive pulmonary hemorrhage or 
hemoptysis has been observed primarily in patients with NSCLC.  Life-threatening and fatal 
hemoptysis was identified as a Avastin-related adverse event in NSCLC trials.  Tumor-
associated hemorrhage was observed in phase I and phase II Avastin studies. Six serious events, 
of which 4 had fatal outcome, were observed in a phase II trial of patients with non-small cell 
lung cancer receiving Avastin. These events occurred suddenly and presented as major or 
massive hemoptysis in patients with either squamous cell histology and/or tumors located in the 
center of the chest in close proximity to major blood vessels. In five of these cases, these 
hemorrhages were preceded by cavitation and/or necrosis of the tumor. Tumor-associated 
hemorrhage was also seen rarely in other tumor types and locations, including central nervous 
system (CNS) bleeding in a patient with hepatoma with occult CNS metastases and continuous 
oozing of blood from a thigh sarcoma with necrosis. 
 
 
GI hemorrhages, including rectal bleeding and Melena have been reported in patients 
with CRC, and have been assessed as tumor-associated hemorrhages. 
 
Mucocutaneous Hemorrhage:  Across all Avastin clinical trials, mucocutaneous 
hemorrhage has been seen in 20%-40% of patients treated with Avastin. These were most 
commonly NCI-CTC grade 1 epistaxis that lasted less than 5 minutes, resolved without medical 
intervention and did not require any changes in Avastin treatment regimen. There have also been 
less common events of minor mucocutaneous hemorrhage in other locations, such as gingival 
bleeding and vaginal bleeding.  
 
Reversible Posterior Leukoencephalopathy Syndrome:  There have been rare reports 
of Avastin-treated patients developing signs and symptoms that are consistent with RPLS, a rare 
neurologic disorder that can present with the following signs and symptoms (among others):  
seizures, headache, altered mental status, visual disturbance, or cortical blindness, with or 
without associated hypertension.  Brain imaging is mandatory to confirm the diagnosis of RPLS.  
In patients who develop RPLS, treatment of specific symptoms, including control of 
hypertension, is recommended along with discontinuation of Avastin.  The safety of reinitiating 
Avastin therapy in patients previously experiencing RPLS is not known (Glusker et al. 2006; 
Ozcan et al. 2006). 
 
Congestive heart failure: CHF has been reported in Avastin clinical trials and may be 
increased in incidence in patients with prior exposure to anthracyclines or prior irradiation to the 
chest wall.  In a phase III trial (AVF2119g) of capecitabine with or without Avastin for 
metastatic breast cancer, 7 subjects (3.1%) who received capecitabine plus Avastin developed 
clinically significant CHF compared with 2 subjects (0.9%) treated with capecitabine alone; of 
note, all subjects in this trial had had prior anthracycline treatment.  In addition, 2 subjects had a 
left ventricular ejection fraction < 50% at baseline and 2 others had prior left chest wall 
irradiation.  A recently published phase II study in subjects with refractory acute myelogenous 
leukemia reported 5 cases of cardiac dysfunction (CHF or decreases to <40% in left ventricular 
ejection fraction) of 48 subjects treated with sequential cytarabine, mitoxantrone, and Avastin.  
All but one of these subjects had significant prior exposure to anthracyclines as well (Karp et al., 
2004).  Other studies are ongoing in this patient population.  Patients receiving anthracyclines or 
 
 

 
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with prior exposure to anthracyclines should have a baseline MUGA or ECHO with a normal 
ejection fraction. 
 
In a randomized, Phase III trial of patients with previously untreated metastatic breast 
cancer (E2100), the incidence of LVEF decrease (defined as NCI-CTC Grade 3 or 4) in the 
paclitaxel + Avastin arm was 0.3% for the paclitaxel alone arm. 
 
No information is available on patients with preexisting CHF of New York Heat 
Association (NYHA) Class II-IV at the time of initiating Avastin therapy, as these patients were 
excluded from clinical trials. 
 
 
Two additional studies investigated concurrent administration of anthracyclines and 
Avastin.  In 21 patients with inflammatory breast cancer treated with neoadjuvant docetaxel, 
doxorubicin, and Avastin, no patients developed clinically apparent CHF; however, patients had 
asymptomatic decreases in LVEF to < 40% (Wedam et al. 2004).  In a small Phase II study in 
patients with soft tissue sarcoma, 2 of the 17 patients treated with Avastin and high-dose 
doxorubicin (75 mg/m2) developed CHF (one Grade 3 event after a cumulative doxorubicin dose 
of 591 mg/m2, one Grade 4 event after a cumulative doxorubicin dose of 420 mg/m2); an 
additional 4 patients had asymptomatic decreases in LVEF (D’Adamo et al. 2004). 
 
 
Other studies in patients with various tumor types and either a history of anthracycline 
exposure or concomitant use with Avastin are ongoing. 
 
 
Patients receiving concomitant anthracyclines or with prior exposure to anthracyclines 
should have a baseline MUGA scans or echocardiograms (ECHOs) with a normal LVEF. 
 
 
Neutropenia:  Increased rates of severe neutropenia, febrile neutropenia, or infection 
with severe neutropenia (including some fatalities) have been observed in patients treated with 
some myelotoxic chemotherapy regimens plus Avastin in comparison to chemotherapy alone 
(Sandler et al. 2006). 
 
Additional Adverse Events: See the Avastin Investigator Brochure for additional details 
regarding the safety experience with Avastin. 
 
1.4 
Hypothesis for This Study 
 
Preclinical and clinical data (described in the background of this protocol) have demonstrated 
that up-regulation of tumor cell VEGF is an important mechanism to subvert estrogen 
dependence in hormone responsive breast cancer resulting in reduced therapy response or tumor 
resistance to hormonal therapy; thus, we hypothesize that the combination of an anti-VEGF 
agent (Avastin, an anti-VEGF monoclonal antibody) and hormonal therapy should be more 
effective than hormonal therapy alone for the treatment of breast cancer. 
 
1.5 
Study Rationale 
 
 
 

 
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Despite the enormous advances in hormonal therapy for breast cancer, impaired response and 
development of resistance to endocrine manipulation is at the core of the problem and must be 
overcome to make additional progress; thus, a number of endocrine resistant modulators are now 
under development (e.g., signal transduction inhibitors, agents to down-regulate VEGF, etc).  
Developing different combination of agents is a challenge because we still do not have a clear 
understanding of the molecular basis of resistance.  
 
The development of anti-VEGF therapy to overcome resistance to endocrine therapies is of 
potentially great importance.  The unique mechanism of action, limited toxicity and preclinical 
and clinical data generated with Avastin, and the pre-clinical and clinical observations by Kern, 
Berns and Qu demonstrating correlation between VEGF expression and response to hormonal 
therapy, support the hypothesis that VEGF expression by breast cancers is a potential mechanism 
for estrogen independence of breast tumors.  Interference with VEGF activity would thus 
improve and/or restore response anti-estrogen drug therapy.  We therefore hypothesize that the 
efficacy of hormonal therapy in women with ER+ and/or PR+ breast cancer can be 
augmented by means of Avastin, a recombinant humanized antibody to vascular 
endothelial cell growth factor.  As described below, neoadjuvant therapy is a very 
attractive model to evaluate the hypothesis. 
 
2.0 
OBJECTIVES 
 
2.1 
Primary 
 
In post-menopausal patients with pathologically confirmed invasive ductal carcinoma or invasive 
lobular carcinoma of the breast (T2, T3, T4a-c, N0-2, and M0, excluding patients with T4d or 
inflammatory breast cancer) whose tumors are estrogen receptor and/or progesterone receptor 
positive, Her-2-neu negative, we will determine pathological complete response to a maximum 
of 24 weeks of neoadjuvant therapy using a combination of letrozole (Femara®) and Avastin  or 
letrozole alone.  
 
2.2 
Secondary 
 
1. 
Determine the clinical objective response rate to a maximum of 24 weeks of neoadjuvant 
therapy using letrozole and Avastin or letrozole alone.  
 
2. 
Determine the tolerability and toxicity of the combination of letrozole and Avastin 
compared to letrozole alone. 
 
3. 
Evaluate specific biomarkers for prognostic value and as markers for response/resistance to 
the combination of letrozole and Avastin or letrozole alone.   
 
3.0 
STUDY DESIGN 
 
3.1 
Description of the Study 
 
 
 

 
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This will be an open label, randomized, Phase II study of the combination of Letrozole and  
Avastin versus Letrozole alone in post-menopausal patients with newly diagnosed and 
pathologically confirmed invasive breast cancer (T2, T3, T4a-c, N0-2, and M0), estrogen 
receptor and/or progesterone receptor positive, Her-2-neu negative (0, 1 or 2 + by IHC or non-
amplified by FISH). Patients with inflammatory breast cancer will not be included (T4d).  The 
primary objective for this randomized phase II trial is to estimate the pathological complete 
response rates, defined as the percentage of patients with a complete pathological response at the 
time of surgery, for each of the two arms in the trial (see Section 11 – Statistical Section).    
 
Patients must be post-menopausal (no peri- or pre-menopausal) and 50 years or older, have 
measurable disease by mammogram and/or ultrasound (in special cases with a clear clinical 
indication, dedicated breast MRI may be used – not required per protocol), and have a 
performance status of ECOG 0 or 1. Patients previously treated or patients with no measurable 
disease or patients with metastatic disease will be excluded. 
 
Patients will be registered through the Clinical Trials Network (CTN) Office of the UAB 
Comprehensive Cancer Center where the CTN Manager will register the patient in the trial  (will 
randomize and complete the randomization forms).  Monthly accrual will be communicated to 
the Breast Cancer Research Consortium and Genentech.  Within eight weeks before the initiation 
of the therapy patients will have a PET/CT scan or [CT of the chest, abdomen, pelvis,  with a 
bone scan].    Patients with metastatic disease will not be enrolled in the trial.  Additionally, 
blood counts, UA, urinary protein/creatinine ratio (24-hour urine collection if indicated), INR 
and CMP will be obtained within 4 weeks before initiation of therapy.  Echocardiogram and 
ultrasound of the breast within 8 weeks before therapy will be also obtained, and they will be 
used as baseline evaluations.  Core needle biopsies will be performed in order to provide 
adequate tissue for standard diagnosis (H&E, ER/PR receptors, Her-2-neu status) as well as to 
provide tissue for the biomarker assays (tumor blocks and fresh tissue).  If the patient agrees to 
consent, a core needle biopsy of the upper quadrant of the unaffected breast will be obtained for 
comparison of biomarkers. 
 
Patients with palpable axillary lymph nodes or patients with FNA positive axillary lymph nodes 
will be treated as having at least N1 disease and are not required to undergo sentinel lymph node 
biopsy prior to initiation of neoadjuvant therapy; axillary lymph node dissection will be 
performed in those patients at the time of definitive surgery.  Sentinel lymph node biopsy is to be 
performed at the discretion of the treating physician according to institution  protocol. A sentinel 
node procedure will not be required on non-palpable nodes before initiation of protocol therapy. 
However, if a sentinel node procedure is performed for non-palpable modes, then Letrozole can 
be started immediately post sentinel node procedure with Avastin being able to begin 2 weeks 
post sentinel node procedure.  Post-surgical adjuvant therapy will be administered at the 
investigator’s discretion (chemotherapy is allowed). 
 
Patients meeting the eligibility criteria and who have signed the consent form will start Letrozole 
2.5 mg PO a day and Avastin 15 mg/kg IV every 3 weeks or Letrozole 2.5 mg PO alone.  At six 
weeks patients will have the first evaluation of response with physical exam, and ultrasound of 
the breast.  In addition, core biopsies will be obtained for evaluation of biomarkers.  Patients 
with objective response or stable disease in the first evaluation (physical evaluation and 
 
 

 
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ultrasound) according to the RECIST criteria will continue the same regimen with restaging 
every 6 weeks for a total of 24 weeks or until progression is observed.  If during the second 
evaluation (12 weeks) an objective response or stable response is observed, the patient will 
complete therapy, but if during the 12 week evaluation the patient has disease progression the 
patient will be removed from the protocol and will be treated at the investigator’s discretion.  If a 
patient has a clinical response with tumor size decreasing to less than 2 cm, a metal coil will be 
placed within the tumor to aid in identification of the primary site at the time of the operation.  
Patients with disease progression according to the RECIST criteria will be removed from the 
study and will be treated at the discretion of the treating physician.  Definitive surgery of the 
primary tumor will be performed at the discretion of the surgeon no sooner than 4 weeks after 
the last Avastin dose (fourth evaluation on week 24).  Patients will continue letrozole during 
those 4 weeks before the surgery.  No dose reductions are planned. 
 
In the absence of treatment delays due to adverse events, treatment may continue until one of the 
following criteria applies: 24 weeks of therapy, disease progression, intercurrent illness that 
prevents further administration of treatment, any toxicity that results in a treatment delay of > 3 
weeks, unacceptable grade 3 or 4 non-hematologic adverse events(s), patient decides to 
withdraw from the study for any reason, or general or specific changes in the patient's condition 
render the patient unacceptable for further treatment in the judgment of the investigator. 
 
