Clinical trial NCT01990352
Conditions: Breast Cancer
Sponsor: University of Arizona
Start date: 2013-11-05; Completion date: 2022-12-31
Enrollment: 24 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Primary Objective: To evaluate if low BRCA1 protein expression has a preferential effect on response when metastatic breast cancer patients are treated with DNA damaging chemotherapy agent, compared to historical controls

Secondary Objective: To evaluate if low BRCA1 protein expression has a preferential effect on tumor progression when metastatic breast cancer patients are treated with DNA damaging chemotherapy agent, compared to historical controls

Detailed description:
DNA is constantly subject to damage from exposures including drugs (including chemotherapy), sunlight and aging. Certain genes in the human body help repair this damage (i.e. BRCA1 and other genes). The purpose of this study is to find out if BRCA1 protein expression has a role in response to chemotherapy drugs.

Arms: arm 1: The enrolled patients will be treated with pegylated liposomal doxorubicin at 30mg/m2 every 21 days.
Interventions: intervention 1: Pegylated liposomal doxorubicin
Intervention descriptions: intervention 1: Doxil will be administered intravenously at 30mg/m2 on days 1 of a 21 day cycle.

Protocol documents:
Prot_SAP_000.pdf: 
Revision 3, February 15, 2015 
Page 1 
 
The University of Arizona Cancer Center 
Hematology Oncology Section 
Breast Cancer Program 
 
 
 
 
A single arm Phase II trial to assess association of BRCA1 protein expression with 
overall response rate in patients with metastatic breast cancer on pegylated liposomal 
doxorubicin 
 
Original Protocol Date: 9 August 2013 
Revision 1 date: August 16, 2013 
Revision 2 date: March 24, 2014 
Revision 3 date: February 15, 2015 
 
 
 
 
 
 
Principal investigator: 
 
Pavani Chalasani, M.D, MPH 
Assistant Professor Medicine 
The University of Arizona Cancer Center 
1515 N. Campbell Avenue, PO Box 245024 
Tucson, AZ 85724-5024 
P- (520) 626-0191 
F- (520) 626-2225 
pchalasani@uacc.arizona.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Revision 3, February 15, 2015 
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Title of Study: A single arm Phase II trial to assess association of BRCA1 protein expression 
with overall response rate in patients with metastatic breast cancer on pegylated liposomal 
doxorubicin 
 
Primary Objective: To evaluate if low BRCA1 protein expression has a preferential effect on 
response when metastatic breast cancer patients are treated with a DNA damaging 
chemotherapy agent.  
 
Secondary Objective: To evaluate if low BRCA1 protein expression has a preferential effect on 
tumor progression when metastatic breast cancer patients are treated with a DNA damaging 
chemotherapy agent.  
 
Primary Endpoint: To correlate BRCA1 protein expression, as measured by IHC with overall 
response rate for metastatic breast cancer patients treated with pegylated liposomal 
doxorubicin. The overall response rate is defined as the percentage of evaluable patients who 
achieve complete response (CR) or partial response (PR) as measured by RECIST 1.1 on CT 
or PET/CT as the best overall response 
 
Secondary Endpoint: To correlate BRCA1 protein expression, as measured by IHC with 
median progression free survival for metastatic breast cancer patients treated with pegylated 
liposomal doxorubicin. The progression free survival is  measured as the times from the start of 
pegylated liposomal doxorubicin to the time the patient is first recorded as having disease 
progression or die 
 
Background information:  
It is well known that women with germline mutations in BRCA1 or BRCA2 are at 
increased risk for developing various malignancies1. Defective homologous recombination, 
which occurs as a result of these mutations, impairs the normal repair mechanism for DNA 
double stranded breaks (DSB)2. Alternative error-prone, potentially mutagenic DNA repair 
mechanisms like non-homologous end joining (NHEJ) and single stranded annealing (SSA) take 
over and lead to genomic instability3. This “homologous recombination deficiency” (HRD) may 
be a critical pathway in development of cancers, and also make the affected cells more 
susceptible to DNA damaging drugs like alkylators, anthracyclines and platinum agents3. 
Somatic mutations in BRCA1 and BRCA2 do not occur frequently in sporadic breast cancer4 but 
potentially any (somatic) inactivation of the genes could result in loss of their phenotypic 
expression5. A phenomenon called “BRCAness” has been reported in sporadic cancers that do 
not have the germline mutations in BRCA1 or BRCA2, but display similar inactivation of the 
BRCA related genes and defective HRD.  The reported incidence in the literature ranges from 
15% to 50%6, 7 . The incidence varies with the histology, with higher percentage of triple 
negative tumors expressing “BRCAness” signature (30-60%)8 compared to hormone receptor 
positive tumors (5-20%)6, 7. Detecting this underlying tumor cell defects in effective DNA repair 
could provide crucial information in attempting to “personalize” treatment for breast cancer 
patients, directing DNA targeted therapy to such patients.  
There have been published studies assessing HRD (primarily involving BRCA1 and 
BRCA2) in breast cancer and associating it with response to neoadjuvant chemotherapy. 
Rodriguez et al reported higher sensitivity to anthracycline-based therapies in patients whose 
tissue samples had a defective DNA repair gene signature9. In a study with similar design by 
Lips et al10, about half of triple negative tumors were found to have BRCA1 inactivation but it 
was only modestly predictive of response. However, in ER positive tumors a BRCA2 like-aCGH 


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was found in a surprising proportion of this neoadjuvant population (43%) and was strongly 
predictive of excellent response to chemotherapy. Vollenbergh et al reported association of 
BRCA1 signature and benefit from high dose platinum-based chemotherapy11. They reported 
improved recurrence free survival and overall survival for patients who had BRCA1 like aCGH 
when they received high-dose platinum treatment.  Currently, there is no standard assay to 
detect HRD. Some early efforts have been reported in the literature3, 13, 14, 15 including array 
comparative genomic hybridization (aCGH), RT-PCR, multiplex ligation-dependent probe 
amplification and immunohistochemistry (IHC). The most frequently used and reported assay is 
DNA based aCGH to assess copy number variation of BRCA1 or BRCA2 or both. All the 
published studies have defined “BRCA1 like signature” independently and currently, there is no 
standardized definition. Moreover, DNA or RNA based approaches require fresh frozen tissue 
for optimal results, expertise in genomic analysis and interpretation (which is not readily 
available in non-academic setting) and time for tissue analysis and interpretation. To date, those 
techniques are not yet ready for utilization on a day to day basis where decisions regarding 
treatment are time-sensitive. IHC can be rapidly translated into current clinical practice due to 
the ability to use FFPE tissue which is readily available in all academic and community 
practices. Furthermore, after an IHC method has been validated it can be replicated in standard 
clinical testing laboratories and easily added to the standard breast diagnostic IHC panel (ER, 
PR, HER2 and Ki 67). 
         In collaboration with the Tissue Core and Cellular/Molecular Analysis core (TACMASS) at 
the University of Arizona Cancer Center we have validated a BRCA1 IHC assay, which can 
detect BRCA1 protein expression in breast tumors samples. Pegylated liposomal doxorubicin is 
frequently used in the salvage treatment of patients with metastatic breast cancer16. It is a DNA 
damaging chemotherapy agent which has been reported to have a 25% overall response rate 
and 2.5 months median progression free survival in the salvage treatment of metastatic breast 
cancer17. It has been shown to be safe in patients who have received prior anthracycline 
treatment.  
          As far as we are aware, there have not been any studies or reports evaluating BRCA1 
expression prospectively in the context of relative benefit from DNA damaging agents. We 
propose such a trial in which patients with metastatic breast cancer who will receive pegylated 
liposomal doxorubicin as a salvage treatment.  The goal is to discern whether there appear to 
be effects on relative benefit from this agent, which can be determined by assaying BRCA1 
protein expression on tumor specimens available from the patient. 
 
Study Design: This study is a prospective single arm Phase II trial to assess the association of 
BRCA1 protein expression with overall response rate in patients with metastatic breast cancer 
treated with pegylated liposomal doxorubicin 
 
Study Centers:  University of Arizona Cancer Center, University of New Mexico Cancer Center 
 
Number of Patients: We plan to consent 50 to 60 patients with the goal of accruing a total of 
50 patients into this prospective single arm, Phase II trial.  
 
Main Criteria for Inclusion/Exclusion:  
To be eligible for inclusion, each patient must have: 
• 
Metastatic breast cancer and have formalin-fixed, paraffin–embedded tumor 
available for testing BRCA1 protein expression 
• 
Adults over 18 years of age  
• 
Have resolution of all acute toxic effects of any prior chemotherapy or radiotherapy 
to NCI CTC grade ≤ 1 prior to study registration. 