3.2 
Rationale for Study Design 
 
Neoadjuvant endocrine therapy is an appealing context to conduct research in this area because 
clinical outcomes can be obtained within a few months of treatment, and repeated images and 
tumor sampling for biomarker analysis can be readily achieved.  There is now substantial 
evidence that the responses seen in the neoadjuvant setting can be used to accurately predict 
disease free survival, dramatically shortening the typical time it takes to derive information from 
a clinical trial of a novel agent/regimen from years to 4-6 months. 
 
In this trial, we will use a novel combination of hormonal agent (letrozole) and an anti-VEGF 
monoclonal antibody (Avastin) in the neoadjuvant setting based on pre-clinical and clinical 
observations previously described in the background and the data will be compared with that 
obtained from patients treated with letrozole alone.  In choosing this combination we hope the 
combination will be synergistic and will demonstrate higher efficacy than the use of the 
hormonal agent alone.  We will investigate modulation of breast cancer biomarkers during 
neoadjuvant therapy for newly diagnosed breast cancer (see appendix I and section 6.2 for a 
detailed description). Tissue will be obtained before initiation of therapy, and 6 weeks after day 
0 of therapy. Specific markers of interest in the paraffin embedded tissue include: marker of 
proliferation (Ki-67), apoptotic markers (Tunnel, caspase 3, bcl-2 and bax), and a novel 
prognostic factor (Zinc finger protein GKLF/KLF-4, a novel oncogene recently described by 
investigators at UAB).  In addition, we will plan on coding, processing, and banking fresh frozen 
tissue; this stored tissue will be transferred to Dr. Charles Perou (University of North Carolina at 
Chapel Hill).  Tissue stored in this fashion will be used for examination of differential gene 
expression patterns. In addition, circulating tumor cells will be evaluated before and after 
therapy (These assays will be performed by Dr. Hope Rugo, University of California at San 
Francisco).   
 
 

 
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Although the majority of the clinical trials published at this time have used neoadjuvant 
hormonal therapy for a period of 3 to 4 months before surgery, we have decided to allow the 
patients enrolled in this trial to receive therapy for up to 6 months based on: 1. the published data 
(described in the background) which suggested that extended therapy with an AI in the 
neoadjuvant setting for up to 12 months is safe with continued tumor regressions, and that 
patients whose tumors respond to letrozole after 3 months can expect a further reduction in 
tumor volume with continued therapy; 2. The fact that tumor response to biological agents is 
slow; and 3. The safety rule included in this trial in which patients who have not had a response 
at the 12 week evaluation will be taken off the study. In addition, the Avastin dose of 15 mg/kg 
every three weeks was selected based on: 1. previous clinical studies in which the dose 
demonstrated to be safe and effective, 2. Pharmacokinetic data which demonstrated that this dose 
is pharmacologically equivalent to the 10 mg/kg every two weeks.  
 
We believe this approach will allow a relatively rapid assessment of a novel regimen 
(translational research) and aid in identifying surrogate endpoint markers which will 
enhance our ability to select patients who are likely to benefit from these interventions.  
 
3.3 
Outcome Measures  
 
3.3.1 Primary Outcome Measures 
 
Pathological complete response is defined as no evidence of residual invasive tumor in the breast 
or axillary lymph nodes or only residual ductal carcinoma in-situ.  
 
3.3.2 Secondary Outcome Measures 
 
The clinical response will be based on the Response Evaluation Criteria in Solid Tumors 
(RECIST Criteria) described in Appendix H.   
 
4.0 
SAFETY PLAN 
Based on the toxicity profile of Avastin (see section 1.3), a number of measures will be taken to 
ensure the safety of the patients enrolled in this trial.  These measures will be addressed through 
exclusion criteria (see section 5.3) and routine monitoring as follows:  
 
Patients enrolled in the trial will be carefully monitored during the entire treatment period and 
will be followed as is appropriate.  Safety evaluations will consist of medical interviews, 
collection of adverse events, PE, VS, and laboratory measurements (see Section 8).  Patients will 
be evaluated for adverse events (all grades), serious adverse events, and adverse events requiring 
study drug interruption or discontinuation at each study visit for the duration of the participation 
in the study.  Patients discontinued from treatment for any reason will be evaluated 
approximately 30 days (28-42 days) after the last dose of Avastin (see section 8.3).  Specific 
monitoring procedures are as follow:  
 
 
 

 
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• Hypertension will be monitored through routine evaluation of blood pressure prior to each 
Avastin treatment.  Optimal control of blood pressure according to standard public health 
guidelines is recommended for patients on treatment with or without Avastin.   
• In patients with bleeding, hemostasis evaluation should be performed as clinically indicated.   
• Proteinuria will be monitored through regular urinalysis and urinary protein/creatinine ratio 
or dipstick at least every 6 weeks (before the administration of Avastin).  
• Patients who have an ongoing Avastin-related Grade 4 or serious adverse event at the time of 
discontinuation from study treatment will continue to be followed (see Section 7). 
• If patients on treatment with Avastin require major surgery or choose elective major surgery, 
it is recommended that Avastin be held for 4-8 weeks prior to the surgical procedure. Patients 
undergoing a major surgical procedure should not begin/restart Avastin until 4 weeks after 
that procedure (in the case of high risk procedures such as liver resection, thoracotomy, or 
neurosurgery, it is recommended that chemotherapy be restarted no earlier than 6 weeks and 
that Avastin be restarted no earlier than 8 wk after surgery). 
 
Please see Section 7 for detailed instructions for the management of study drug–related 
toxicities. 
4.1 
Protocol Management and Oversight of Participating Affiliate Sites 
 
Dr. Andres Forero functions as the sponsor of the trial at UAB and at each affiliate.  Each 
affiliate will utilize their respective IRBs of record.  Appendix J provides guidelines for off-site 
conduct of protocols sponsored by UAB Cancer Center faculty. 
 
5.0 
STUDY SUBJECTS 
 
5.1 
Subject Selection and Registration 
 
At the time of study entry, the patient will be evaluated by a multidisciplinary team of medical 
oncologists, radiation oncologists, and surgical oncologists.  Before registration investigators are 
required to indicate the type of operation they intend to perform (lumpectomy or mastectomy).  
Patients will be consented by the investigator and the coordinator of the trial. 
 
Patients will be registered through the Clinical Trials Network (CTN) Office of the UAB 
Comprehensive Cancer Center where the CTN Manager will register the patient in the protocol, 
complete the randomization forms, and notify the sponsor-investigator.  Prior to any treatment, 
patients will be randomized and registered with the CTN Manager (Pam Dixon, Tel: 205-975-
5387; Fax: 205-975-9875; Email: pam.dixon@ccc.uab.edu), between 8:00 A.M. and 4:30 P.M. 
central time, Monday through Friday. 
 
5.2 
Inclusion Criteria 
 
All patients must meet the following criteria to be eligible for study entry: 
 
 

 
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5.2.1  Pathologically confirmed invasive ductal carcinoma or invasive lobular carcinoma of the 
breast, T2-T3 / T4a-c / N0-2 / M0, with positive estrogen and/or progesterone receptors, and 
Her-2-neu negative.  Patients with inflammatory breast cancer will not be included (T4d).  
Patients previously treated patients with no measurable disease or patients with metastatic 
disease will be excluded.  
 
5.2.2  Give written informed consent prior to study specific screening procedures, with the 
understanding that the patient has the right to withdraw from the study at any time, without 
prejudice (see Appendix B – consent form). 
 
5.2.3 Patients must be postmenopausal, defined as one of the following: 
• 
Patients > 50 years of age with no spontaneous menses for at least 12 months, 
• Bilateral oophorectomy 
 
5.2.4  Be ambulatory (outpatient) and have an ECOG PS <1 (Appendix F). 
 
5.2.5  Patients must have measurable disease by mammogram and/or breast ultrasound (in 
special cases a dedicated breast MRI may be clinically indicated). The target lesion must not 
have been previously irradiated. 
5.2.6 No prior chemotherapy. 
 
5.2.7  Patients must have adequate organ and marrow function as defined as follows:  absolute 
neutrophil count > 1,500/mm3, hemoglobin > 8.0 g/dl, platelets > 75,000/mm3, total bilirubin < 2 
mg/dl, serum creatinine < 2 mg/dl, Transaminases (AST, ALT) may be up to 2 x institutional 
upper limit of normal.  In addition < 1 gr of protein in 24 hr urine collection and urine 
protein/creatinine ratio < 1.0 (see Appendix E).  No evidence of proteinuria as evidenced by 
either urine protein/creatinine ratio <1 or urine proteinuria greater than 2+.  Patients who have ≥ 
2+ proteinuria should have a 24-hour urine collection and must demonstrate ≤ 1g of protein in 24 
hours to be eligible. 
 
5.2.8 No life threatening parenchymal disease or rapidly progressing disease warranting 
cytotoxic chemotherapy. 
 
5.2.9 Hypertension must be controlled (<150/100 mmHg). 
 
5.2.10 Ejection Fraction > 50% by echocardiogram/MUGA. (LVEF greater than 75% at 
baseline should be reviewed and/or the test repeated as it may be falsely elevated). 
 
5.2.11 No history of thrombosis during the previous 12 months. 
 
5.3  
Exclusion Criteria 
 
5.3.1 Current, recent (within 4 weeks of the first infusion of this study), or planned participation 
in an experimental drug study other than this sponsor-investigator Avastin cancer study. 
 
 

 
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5.3.2 Uncontrolled high blood pressure (>150/100 mmHg). 
 
5.3.3 Prior history of hypertensive crisis or hypertensive encephalopathy 
 
5.3.4 Unstable angina 
 
5.3.5 New York Heart Association (NYHA) Grade III or greater congestive heart failure (see 
Appendix G) 
 
5.3.6 History of myocardial infarction or unstable angina within 12 months  
 
5.3.7 History of stroke or TIA  
 
5.3.8 Clinically significant peripheral vascular disease 
5.3.9 History of a bleeding disorder 
 
5.3.10 Presence of central nervous system or brain metastases 
 
5.3.11 Major surgical procedure, open biopsy, or significant traumatic injury within 28 days 
prior to Day 0, anticipation of need for major surgical procedure during the course of the study 
 
5.3.12 Minor surgical procedures (excluding fine needle aspirations or core biopsies) within 5 
days prior to Day 0 
 
5.3.13 Pregnant (positive pregnancy test) or lactating 
 
5.3.14 Urine protein: creatinine ratio ≥1.0 at screening and patients demonstrating > 1 gr of 
protein in 24 hr urine collection within 4 weeks prior to study entry will not participate in the 
trial. 
 
5.3.15 History of abdominal fistula, gastrointestinal perforation, or intra-abdominal abscess 
within 6 months prior to Day 1 
 
5.3.16 Serious, non-healing wound, ulcer, or bone fracture 
 
5.3.17 Unwilling or unable to comply with the protocol for the duration of the study. 
 
5.3.18 Psychiatric illness/social situations that would limit compliance with study requirements.  
 
5.3.19 History of another malignancy within the last five years except non-melanoma skin 
cancer and carcinoma in–situ of uterine cervix.  Second primary breast cancers are allowed 
regardless of the number of years since they were first diagnosed. 
 
5.3.20 Patients with metastatic disease. 
 
 

 
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5.3.21 Life expectancy of less than 12 weeks 
 
5.3.22 Known hypersensitivity to any component of Avastin 
 
6.0 
STUDY DESIGN 
 
6.1 
Treatment Plan 
 
This will be an open label, randomized, Phase II study of the combination of Letrozole and 
Avastin versus letrozole alone in patients with newly diagnosed breast cancer (randomization 
2:1).  It is anticipated that with 50 evaluable patients receiving the combination therapy and 25 
evaluable patients receiving letrozole as a single agent (total 75 evaluable patients).  Accrual is 
expected to be completed in 24 months (see Section 11 on statistics).  Patients meeting the 
eligibility criteria and who have signed the consent form will start Letrozole 2.5 mg PO daily 
alone or in combination with Avastin 15 mg/kg IV every 3 weeks for 24 weeks.  After 
neoadjuvant therapy, patients will undergo surgical treatment and will receive adjuvant therapy 
according to the treating physician.  It is anticipated an accrual rate of 4 patients a month.  
 
6.2 
Biopsy Specimens and Research Blood Samples  
(See Appendix I for detailed description of sample timing, processing and shipping) 
 
Core biopsies will be obtained before initiation of therapy and 6 weeks after initiation of therapy.  
Biopsy samples will preferably be obtained using a 14-18 gauge core needle; at least two core 
biopsies will be obtained for frozen research biopsies and two for the preparation of the paraffin 
blocks.  If blocks from original diagnosis (before enrolling in the trial) are available, they will be 
obtained and then only samples for the frozen research biopsies will be obtained.  Processing of 
the specimens is described in Appendix I.  Paraffin blocks will be sent to UAB while frozen 
research biopsies will be sent to Dr. Charles Perou (Departments of Genetics and Pathology, 
Lineberger Comprehensive Cancer Center, University of North Carolina Chapel Hill, NC). 
 
Tissue sections obtained as described in Appendix I from paraffin-embedded material will be 
sectioned and stained for a marker of proliferation (Ki67), apoptosis markers (TUNEL, caspase 3 
bcl-2, bax), and a novel prognostic factor (zinc finger protein GKLF/KLF4, a novel oncogene 
recently described by investigators at UAB) by conventional IHC. Frozen research biopsies will 
be used for gene expression array analysis and will be performed as previously described (97). 
Briefly, total RNA will be isolated from the research biopsies. Dr. Perou has successfully 
analyzed specimens using tissue from ½ of a core biopsy (approximately 5 mg tissue). The 
isolated RNA from the breast tumors will be reverse transcribed to cDNA using the 
incorporation of a fluorescently tagged “red” dye (Cy5). A "common reference" sample cDNA 
will be prepared from RNA pooled from 11 diverse human cell lines, and fluorescently tagged 
with a “green” dye (Cy3). The red-tagged cDNA from the breast tumor will be hybridized on a 
microarray with the green-tagged "common reference". The ratio of fluorescence intensity at 
each gene on the array will be measured for the data analysis. The DNA microarray will be 
obtained from UNC/LCCC Genomics Core Facility. The analysis of gene expression profiles 
based on the DNA microarray data will be done using statistical analysis and hierarchical 
 
 

 
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clustering. Hierarchical clustering analysis allows individual tumors to group together based on 
common and recurrent gene expression patterns. Identification of the breast cancer subtype using 
previously established criteria (97) and comparison of changes in profile induced by the research 
agents will be performed.  
 
Gene expression Profiling Analyses.  In this study, patients will be randomized to receive 
letrozole, or letrozole plus Avastin.  To examine the possibility that gene expression patterns 
might be able to identify those patients who would gain a benefit from Avastin, we propose to 
perform DNA microarray analyses on the pretreatment biopsies and perform supervised analyses 
to identify gene patterns that might correlate with response to Avastin.  We will perform an 
exploratory study of the available microarray data in order to identify a set(s) of genes that are 
predictive of response or resistance to Avastin.  The clinical endpoint used will be pCR vs. no 
pCR, and clinical response classified as SD+PD vs. CR+PR measured after the first phase of 
treatment, and after all neoadjuvant treatments are complete.  To predict response, the expression 
data for the pre-treatment samples will be used and the “supervising parameter” will be 
pathologic complete response (pCR).  Four statistical classification methods are used to develop 
predictors of response when using the pre-treatment gene expression data: a k-Nearest Neighbor 
Classifier (k-NN with k=1, 3, 5, or 7) with either Euclidean distance or one-minus-Spearman-
correlation as the distance function, and a Class Nearest Centroid (CNC) classifier with either 
Euclidean distance or one-minus-Spearman-correlation as the distance function.  
 
To evaluate the gene expression predictor accuracy, each of the four prediction methods 
undergoes 10-fold cross validation (CV) where in a given round of CV, each predictor uses n 
genes (with the n genes selected using the ratio of between-group to within-group sums of 
squares {105}) obtained from training on 90% of the samples, which are then used to make 
predictions on the remaining 10%.  This procedure is repeated 9 more times such that every 
sample is “left out” exactly once.  The prediction accuracies on the withheld samples for each of 
the 10 iterations are averaged and this average prediction accuracy recorded for each prediction 
method with each n gene set. n is increased and decreased for many subsequent rounds of CV.  
For each supervising parameter/response variable, the set of n genes that gives the highest 
average prediction accuracy during CV is determined and reported for each prediction method.  
Using this approach, we have created centroid predictors that could classify tumors (1) into 
intrinsic subtypes that predict outcomes and show high concordance with the GHI RS and NKI 
70-gene profiles, (2) according to novel patterns of estrogen-regulated genes that predict 
outcomes in tamoxifen-treated patients, and (3) according to lymph node metastasis status.  
Thus, we will be able to determine if gene expression patterns are associated with response to 
Avastin.  Lastly, we will also compare pretreatment samples versus during treatment, and post 
treatment samples if they exist, in order to identify a set of genes, or sets of genes, that change in 
response to the administration of letrozole, and the addition of letrozole and Avastin. 
 
Circulating tumor cells (CTC) from whole blood will be processed for immunohistochemistry 
and RNA-based studies and compared to results obtained using the automated Veridex method 
for enumeration of circulating tumor cells.  
 
Patients will be allowed to start Avastin therapy next day after core biopsies. 
 
 
 

 
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7.0 
STUDY MEDICATION  
 
7.1  
Avastin Dosage and Formulation 
 
Avastin will be supplied by Genentech as a clear to slightly opalescent, colorless to pale brown, 
sterile liquid concentrate for parenteral administration.  Avastin will be supplied in 5 cc (100 
mg), 20 cc (400 mg), and 50 cc (1000 mg) glass vials with a 4 ml, 16 ml, or 40 ml of Avastin, 
respectively (all at 25 mg/ml).  The formulation contains sodium phosphate, trehalose, 
polysorbate 20, and sterile water for injection, USP.  Vials contain no preservative and are 
suitable for single use only.  The monoclonal antibody (Avastin) being administered is the 
commercially marketed drug (Avastin). For further details and molecule characterization, see the 
Avastin Investigator Brochure. 
 
The dose of Avastin administered in this study is 15 mg/kg given by intravenous infusion once 
every 3 weeks for up to 24 weeks.  The patient’s weight at screening will be used to determine 
the dose of Avastin to be used for the duration of the study; however, if a patient’s weight 
changes by 10% during the study, the dose of Avastin will be recalculated.  Treatment will be 
administered on an outpatient basis. 
 
7.1.1 Avastin Administration 
 
Avastin will be diluted in 0.9% sodium chloride injection, USP, to a total volume of 100 ml.  
Avastin will be administered as a continuous IV infusion.  Anaphylaxis precautions should be 
observed during the study drug administration.  The initial Avastin dose should be delivered over 
90±15 minutes.  If the first infusion is tolerated without infusion-associated adverse events, the 
second infusion may be delivered over 60±10 minutes.  If the 60-minute infusion is well 
tolerated, all subsequent infusions may be delivered over 30± 10 minutes.  A rate-regulating 
device should be used for all Avastin infusions.  When the Avastin IV bag is empty, an 
additional 50 ml of 0.9% sodium chloride injection should be added to the IV bag and the 
infusion should be continued for a volume equal to that of the tubing to ensure complete delivery 
of the Avastin.  An alternative method of flushing the infusion line would be to replace the 
empty Avastin infusion bag with a 50 ml bag of 0.9% Sodium Chloride Injection and infuse a 
volume equal to that of the tubing to ensure complete delivery of the Avastin.  This additional 
saline flush is not included in the specified infusion times. 
 
If a patient experiences Avastin infusion-associated adverse events, the patient may receive pre-
medication at the investigator’s discretion prior to the next Avastin infusion.  If pre-medication 
is required, the infusion time may not be decreased for the subsequent infusion.  However, if the 
next infusion is well tolerated with pre-medication, the subsequent infusion time may then be 
decreased by 30 minutes per infusion to a minimum infusion time of 30±10 minutes as long as 
the patient continues to receive the same pre-medication.  If a pre-medicated patient experiences 
infusion-associated adverse events with the 60-minute infusion, all subsequent doses should be 
given over 90±15 minutes.  Similarly, if a pre-medicated patient experiences infusion associated 
adverse events with the 30-minute infusion, all subsequent doses should be given over 60±10 
minutes. 
 
 
 

 
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Anaphylaxis Precautions:  
Anaphylaxis precautions should be observed during Avastin administration.  The patient’s blood 
pressure and heart rate should be monitored every 15 minutes during the infusion.  Emergency 
agents including oxygen, oral and endotracheal airways, intubation equipment, epinephrine, 
antihistamines and corticosteroids should be available.  In the event of a suspected anaphylactic 
reaction during infusion of Avastin, the infusion should be interrupted for subjects who develop 
dyspnea or clinically significant hypotension. Subjects who experience a NCI CTCAE v. 3.0 
Grade 3 or 4 allergic reaction / hypersensitivity, adult respiratory distress syndrome, or 
bronchospasm (regardless of grade) will be discontinued from Avastin treatment. 
 
The infusion should be slowed to 50% or less or interrupted for subjects who experience any 
infusion-associated symptoms not specified above. When the subject’s symptoms have 
completely resolved, the infusion may be continued at no more than 50% of the rate prior to the 
reaction and increased in 50% increments every 30 minutes if well tolerated. Infusions may be 
restarted at the full rate during the next cycle. Administer antihistamines, epinephrine, or other 
medications at the investigator’s discretion. 
 
Avastin Infiltration:  
Should infiltration of the Avastin infusion occur the following steps are to be taken:  Discontinue 
the IV.  If a significant volume of the Avastin infusion remains, restart the IV and complete the 
infusion.  Treat the infiltration according to institutional guidelines for infiltration of a non-
caustic agent. 
 
7.1.2 Avastin Storage 
 
Upon receipt of Avastin, vials are to be refrigerated at 2°C-8°C (36°F-46°F) and should remain 
refrigerated until just prior to use.  DO NOT FREEZE. DO NOT SHAKE.  Opened vials must be 
prepared for use within 8 hours.  Vials should be protected from light. VIALS ARE FOR 
SINGLE USE ONLY.  Vials used for one patient may not be used for any other patient.  Once 
Avastin has been added to a bag of sterile saline, the solution must be administered within 8 
hours. 
 
 
 

 
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7.1.3  Avastin Dose Modification and Toxicity Management 
 
There are no reductions in the Avastin dose.  If adverse events occur that require holding 
Avastin, the dose will remain the same once treatment resumes.  Any toxicities associated with 
or possibly associated with Avastin treatment should be managed according to standard medical 
practice. Avastin has a terminal half-life of 2 to 3 weeks; therefore, its discontinuation results in 
slow elimination over several months. There is no available antidote for Avastin. Patients 
randomized in the combination arm will receive a total of 9 cycles of Avastin even if delays 
occur between doses; for these patients, the total time of participation in the trial may be longer 
than 24 weeks. For these patients requiring Avastin infusions delays, evaluations scheduled by 
weeks will be changed to evaluations by cycle as follow: 
 
 
Week 0 or cycle 1 
 
 
Week 3 or cycle 2 
 
 
Week 6 or cycle 3 
 
 
Week 9 or cycle 4 
 
 
Week 12 or cycle 5 
 
 
Week 15 or cycle 6 
 
 
Week 18 or cycle 7 
 
 
Week 21 or cycle 8 
 
 
Week 24 or cycle 9 
 
Adverse events requiring delays or permanent discontinuation of Avastin are listed in Table 1.  
Regardless of the reason for holding study drug treatment, the maximum allowable length of 
treatment interruption is 2 months. 

 
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Table 1:  Avastin Dose Management Due to Adverse Events 
 
Event 
Action to be Taken 
Hypertension 
No dose modifications for grade 1/2 events 
Grade 3 
If not controlled to < 150/100 mmHg even with adjustments of the 
medications, discontinue Avastin. 
Grade 4 (including 
RPLS (confirmed by 
MRI) or hypertensive 
encephalopathy) 
Discontinue Avastin 
Hemorrhage 
No dose modifications for grade 1/2 nonpulmonary and non-CNS events 
Grade ≥ 2 pulmonary or 
CNS hemorrhage 
Discontinue Avastin 
Grade 3  nonpulmonary 
and non-CNS 
hemorrhage 
Subjects who are also receiving full-dose anticoagulation will be 
discontinued from receiving Avastin.   
All other subjects will have study treatment held until all of the 
following criteria  are met: 
• The bleeding has resolved and hemoglobin is stable. 
• There is no bleeding diathesis that would increase the risk 
of therapy. 
• There is no anatomic or pathologic condition that 
significantly increases the risk of hemorrhage recurrence. 
Subjects who experience a repeat Grade 3 hemorrhagic event will be 
discontinued from receiving Avastin. 
Grade 4 nonpulmonary 
or non-CNS hemorrhage 
Discontinue Avastin 
Grade 1 pulmonary or 
CNS hemorrhage 
 
 
 
 
 
 
 
 
Grade 2, 3, or 4 
pulmonary or CNS 
hemorrhage 
Subjects who are also receiving full-dose anticoagulation will be 
discontinued from receiving Avastin.   
All other subjects will have study treatment held until all of the 
following criteria  are met: 
• The bleeding has resolved and hemoglobin is stable. 
• There is no bleeding diathesis that would increase the risk 
of therapy. 
• There is no anatomic or pathologic condition that 
significantly increases the risk of hemorrhage recurrence. 
 