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• 
Be informed of the investigational nature of this study and provide written informed 
consent in accordance with institutional and federal guidelines prior to study 
specific screening procedures 
• 
Be willing and able to comply with the treatment plan, scheduled clinic visits, 
laboratory and oncological tests and other study procedures 
• 
Have a ECOG performance status of 0 – 2 
• 
Measurable disease by CT by RECIST 1.1 to evaluate response. Patients with 
bone only lesions are also eligible to enroll. Criteria to monitor for response will be 
based on non-target lesions per RECIST criteria 
• 
Adequate bone marrow function defined as platelets ≥ 100 X 109 cells/L, 
neutrophils ≥ 1.5 x 109 cells/L, white blood cells (WBC) ≥ 3.0 x 109 cells/L and a 
hemoglobin ≥ 90 gm/L 
• 
Liver function tests (AST and/ or ALT) should be ≤ 2 x upper limit of normal (ULN, 
defined as per laboratory where blood testing is done), total biirubin ≤ 1.5 x ULN 
(except for patients with liver metastases, ALT and/or ALT ≤ 5 times the upper limit 
of normal is accepted) 
• 
Patients who are already on pegylated liposomal doxorubicin will be eligible to 
participate in this study as long as they meet the other inclusion/exclusion criteria 
and can sign informed consent  
 
Exclusion CriteriaThe following will exclude patients from study participationMyocardial 
infarction within 6 months of registrationBrain metastases unless documented to be 
controlled post completion of local therapy (surgery and/or radiation therapy) for at least 
four weeks prior to registration Pregnant or breast feeding women. Women with child 
bearing potential must use effective measures to prevent pregnancy while receiving 
pegylated liposomal doxorubicin Have a concurrent active non-breast malignancy except 
for non-melanoma skin cancerHave a concurrent active non-breast malignancy except 
for non-melanoma skin cancerHer2 positive tumors as defined by FDA guidelines (3+ 
immunohistochemical staining, defined as uniform, intense membrane staining of more 
than 10% of invasive tumor cells, and for cases with 2+ staining showing gene 
amplification by FISH, expressed as a ratio of more than 2 when comparing HER-2 gene 
and chromosome 17 fluorescent signals)18,19 
 
Intervention: Patients with metastatic breast cancer treated at the University of Arizona Cancer 
Center and University of New Mexico Cancer center will be enrolled in this trial. Patients whose 
treatment for their tumor includes liposomal doxorubicin are eligible to take part in this study. 
Their primary tumors will be obtained for evaluation of BRCA1 protein expression. When 
primary tumor is not available, paraffin embedded metastatic tumor will be tested for BRCA1 
protein expression.  Patients will receive pegylated liposomal doxorubicin intravenously at 
30mg/m2 every 21 days18.  
 
Duration of Intervention: Patients will get a baseline PET/CT or CT chest/abdomen/pelvis 
scan prior to receiving pegylated liposomal doxorubicin. We will repeat the same imaging scan 
at 9-12 weeks to evaluate response by RECIST 1.1. Pegylated liposomal doxorubicin will be 
stopped if there is evidence of disease progression. Also, it will be discontinued if there is 
development of persistent ≥ grade 3 fatigue or non-reversible ≥ grade 4 hematological or renal 
or liver abnormalities or if the patient chooses. 
 
 
 


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Schema 
 
Statistical Methods: This study is a prospective single arm Phase II trial to assess the 
association of BRCA1 protein expression to overall response rate when metastatic breast 
cancer patients are treated with pegylated liposomal doxorubicin. We plan to consent 50 to 60 
patients with the goal of enrolling a total of 50 patients to this study.  It is anticipated that 30% of 
patients will have low BRCA1 protein expression and therefore anticipate approximately a 35 to 
15 patient split for intact BRCA1 to low BRCA1 protein expression. 
 
Definition of primary endpoint: Response rate is defined as the percentage of evaluable 
patients who achieve complete response (CR) or partial response (PR) as measured by 
RECIST 1.1 on CT or PET/CT as the best overall response.  
 
Definition of secondary endpoints: Progression free survival will be measured as the times 
from the start of pegylated liposomal doxorubicin to the time the patient is first recorded as 
having disease progression or dies. If a patient does not progress or die while being followed via 
tumor assessment, progression-free survival will be censored at the time of last disease 
assessment.  
 
Sample size determination 
 
Low BRCA1 protein expression:  Since it is expected only 30% of patients will have low 
BRCA1 protein expression, we will accrue 15 patients to this arm and estimate the response 
rate among these patients. With a sample size of 15 we will be able to estimate the response  
rate of 50% in patients with low BRCA1 protein expression with a 95% confidence interval width 
of ± 25%.  
  
Normal BRCA1 protein expression:  A Simon two-stage design will be used in which a 10% 
response rate is considered not promising, a 30% response rate is considered promising, and 
the probabilities of a type I error (falsely accepting a non-promising therapy) and type II error 


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(falsely rejecting a promising therapy) are set at 0.10 and 0.10, respectively20.  In this scenario, 
the maximum trial size would be 35 patients.  In the first stage of this design, 12 patients with 
intact BRCA1 expression will be accrued. If at least two responses are observed among these 
12 patients, then an additional 23 patients will be accrued to the second stage.  If 0 or 1 
response is observed among the initial 12 patients, then accrual to this cohort will be terminated 
and declared negative.  At the end of the study if 6 or more patients in this cohort respond then 
pegylated liposomal doxorubicin will be considered worthy of further study in this cohort. This 
design yields at least 90% probability of a positive result if the true response rate is at least 30% 
and a 90% probability of a negative result if the true response rate is 10%.   
 
Analytic plan for primary objective:  
 
Low BRCA1 protein expression:  The number and percentage of patients falling into response 
category will be tabulated. The estimate of the response rate will be presented with 2-sided 95% 
exact binomial confidence intervals. We anticipate this data to be hypothesis generating and 
will form the basis for a future prospective clinical trial enrolling larger number patients to 
evaluate if low BRCA1 expression, as determine by IHC, is predictive of response to treatment 
with DNA damaging agents. 
 
Normal BRCA1 protein expression:  The estimate of the response rate will be presented with 
2-sided 95% exact binomial confidence intervals. If 5 or fewer patients with response are 
observed among the 35 patients, then there will be no further investigation of the treatment. If 6 
responses are observed out of 35 patients, there is 80% confidence that the true response rate 
>11.2%.   
  
Analytic plan for secondary objective:  
The progression free survival in patients with low BRCA1 protein expression and intact BRCA1 
protein expression will be analyzed by Kaplan-Meier methodology. The results will be 
summarized by 25th, 50th  (median), and 75th percentiles with associated 2-sided 95% 
confidence intervals, 
 
Interim Analysis 
 
Low BRCA1 protein expression: No interim analysis will be conducted 
 
Normal BRCA1 protein expression: An interim analysis for futility will be conducted in this 
group. The response rate will be used as the endpoint for the interim analysis for this group. The 
interim analysis will be performed when the first 12 treated patients are accrued. Based on 
Simon’s 2-stage design, if less than 2 responders are observed out of these initial 12 evaluable 
patients in normal BRCA1 protein expression, patient enrollment will be discontinued into this 
arm. Otherwise patient enrollment will continue until a total of 35 patients evaluable for response 
are enrolled. If only 1 response is observed out of 12 patients, there is 80% confidence that the 
true response rate <22.1%.   
 
Feasibility Issues:  Patients with metastatic breast cancer (exclude Her-2/neu patients) whose 
primary tumor tissue is available for assessing defective DNA repair will be identified by the 
medical oncologists at the University of Arizona Cancer Center  and the University of New 
Mexico to be included and treated with pegylated liposomal doxorubicin in the salvage setting. 
During the past year we have treated more than 25 patients with metastatic breast cancer with 
liposomal doxorubicin. We anticipate to complete accrual of 50 patients in 2 years. If we do not 


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accrue as expected after year 1, we plan to expand enrollment to other institutions we are 
currently collaborating (University of Washington and University of New Mexico). 
 
 
 
TABLE OF CONTENTS 
 
                                                                                                                                                          Page 
Protocol Synopsis 
 
2 
1. Introduction 
1.1. 
Breast Cancer 
1.2. 
Background 
1.3. 
Study Population 
 
9 
2. Trial Objectives 
 
12 
3. Trial Design 
3.1. 
Study Endpoints 
3.2. 
Study Design 
3.3. 
Study Duration and Follow up 
 
12 
4. Selection and Withdrawal of Patients 
4.1. 
Inclusion Criteria 
4.2. 
Exclusion Criteria 
4.3. 
Study Exit 
4.4. 
Study Patient Identification 
 
13 
5. Study Procedures 
5.1. 
Pretreatment 
5.2. 
Patient Registration 
5.3. 
Treatment Phase 
5.4. 
Follow Up 
 
15 
6. Criteria for Evaluation 
6.1. 
Evaluation Criteria Definitions 
 
16 
7. Study Drug Information 
 
17 
8. Study Statistics 
8.1. 
Statistical Methods 
8.2. 
Analytic Plan for Primary Objective 
8.3. 
Analytic Plan for Secondary Objective 
 
20 
9. Data and Safety Monitoring Plan 
 
21 
10.  Data Submission Schedule 
 
24 
11. Special Instructions 
 
24 


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12. Ethics 
 
25 
13. Bibliography 
 
26 
14. Appendices 
 
28 
 
List of Appendices 
 
Appendix 1 
Eastern cooperative oncology Group (ECOG) Performance Status 
Appendix 2 
Response evaluation 
Appendix 3 
Investigator’s statement 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1. Introduction 
 
1.1. Breast Cancer    
 
Breast cancer is a heterogeneous disease with different clinico-pathological features, 
responses to treatment and prognoses. Progress has been made in the development of 
targeted therapies (for example HER-2/neu targeting monoclonal antibodies and small 
molecule inhibitors) that have made a substantial improvement in both response rates 
and survival. Despite these  clinical advances there are still 40,000 women who die in 
the US of breast cancer each year.1 Therefore, there is a continuing need to search for 
additional therapeutic strategies. All patients with metastatic breast cancer eventually 
receive treatment with chemotherapy. In the available menu of chemotherapy agents, 
clinicians frequently select therapy based on the patient’s treatment history, prior 
response rates to similar classes of drugs, toxicity profiles, patient preference, etc. 
Currently, there is no proven clinical predictive marker for selecting the most efficacious 
chemotherapy agent outside the setting of the targeted therapies i.e. HER2 or ER 
targeted antitumor agents.  Better methods for choosing the most efficacious treatment 
would spare patients undue toxicity and lead to improved response rates. 
 