Discontinue Avastin 
Venous Thrombosis 
No dose modifications for grade 1/2 events 
 
 

 
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Table 1:  Avastin Dose Management Due to Adverse Events 
 
Event 
Action to be Taken 
Grade 3/ Asymptomatic 
Grade 4 
Hold study drug treatment.  If the planned duration of full-dose 
anticoagulation is <2 weeks, study drug should be held until the full-
dose anticoagulation period is over.  If the planned duration of full-
dose anticoagulation is >2 weeks, Avastin  may be resumed during 
the period of full-dose anticoagulation if all of the following criteria  
are met: 
• The subject must have an in-range INR (usually between 2 
and 3) on a stable dose of warfarin (or other anticoagulant) 
prior to restarting study drug treatment. 
• The subject must not have had a Grade 3 or 4 hemorrhagic 
event while on anticoagulation. 
• The subject must not have had evidence of tumor 
involving major blood vessels on any prior CT scan. 
Symptomatic Grade 4 
Discontinue Avastin. 
Arterial Thromboembolic event 
(Angina, myocardial infarction, transient ischemic attack, cerebrovascular accident, and any 
other arterial thromboembolic event) 
 Any grade 
Discontinue Avastin 
Congestive Heart Failure (Left ventricular systolic dysfunction) 
No dose modifications for grade 1/2 events 
Grade 3 
Hold Avastin until resolution to Grade ≤ 1 
Grade 4 
Discontinue Avastin 
Proteinuria 
No dose modifications for grade 1/2 events 
Grade 3 
(UPC> 3.5,  
24- hr urine >3.5 g/24 hr)   
 
Hold Avastin treatment until ≤ Grade 2, as determined by either UPC 
ratio ≤ 3.5 or 24 hr collection ≤ 3.5 g 
 
Grade 4 (nephrotic 
syndrome) 
Discontinue Avastin 
GI Perforation 
 
Discontinue Avastin (Any grade) 
Fistula 
 
Any grade (TE 
fistula 
 
Grade 4 fistula 
 
Discontinue Avastin 
 
Discontinue Avastin 
Bowel Obstruction 
Grade 1 
 
Continue patient on study for partial obstruction NOT requiring 
medical intervention. 
 
 

 
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Table 1:  Avastin Dose Management Due to Adverse Events 
 
Event 
Action to be Taken 
Grade 2 
Hold Avastin for partial obstruction requiring medical intervention.  
Patient may restart upon complete resolution. 
Grade 3/4 
Hold Avastin for complete obstruction.  If surgery is necessary, patient 
may restart Avastin after full recovery from surgery, and at 
investigator’s discretion. 
Wound dehiscence 
requiring medical or 
surgical therapy 
Discontinue Avastin 
Reversible Posterior 
Leukoencephalopathy 
 
Any grade 
(confirmed by 
MRI) 
 
 
Discontinue Avastin 
Other Unspecified Avastin-Related Adverse Events 
Grade 3 
 
Grade 4 
Hold  Avastin until recovery to ≤ Grade 1 
 
Discontinue Avastin 
 
7.2 
Letrozole 
Letrozole (Femara®) is the hormonal agent that will be used in this trial.  Patients will receive 
2.5 mg PO daily until 1 day before surgery.  Dose of the hormonal agent will not be modified for 
this protocol.  Adverse events associated with these medications are provided in the package 
inserts (www.fda.gov/cder/foi/label/2001/20726S006LBL.PDF). The hormonal agent is 
commercially available and will not be provided by the study. 
 
7.3 
Other Concomitant Medications 
 
The reason(s) for treatment dosage, and dates of treatment should be recorded in the source 
documents.  Concomitant medications should be reported to the investigator and will be recorded 
as instructed in the study specific case report forms.  Patients who experience Avastin-related 
temperature elevations or other infusion-related symptoms may be treated symptomatically with 
acetaminophen, dyphenhydramine, meperidine, or other medications as clinically indicated, 
including < 48 hours of treatment with corticosteroids.  Patients should receive full supportive 
care, including transfusions, antibiotics, anti-emetics, etc. when appropriate.  Pre-trial supportive 
care will continue. 
 
Unless there is a contraindication, patients with a history of atherosclerosis or at high risk for 
atherosclerosis should be taking aspirin (81 mg PO a day) and/or clopidrogel (75 mg PO a day) 
to prevent arterial thrombosis.  Unless there is a contraindication, patients with a central venous 
line should be receiving warfarin (1 mg PO a day) to prevent venous thrombosis.  Patients 
 
 

 
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receiving warfarin 1 mg a day should have their INR checked every week for 4 weeks to make 
sure that it does not increase to > 1.5.   
 
The following therapies are excluded during active protocol therapy: radiotherapy, hormonal 
therapy different from the agent prescribed in the protocol, chemotherapy, and cancer 
immunotherapy or other biologic agents. 
 
8.0 
CLINICAL AND LABORATORY EVALUATIONS 
 
Patients will be monitored for safety and tolerability during and after the treatment period.  
Patients will undergo an assessment of tumor status at baseline (screening) and at the completion 
of the first 6 weeks of therapy.  Further tumor status evaluation will be done every 6 weeks until 
disease progression or for 24 weeks.  If there is any doubt about whether disease progression has 
occurred based on clinical evaluation or images, treatment on study should continue until the 
next tumor assessment, as appropriate for patient care. 
 
8.1 
Pre-Treatment Evaluations 
 
Informed consent will be obtained before study-specific screening evaluations are performed.  
Screening evaluations must be performed within 8 weeks prior to day 0 (except blood and urine 
tests which should be obtained within 4 weeks).  Results of tests or examinations done as 
standard of care prior to obtaining informed consent and within 56 days prior to day 0 may be 
used rather than repeating tests.  
 
The following evaluations and procedures will be performed during the screening period:  
 
-  Signed informed consent. 
 
-  Clinical evaluations: medical history, demographics, weight and height, complete physical 
examination, vital signs and performance status.   
 
- 
Laboratory assessment: UA, urinary protein/creatinine ratio (24-hour urine collection if 
indicated), CBC with differential and platelet count, serum chemistries (glucose, BUN, 
creatinine, total protein, albumin, bilirubin, calcium, alkaline phosphatase, AST, and ALT), 
serum electrolytes (Na, K,Cl), INR.  
- 
Core needle biopsy of the tumor and of the unaffected breast (optional); samples for 
circulating tumor cells (Appendix I). 
 
-  Sentinel lymph node biopsy when required.  
 
- 
Estrogen and Progesterone receptor status.  
- 
Her-2-neu by immunohistochemistry (Negative: IHC 0, 1 or 2+ or non-amplified by FISH). 
 
- 
Tumor assessments: Mammogram and breast ultrasound are required.  PET/CT scan or [CT 
scan of chest, abdomen,  pelvis,  with bone scan] will be obtained.  If the scan shows disease 
outside the breast and/or the axilla, the patient will not be enrolled in the trial.   
 
 

 
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- 
Evaluation of the ejection fraction with an echocardiogram/MUGA. 
 
-  Concomitant medication assessment. 
 
-  Patients who have satisfied basic eligibility criteria will be assigned with a study patient 
number and the patient can proceed to be consented. After screening procedures are 
completed, the patient will be randomized. 
 
8.2 
Evaluations During Treatment 
The following evaluations and procedures will be performed during the treatment period:  (All 
assessments should be performed within 5 working days of the protocol-specified date). 
 
-  Physical examination, vital signs, weight, and performance status on weeks 6, 12, 18, and 24 
or until disease progression.     
 
- 
For patients randomized to the Avastin arm: urine protein/creatinine ratio or urine dipstick 
before every administration of Avastin (every 6 weeks).  Patients found to have > 2+ or 
greater proteinuria and/or urine protein/creatinine ratio > 1.0 must undergo a 24-hour urine 
collection Patients who develop grade 3 proteinuria during treatment will not receive 
additional doses of Avastin unless the proteinuria improves to grade 2 or better and the 
patient has not missed more than 2 scheduled sequential doses (see Appendix F).  
 
- 
Laboratory assessments: CBC with differential and platelet count, serum chemistries 
(glucose, BUN, creatinine, total protein, albumin, bilirubin, calcium, alkaline phosphatase, 
AST, and ALT), and serum electrolytes (Na, K, Cl) on weeks 1, 3, 6, 9, 12, 15, 18, 21, and 
24 for the combination arm and weeks 1, 6, 12, 18, and 24 or until disease progression is 
documented in the Letrozole alone arm.  The last evaluation is at 24 weeks.  
 
- 
Core needle biopsy of the tumor and of the unaffected breast (optional) at week 6 only; 
samples for evaluation of circulating tumor cells (Appendix I). 
  
- 
Tumor assessments: Ultrasound of the breast will be obtained on week 6, 12, 18, and 24.  
Mammogram will be obtained on week 24.  Additional PET scans or CT of the chest, 
abdomen, and pelvis, or bone scans are not required except as clinically indicated.    (see 
Appendix H for RECIST criteria).   
 
- 
Evaluation of the ejection fraction with an echocardiogram/MUGA (Week 24 only) 
 
-  Surgical evaluation at the end of neoadjuvant therapy.  There must be at least four weeks 
between the last dose of Avastin and surgery. Patients will continue letrozole until the day 
before surgery.  
 
- 
Concomitant medication assessment. 
 
 
 

 
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- 
Assessment of adverse events (Appendix C and section 13).  
 
8.3 
Post-Treatment Evaluations 
 
Patients will complete participation in the protocol at the time of surgery and will not be 
followed unless the patient requires following of adverse events. The following evaluations and 
procedures will be performed at early termination:  
 
- 
Clinical evaluations: Weight, physical examination (complete), vital signs and performance 
status.  There must be a complete and clear documentation of objective response or disease 
progression using the RECIST criteria.  Reason(s) for discontinuation must be documented. 
 
- 
Laboratory assessment: UA (subjects found to have > 2+ or greater proteinuria and/or urine 
protein/creatinine ratio > 1.0 must undergo a 24-hour urine collection), CBC with differential 
and platelet count, serum chemistries (glucose, BUN, creatinine, total protein, albumin, 
bilirubin, calcium, alkaline phosphatase, AST, and ALT), and serum electrolytes (Na, K, Cl).  
 
- 
Tumor assessments: Mammogram and breast ultrasound.  Spiral CT scans of chest, abdomen, 
and pelvis, bone scan, and any other procedure are not required per protocol but will be 
obtained if clinically indicated.  
 
-  Concomitant medication assessment. 
 
-  Assessment of adverse events (Appendix C and section 13).  If the patient has an ongoing 
toxicity, the patient should be followed until resolution or stabilization.  Serious and non-
serious adverse events occurring within 30 days of day 0 of the last treatment should be 
reported on the appropriate adverse event CRF.  Patients with ongoing hypertension at 
termination visit will have their BP and use of anti-hypertensive medications monitored 
every 4 months for up to 1 year or until BP returns to normal limits.  Patients who have 
ongoing proteinuria (grade 3) at the termination visit will be monitored with 24-hour urine 
collection every 4 months for up to 1 year or until urine protein improves to grade 2.   
 
9.0 
SUBJECT DISCONTINUATION 
 
Patients may withdraw from this study at any time.  Any patient who withdraws will be 
encouraged to return to the study center for the post-dose evaluations.  The termination visit 
consists of all evaluations scheduled for the termination visit.  The primary reason(s) for 
discontinuation must be recorded on the appropriate CRF page.  The PI may discontinue a 
patient from treatment.  Reasons may include, but are not limited to, the following: clinically 
significant deterioration, noncompliance, persistent grade 3 or 4 adverse event or any significant 
adverse event that compromises the patient’s ability to participate in the study, requirement of a 
significant surgical procedure or radiation therapy during the treatment period of the study, 
investigator’s determination that it is not in the patient’s best interest to continue participation, 
proteinuria greater than 2 gr in 24 hours, grade 4 hypertension, nephrotic syndrome, grade 2 or 
higher pulmonary or CNS hemorrhage and any other grade 4 hemorrhage, grade 4 venous 
thromboembolic event, any grade arterial thromboembolic event, grade 4 congestive heart 
 
 

 
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failure, gastrointestinal perforation, wound dehiscence requiring medical or surgical 
intervention, development of brain metastases, pregnancy.   
 
Patients who have an ongoing Avastin-related Grade 4 or serious adverse event at the time of 
discontinuation from study treatment will continue to be followed until resolution of the event or 
until the event is considered irreversible (see Section 7.1.3). 
 
10.0 
STUDY DISCONTINUATION 
 
The principal investigator and/or Genentech and/or the Breast Cancer Research Consortium have 
the right to terminate the study for the following reasons: the incidence or severity of adverse 
events indicates potential health hazard for patients, subject enrollment is unsatisfactory, or data 
recording is inaccurate or incomplete. 
 
11.0 
STATISTICAL METHODS 
 
11.1 
Sample Size Determination 
 
The design utilized will be an open label phase II randomized trial of neoadjuvant Avastin and 
letrozole or single agent letrozole in post-menopausal patients with new diagnosed stage II/III, 
ER/PR positive, Her-2 negative breast cancer.  The randomization will be 2:1 combination 
versus single agent letrozole with a total of 75 patients (50 receiving the combination and 25 
receiving single agent.  The intent of the trial is not a comparison of the two arms of the study 
but rather to estimate the pathologic complete remission rate (pCR) of the combination therapy.  
Previous studies have demonstrated that the pCR rate of single agent letrozole is <1% (44 and 
recent analysis by Ellis, et al, J. Natl. Cancer Inst 2008, 100:1380-1388).  A recent pilot trial of 
neoadjuvant therapy of this population using Avastin and letrozole had a pCR of 12% (breast 
and nodes) and breast pCR of 16%.  In order to achieve a more accurate analysis of pCR rate, 
this trial in 50 patients would have standard errors of + 0.052 and using the Blyth-Still-Cosella 
method provide a pCR rate and two-sided confidence interval of 5.4% - 23.3% based on the pilot 
study of 12% pCR.   The single agent letrozole 25 patient arm with projected <1% pCR rate 
would be expected to have no pCR events and the probability of observing one pCR event would 
be 22%.  The purpose of the single agent letrozole arm is to characterize the pCR response as 
similar to the prior (historical control) trials of single agent neoadjuvant letrozole. 
 
 
 

 
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11.2 
Statistical Analysis 
 
Descriptive statistics will be used to summarize baseline characteristics of all patients by 
randomized arm in order to assess relative comparability of the two arms of the study. Mean, 
standard deviation and range will be summarized for continuous variables, and frequency 
(percentages) will be calculated for categorical variables. The difference between the 
pathological complete responses and clinical responses will also be determined along with 95% 
exact confidence intervals about these differences in proportions.   
 
Descriptive statistics will be calculated to summarize biomarker values from biopsies during pre- 
and post-therapy (6 weeks) for all patients.  The change between pre- and post-therapy for each 
biomarker will also be calculated and summarized for each arm together with 95% confidence 
intervals. All toxicities will be recorded and summarized by calculating frequencies and 
proportions of toxicity grades for each arm. 
 
12. 
ADMINISTRATIVE RULES OF THE PROTOCOL 
 
12.1 
Compliance with the Protocol and Protocol Revisions 
 
The study must be conducted as described in this approved protocol. Should amendments to the 
protocol be required, the amendments will be originated and documented by the Principal 
Investigator at UAB. The written amendment will be sent to investigators and must be submitted 
to the IRB at the investigator's site for approval. All revisions to the protocol will be provided to 
Genentech by UAB. The Investigator should not implement any deviation or change to the 
protocol without prior review and documented approval/favorable opinion from the IRB of an 
Amendment, except where necessary to eliminate an immediate hazard(s) to study patients. 
Documentation of approval signed by the chairperson or designee of the IRB(s) must be sent 
tothe UAB Clinical trial Network Coordinator (Pam Dixon fax 205-975-9875). If the revision is 
an Administrative Letter, Investigators must inform their IRB(s)/IEC(s). 
 
12.2 
Informed Consent 
 
The Investigator must ensure that patients or their legally acceptable representatives are clearly 
and fully informed about the purpose, potential risks and other critical issues regarding clinical 
trials in which they volunteer to participate. Preparation of the consent form is the responsibility 
of the Investigator and must include all elements required by CFR 21 Part 50.25 and the local 
IRB. 
 
12.3 
Records and Reports 
 
An Investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the investigation on each 
individual treated with the investigational product or entered as a control in the investigation. 
 
 
 

 
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12.4 
Study Management 
 
12.4.1 Institutional Review Board (IRB) Approval and Consent 
 
Before study initiation, the Investigator must have written and dated approval/favorable opinion 
from the IRB for the protocol, consent form, patient recruitment materials/process (e.g., 
advertisements), and any other written information to be provided to patients.  The Investigator 
should provide the IRB with reports, updates, and other information (e.g., Safety Updates, 
Amendments, Administrative Letters) according to regulatory requirements or Institution 
procedures. Copies of the initial IRB approval as well as annual re-approvals must be submitted 
to UAB. UAB will provide copies of IRB approval to Genentech. 
 
It is expected that the IRB will have the proper representation and function in accordance with 
federally mandated regulations. In obtaining and documenting informed consent, the investigator 
should comply with the applicable regulatory requirement(s), and should adhere to Good 
Clinical Practice (GCP) and to ethical principles that have their origin in the Declaration of 
Helsinki. Before recruitment and enrollment onto this study, the patient will be given a full 
explanation of the study and will be given the opportunity to review the consent form. Each 
consent form must include all the relevant elements currently required by the FDA Regulations 
and local or state regulations. Once this essential information has been provided to the patient 
and the investigator is assured that the patient understands the implications of participating in the 
study, the patient will be asked to give consent to participate in the study by signing an IRB-
approved consent form. 
 
Prior to a patient’s participation in the trial, the written informed consent form should be signed 
and personally dated by the patient or the patient’s legally acceptable representative, and by the 
person who conducted the informed consent discussion. Documentation of IRB approval must be 
on file before any patient can be registered. Registration begins with signed informed consent. 
 
12.4.2 Study Monitoring 
 
Data from every site will be captured on manual CRFs, and supporting documents will be faxed 
to UAB.  Personnel from the University of Alabama at Birmingham will monitor the trial and 
may periodically visit the investigative site to ensure proper conduct of the trial and proper 
collection of the data. The investigators at each site will allow the monitor to review all source 
documents used in the preparation of the case reports.  
 
12.4.3 Required Documentation 
 
Before the study can be initiated at any site, the following documentation must be provided to 
the UAB Clinical Trials Network Office: 
 
• A copy of the official IRB approval letter for the protocol and informed consent 
• IRB membership list 
• CVs and medical licensure for the principal investigator and any associate investigators who 
will be involved in the study 
 
 

 
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• A copy of the IRB-approved consent form and HIPAA authorization 
• CLIA Laboratory certification and institution lab normal values 
 
In addition, an executed subcontract must be in place at UAB.  A separate agreement with 
Genentech may be required for Avastin shipment. 
 
12.4.4 Adherence to the Protocol 
 
Except for an emergency situation in which proper care for the protection, safety, and well-being 
of the study patient requires alternative treatment, the study shall be conducted exactly as 
described in the approved protocol. Any deviation from the protocol must have prior approval by 
the Principal Investigator and must be recorded and explained. 
 
12.4.5 Record Retention 
 
Study documentation includes all Case Report Forms, data correction forms or queries, source 
documents, Sponsor-Investigator correspondence, monitoring logs/letters, and regulatory 
documents (e.g., protocol and amendments, IRB correspondence and approved signed patient 
consent forms). Source documents include all recordings of observations or notations of clinical 
activities and all reports and records necessary for the evaluation and conduction of the clinical 
research study. Government agency regulations and directives require that all study 
documentation pertaining to the conduct of a clinical trial must be retained by the study 
investigator. In the case of a study with a drug pursuing regulatory approval and marketing, these 
documents shall be retained for at least two years after the last approval of marketing application 
in an International Conference on Harmonization (ICH) region. In all other cases, study 
documents should be kept on file until three years after the completion and final study report of 
this investigational study. If the Investigator withdraws from the study (e.g., relocation, 
retirement), the records shall be transferred to a mutually agreed upon designee (e.g., another 
Investigator, IRB). Notice of such transfer will be given in writing to Genentech. Records of the 
patient’s participation in this study will be kept confidential so far as permitted by law.  
However, the patient’s doctor and his/her staff, representatives of Genentech, representatives of 
the Breast Cancer Research Consortium, the U.S. Food and Drug Administration, and the IRB 
will be able to inspect patient records and have access to confidential information which 
identifies the patient by name.  Any publication of data will not identify the patient by name.  
Should the patient’s medical record need to be reviewed by a foreign regulatory agency, a 
member of the IRB staff will observe the review so that the record is not removed, copied, or 
identifiable information recorded in any manner.  
 
12.4.6 Obligations of Investigators 
 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of Helsinki. 
The Principal Investigator is responsible for personally overseeing the treatment of all study 
patients. The Principal Investigator must ensure that all study site personnel, including 
subinvestigators and other study staff members, adhere to the study protocol and all 
 
 

 
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FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and after study 
completion. 
 
The Principal Investigator at each institution or site will be responsible for ensuring that all the 
required data will be collected and entered onto the Case Report Forms. Periodically, monitoring 
visits will be conducted and the Principal Investigator will provide access to his/her original 
records to permit verification of proper data entry. At the completion of the study, all case report 
forms will be reviewed by the Principal Investigator and will require his/her final signature to 
verify the accuracy of the data. 
 
13.0 
ASSESSMENT OF SAFETY 
 
13.1 
Adverse Event Reporting and Definitions (See also Appendix D) 
 
In the event of an adverse event the first concern will be for the safety of the subject.   
 
13.1.1 Adverse Events (AEs) 
 
Adverse events should be followed to resolution or stabilization, and reported as SAEs if they 
become serious. This also applies to patients experiencing AEs that cause interruption or 
discontinuation of investigational product or those experiencing AEs that are present at the end 
of their participation in the study. Such patients should receive post-treatment follow-up as 
appropriate as it has been described throughout the protocol. If an ongoing AE changes in its 
severity or in its perceived relationship to study drug, a new AE should be completed.  
 
13.1.2 Serious Adverse Events (SAEs) 
 
SAEs require expeditious handling and reporting to UAB to comply with regulatory 
requirements. Investigators are required to report within 24 hours of investigator’s knowledge 
(MedWatch Form 3500 – Appendix D) to the principal investigator (UAB – Andres Forero), 
who will report to the FDA, UAB IRB and Genentech Drug Safety, ANY serious treatment 
emergent adverse event (SAE) as soon as possible. Affiliate SAEs must be reported to local IRB 
according to their institutional guidelines. All SAEs must be collected and reported until 30 days 
following patient discontinuation of dosing; if only limited information is initially available, 
follow-up reports are required. The original SAE form must be kept on file at the study site. 
 
A SAE is any sign, symptom or medical condition that emerges during Avastin treatment or 
during a post-treatment follow-up period that (1) was not present at the start of Avastin treatment 
and it is not a chronic condition that is part of the patient’s medical history, OR (2) was present 
at the start of Avastin treatment or as part of the patient’s medical history but worsened in 
severity and/or frequency during therapy, AND that meets any of the following regulatory 
serious criteria: 
 
• Results in death  
• Is life-threatening  
• Requires or prolongs inpatient hospitalization  
 
 

 
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• Is disabling  
• Is a congenital anomaly/birth defect  
• Is medically significant or requires medical or surgical intervention to prevent one of the 
outcomes listed above. 
 
13.2 
Reporting of Serious Treatment Emergent Adverse Events  
 
All SAEs should be recorded on a MedWatch 3500 Form (see Appendix D - can be accessed at: 
https://www.accessdata.fda.gov/scripts/MedWatch) and faxed to UAB: 
Pam Dixon 
Phone: 205-975-5387 
Fax: 205-975-9875 
 
And 
Andres Forero 
Phone: 205-934-7167 
Fax: 205-975-6911 
 
Once forms are reviewed, UAB will report to: 
 
MedWatch 
5600 Fishers Lane 
Rockville, MD 20852-9787 
Fax: 1-800-FDA-0178 (1-800-332-0178) 
 
And 
 
Genentech Drug Safety  
Fax: 
(650) 225-4682 or (650) 225-5288 
 
And: 
 
UAB IRB  
 
MedWatch 3500a Reporting Guidelines: 
In addition to completing appropriate patient demographic and suspect medication information, 
the report should include the following information within the Event Description (section 5) of 
the MedWatch 3500 form: 
 
• Treatment regimen (dosing frequency, combination therapy) 
• Protocol description (and number, if assigned) 
• Description of event, severity, treatment, and outcome, if known 
• Supportive laboratory results and diagnostics 
• Investigator’s assessment of the relationship of the adverse event to each investigational 
product and suspect medication 
 
 

 
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Follow-up information:   
Additional information may be added to a previously submitted report by any of the following 
methods: 
 
• Adding to the original MedWatch 3500a report and submitting it as follow-up 
• Adding supplemental summary information and submitting it as follow-up with the 
original MedWatch 3500a form  
• Summarizing new information and faxing it with a cover letter including subject 
identifiers (i.e. D.O.B., initials, subject number), protocol description and number, if 
assigned, suspect drug, brief adverse event description, and notation that additional or 
follow-up information is being submitted  (The subject identifiers are important so that 
the new information is added to the correct initial report) 
 
Occasionally the principal investigator and/or Genentech may contact the reporter for additional 
information, clarification, or current status of the subject for whom an adverse event was 
reported.  
 
Assessing Causality: 
Investigators are required to assess whether there is a reasonable possibility that Avastin caused 
or contributed to an adverse event.  The following general guidelines may be used. 
Yes: 
if the temporal relationship of the clinical event to Avastin administration makes a causal 
relationship possible, and other drugs, therapeutic interventions or underlying conditions do not 
provide a sufficient explanation for the observed event. 
 
No: 
if the temporal relationship of the clinical event to Avastin administration makes a causal 
relationship unlikely, or other drugs, therapeutic interventions or underlying conditions provide a 
sufficient explanation for the observed event. 
 
 

 
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14. 
REFERENCES/BIBLIOGRAPHY 
1.  
 American Cancer Society. Cancer facts and figures. Atlanta, GA: American Cancer 
Society, 2005.  
2.  
 Forbes JF. The control of breast cancer: the role of tamoxifen. Semin Oncol 
1997;24(1):S15–9. 
3. 
 Beatson GT. On the treatment of inoperable cases of carcinoma of the mamma; 
suggestions for a new method of treatment with illustrative cases. Lancet 1896; ii: 104–7. 
4.  
 Morgan LR Jr, Schein PS,Woolley PV, et al. Therapeutic use of tamoxifen in advanced 
breast cancer: correlation with biochemical parameters. Cancer Treat Rep 1976; 
60:1437–43. 
5.  
 Henderson IC. Endocrine therapy of metastatic breast cancer. Breast Dis 1991; 2:559–
603. 
6.  
 Jordan VC. The role of tamoxifen in the treatment and prevention of breast cancer. Curr 
Probl Cancer 1992; 16:129–76. 
7. 
Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for early breast cancer: an 
overview of the randomized trials. Lancet 1998; 351:1451–67. 
8.  
Current Trials Working Party of the Cancer Research Campaign Breast Cancer Trials 
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APPENDIX A 
 
Study Flowchart 
 
 
 
 
 
 

 
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a.  Prior to each dose of Avastin. 
 