1.2. Background  
 
It is established that women with germline mutations in breast cancer 1, early onset 
gene (BRCA1) or breast cancer 2, early onset gene (BRCA2) are at increased risk for 
developing breast and ovarian cancers.2 In addition, there is a higher risk for pancreatic, 
prostate and male breast cancer.2 This is thought to be related to the roles BRCA1 and 
BRCA2 genes play in DNA repair. DNA damage activates cell cycle check points and 
recruitment of DNA repair machinery. In cells deficient in the BRCA1 or BRCA2 genes 
there is defective DNA repair of double-stand DNA breaks (DSB) through homologous 
recombination (HR), a conservative DNA repair mechanism with a high degree of 
fidelity. Alternative error-prone, potentially mutagenic DNA repair mechanisms like non-
homologous end joining (NHEJ) and single stranded annealing (SSA) compensate for 
this loss, but lead to genomic instability.3 The relative roles of BRCA1 and BRCA2 in 
repair of DNA DSB have been explored and better defined over the past two decades. 
BRCA1 is a critical organizing molecule that has been linked to a range of cellular 
processes beyond DNA repair, including transcriptional regulation and chromatin 
remodeling. BRCA2 function in HR is primarily via regulation of RAD51 activity.4 BRCA2 
regulates RAD51 recombinase, which is a critical step in strand invasion and homology 
directed repair.4  
 
Germline mutation in one BRCA1or BRCA2 allele is sufficient to predispose for cancer 
development.5 There is a loss of heterozygosity, with loss of the normal allele while 
retaining the mutant allele, in the tumor tissue of susceptible individuals, suggesting that 
the genes may have a role as tumor suppressors.6,7 Somatic mutations in BRCA1 or 
BRCA2 do not occur frequently in sporadic (or non-familial) breast cancer8 but 
potentially any (somatic) inactivation of the genes could result in phenotypic 
suppression of BRCA, 9 a phenomenon called “BRCAness” (or more properly 
“BRCAlessness”).  This phenomenon has been reported in sporadic cancers which do 
not have germline mutations in BRCA1 or BRCA2, but display similar inactivation of the 
BRCA related genes and consequently have defective HR.3  
 
Preferential effect of chemotherapy in relation to BRCA1 or BRCA2 expression 


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The key roles BRCA1 and BRCA2 genes play in DNA repair are through HR, thus cells 
deficient in these proteins could be more sensitive to chemotherapy agents which 
produce DNA damage. DNA injury can be induced by strand breaks through failure to 
reseal cleavable complexes in strand passage or by intercalation with base pairs (i.e. 
anthracyclines) or by DNA adduct formation (i.e. alkylators and platinating agents) with 
subsequent intra- or interstrand DNA crosslinks and resultant double strand breaks.10 
Most published clinical studies assessing homologous recombinant deficiency (HRD) 
involve BRCA1 signatures and are retrospective. 
 
Rodriguez et al reported higher sensitivity to anthracycline-based therapies in patients 
whose tumor samples had a defective DNA repair gene signature (determined by 
quantitative RT-PCR in a 69 gene low density array).11 In a study with similar design by 
Lips et al, approximately 50% of triple negative tumors were found to have BRCA1 
inactivation (determined by aCGH, mRNA levels, or BRCA1 promoter methylation) but it 
was only modestly predictive of response.12 Vollenbergh et al reported association of 
BRCA1 like signature (determined by aCGH) to be associated with a higher recurrence 
free survival and overall survival after high dose platinum-based chemotherapy.13 One 
reported prospective trial of 28 patients reported higher pathological complete 
responses after neoadjuvant cisplatin, in patients with BRCA1 mutations, low BRCA1 
mRNA levels, or BRCA1-promoter methylation.14  
 
DNA damaging chemotherapy can potentially influence expression of BRCA1 protein in 
treated tumor cells as a potential mechanism of resistance. There is currently only 1 
report in the literature assessing change in expression of BRCA1 in primary and 
metastatic tumors.21 This was assessed in 9 BRCA1 germline mutation patients with 
ovarian tumors who were treated with a platinum-containing chemotherapy regimen. 
Their primary and metastatic tumor specimens were evaluated for BRCA1 mutations by 
DNA sequencing and BRCA1 protein expression by IHC. Four of the specimens 
showed reversal of mutations by DNA sequencing however clinical implication of that 
change was unclear. In addition this data only included patients with germline mutations 
and not in sporadic tumors which have an acquired loss of BRCA1. Currently, there is 
no data suggesting that acquired BRCA1 loss can be reversed with treatment. We 
propose to determine if reversal of BRCA1 protein expression is observed after DNA 
damaging chemotherapy as a secondary aim. 
 
Detecting “BRCAness” 
 
One area of controversy is the best method for detecting “BRCAness” in tumor 
samples.  Several efforts to identify a “BRCAness” signature have been reported using 
different methodologiesincluding array comparative genomic hybridization (aCGH), 
quantitative real time- polymerase chain reaction (RT-PCR), multiplex ligation-
dependent probe amplification (MPLA) and immunohistochemistry (IHC).13-
15Techniques have been reported using frozen or formalin-fixed tumor tissue. As all the 
published studies have defined a “BRCA1 like signature” independently there is 
currently no standardized definition. Moreover, DNA or RNA based approaches require 
fresh frozen tissue for optimal results, expertise in those fields for interpretation (which 
is not readily available in non-academic setting) and significant time for tissue analysis 
and interpretation. Consequently, none of these methodologies are readily conducive 
for utilization by the clinician who is required to make time-sensitive treatment 
decisions. IHC can be rapidly translated into current clinical practice as both community 


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and academic pathologists utilize IHC on a routine basis from FFPE tissue and are 
competent in performing the assays and interpreting the results. Furthermore, IHC 
methods can be readily validated and replicated in standard clinical laboratories and 
added to the standard breast cancer diagnostic IHC panel (ER, PR, HER2, and Ki 67).   
 
Preliminary data: 
 
In collaboration with the Tissue Core and Cellular/Molecular Analysis core (TACMASS) 
at the University of Arizona Cancer Center (UACC) we have validated a BRCA1 IHC 
assay in breast tumor samples. Antigen retrieval techniques and antibody titers have 
been optimized using the Genscript A00490 Rb polyclonal antibody. We retrospectively 
analyzed 45 breast tumor samples, 37 of which were triple negative tumors. Low 
BRCA1 protein expression was found in 43% of the triple negative tumors. This result is 
in concordance with other reported series of BRCA1 signatures using DNA and/or RNA 
based approaches. Clinical information was available in 37 patients. A total of 21 
patients received anthracycline based chemotherapy of which 13 had intact BRCA1 and 
8 had low BRCA1 protein expression. Fewer breast cancer recurrences were seen in 
patients with low BRCA1 protein expression (2/8) compared to those with intact BRCA1 
protein expression (6/13). However, in this small sample of assessed tumors, 
chemotherapy was administered at different time points (neoadjuvantly or adjuvantly) 
and follow up was not standardized in all patients. Nevertheless, these results support 
our hypothesis that low BRCA1 protein expression predicts increased sensitivity to 
treatment with DNA damaging chemotherapy.  
 
DNA damage leads to the early activation of BRCA1 where it plays a vital role in the 
homologous recombination (HR) repair of double stranded DNA.  Unlike other 
mechanisms of DNA repair, BRCA1 mediated HR tends to be conservative and error 
free. Thus cells deficient in BRCA1 protein are uniquely susceptible to 
agents/mechanisms which cause DNA damage due to their inability to repair through 
HR.  We propose to test the clinical utility of BRCA quantification by 
immunohistochemical (IHC) analysis for predicting response to chemotherapy in the 
prospective setting. Our hypothesis is that low BRCA1 protein expression in primary 
breast tumors of metastatic breast cancer patients predicts improved response to DNA 
damaging chemotherapy in the metastatic setting. 
 
Methodology: 
 
IHC staining for BRCA1: Tumor samples will be reviewed to confirm adequate sample 
for staining. Tissues sections will be stained for BRCA1 by Genscript A00490 Rb 
polyclonal clone antibody using the Benchmark Ultra automated stainer (Ventana 
Medical Systems Inc, Tucson, Arizona).  Low BRCA1 protein expression will be defined 
as ≤5% of cells staining with 1+ intensity of the tumor nucleus (i.e. nuclear long score ≤ 
5)16. Intact BRCA1 protein expression will be defined as ≥10% of cells staining with 1+ 
intensity or ≥5% of cells staining with 2+ intensity of their nucleus (i.e. nuclear long 
score of ≥10). Nuclear long score of 6-9 i.e. 5-10% of cells staining with 1+ or <5% of 
cells stain with 2+ intensity, will be defined as indeterminate status. 
 
Quantitation of IHC: Two Board Certified pathologists (Raymond B. Nagle,MD, PhD 
and Lauren Lebeau, MD) will evaluate each case using a semi quantitative histologic 
scoring method as previously described.19 Briefly, staining intensity for neoplastic cell’s 
nucleus will be scored as:  0 negative, 1 weak, 2 moderate and 3 intense.  In addition, 


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the percentage of positive neoplastic cells will be evaluated.  The overall scores will be 
calculated by multiplying the intensity by the corresponding percentage of positive cells, 
resulting in a values ranging from 0 to 300. Low BRCA1 protein expression will be 
defined as nuclear long score of ≤ 5, intact BRCA1 as nuclear long score of ≥ 10, 
indeterminate BRCA1 expression as nuclear long score of 6-9. 
 
Chemotherapy agent: Pegylated liposomal doxorubicin is frequently used in the 
salvage treatment of patients with metastatic breast cancer. It is a DNA damaging 
chemotherapy agent which has been reported to have a 25% overall response rate and 
2.5 months median progression free survival in the salvage treatment of metastatic 
breast cancer16. It has been shown to be safe in patients who have received prior 
anthracycline treatment17.  
 
 
1.3. Study Population 
This study will enroll patients with HER2 negative metastatic breast cancer whose 
treatment includes liposomal doxorubicin.. The enrolled patients will be treated with 
pegylated liposomal doxorubicin at 30mg/m2 every 21 days 
 
2. Trial Objectives 
 
Primary Objective: To evaluate if low BRCA1 protein expression has a preferential effect 
on response when metastatic breast cancer patients are treated with DNA damaging 
chemotherapy agent. 
 
Secondary Objective: To evaluate if low BRCA1 protein expression has a preferential 
effect on tumor progression when metastatic breast cancer patients are treated with DNA 
damaging chemotherapy agent. 
 
 
3. Trial Design 
 
3.1. Study Endpoints 
 
Primary Endpoint: To correlate BRCA1 protein expression, as measured by IHC with 
overall response rate for metastatic breast cancer patients treated with pegylated 
liposomal doxorubicin. . The overall response rate is defined as the percentage of 
evaluable patients who achieve complete response (CR) or partial response (PR) as 
measured by RECIST 1.1 on CT or PET/CT as the best overall response 
 
Secondary Endpoint: To correlate BRCA1 protein expression, as measured by IHC 
with median progression free survival for metastatic breast cancer patients treated with 
pegylated liposomal doxorubicin. The  progression free survival is  measured as the 
times from the start of pegylated liposomal doxorubicin to the time the patient is first 
recorded as having disease progression or dies. 
 
3.2. Study Design This study is a prospective single arm Phase II trial to assess the 
association of BRCA1 protein expression with overall response rate in patients with 
HER2 negative metastatic breast cancer treated with pegylated liposomal doxorubicin. 
Patients who qualify for enrollment into the study will receive pegylated liposomal 
doxorubicin as a single agent intravenous at 30mg/m2 every 21 days 


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3.3. Study Duration and Follow up 
 
All patients will be treated with pegylated liposomal doxorubicin until evidence of tumor 
progression. Patients will get a baseline CT or PET/CT scan prior to receiving pegylated 
liposomal doxorubicin. Repeat imaging at week 9 -12 (± 7 days) will be performed to 
evaluate response. Subsequent imaging schedule will be left to the treating physician’s 
discretion. All patients will be treated until documented disease progression, intolerable 
toxicity or withdrawal of consent. 
 
4. Selection and Withdrawal of Patients 
 
4.1. Inclusion Criteria:  
To be eligible for inclusion, each patient must have: 
4.1.1 Metastatic breast cancer and have formalin-fixed, paraffin–embedded tumor 
available for testing BRCA1 protein expression 
4.1.2 Adults over 18 years of age  
4.1.3 Have resolution of all acute toxic effects of any prior chemotherapy or 
radiotherapy to NCI CTC grade ≤ 1 prior to study registration. 
4.1.4 Be informed of the investigational nature of this study and provide written 
informed consent in accordance with institutional and federal guidelines prior to 
study specific screening procedures 
4.1.5 Be willing and able to comply with the treatment plan, scheduled clinic visits, 
laboratory and oncological tests and other study procedures 
4.1.6 Have a ECOG performance status of 0 – 2 
4.1.7 Measurable disease by CT by RECIST 1.1 to evaluate response. Patients with 
bone only lesions are also eligible to enroll. Criteria to monitor for response will 
be based on non-target lesions per RECIST criteria 
4.1.8 Adequate bone marrow function defined as platelets ≥ 100 X 109 cells/L, 
neutrophils ≥ 1.5 x 109 cells/L, white blood cells (WBC) ≥ 3.0 x 109 cells/L and a 
hemoglobin ≥ 90 gm/L 
4.1.9 Liver function tests (AST and/ or ALT) should be ≤ 2 x upper limit of normal 
(ULN, defined as per laboratory where blood testing is done), total bilirubin ≤ 1.5 
x ULN (except for patients with liver metastases, ALT and/or ALT ≤ 5 times the 
upper limit of normal is accepted) 
4.1.10 Patients who are already on pegylated liposomal doxorubicin will be eligible to 
participate in this study as long as they meet the other inclusion/exclusion criteria 
and can sign informed consent  
 
4.2. Exclusion Criteria 
The following will exclude patients from study participation: 
4.2.1. Myocardial infarction within 6 months of registration 
4.2.2. Brain metastases unless documented to be controlled post completion of local 
therapy (surgery and/or radiation therapy) for at least four weeks prior to 
registration 
4.2.3. Pregnant or breast feeding women. Women with child bearing potential must use 
effective measures to prevent pregnancy while receiving pegylated liposomal 
doxorubicin  
4.2.4. Have a concurrent active non-breast malignancy except for non-melanoma skin 
cancer 


Revision 3, February 15, 2015 
Page 14 
 
4.2.5. Her2 positive tumors as defined by FDA guidelines(3+ immunohistochemical 
staining, defined as uniform, intense membrane staining of more than 10% of 
invasive tumor cells, and for cases with 2+ staining showing gene amplification by 
FISH, expressed as a ratio of more than 2 when comparing HER-2 gene and 
chromosome 17 fluorescent signals)18,19 
 
 
4.3. Study Exit 
 
Patients will continue with study visits until study exit. Patients will exit the study at 
tumor progression or withdrawal. Each patient has the right to withdraw from the 
study at any time without prejudice. Should a patient withdraw from the study prior to 
disease progression, the reason(s) must be stated on the case report form. All 
procedures (vital signs, laboratory evaluations, physical examination, tumor 
response, adverse events and concomitant medications) should be performed at the 
study exit visit. All enrolled patients will be followed for 30 days after week 9 visit. 
 
Patients may be withdrawn from the study early due to:  
a. Development of toxicity which in the Investigator’s judgment precludes 
further study participation  
b. Significant protocol violations or noncompliance on the part of the patient 
or Investigator  
c. Discontinuation, in the judgment of the Investigator, is in the patient’s best 
interest  
d. The patient is beginning another treatment  
e. Refusal of the patient to continue treatment or follow-up  
f. 
Loss to follow-up  
g. Pregnancy  
 
4.4. Patient Registration 
 
Data for patients enrolled on interventional trials must be entered per current 
practices into the Oncore electronic clinical trials management system.  
 
University of Arizona Cancer Center 
 
Patients must be registered prior to initiation of treatment. Patients will be registered 
through a Breast Team Clinical Research Coordinator (CRC) from 8:00 a.m. to 5:00 
p.m., Mountain Standard Time, Monday through Friday, (excluding holidays) 
 
University of New Mexico Cancer Center 
 
Patients must be registered prior to initiation of treatment. Prior to registration, 
eligibility criteria must be confirmed with the UNM CC Clinical Research Office. If the 
patient meets the eligibility criteria he/she will be assigned a unique patient study 
number by the Clinical Research Office as further detailed below. 
The patient will be registered in the Oncore System using the study number. 
 
4.5. Study Patient Identification 
 


Revision 3, February 15, 2015 
Page 15 
 
Patients who have been consented and are undergoing study screening will be 
identified on study-related documentation and forms by their initials (first/middle and 
last name initials). All patients treated on this trial will be identified by their study 
initials and a unique study identification number. A unique study number will only be 
assigned to patients who meet the eligibility requirements and have completed the 
screening visit and are registered for treatment. The unique number will begin with 
the following prefix: BD. The prefix will be followed by the patient identification 
number beginning with # 001 in each cohort. These numbers will be issued to 
patients sequentially and no patient identification numbers will be re-assigned in the 
event that the subject withdraws from protocol treatment. 
 
Patients enrolled at University of New Mexico Cancer Center will be identified by 
prefix NM to help identify patients enrolled at that site. The prefix will be followed by 
the patient identification number beginning with # 001 in each cohort. These 
numbers will be issued to patients sequentially and no patient identification numbers 
will be re-assigned in the event that the subject withdraws from protocol treatment 
 
5. Study Procedures 
 
5.1. Pretreatment 
 
Patients will be consented and evaluated for participation based on the following 
procedures which should be performed within 28 days of registration  
 
• Physical exam  
• Complete blood count with absolute neutrophil count and differential 
• Liver function panel to include total bilirubin 
• Serum Creatinine 
• Staging studies by the same method that will be used throughout the study (per 
treating physician discretion): 
• Such as CT of the chest, abdomen and pelvis or 
• PET/CT or 
• Bone scan 
 
5.2.  Patient Registration 
 
See section 4.4 above 
 
 
Registration Guidelines 
Before a subject participates in the trial, the investigator or delegate is responsible for 
obtaining written informed consent after adequate explanation of the aims, methods, 
anticipated benefits, subject responsibilities and potential hazards of the study and 
before any protocol-specific screening procedures or any study required medications 
are administered. All patients must be registered before the start of treatment.  
 