Screening 
Treatment 
Termination 
End of Study Visit 
 
Days -56 to -
1 
At each Avastin dose 
Week 24 
 
Signed Informed Consent 
X 
 
 
 
Medical History/Demographics 
X 
 
 
 
Weight 
X 
Xa
X 
 
Height 
X 
 
 
 
Physical Exam 
X 
Xi
X 
 
Vital Signs 
X 
Xb
X 
 
ECOG Performance Status 
X 
Xa
X 
 
Core Needle Biopsies  
[tumor and normal breast (optional)] 
X 
X (only on week 6) 
 
 
UAk, protein/creatinine ratioc, i (24 hr 
urine collection if indicated) or urine 
dipstickk 
Xk 
Xi 
X 
 
Urine Proteinc 
Xk
X
X 
 
CBC (complete)l 
Xk 
X 
X 
 
Circulating Tumor Cellsg 
X
X
X 
 
Serum Chemistries/ Electrolytesl 
Xk 
X 
X 
 
INR 
Xk 
 
 
 
FSHd 
X 
 
 
 
Mammogram 
X 
 
X 
 
Breast Ultrasounde 
X 
X (every 6 weeks)  
X 
 
PET/CT scan or [CT of the chest, 
abdomen, pelvis, with Bone Scan] 
X 
 
 
 
Ejection Fraction – 
Echocardiogram/MUGAh 
X
 
X 
 
Sentinel LN biopsy as per institution 
protocol 
X 
 
 
 
Avastin Administration 
 
X 
X 
 
Hormonal Therapyf 
 
X
 
 
Concomitant Medications 
X 
X 
X 
 
Adverse Events 
X 
X 
X 
Xj 
Surgery 
 
 
 
X 
Pathology Report 
 
 
 
X 
b. 
Record complete vital signs before and after the Avastin infusion. BP and HR every 15 minutes during Avastin administration. 
c. 
See Appendix E. 
d. 
If needed to confirm post-menopausal status. 
e. 
First evaluation at 6 weeks; then every 6 weeks until week 24 or tumor progression. 
f. 
Daily by mouth until surgery -1 day or disease progression (Letrozole, 2.5 mg PO a day). Letrozole can start immediately post 
 
sentinel node biopsy procedure 
g. 
See Appendix I. 
h. 
LVEF calculated only by echocardiogram/MUGA. 
i. 
Weeks 6, 12, 18, 24. 
j. 
Follow according to Section 8.3. 
k. 
U/A, protein/creatinine ratio (24 hr urine collection if indicated) or urine dipstick within 4 weeks of Day 0. 
l.   For the Letrozole arm alone; to be done every 6 weeks.

 
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APPENDIX B 
 
NCI Common Toxicity Criteria, Version 3.0 
http://www.ctep.cancer.gov/forms/ctcaev3.pdf 
 
                                                         
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APPENDIX C 
 
FDA MedWatch 3500 Form  
http://www.accesdata.fda.gov/scripts/MedWatch 
 
                                                         
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SAE Reporting 
 
 
 SAE reporting period begins at the signing of informed consent. 
 
 All SAEs, regardless of attribution must be reported to UAB by fax within 24 hrs of 
investigator’s knowledge: 
 
 MedWatch Form 3500 must be submitted, regardless of completeness of 
information. 
 
 Fax to Pam Dixon at 205-975-9875. 
 
 If only limited information is available, follow-up reports are required. 
 
 Affiliate SAEs must be reported to local IRB according to their institutional 
guidelines. 
 
 UAB is responsible for submitting all SAE MedWatch forms to FDA. 
 
 UAB  is responsible for submitting all MedWatch forms to Genetech. 
 
 All SAEs must be collected and reported until 30 days following patient 
discontinuation of dosing. 
 
 If only limited information is initially available, follow-up reports are required.  
 
 The original SAE form must be kept on file at the study site. 
                                                         
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APPENDIX D 
 
Evaluation of Proteinuria 
 
                                                         
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EVALUATION OF PROTEINURIA 
 
At Screening 
 
- Urinalysis is required for all subjects 
- Urinary protein/creatinine ratio is required for all subjects 
- 24 hour urine collection if indicated 
 
Subjects must demonstrate < 1g of protein or a urinary protein/creatinine ratio < 1.0 to allow 
participation in the study. 
 
During Study (every 6 weeks for patients randomized to the Avastin arm) 
 
 
- Urinalysis with urinary protein/creatinine ratio or urine dipstick is required for all subjects 
  
Subjects found to have ≥ 2+ proteinuria by urinalysis or dipstick and/or a urine protein/creatinine ratio > 
1.0 must undergo a 24 hour urine collection for quantitation of protein and determination of creatinine 
clearance (urine volume required) 
Proteinuria 
Grade 3 protein/24 hour 
If urinalysis indicates > 2+ protein, perform 24-hr urine 
collection for quantitation of protein. If Grade 3 
proteinuria discontinue Avastin until proteinuria grade 2 
or better. 
Grade 4 (nephrotic syndrome) 
Discontinue the subject from Avastin. 
 
Study Termination 
 
- Urinalysis with urinary protein/creatinine ratio or urine dipstick is required for all subjects 
is required for all subjects 
 
Subjects found to have  ≥ 2+ proteinuria by urinalysis and/or urine protein/creatinine ratio > 1.0 must 
undergo a 24 hour urine collection for quantitation of protein and determination of creatinine clearance 
(urine volume required)  
 
Follow-Up 
 
For subjects who have ongoing grade 3 protein/24 hour urine at the termination visit will be monitored 
by 24 hour urine collection every 4 months for up to 1 year or until total 24 hour urine protein improves 
to grade 2 or better of protein/24 hour, whichever comes first. 
 
 
 
                                                         
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PROCEDURE FOR OBTAINING A URINE PROTEIN / CREATININE RATIO 
 
1)  Obtain at least 4 ml of a random urine sample (does not have to be a 24 hour urine)  
2)  Determine protein concentration (mg/dL)  
3)  Determine creatinine concentration (mg/dL)  
4)  Divide #2 by #3 above: urine protein / creatinine ratio = protein concentration (mg /dL) / 
creatinine concentration (mg /dL) 
 
The UPC directly correlates with the amount of protein excreted in the urine per 24 hrs (i.e. a 
UPC of 1 should be equivalent to 1g protein in a 24hr urine collection) 
 
Protein and creatinine concentrations should be available on standard reports of urinalyses, not 
dipsticks.  If protein and creatinine concentrations are not routinely reported at an Institution, 
their measurements and reports may need to be requested. 
 
                                                         
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APPENDIX E 
 
ECOG Performance Status Scale 
 
 

 
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ECOG PERFORMANCE STATUS SCALE 
 
 
GRADE 
DESCRIPTION 
0 
Full activity, able to carry on all pre-disease 
performance without restriction 
 
1 
Restricted in physically strenuous activity but 
ambulatory and able to carry out work of a light 
or sedentary nature, e.g., light housework or 
office work. 
 
2 
Ambulatory and capable of all self-care but 
unable to carry out any work activities.  Up and 
about >50% of waking hours. 
 
3 
Capable of only limited self-care, confined to a 
bed or chair >50% of waking hours. 
 
4 
Completely disabled.  Cannot carry on any self-
care.  Totally confined to bed or chair. 
 
5 
Dead 
 
 
                                                         
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APPENDIX F 
 
NYHA GUIDELINES 
http://www.hcoa.org/hcoacme/chf-cme/chf00070.htm 
 
 

 
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NEW York Heart Association (NYHA) Classification: 
 
 
A functional and therapeutic classification for prescription of physical activity for cardiac 
patients. 
 
• 
Class I: patients with no limitation of activities; they suffer no symptoms from ordinary 
activities. 
• 
Class II: patients with slight, mild limitation of activity; they are comfortable with rest or 
with mild exertion. 
• 
Class III: patients with marked limitation of activity; they are comfortable only at rest. 
• 
Class IV: patients who should be at complete rest, confined to bed or chair; any physical 
activity brings on discomfort and symptoms occur at rest. 
 
 
 
 
 
                                                         
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APPENDIX G 
 
RECIST Criteria 
 
J Natl Cancer Ins; 92 (3): 205, 2000 
 
http://ctep.cancer.gov/guidelines/recist.html 
 
 
                                                         
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APPENDIX H 
 
Blood and Biopsy Procedures 
 
 
                                                         
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UAB 0648 - Blood Draw and Biopsy Schedule 
Paraffin blocks to UAB, Frozen biopsies to UNC, all other blood to UCSF 
 
Collect in Specified Tubes 
Pre-Therapy 
Day -56 to Day 1 
Post-Therapy 
6 week 
Post-Therapy 
Week 18 
Post-Therapy 
Week 24 
10 ml EDTA – Lavender Top 
X 
X 
X 
X 
22.5 ml – Cell Saver 
X 
X 
X 
X 
Research Biopsies 
X 
X 
 
 
 
A. TO UCSF 
 
Whole blood samples for circulating tumor cell (CTC) analysis will be obtained at: 
  
1. Pre-therapy;  
2. On weeks 6, 18, and 24 during therapy   
 
The following samples will be collected at each time point listed above: 
 
1. Pre-therapy: 10 ml of whole blood (1-10 ml tubes, 2-4 ml tubes) collected in a 
lavender top EDTA tubes and 22.5 ml collected into three Cell Saver tubes 
(provided to each site). 
2. At subsequent time points:  10 ml of whole blood  in lavender top EDTA tubes 
and 22.5 ml collected into three Cell Saver tube (provided to each site). 
 
All samples will be labeled with an indelible marker to include: 
 
1. The study identification number (UAB 0648);  
2. The subject’s study number; and  
3. The date of collection. 
 
Shipping samples as diagnostic specimens must be in compliance with IATA packing 
instruction 650 (see below). Ship whole blood samples at room temperature 
overnight in appropriate containers. Blood samples may be drawn Monday 
through Thursday, but not on Fridays or the day before a holiday.  Any samples 
collected on Thursday should be collected early in the day to ensure they are sent the 
same day for overnight shipment.  Notification that a sample will be sent should 
occur on the day of shipping or before if possible by email to: jscott@cc.ucsf.edu and 
hrugo@medicine.ucsf.edu.  Blood should be shipped attention to: 
 
Park Laboratory 
2340 Sutter Street, 4th floor, Room S471 
San Francisco CA   94115 
(Phone: 415/514-3969) 
 
                                                         
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UAB Pam Dixon (pam.dixon@ccc.uab.edu or 205-975-5387) will inform UCSF that 
all regulatory documents have been received and are in order.  Then UCSF will 
provide cell saver tubes to each site.  Ten (10) tubes will be shipped to sites upon said 
 
 
 
 

 
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notification.  Requests for additional cell saver tubes when initial shipment of tubes is 
exhausted should be directed to Hope Rugo (hrugo@medicine.ucsf.edu or 415/353-
7428) and Lauren Metzroth (Lauren.Metzroth@ucsfmedctr.org or 415/885-7213).  
The study coordinator for UCSF will be the secondary contact (in lieu of Lauren 
Metzroth) when that contact information is made available. 
 
B. To UAB - Paraffin blocks (all patients) 
 
Paraffin blocks for immunohistochemistry studies will be obtained at: 
  
1. Pre-therapy;  
2. At week 6 
 
Fixed formalin paraffin embedded blocks (FFPE) will be obtained from the original 
diagnostic specimen from all patients.  These blocks will be shipped by the 
registering institution to UAB for sectioning.  Approximately fifteen sections will be 
cut from these blocks, and the blocks will be returned to registering institution.  If the 
registering institution is not able to provide tumor blocks for sectioning, please 
contact Kathy Sexton at the University of Alabama at Birmingham Comprehensive 
Cancer Center’s Tissue Procurement Core Facility (sexton@uab.edu; phone 205-934-
6071) for procedures to obtain tumor slides. 
 
All specimen blocks will be labeled with an indelible marker to include: 
1. The study identification number (UAB 0648); 
2. The subject’s study number; and  
3. The date of acquisition., and 
4. Timepoint of sample acquisition (pre-therapy, 6 week evaluation) 
 
Shipping samples as diagnostic specimens must be in compliance with IATA packing 
instruction 650 (see below).  Wrap FFPE blocks in bubble wrap and place in zip lock 
bags. Have the cassettes and bags appropriately labeled and include a specimen 
tracking form with shipment.  Ship the slides by overnight carrier at ambient 
temperature.  Samples may be sent monthly on the Monday or Tuesday of a non-
holiday week.  The receiving laboratory should be notified that a shipment is being 
sent by email to sexton@uab.edu (phone 205-934-6071).  The shipping address is: 
 
 
University of Alabama Comprehensive Cancer Center 
Tissue Procurement Core Facility 
703 South 19th Street 
Zeigler Research Building, Room 449 
Birmingham, AL 35294 
(Phone: 205-934-6071) 
C. To UNC - Frozen research biopsies (all patients) 
Preferably, at least 2 core biopsies will be obtained at: 
1. Pre-therapy;  
2. At week 6 
                                                         
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Biopsy samples, preferably obtained with a 14-18 gauge core needle, should be flash 
(snap) frozen (close cap tightly), ideally within 5 minutes but not greater than 30 
minutes, and stored in liquid nitrogen or at -80°C until shipping. If research biopsies 
prove inadequate, up to two additional biopsies may be requested. 
 