 
5.3. Treatment Phase 
 
The following tests and observations will be performed during the treatment phase of 


Revision 3, February 15, 2015 
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the study.  
 
Test                                                                                                               Interval 
Physical exam 
No less than every 8 weeks 
Complete blood counts and ANC and platelet 
count 
No less than every 4 weeks 
Liver function panel to include total bilirubin 
No less than every 4 weeks 
Serum creatinine 
No less than every 4 weeks 
Repeat disease assessment with physical exam 
and repeat imaging test that showed measured 
pretreatment 
No less than every 9 weeks 
 
5.4. Follow Up 
 
Patients will be followed according to the institutional standard guidelines. We will only 
follow patients who have experienced grade 3 or 4 adverse event or serious adverse 
event that are possible/probably or definitely related to the study required medication for 
30 days after the week 9 visit. 
6. Criteria for Evaluation 
The primary endpoint is the clinical response rate (complete response + partial response, 
see 6.3.4.1).  Other efficacy measures are progression-free survival.  
Evaluation Criteria Definitions 
6.1. Treatment related toxicity is an adverse effect that is clearly related to the 
chemotherapy regimen. 
 
6.2. 
 Clinical response:  Will be evaluated using the following definitions: 
 
6.2.1. Baseline assessment:   
 
To assess response, it is necessary to estimate the tumor burden at baseline 
before the start of treatment.  Subsequent measurements will be compared to this.  
Measurable disease is defined by the presence of at least one lesion that can be 
accurately measured in at least one dimension (longest diameter recorded) as > 
2.0 cm with conventional techniques or as > 1.0 cm by spiral CT scan.  All lesions 
that do not fit these criteria are considered to be non-measurable disease.  All 
measurable lesions up to a maximum of five lesions per organ and ten lesions in 
total should be identified as target lesions and be recorded and measured at 
baseline.  Target lesions should be selected on the basis of their size (those with 
longest diameter) and their suitability for accurate repeated measurements.  A sum 
of the longest diameter for all target lesions will be calculated and reported as the 
baseline sum.  This will be used as the reference for characterizing the objective 
tumor response 
 


Revision 3, February 15, 2015 
Page 17 
 
6.2.2. Tumor assessments will be made during screening and then at 9 (± 7 days) 
weeks to evaluate response rate. There after the use of further imaging for tumor 
assessment will be left to the treating physicians discretion 
 
6.2.3. Evaluation of target lesions 
 
Tumor response will be evaluated as follows (see appendix 2, Recist 1.1) 
 
 
 
 
 
 
6.2.4 
Evaluation 
of 
best 
overall 
response:  
The 
best 
overall 
response 
is 
the 
best 
response 
recorded 
from the start 
of 
treatment and 
repeat 
imaging done 
at 
9 
-12 
weeks (± 7 
days) 
 
6.2.5 Response rate:  The clinical response rate is the percentage of evaluable patients 
who achieve complete response (CR) or partial response (PR) as the best overall 
response. 
 
6.2.6 Progression-free survival: will be measured as the time from the start of 
pegylated liposomal doxorubicin to the time the patient is first recorded as having 
disease progression or dies.  If a patient does not progress or die while being followed 
via tumor assessment, progression-free survival will be censored at the time of last 
disease assessment. 
 
7. 
Study Drug Information 
 
Pegylated Liposomal Doxorubicin (Doxil) 
 
Doxil® (doxorubicin HCl liposome injection) is doxorubicin hydrochloride (HCl) encapsulated 
in STEALTH® liposomes forintravenous administration. Doxorubicin is a cytotoxic 
anthracycline antibiotic isolated from Streptomyces peucetius var. caesius. Doxorubicin HCl, 
is 
the 
established 
name 
for 
(8S,10S 
)-10-[(3-amino-2,3,6-trideoxy-_-L-lyxo-
hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-
naphthacenedione. Doxil will be obtained from commercial sources and administered on 
days 1 of 21 day cycle (this will be done as standard of care). This drug will be administered 
as directed by their package inserts or standard of care. Doxil will be administered 
intravenously at 30mg/m2. 
 
Dose Modification Guidelines 
 
Target lesions Non-target 
lesions 
New 
lesions 
Overall 
response 
CR 
CR 
No 
CR 
CR 
Non-CR/non-PD 
No 
PR 
CR 
Not evaluated 
No 
PR 
PR 
Non-PD or not 
evaluated 
No 
PR 
SD 
Non-PD  or not 
evaluated 
No 
SD 
Not all 
evaluated 
Non-PD 
No 
NE 
PD 
Any 
Yes or no 
PD 
Any 
PD 
Yes or no 
PD 
Any 
Any 
Yes 
PD 


Revision 3, February 15, 2015 
Page 18 
 
Patients will be carefully monitored for toxicity. Adverse reactions, such as Hand Foot 
Syndrome (HFS), hematologic toxicities, and stomatitis will be managed by dose delays and 
adjustments. Following the first appearance of a Grade 2 or higher adverse reactions, the 
dosing will be adjusted or delayed as described in the following tables. Once the dose has 
been reduced, it will not be increased at a later time. After a dose modification, if treatment 
is continued, it will return to the original dose interval of every 21 days. 
 
Recommended Dose Modification Guidelines 
 
Table 1: Dose Adjustment for Hand-Foot Syndrome (HFS) 
 
            Toxicity Grade                                                                              Dose Adjustment 
 
1-mild erythema, swelling, or 
desquamation not interfering with daily 
activities 
 
Redose unless patient has experienced 
previous Grade 3 or 4 HFS. If so, delay up 
to 2 weeks and decrease dose by 25%. 
Return to original dose interval. 
 
 
2- erythema, desquamation, or swelling 
interfering with, but not precluding normal 
physical activities; small blisters or 
ulcerations less than 2 cm in diameter 
 
 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. If after 2 weeks 
there is no resolution, DOXIL should be 
discontinued. If resolved to Grade 0-1 
within 2 weeks, and there are no prior 
Grade 3-4 HFS, continue treatment at 
previous dose and return to original dose 
interval. If patient experienced previous 
Grade 3-4 toxicity, continue treatment with 
a 25% dose reduction and return to original 
dose interval. 
 
 
3- blistering, ulceration, or swelling 
interfering with walking or normal daily 
activities; cannot wear regular clothing 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. Decrease dose by 
25% and return to original dose interval. If 
after 2 weeks there is no resolution, DOXIL 
should be discontinued. 
 
 
4- diffuse or local process causing 
infectious complications, or a bedridden 
state or hospitalization 
 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. Decrease dose by 
25% and return to original dose interval. If 
after 2 weeks there is no resolution, DOXIL 
should be discontinued. 
 
 
 
Table 2: Dose Adjustments for Stomatitis 
 
Toxicity Grade                                                                                     Dose Adjustment 


Revision 3, February 15, 2015 
Page 19 
 
1-painless ulcers, erythema, or mild 
soreness) 
 
 
Redose unless patient has experienced 
previous Grade 3 or 4 HFS. If so, delay up 
to 2 weeks and decrease dose by 25%. 
Return to original dose interval. 
 
 
2-painful erythema, edema, or ulcers,but 
can eat 
 
 
 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. If after 2 weeks 
there is no resolution, DOXIL should be 
discontinued. If resolved to Grade 0-1 
within 2 weeks, and there are no prior 
Grade 3-4 HFS, continue treatment at 
previous dose and return to original dose 
interval. If patient experienced previous 
Grade 3-4 toxicity, continue treatment with 
a 25% dose reduction and return to original 
dose interval. 
 
 
3-painful erythema, edema, or ulcers, and 
cannot eat 
 
 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. Decrease dose by 
25% and return to original dose interval. If 
after 2 weeks there is no resolution, DOXIL 
should be discontinued. 
 
 
4-requires parenteral or enteral support) 
 
Delay dosing up to 2 weeks or until 
resolved to Grade 0-1. Decrease dose by 
25% and return to original dose interval. If 
after 2 weeks there is no resolution, DOXIL 
should be discontinued. 
 
 
Table 3: Dose Adjustments for Hematological Toxicity 
 
 
Situation  
Doxil Dose  
First episode of febrile neutropenia (grade 3 or 4) 
75%  
Second episode of febrile neutropenia (grade 3 or 4) 
Discontinue  
Grade 4 thrombocytopenia or bleeding associated with 
thrombocytopenia  
75%  
 
In addition to the above dose adjustments, subjects experiencing febrile neutropenia secondary 
to Doxil may receive G-CSF therapy in association with subsequent chemotherapy doses.  
 
Table 4: Dose Adjustments for Other Toxicities  
 
 NCI CTC Grade  
Doxil Dose  
0 - 2  
100%  
3 (except alopecia)  
75%  
4  
Hold until resolution of toxicity 


Revision 3, February 15, 2015 
Page 20 
 
 
8. Study Statistics 
 
8.1. Statistical Methods: This study is a prospective single arm Phase II trial to assess 
association of BRCA1 protein expression with overall response rate in patients with 
metastatic breast cancer on pegylated liposomal doxorubicin in patients with metastatic 
breast cancer. We plan to consent 50 to 60 patients with the goal of enrolling a total of 
50 patients, with adequate primary tumor tissue for BRCA1 protein expression, into this 
study.  It is estimated that 30% of patients will have low BRCA1 protein expression19 
and therefore anticipate approximately a 35 to 15 patient split for normal BRCA1 to low 
BRCA1 expression status. 
 
Recruitment and tissue analysis: Patients will be consented and after enrollment of the 
first 12 patients, a batch analysis of their tumors will be performed to analyze tumor 
BRCA1 protein expression. This will allow us to assign the patients to their respective 
cohorts (Normal BRCA1 protein expression and Low BRCA1 protein expression). This 
will be done until we have 12 patients in the normal BRCA1 protein arm. After that, an 
interim analysis will be performed for the normal BRCA1 arm and stopping rule will be 
applied as mentioned in the study design 
8.2. Analytic plan for primary objective:  
 
Definition of primary endpoint: Response rate is defined as the percentage of 
evaluable patients who achieve complete response (CR) or partial response (PR) as 
measured by RECIST on imaging as the best overall response.  
Low BRCA1 protein expression:  The number and percentage of patients falling into response 
category will be tabulated. The estimate of the response rate will be presented with 2-sided 95% 
exact binomial confidence intervals. We anticipate this data to be hypothesis generating and 
will form the basis for a future prospective clinical trial enrolling larger number patients to 
evaluate if low BRCA1 expression, as determine by IHC, is predictive of response to treatment 
with DNA damaging agents. 
 
Normal BRCA1 protein expression:  The estimate of the response rate will be presented with 
2-sided 95% exact binomial confidence intervals. If 5 or fewer patients with response are 
observed among the 35 patients, then there will be no further investigation of the treatment. If 6 
responses are observed out of 35 patients, there is 80% confidence that the true response rate 
>11.2%.   
 
 
Interim analysis:  
Low BRCA1 protein expression: No interim analysis will be conducted 
 
Normal BRCA1 protein expression: An interim analysis for futility will be conducted for this 
arm. The response rate will be used as the endpoint for the interim analysis. The interim 
analysis will be performed when the first 12 treated patients are accrued to normal BRCA1 
protein expression arm. Based on Simon’s 2-stage design, if less than 2 responders are 
observed out of these initial 12 evaluable patients, patient enrollment will be discontinued into 
this arm. Otherwise patient enrollment will continue until a total of 35 patients evaluable for 
response are enrolled. If only 1 response is observed out of 12 patients, there is 80% 
confidence that the true response rate <22.1%.   
 


Revision 3, February 15, 2015 
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If the normal BRCA1 protein expression arm is closed to accrual, we will continue to 
consent patients who are being treated with pegylated liposomal doxorubicin. As the 
expression of BRCA1 protein is anticipated to be low in only 30% of patients, we will 
continue to consent patients and analyze their tumors and only those with low BRCA1 
protein expression will be enrolled into the arm. We anticipate consenting total of 50-60 
patients to enroll 15 patients into the low BRCA1 protein expression arm. 
 
 
8.3. Analytic plan for secondary objective:  
 
Definition of secondary endpoints: Progression free survival will be measured as the 
times from the start of pegylated liposomal doxorubicin to the time the patient is first 
recorded as having disease progression or dies. If a patient does not progress or die 
while being followed via tumor assessment, progression-free survival will be censored 
at the time of last disease assessment.  
 
We will calculate the median progression free survival in patients with low BRCA1 and 
normal BRCA1 protein expression. Median progression free survival will be estimated 
by Kaplan-Meier methodology. 
 
9. Data and Safety Monitoring Plan 
 
Data and Safety Monitoring Plan for Patients Enrolled at the University of Arizona 
 
The University of Arizona Cancer Center Data and Safety Monitoring Board (DSMB) 
reviewed the protocol and determined this study does not require DSMB oversight or routine 
monitoring by the QA/QC program. Principal investigator will be responsible for monitoring 
and reporting any changes/ adverse events to IRB. PI will be responsible all the study 
related activities and will report annually to IRB. 
 
The Principal Investigator will ensure the accuracy, completeness, legibility and timeliness of 
the data reported in the Case Report Form (CRF). Source documentation supporting the 
CRF data will indicate the subject’s participation in the trial and will document the dates and 
details of study procedures, adverse events, and patient status.  
 
Case report forms, which include the inclusion/exclusion criteria form, adverse event forms 
and serious adverse event forms should be completed with a black ball-point pen or typed. 
Corrections to the forms should not obscure the original entry and should be made by 
striking the incorrect information with a single line. Each strike should be accompanied by 
the initials of the corrector and the correction date. All subject forms and study files will be 
stored in a secure area limited to authorized staff. 
 
Note: Routine monitoring of regulatory documents and test article will be conducted at least 
annually.  
 
Data and Safety Monitoring Plan for Patients Enrolled at the University of New Mexico 
 
The University of New Mexico Cancer Center (UNM CC) places a high priority on ensuring 
the safety of patients participating in clinical trials. All clinical trials require monitoring 
commensurate with the degree of risk involved in participation of studies. Clinical Research 


Revision 3, February 15, 2015 
Page 22 
 
Office Standard Operating Procedures (SOP’s) detail functions and processes found in this 
plan. SOPs are available at http://hsc.unm.edu/UNM CC/intranet/ctoforms.asp. 
 
Data and safety monitoring activities for each study continue until all patients have 
completed their treatment and all patients are beyond the time point at which study-related 
adverse events would likely be encountered. The UNM CC has implemented a process for 
routine, real time data monitoring and safety review of Investigator Initiated trials which 
takes into account the Essential Elements of the National Cancer Institute (NCI) guidelines, 
the FDA monitoring regulations, Good Clinical Practice Guidelines and other DSM plans and 
programs approved by the NCI. These are outlined in the UNM CC Data and Safety 
Monitoring Plan, or DSMP. 
 
In addition to complying with NIH/NCI guidelines, the UNM CC DSMP complies with, the 
University of New Mexico Health Sciences Center Human Research Protections Office 
(HRPO) guidelines for safety and data monitoring which can be found at: 
http://hsc.unm.edu/som/research/hrrc/PoliciesGuidelines.shtml). 
 
The DSMP is distinct from, and complements, the activities of the Protocol Monitoring & 
Review Committee (PRMC) and the Clinical Protocol Data Management (CPDM) functions 
of UNM CC. The most current version is maintained on file with the Human Research 
Review Committee, and is therefore not included in this protocol as an Appendix. 
 
De-identified study results, in the form of glass slides, digitized pathology and radiology 
images and Microsoft Word documents and Microsoft Excel databases, will be stored for 10 
years.  All study results will be stored in locked offices and on password protected 
computers. During that time, PIs at the University of Arizona and the University of New 
Mexico will have access to the de-identified data for the purposes of analysis and 
publication.  
 
Process to implement study closure when significant risks or benefits are identified: 
Stopping rules will be based on Simon’s optimal design (Simon, R.  “Optimal Two-Stage 
Design for Phase II Clinical Trials”.  Controlled Clinical Trials, 101-10, 1989, Elsevier 
Science Publishing Company, N.Y., N.Y.) which minimizes the expected sample size under 
the null response rate.20 In the patients with normal BRCA1 protein expression if at least two 
responses are observed among these 12 patients, then an additional 23 patients will be 
accrued to the second stage.  If 0 or 1 response is observed among the initial 12 patients, 
then accrual to this cohort will be terminated and declared negative.  At the end of the study 
if 6 or more patients in this cohort respond then pegylated liposomal doxorubicin will be 
considered worthy of further study in this cohort. This design yields at least 90% probability 
of a positive result if the true response rate is at least 30% and a 90% probability of a 
negative result if the true response rate is 10%. 
 
Description of adverse events and reporting procedures: 
 
Adverse Event 
 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and that does not necessarily 
have a casual relationship with this treatment. An AE can therefore be any unfavorable and 
unintended sign, symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not related to the medicinal (investigational) product.  


Revision 3, February 15, 2015 
Page 23 
 
 
Adverse events of grade 3 or 4 that are possible/probably or definitely related to the study 
required medication or serious adverse events will be recorded on the UMC adverse events 
record form and reviewed by the Principal Investigator. These same events will be reported 
in the case report form. 
 
All adverse events will be classified using either the MedDRA term or NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 and will address: 
• 
Grade 
• 
Relationship to study drug(not related, unlikely, possible, probable, definitely) 
• 
Causality other than study drug (disease related, concomitant medication related, 
intercurrent illness, other)   
• 
Date of onset, date of resolution 
• 
Frequency of event (single, intermittent, continuous) 
• 
Event outcome (resolved, ongoing, death) 
• 
Action taken (none, held, dose reduced, discontinued, medication given) 
 
Serious Adverse Events 
 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose:  
• 
Results in death 
• 
Is life-threatening 
• 
Requires in-patient hospitalization or prolongation of an existing hospital stay 
• 
Results in disability persistent or significant disability/incapacity, or 
• 
Is a congenital anomaly/birth defect.  
 
Note: Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the patient or subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above. 
 
All SAEs which meet the criteria for a reportable event will be reported in writing to the FDA 
and to the University of Arizona Human Subjects Protection Program within 10 working days 
with the exceptions of unexpected death or life-threatening even, which should be reported 
within 5 working days after learning of the event.  
Non-local Unanticipated Problem Involving Risks to Subjects or Others, which is any 
information that meets all three of the following criteria:  
i) 
Unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent; and (b) the characteristics of the 
subject population being studied. A harm is “unexpected” when its specificity and 
severity are not accurately reflected in the consent document.  
ii) Related or possible related due to participation in this research (possible related means 
that the outcome may have been caused by the procedures involved in the 
research); and  
iii) Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. A harm is “at least probably related to the Human Research procedures” 
if in the opinion of the investigator, the research procedures more likely than not 
caused the harm.  