All cryovials (NALGENE® Cryoware™ 5000-0000) will be labeled with an indelible 
marker to include: 
 
1. The study identification number (UAB 0648);  
2. The subject’s study number;  
3. The date of acquisition; AND 
4. Timepoint of sample acquisition (pre-therapy, 6 week evaluation). 
 
Each subject’s biopsy specimen’s cryovials should be placed in an individual ziploc 
bag with the same identifying information written on the ziploc bag. Samples should 
be shipped on dry ice with a specimen tracking form in an approved container using 
overnight shipping.  The amount of dry ice to use for each shipment will depend on 
how many specimens are included.  The box should be full.  The container for 
shipping on dry ice is 12.25 inches on the inside.  If, for example, you have 3 baggies 
with 2 to 3 vials each, then you would put 5-6 inches of dry ice on the top and 
bottom.  Remember the net weight of dry ice included in the shipping container must 
be declared on the outside of the container.  Samples may be sent monthly or 
quarterly, depending on quantity, on the Monday or Tuesday of a non-holiday week.  
The receiving laboratory should be notified that a shipment is being sent by 
contacting Xiaping He (Xiaping@med.unc.edu).  The shipping address is: 
 
Charles Perou (attention Xiaping He) 
Lineberger Comprehensive Cancer Center 
102 Mason Farm Road 
CB# 7295 Room 12-020 
Chapel Hill, NC 27599-7925 
(Phone: 919-843-5717) 
 
 
Preparing and Shipping Tissue in Frozen Cryovials 
 
Snap Freezing: 
Do not Cut the tissue and simply ship as is. Place each core of tissue into a labeled 
individual 2.0 ml   NALGENE® Cryoware™ cryovials.  The label should contain the 
UAB study number (UAB 0648) followed by the subject’s study number, as well as 
the date the specimen was collected.  Do not place the patient’s name or medical 
record number on the label.  The container will become very cold.  Do not handle 
with unprotected hands.  Safety goggles should be use throughout the procedure. 
Snap freeze samples in liquid nitrogen.  If not available, you may use an alcohol/dry 
ice bath.   Fill pan about 1-2 inches deep with methanol.  The depth should be enough 
to cover the height of the vial.  Slowly add crushed dry ice until the boiling stops.  
                                                         
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The bath is now read for use.  Drop the sealed vials directly into the liquid nitrogen 
(LN2) carefully.  Leave the vials in the liquid nitrogen for at least one minute.  Once 
frozen, transfer the samples using tongs or a large spoon to remove the vials from the 
liquid nitrogen and transfer to a –80 °C freezer until shipped.   
 
Shipping Frozen Tissue: 
 
Shipping frozen tissue on dry ice must be in compliance with IATA packing 
instruction 904 (see below).  Dry ice is considered dangerous goods and must always 
be declared by marking the box appropriately with labeling and documentation 
according to Class 9 procedures. Proper training is required under federal and/or state 
regulations to handle dangerous goods.  All persons responsible for shipping 
dangerous goods must comply with all federal regulations, including but not limited 
to the specific training requirements of 49 CFR (172.700-172.704). 
 
Specimens should be shipped in dry ice safe containers, such as styrofoam (not metal) 
and using a secondary packaging (e.g. styrofoam box inside cardboard box). For 
shipping specimens on dry ice the “Special Handling” box of the Airbill (FedEx) 
should be used.  Proper shipping name box should be checked, the Class Number 9 
(misc.) is printed on the form – fill in amount of dry ice, the UN Number (UN1845) 
should be on the form, and mark “yes (Shippers declaration not required)”. 
 
D. IATA Packing Instructions 
 
650 GENERAL REQUIREMENTS 
Shippers of diagnostic specimens where a relatively low probability exists that 
infectious substances are present must comply with Packaging Instruction 650 of 
these regulations. Diagnostic specimens being transported for the purpose of initial 
diagnosis may be considered to fall under this category where a low probability exists 
that infectious substances are present. The shipper must also ensure that shipments 
are prepared in such a manner that they arrive at their destination in good condition 
and that they present no hazard to persons or animals during shipment.  
Inner packaging contains: 
• A watertight primary receptacle(s)- for diagnostic specimens the maximum 
quantity must not exceed 500mL 
• A watertight secondary packaging -the maximum quantity per outer packaging for 
diagnostic specimens must not exceed 4L 
• An absorbent material- must be placed between the primary receptacle and the 
secondary packaging. No absorbent material is required when shipping solid 
substances. 
 
If multiple primary receptacles are placed in a single secondary packaging they must 
be wrapped individually or for those transported in liquid nitrogen, separated and 
supported to ensure that contact between them is prevented.  The absorbing material, 
                                                         
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for example cotton or wool, must be sufficient to absorb the entire contents of all 
primary receptacles. 
 
• An outer packaging of adequate strength for its capacity, weight and intended use. 
 
The primary receptacle or the secondary packaging used for liquid diagnostic 
specimens must be capable of withstanding, without leakage, an internal pressure 
which produces a pressure differential of not less than 95 kPa (0.95 bar, 13.8lb/in2) in 
the range of -40.oC to + 55.oC(-40.oF to 130.oF). It is not necessary for the primary 
or secondary packaging to be capable of withstanding 95 kPa pressure differential 
when solid diagnostic specimens are being shipped. 
Packages consigned as freight must be at least 100 mm (4 in) in the smallest overall 
external dimension. 
 
An itemized list of contents must be enclosed between the secondary packaging and 
the outer packaging. Each package and the "Nature and Quantity of Goods" box of 
the airbill must show the text "DIAGNOSTIC SPECIMEN PACKED IN 
COMPLIANCE WITH IATA PACKING INSTRUCTION 650" (example below). A 
Shipper's Declaration for Dangerous Goods is not required. 
 
650 SPECIFIC REQUIREMENTS 
Substances shipped at ambient temperatures or higher- Primary receptacles include 
those of glass, metal or plastic.  Positive means of ensuring a leak-proof seal, such as 
heat seal, skirted stopper or metal crimp seal must be provided.  If screw caps are 
used these must be reinforced with adhesive tape or parafilm. 
 
Substances shipped on dry ice must be placed outside the secondary packaging(s) or 
alternatively in an overpack with one or more completed packagings.  Interior support 
must be provided to secure the secondary packaging(s) in the original position after 
the dry ice has dissipated.  The packaging must be leak-proof. The outer packaging 
must permit the release of carbon-dioxide gas. The primary receptacle must maintain 
its containment integrity at the temperature of the refrigerant as well as at the 
temperatures and pressure of air transport to which the receptacle could be subjected 
if refrigeration were to be lost. New shipping regulations effective January 1, 2005: 
Package must have both the shipper and recipient’s name, address and phone number. 
                                                         
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Package must have the new UN3373 symbol and the words “Diagnostic Specimen” 
adjacent to that symbol; keep the biohazard sticker in place as well. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
labeled box should be inserted into the FedEx Diagnostic Shipping Bag as usual for 
shipment via FedEx. 
 
                                                         
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904 GENERAL REQUIREMENTS 
Carbon dioxide, (dry ice, LN2), when offered for transport by air, must be in 
packaging designed and constructed to permit the release of carbon dioxide gas and 
to prevent a build-up of pressure that could rupture the packaging. 
 
The net weight of the Carbon dioxide, solid (dry ice) must be marked on the outside 
of the package. 
 
Arrangements between shipper and operators must be made for each shipment to 
ensure ventilation safety procedures are followed.  When a Shipper's Declaration is 
not required, the information as required by 8.2.3 for dry ice (LN2) must be contained 
in the "Nature and Quantity of Goods" box on the airbill, excluding the packing 
instruction number and packing group. 
 
                                                         
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APPENDIX I 
 
IMPLEMENTATION PLAN FOR OFF-SITE 
CONDUCT 
OF UAB COMPREHENSIVE CANCER CENTER 
INVESTIGATOR-INITIATED PHASE I AND II 
STUDIES  
 
 

 
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IMPLEMENTATION PLAN FOR OFF-SITE CONDUCT 
OF UAB COMPREHENSIVE CANCER CENTER 
INVESTIGATOR-INITIATED PHASE I AND II STUDIES 
 
CLINICAL TRIALS NETWORK (CTN) 
 
1.0 
INTRODUCTION 
 
The Clinical Trials Network (CTN) of the UAB Comprehensive Cancer Center (CCC) 
coordinates investigator-initiated clinical trials under Good Clinical Practice conditions at 
CTN sites to achieve timely study subject enrollment and to provide subjects at CTN 
sites with access to CCC investigator-initiated phase I and II studies.  The CTN sites are 
required to have 1) an experienced staff of investigators, research nurses, and data 
managers; 2) a designated IRB and significant experience in undertaking clinical trials; 
and 3) accrual of at least five study subjects annually into CCC investigator-initiated 
CTN site studies, as well as successful completion of monitoring and annual audits.  CTN 
sites must adhere to guidelines in the CCC Data Safety and Monitoring Plan (DSMP). 
 
2.0 
ADMINISTRATION OF THE CLINICAL TRIALS NETWORK 
 
Cancer Center Director (Acting) 
Dr. Edward Partridge
 Cancer Center Associate Director for Clinical Research (Acting)
Dr. Albert LoBuglio 
Clinical Trials Network Director 
Dr. Mansoor Saleh
                                                         
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Clinical Trials Coordinating Center Manager 
Mrs. Pam Dixon
CLINICAL TRIALS 
NETWORK SITES
 
CLINICAL 
TRIALS 
NETWORK 
 
 
 
 

 
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Overall responsibility for the CTN lies with the Director of the CCC with primary 
oversight by the CCC Associate Director for Clinical Research.  The Director of the CTN 
coordinates the overall efforts of the CTN, while day-to-day activities are supervised by 
the CTN Coordinating Center Manager.  
 
2.1 
Current CTN Personnel 
 
• CTN Director:  
 
 
 
Mansoor Saleh, MD 
• CTN Coordinating Center Manager:   
Pam Dixon, RN, BSN, OCN, 
 
CCRP 
 
 
3.0 
IMPLEMENTATION PLAN FOR CTN SITE STUDIES 
 
The P.I. will submit the phase I and/or phase II studies to the UAB IRB as identified 
protocols to be carried out at affiliate site (CTN).  Once approved by the UAB IRB, the 
affiliate site P.I. will proceed with affiliate site IRB approval.  The UAB P.I. will provide 
UAB IRB with an amendment to the approved UAB protocol which will provide the 
affiliate site IRB approval, consent documents and proposed date of initiation of the trial 
at the affiliate site.  When the amendment is approved by the UAB IRB, the affiliate site 
can initiate patient accrual. 
 
Serious Adverse Events (SAEs) are reported by the CTN site Lead Investigator within 24 
hours to the CTN Coordinating Center Manager by email (pam.dixon@ccc.uab.edu) or 
by fax (205) 975-9875.  The 24 hour paging number for the CTN Coordinating Center 
Manager is (205) 934-3411, beeper #5904.  The CTN Manager is then responsible for 
reporting SAEs to the UAB IRB and protocol P.I. in accordance with study-specific 
requirements. SAEs occurring at CTN sites are reported to the UAB IRB as “non-UAB” 
events.  
 
The CTN Coordinating Center Manager also ensures that the following are 
accomplished: 
 
• 
Develop and implement plans for monitoring and auditing CTN site studies  
• 
Provide initial protocol and consent documents to CTN sites 
• 
Coordinate CTN site initiation visits / teleconferences  
• 
Receive and evaluate documents from CTN sites and determine the need for 
further evaluation 
• 
Submit required regulatory documentation to the UAB IRB 
• 
Generate study-specific queries and develop resolutions 
• 
Perform monitoring at CTN sites 
• 
Provide for needed fiscal management including development of CTN site 
budgets and ensure sub-contracts are negotiated 
• 
Provide for distribution of study-specific funds to CTN sites. 
 
                                                         
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4.0 
CTN SITE REQUIREMENTS 
 
4.1 
Personnel  
 
A Lead Site Investigator is identified for each CTN site study. Adequate CTN site 
staff is provided for administration, data management, and research nurse 
activities.  The administrative staff provides clerical assistance to support CTN 
site studies.  The data management staff collects and manages necessary research 
data on study subjects enrolled onto CTN site studies.  The research nurses 
coordinate the study subjects’ care in conjunction with study-specific 
requirements to ensure patient safety, continuity of care and to provide ongoing 
assessment of the impact of the study on the CTN site and on patient resources.  
The CTN site staff has expertise in study management and data collection.  
Training in necessary computer applications, Human Subject Research, and 
HIPAA regulations is provided and documentation of this training is provided to 
the CTN Coordinating Center Manager.  CTN site staff cooperates in all data 
management activities with the CTN Coordinating Center Manager, including 
participation in monitoring and audit activities. 
 