Revision 3, February 15, 2015 
Page 24 
 
Patients will be followed according to the institutional standard guidelines. We will only 
follow patients who have experienced grade 3 or 4 adverse event or serious adverse event 
that are possible/probably or definitely related to the study required medication. 
 
Plan for assuring data accuracy and protocol compliance: 
Routine study activity and safety information will be reported to the IRB once a year, or more 
frequently if requested. These reports will include: 
• 
Study activity, cumulative and for the period under review; 
• 
Safety (narrative description on non-serious and serious adverse events); 
• 
Predetermined protocol early stopping rules for efficacy/futility;    
• 
Monitoring and protocol compliance; 
• 
Comments;  
• 
Attachments (AE data reviewed by the PI to compile the report, SAE letters and 
reports, results of any review(s), applicable correspondence with the IRB or other 
regulatory agencies.  
 
Data, safety and study progress will be reported to: 
• 
Human Subjects Protection Program (IRB) at least annually; 
• 
Sponsor (if applicable) at least every six months. 
 
Identification of the sponsor or funding agency, as applicable: 
The PI will immediately notify; in writing, the funding agency, if applicable, any action 
resulting in a temporary or permanent suspension of the study. 
 
10. Data Submission Schedule 
 
Data forms must be completed for all subjects registered to the study. 
Electronic case report forms will be completed in the OnCore system. 
 
 
11. Special instructions 
 
Patients will be consented for acquiring their primary tumor tissue to evaluate for BRCA1 
protein expression. Formalin-fixed paraffin-embedded (FFPE) tissue will be accessed from 
the site where they had surgery for their primary tumor. If their primary surgery was at UMC 
we will request department of pathology for their tumor blocks. If they had surgery at an 
outside institution we will request their tumor blocks. Patients will need to sign a release of 
tissue form. We will use the standard tissue requisition form used for University of Arizona 
Cancer Center. 
 
Patient tissue in the form of a tumor block is preferred. However, if tumor block is not 
available, 10 unstained slides will be submitted.   
 
For patients participating at the University of New Mexico Cancer Center, it will be confirmed 
that tissue (in the form of tumor block or 10 unstained slides) is available from either primary 
or metastatic site prior to registration.   
 
Once their tumor blocks / slides have been accessed they will be sent via Fed-Ex to the 
University of Arizona Cancer Center for further analysis.  
 


Revision 3, February 15, 2015 
Page 25 
 
Yrma Burrel, CRC 
University of Arizona Cancer Center 
3838 N. Campbell Ave. 
Tucson, Arizona 85719 
520-694-9081 office   Pager: 8303 
520-694-9092 fax 
yburrel@uacc.arizona.edu 
 
 
12.  Ethics  
 
The trial will be conducted in accordance with the Declaration of Helsinki for biomedical 
research involving human subjects and local regulatory requirements.  
 
Ethical Principles  
This study will be conducted in accordance with Title 21 of the Code of Federal Regulations 
(CFR).  Specifically, this study is based on adequately performed laboratory and animal 
experimentation; the study will be conducted under a protocol reviewed by an Institutional 
Review Board; the study is to be conducted by scientifically and medically qualified persons; 
the benefits of the study are in proportion to the risks; the rights and welfare of the patients 
will be respected; the physicians conducting the study will ensure that the hazards do not 
outweigh the potential benefits; the results to be reported will be accurate; patients will give 
their informed consent and will be competent to do so and not under duress; and the study 
will comply with the ethical principles in Title 21 of the CFR.  
 
Informed Consent  
This study will be conducted in full compliance with the informed consent regulations in 21 
CFR 50. The Sponsor-Investigator is responsible for ensuring that written informed consent 
from potential patients is obtained prior to performing any trial tests or assessments required 
by the protocol.  
Informed consent, University of Arizona  
 
A copy of the fully executed informed consent form (PHI authorization form and ancillary 
consent forms if applicable), is given to the subject. One copy is placed in the subject’s 
medical record, another copy is placed in the research chart, and the originals are filed by 
the protocol number in room 2111 at UACC North Campus. 
 
Informed consent, University of New Mexico  
 
A copy of the fully executed informed consent form will be given to the subject. One copy is 
placed in the subject’s medical record, another copy is placed in the research chart, and the 
originals are filed by protocol number in the Clinical Trails Office at the New Mexico Cancer 
Care Alliance, UNM Cancer Center Admin. Bldg. 2nd Floor.  
 
 
Institutional Review Board  
This study will be conducted in full compliance with the Institutional Review Board (IRB) 
regulations in 21 CFR 56, in accordance with the Declaration of Helsinki. This protocol will 
not be initiated unless it and the informed consent form have been reviewed and approved 
by, and remains open to continuing review by, an IRB meeting the requirements of 21 CFR 
56. The IRB shall review and have the authority to approve, require modification in (to 


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secure approval), or disapprove the protocol. The IRB shall notify the Investigator and the 
institution in writing of its decision. The IRB shall require that the information given to 
patients as part of the informed consent is in accordance with 21 CFR 50.25. The IRB shall 
conduct continuing reviews of the protocol at intervals appropriate to the degree of risk, but 
not less than once per year. At the completion or early termination of the trial, a final report 
should be sent to the IRB by the Investigator. The Investigator is obligated to maintain an 
IRB correspondence file.  
 
Confidentiality of Patient Data 
The investigator must ensure that patient confidentiality will be maintained. Patients will be 
identified by initials and a protocol-assigned patient number as described in section 4.5. 
Permission for direct access to patient data will be sought in writing for the patient by the 
investigator as part of the informed consent procedure. The patient will be informed that all 
clinical information is confidential, but that the IRB, and regulatory authorities may inspect 
these records. 
 
 
 
 
 
 
 
 
13. 
Bibliography 
 
1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: a cancer journal for 
clinicians. Jan 2013;63(1):11-30 
2. Wooster R, Weber BL. Breast and ovarian cancer. The New England journal of 
medicine. Jun 5 2003;348(23):2339-2347 
3. Turner N, Tutt A, Ashworth A. Hallmarks of 'BRCAness' in sporadic cancers. Nat Rev 
Cancer. Oct 2004;4(10):814-819 
4. Turner N, Tutt A, Ashworth A. Targeting the DNA repair defect of BRCA tumours. 
Current opinion in pharmacology. Aug 2005;5(4):388-393. 
5. Venkitaraman AR. Functions of BRCA1 and BRCA2 in the biological response to DNA 
damage. Journal of cell science. Oct 2001;114(Pt 20):3591-3598 
6. Collins N, McManus R, Wooster R, et al. Consistent loss of the wild type allele in breast 
cancers from a family linked to the BRCA2 gene on chromosome 13q12-13. Oncogene. 
Apr 20 1995;10(8):1673-1675 
7. Cornelis RS, Neuhausen SL, Johansson O, et al. High allele loss rates at 17q12-q21 in 
breast and ovarian tumors from BRCAl-linked families. The Breast Cancer Linkage 
Consortium. Genes, chromosomes & cancer. Jul 1995;13(3):203-210. 
8. Rahman N, Stratton MR. The genetics of breast cancer susceptibility. Annual review of 
genetics. 1998;32:95-121. 
9. Rhiem K, Todt U, Wappenschmidt B, Klein A, Wardelmann E, Schmutzler RK. Sporadic 
breast carcinomas with somatic BRCA1 gene deletions share genotype/phenotype 
features with familial breast carcinomas. Anticancer research. Sep 2010;30(9):3445-
3449. 
10. Quinn JE, Kennedy RD, Mullan PB, et al. BRCA1 functions as a differential modulator of 
chemotherapy-induced apoptosis. Cancer Res. Oct 1 2003;63(19):6221-6228. 


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11. Rodriguez AA, Makris A, Wu MF, et al. DNA repair signature is associated with 
anthracycline response in triple negative breast cancer patients. Breast cancer research 
and treatment. Aug 2010;123(1):189-196. 
12. Lips EH, Mulder L, Hannemann J, et al. Indicators of homologous recombination 
deficiency in breast cancer and association with response to neoadjuvant chemotherapy. 
Ann Oncol. Apr 2011;22(4):870-876. 
13. Vollebergh MA, Lips EH, Nederlof PM, et al. An aCGH classifier derived from BRCA1-
mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-
negative breast cancer patients. Ann Oncol. Jul 2011;22(7):1561-1570. 
14. Silver DP, Richardson AL, Eklund AC, et al. Efficacy of neoadjuvant Cisplatin in triple-
negative breast cancer. Journal of clinical oncology : official journal of the American 
Society of Clinical Oncology. Mar 1 2010;28(7):1145-1153. 
15. Lips EH, Laddach N, Savola SP, et al. Quantitative copy number analysis by Multiplex 
Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer 
regions identifies BRCAness. Breast Cancer Res. 2011;13(5):R107. 
16. Verma S, Dent S, Chow BJ et al: Metastatic breast cancer: the role of pegylated 
liposomal doxorubicin after conventional anthracyclines. Cancer Treat Rev. 2008 
Aug;34(5):391-406 
17. O’Brien MER, Wigler N, Inbar M et al. Reduced cardiotoxicity and comparable efficacy in 
a phase III trial of pegylated liposomal doxorubicin HCL (CAELYXTM/Doxil) versus 
conventional doxorubicin for first-line treatment of metastatic breast cancer. Annals of 
Oncology. 2004;15: 440-449 
18. Birner P, Oberhuber G, Stani J, et al. Evaluation of the United States Food and Drug 
Administration–approved scoring and test system of HER-2 protein expression in breast 
cancer. Clin Cancer Res. 2001;7:1669-1675. 
19. Jacobs TW, Gown AM, Yaziji H, et al. Specificity of HercepTest in determining HER-
2/neu status of breast cancers using the United States Food and Drug Administration–
approved scoring system. J Clin Oncol. 1999;17:1983-1987 
20. Simon R, Optimal two-stage designs for phase II clinical trials, Controlled Clinical Trials, 
1989, 10, 1 - 10. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix 1. Eastern Cooperative Oncology Group (ECOG) Performance Status 
 
Activity Status  
Description  
0  
Asymptomatic, fully active, and 
able to carry on all predisease 
performance without restrictions  
1  
Symptomatic, fully ambulatory but 
restricted in physical strenuous 
activity and able to carry out 
performance of a light or 
sedentary nature, e.g., light 
housework, office work  
2  
Symptomatic, ambulatory and 
capable of self-care but unable to 
carry out any work activities. Up 
and about more than 50% of 
waking hours, but not bedridden  
3  
Symptomatic, capable of only 
limited self care, confined to a 
bed/chair more than 50% of 
waking hours, but not bedridden.  
4  
Completely disabled. Can not 
carry on self-care. Totally 
bedridden.  


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5  
Dead  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix 2. Response Evaluation Criteria in Solid Tumors (RECIST 1.1) 
Eligibility 
• 
Only patients with measurable disease at baseline should be included in protocols where 
objective tumor response is the primary endpoint.  
Measurable disease - the presence of at least one measurable lesion. If the measurable 
disease is restricted to a solitary lesion, its neoplastic nature should be confirmed by 
cytology/histology.  
Measurable lesions - lesions that can be accurately measured in at least one dimension with 
longest diameter ≥ 20 mm using conventional techniques or ≥10 mm with spiral CT scan. 
Non-measurable lesions - all other lesions, including small lesions (longest diameter <20 
mm with conventional techniques or <10 mm), i.e., bone lesions, leptomeningeal disease, 
ascites, pleural/pericardial effusion, inflammatory breast disease, lymphangitis 
cutis/pulmonis, cystic lesions, and also abdominal masses that are not confirmed and 
followed by imaging techniques; and 
• 
All measurements should be taken and recorded in metric notation, using a ruler or calipers. 
All baseline evaluations should be performed as closely as possible to the beginning of 
treatment and never more than 4 weeks before the beginning of the treatment.  
• 
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up.  
• 
Clinical lesions will only be considered measurable when they are superficial (e.g., skin 
nodules and palpable lymph nodes). For the case of skin lesions, documentation by color 
photography, including a ruler to estimate the size of the lesion, is recommended.  
Baseline documentation of “Target” and “Non-Target” lesions 
• 
All measurable lesions up to a maximum of five lesions per organ and 10 lesions in total, 
representative of all involved organs should be identified as target lesions and recorded 
and measured at baseline.  
• 
Target lesions should be selected on the basis of their size (lesions with the longest 
diameter) and their suitability for accurate repeated measurements (either by imaging 
techniques or clinically).  
• 
A sum of the longest diameter (LD) for all target lesions will be calculated and reported as 
the baseline sum LD. The baseline sum LD will be used as reference by which to 
characterize the objective tumor. 
• 
All other lesions (or sites of disease) should be identified as non-target lesions and should 
also be recorded at baseline. Measurements of these lesions are not required, but the 
presence or absence of each should be noted throughout follow-up.  


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Response Criteria 
Evaluation of target lesions 
* Complete Response 
(CR): 
Disappearance of all target lesions 
* Partial Response 
(PR): 
At least a 30% decrease in the sum of the LD of target 
lesions, taking as reference the baseline sum LD 
* Progressive Disease 
(PD): 
At least a 20% increase in the sum of the LD of target 
lesions, taking as reference the smallest sum LD recorded 
since the treatment started or the appearance of one or 
more new lesions 
* Stable Disease (SD): 
Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest 
sum LD since the treatment started 
Evaluation of non-target lesions 
* Complete Response 
(CR): 
Disappearance 
of 
all 
non-target 
lesions 
and 
normalization of tumor marker level 
* Non-CR/Non-PD 
Persistence of one or more non-target lesion(s) or/and 
maintenance of tumor marker level above the normal 
limits 
* Progressive Disease 
(PD): 
Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions 
(1)  
(1) Although a clear progression of “non target” lesions only is exceptional, in such 
circumstances, the opinion of the treating physician should prevail and the 
progression status should be confirmed later on by the review panel (or study 
chair). 
Evaluation of best overall response 
• 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for PD the smallest measurements 
recorded since the treatment started). In general, the patient's best response assignment 
will depend on the achievement of both measurement and confirmation criteria. 
  
Target lesions 
Non-Target lesions 
New Lesions 
Overall response 
CR 
CR 
No 
CR 
CR 
Non-CR/non-PD 
No 
PR 
CR 
Not evaluated 
No 
PR 


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PR 
Non-PD or not all 
evaluated 
No 
PR 
SD 
Non-PD or not all 
evaluated 
No 
SD 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
• 
Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be classified as having 
“symptomatic deterioration”. Every effort should be made to document the objective 
progression even after discontinuation of treatment.  
• 
In some circumstances it may be difficult to distinguish residual disease from normal tissue. 
When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to 
confirm the complete response status. 
Confirmation 
• 
The main goal of confirmation of objective response is to avoid overestimating the response 
rate observed.  In cases where confirmation of response is not feasible, it should be made 
clear when reporting the outcome of such studies that the responses are not confirmed. 
• 
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed 
by repeat assessments that should be performed no less than 4 weeks after the criteria for 
response are first met. Longer intervals as determined by the study protocol may also be 
appropriate.  
• 
In the case of SD, follow-up measurements must have met the SD criteria at least once after 
study entry at a minimum interval (in general, not less than 6-8 weeks) that is defined in the 
study protocol  
Duration of overall response 
• 
The duration of overall response is measured from the time measurement criteria are met 
for CR or PR (whichever status is recorded first) until the first date that recurrence or PD is 
objectively documented, taking as reference for PD the smallest measurements recorded 
since the treatment started. 
Duration of stable disease 
• 
SD is measured from the start of the treatment until the criteria for disease progression are 
met, taking as reference the smallest measurements recorded since the treatment started.  
• 
The clinical relevance of the duration of SD varies for different tumor types and grades. 
Therefore, it is highly recommended that the protocol specify the minimal time interval 
required between two measurements for determination of SD. This time interval should take 
into account the expected clinical benefit that such a status may bring to the population 
under study.  
 


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Response review 
• 
For trials where the response rate is the primary endpoint it is strongly recommended that all 
responses be reviewed by an expert(s) independent of the study at the study’s completion.  
Simultaneous review of the patients’ files and radiological images is the best approach.  
Reporting of results 
• 
All patients included in the study must be assessed for response to treatment, even if there 
are major protocol treatment deviations or if they are ineligible.  Each patient will be 
assigned one of the following categories: 1) complete response, 2) partial response, 3) 
stable disease, 4) progressive disease, 5) early death from malignant disease, 6) early 
death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, 
insufficient data). 
• 
All of the patients who met the eligibility criteria should be included in the main analysis of 
the response rate.  Patients in response categories 4-9 should be considered as failing to 
respond to treatment (disease progression).  Thus, an incorrect treatment schedule or drug 
administration does not result in exclusion from the analysis of the response rate.  Precise 
definitions for categories 4-9 will be protocol specific. 
• 
All conclusions should be based on all eligible patients. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX 3 
  
Investigator Agreement  
 
Protocol No.  
 
Protocol Title: A single arm Phase II trial to assess association of defective DNA repair status 
with overall response rate in patients with HER2 negative metastatic breast cancer on pegylated 
liposomal doxorubicin 
 
By signing below I agree:  
 
1) That my staff and I have read, understand and will adhere to the protocol as written, and that 
any changes to the protocol will be agreed to and approved by the Principal Investigator and the 
Institutional Review Board (IRB) 
 
2) To abide by all obligations stated on the FDA Form 1572 and other documents required by 
regulation;  
 
3) To conduct this study in accordance with the current International Conference on 
Harmonization (ICH) guidance, the Good Clinical Practices (GCP) guidance, the Declaration of 
Helsinki, US FDA regulations and local IRB and legal requirements;  
 
4) To obtain IRB approval of the protocol, any amendments to the protocol, and periodic re-
approval as required, and to keep the IRB informed of adverse events as required by their 
guidelines report the status of the study to them;  
 
5) To ensure that each individual enrolled into the trial, or legally authorized representative, has 
read, understands, and has signed the Informed Consent form;  
 
6) To ensure that I and all persons assisting me with the study are adequately informed and 
trained about the study and the possible adverse events associated with the study required 
medication 
 
7) To make prompt reports of SAEs and deaths to the FDA according to the regulations;  
 
8) To prepare and maintain adequate and accurate case histories to document all observations 
and other data pertinent to the study for each individual enrolled in the clinical trial.  
 
 
 
Investigator Signature Date  
 
 
Investigator Name (Print) 
 
 
 


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