4.2 
Equipment and Facilities 
 
CTN sites have equipment and facilities to conduct CTN site studies. These 
include: 
 
• 
Patient care facilities, equipment, and supplies 
• 
Required information systems 
• 
Equipment and facilities for processing and storage of study-related 
tissues, tissue extracts, and blood products 
• 
The capability to transport study-related tissues, tissue extracts, and 
blood products per study specifications 
• 
Dedicated fax machine for transmitting and receiving study-specific 
correspondence. 
 
The CTN site provides adequate facilities for secure storage of study documents 
and for study monitoring activities.  A designated monitoring area is provided that 
includes a workstation, copier, and fax machine.  The workstation is located in 
proximity to the workstations of the CTN site study-specific research nurse and 
data manager.  A secure facility, with restricted access, is provided for storing 
study drugs, specimens, and other study-specific materials.  Systems to assure 
maintenance of drug receipts and drug dispensing documents and to assure secure 
disposal of sensitive documents are in place.  
                                                         
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CLINICAL TRIALS NETWORK (CTN) 
STANDARD OPERATION PROCEDURES 
 
 
1.0 
PROCEDURES FOR INITIATING AND ACTIVATING CLINICAL 
TRIALS 
 
1.1  
Letter of Understanding 
 
An Initial Study Offering Document addressed to the Lead Investigator at a CTN 
site is developed by the CTN Director.  This document enables the CTN site to 
provide assurances that all responsibilities have been defined and that all 
necessary resources are available to conduct the proposed CTN site study.  The 
document includes a synopsis of the study, enrollment targets, and estimated 
funding.  The document is signed by the Lead Investigator at the CTN site and is 
on file at the CTN Coordinating Center before the study can be initiated at the 
CTN site. 
 
1.2   
Budget Development 
 
Designated personnel at the CTN site coordinate budget development and budget 
negotiations for the study with the CTN Coordinating Center Manager.  
Acceptable payment terms are also addressed by both parties in conjunction with 
any sub-contracting requirements. 
 
1.3   
Regulatory Procedures 
 
Prior to activation of a study at a CTN site, the following documents must be on 
file at the CTN Coordinating Center: 
 
• 
A signed Sub-Contract 
• 
A signed FDA Form 1572, with a current  signed and dated CV for each 
CTN site investigator listed on the form and a copy of the investigators’ 
current medical license 
• 
Current resume and contact information for the study-specific CTN site 
research nurse and study-specific data manager 
• 
Human Subjects Research Training certification for key CTN site study 
personnel 
• 
Documentation of HIPPA Compliance Training for key CTN site study 
personnel 
• 
A copy of all current CTN site laboratory certifications (CAP and / or 
CLIA) and current laboratory reference ranges for all tests specified in 
the study.  Current CV and license of the CTN site laboratory director, if 
listed on the FDA Form 1572 
• 
Name of and contact information for the CTN site’s IRB Chairperson, 
                                                         
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contact information and current CTN site IRB membership roster 
• 
Assurance Identification / CTN site IRB Certification 
• 
CTN site IRB approval of the study protocol and consent form 
• 
CTN site IRB acknowledgement of receipt of the study-specific 
Investigational Brochure 
• 
Any special study-specific approvals such as radiation licensing  
• 
Signed final CTN site budget / contracting documents 
• 
Yearly updates of the above information are required during the course 
of the CTN site study 
 
All documents are mailed to the CTN Coordinating Center: 
 
 
UAB Comprehensive Cancer Center 
 
Clinical Trials Network Coordinating Center 
 
c/o Pam Dixon, RN 
 
1802 6th Avenue South 
 
North Pavilion Room 2523 
 
Birmingham, Alabama 35294 
 
 
 
Tel: 205-975-5387 
 
Fax: 205-975-9875 
 
Email: pam.dixon@ccc.uab.edu 
 
 
CTN sites are responsible for the timely submission of safety reports, protocol 
amendments, protocol exceptions and deviations, ancillary study documentation 
(such as advertisement materials), and annual renewals to their CTN site IRB.  
Copies of CTN site IRB approvals are forwarded to the CTN Coordinating 
Center. 
 
1.4     Initiation  
 
An initiation is conducted the first time a CTN site participates in a CCC 
investigator-initiated study.  The initiation provides instruction and orientation 
and reviews the facilities and study staff to ensure that all study-specific 
requirements are satisfied.  CTN site personnel who must attend the initiation 
include the research nurse, data manager, study investigators, research 
pharmacist, and other study-specific personnel.  
 
During initiation, the CTN Coordinating Center Manager presents: 1)  receipt, 
review and filing of the Investigator’s Brochure; 2) maintaining up-to-date study-
specific protocols; 3) study objectives and design; 4) participant screening log  
and participant identification log; 5) clinical and laboratory evaluations and 
schedule of evaluations; 6) specimen collection, processing, storage, and 
shipping; 7) informed consent process and randomization procedures; 8) missed 
evaluations and protocol deviations and violations; 9) adverse event reporting 
guidelines, procedures and forms for adverse event reporting, and toxicity 
                                                         
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management; and 10) receipt, review, and filing of addendums to the 
Investigator’s Brochure.  Study specific data collection procedures are presented 
including: CRF guidelines; common errors and corrections; CRF update 
procedures; missed visits; data inquires; source documentation; record keeping; 
and disposition of CRFs.  Investigational agent procedures are reviewed 
including: dissemination of information to the CTN site research pharmacist; 
agent storage and accountability; pharmacy guidelines; and quality assurance.  A 
review of the CTN site Lead Investigator’s responsibility is carried out with the 
CTN site Lead Investigator.  The CTN site Regulatory Document Binder is 
reviewed including: CTN site IRB documentation, study-specific approval letter, 
study-specific informed consent, advertisements and participant information 
sheets when applicable, annual renewal, and roster.  Assurance number, 
laboratory certification, laboratory norms, documentation of submission of the 
Investigator’s Brochure, and signature logs are also reviewed.  
 
A CTN site may not enroll a patient on a study until all of the regulatory 
documents have been received and CTN site IRB documentation is reviewed and 
approved by the CTN Coordinating Center manager and UAB IRB. 
 
2.0 
PROCEDURES FOR CONDUCTING CLINICAL TRIALS  
 
All studies conducted at CTN sites must adhere to the study-specific protocol. 
 
2.1 
Patient Registration 
 
The CTN site maintains an accurate screening log for the study and forwards this 
to the CTN Coordinating Center Manager on a monthly basis. 
  
Once a study subject has been consented, screened and deemed eligible for study 
entry by the CTN site, a study-specific study subject eligibility checklist, a copy 
of the dated and signed consent form, and corresponding source documentation 
are faxed to the CTN Coordinating Center Manager for eligibility verification.  
Subsequently, a study-specific number is assigned to the study subject and sent to 
the CTN site.  Finally, a Patient Registration Form is completed and faxed by the 
CTN site to the CTN Coordinating Center Manager.   
 
Queries regarding data accuracy are forwarded from the CTN Coordinating 
Center Manager to the CTN site for clarification or correction.  Once queries are 
addressed by the CTN site, any corrected data forms or copies of corrected source 
documentation are faxed to the CTN Coordinating Center Manager. 
 
                                                         
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2.2 
Study Monitoring 
 
Each study subject is discussed at the weekly CCC Clinical Trials Monitoring 
Committee meeting by the CTN Coordinating Center Manager. 
 
All questions and concerns regarding the conduct of a study at a CTN site are 
directed to the site Lead Investigator who consults with the CTN Director and / or 
the CTN Coordinating Center Manager when necessary.  The P.I. (assisted by the 
CTN Coordinating Center Manager) is the primary contact for study-specific 
questions such as dose modifications, toxicities, and supportive care. The CTN 
Coordinating Center Manager is the primary contact for issues regarding patient 
registration, regulatory documents, completion of CRFs, data collection, and data 
submission.  
 
The CTN office will maintain 100% of the CRF’s with source documentation in 
the form of a shadow chart.  
 
Monitoring of all CTN site studies (25% of patients) will be  conducted “off-site” 
at the CTN Coordinating Center using the CRFs and supporting source documents 
that are transmitted from the CTN site on a monthly basis.   
 
 
Following each monitoring exercise, queries and/or requests for additional 
documentation are generated by the CTN Coordinating Center Manager generally 
within 2 weeks of receipt of the CTN site documents.  Subsequently, the CTN site 
reviews and responds to queries and implements the necessary corrective 
action(s).  Within two weeks, the CTN site submits query responses and, if 
appropriate, a written summary of corrective actions to the CTN Coordinating 
Center Manager.  When necessary, the CTN Coordinating Center Manager 
conducts “on-site” monitoring visits to provide staff education and to assist in 
implementing corrective action at the CTN site. 
 
2.3 
Auditing of CTN Site Studies by the CCC Quality Assurance 
Committee 
 
Audits of all CCC investigator-initiated studies are conducted on a yearly basis, 
within two to three months of the selected study’s UAB IRB renewal date.  Ten 
percent of the patient charts are audited.  Included in this audit are the CTN site 
“shadow charts” maintained at the CTN Coordinating Center.  Following the 
audit of the CTN site “shadow charts”, a summary of the audit findings are 
forwarded to the CTN site IRB, the UAB IRB, the CTN site Lead Investigator, 
the CTN Director and the CCC Associate Director for Clinical Research.  The 
CTN site Lead Investigator, in consultation with the CTN director, has 2 weeks to 
respond to any deficiencies prior to formal UAB IRB action concerning the audit 
findings. 
 
 
                                                         
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2.4 
Data Collection and Submission 
 
Data collection and submission is the responsibility of the CTN site Lead 
Investigator.  Data collection forms are provided by the CTN Coordinating Center 
Manager.  Each CTN site maintains a study-specific research data file (research 
chart) for study subjects enrolled into a study.  The research chart includes 
completed CRFs and copies of all source documentation.  Completed CRFs are 
reviewed, signed and dated by CTN site Lead Investigator.  Any deviations from 
the study protocol are documented in the study subject’s medical record and 
research chart.  Missing data is documented in the research chart.  Copies of the 
completed CRFs, other study-related documents, and source documents are faxed 
to the CTN Coordinating Center Manager on a monthly basis.   
 
Failure to submit the required documents in a timely fashion (within one week of 
the due date) results in a Letter of Notification.  Subsequent failure to comply can 
result in the following: 
 
• Suspension of CTN site study subject accrual until all delinquent data is 
submitted to the CTN Coordinating Center 
• Permanent suspension of study subject accrual to a CTN site 
• Other action determined by unique circumstances. 
 
2.5 
 Adverse Event Reporting 
 
In cancer clinical trials, an Adverse Event (AE) is any unfavorable physical 
sign, symptom, significant laboratory test abnormality or disease that is 
temporally associated with the use of a medical treatment, procedure or 
device.  All AEs must be recorded on study-specific CRFs.  AE information is 
collected from the initiation of the treatment, procedure or use of a study 
device and continues until the treatment, procedure or use of a study device 
is stopped and follow-up is completed.  Severity is graded according to 
Common Toxicity Criteria.  The CTN site Lead Investigator must evaluate 
the severity of each AE, assess causal relationships, determine the action to 
be taken and document the outcome.  The CTN site study-specific research 
nurse is responsible for documenting, filing, and reporting AEs on a weekly 
basis to the CTN Coordinating Center Manager, who in turns reports the AE 
at the next weekly CCC Clinical Trials Monitoring Committee meeting.   
 
A Serious Adverse Event (SAE) is an AE that 1) results in patient hospitalization 
or prolongation of hospitalization; 2) results in persistent or significant disability 
or incapacity; 3) results in death; 4) is a cancer or congenital abnormality or 5) 
results in the development of drug dependence or abuse.  An AE must be 
considered an SAE when the nature or severity of the event is not consistent with 
the current Investigator’s Brochure.  CTN site SAEs must be reported by the CTN 
site Lead Investigator to the CTN Coordinating Center Manager by email or by 
                                                         
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fax.  It is also the responsibility of the CTN site Lead Investigator to report SAEs 
to the CTN site IRB and to submit copies of that report to the CTN Coordinating 
Center Manager.  It is the CTN Coordinating Center Manager’s responsibility to 
report the SAE to the appropriate regulatory agency and / or industry sponsor.   
 
3.0 
PROCEDURES FOR STUDY CLOSURE  
 
3.1 
Closeout  
 
A closeout at the CTN site is conducted by the CTN Coordinating Center 
Manager when a CTN site study reaches its accrual goal or is closed prematurely.  
This includes close out and storage of study subject records and resolution of all 
outstanding regulatory, data management, and pharmacy issues.  The CTN site 
retains study documents pertaining to an investigational agent for two years when 
a marketing application is approved or, if a marketing application is not approved, 
for two years after shipment and delivery of the investigational drug is 
discontinued. 
 
3.2 
Manuscripts, Publications, Press Releases 
 
Any reports or manuscripts describing study results are reviewed by the CTN 
Director and circulated to contributing CTN sites for their review prior to any 
presentation, submission for publication, or press release.  The CTN Director, in 
consultation with the CCC Principal Investigator, determines the time and place 
of any presentation, submission for publication or press release and is the final 
arbiter of authorship.