Neoadjuvant Weekly Paclitaxel and Biomarkers of Therapy Response
Phase: PHASE4
Conditions: Breast Neoplasm Female
Sponsor: University of Wisconsin, Madison

Neoadjuvant weekly paclitaxel and biomarkers of therapy response
NCT03096418
PI: Mark Burkard, MD, PhD
Version Date: 8/23/2019

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 UW16106:  Neoadjuvant weekly paclitaxel and biomarkers of therapy 
response 
Coordinating Center: 
University of Wisconsin Paul P. Carbone Comprehensive 
Cancer Center 
*Principal Investigator:
Name:  
 Mark Burkard, MD, PhD. 
Address: 1111 Highland Ave 
Department of Hematology/Oncology, Room 6059 
Madison, WI 53705-2275 
Telephone: (608) 262-2803 
Fax: (608) 265-0614 
e-mail address:  meburkard@medicine.wisc.edu
Co-Principal Investigator:          Name:   Beth Weaver, PhD. 
 (not directly responsible for patient care) 
Address:  1111 Highland Ave 
Department of CRB, Room 6109 
Madison, WI 53705-2275 
Telephone: (608) 263-3509 
e-mail address:  baweaver@wisc.edu
Co-Investigators: 
Name:      Lee Wilke, MD 
Address: Clinical Science Center-H4/722 
600 Highland Ave. 
Department of General Surgery 
Madison, WI 53792 
Telephone: (608) 265- 5852 
e-mail address:  neuman@surgery.wisc.edu
Name:      Heather Neuman, MD 
Address: 785d Clinical Science Center-H4 
600 Highland Ave. 
Department of General Surgery 
Madison, WI 53792 
Telephone: (608) 265- 5852 
e-mail address:  neuman@surgery.wisc.edu
Name:      Linda Szalkucki APNP, CBCN 
Address: 600 Highland Ave. 
Breast Center 
Madison, WI 53705-2275 
e-mail address: SZALK@surgery.wisc.edu

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Name:      Kari Wisinski, MD 
Address: 1111 Highland Ave. 
Department of Hematology/Oncology, Room 6033 
Madison, WI 53705-2275 
Telephone: (608) 262- 2876 
Fax: (608) 265-0614 
e-mail address:  kbwisinski@medicine.wisc.edu 
 
Name: 
Amye Tevaarwerk, MD 
Address: 1111 Highland Ave. 
Department of Hematology/Oncology, Room 6037 
Madison, WI 53705-2275 
Telephone: (608) 262-2837 
Fax: (608) 265-0614 
 
e-mail address:  at4@medicine.wisc.edu  
 
Name: 
Ruth O’Regan, MD 
Address: K4/552, 5666 CSC    
 
600 Highland Avenue 
Department of Hematology/Oncology, Madison, WI 
53792 
Telephone: (608) 265-9701 
 
e-mail address:  roregan@medicine.wisc.edu 
 
 
 
Name:     Stephanie McGregor, MD, Ph.D. 
 
 
 
 
    (not directly responsible for patient care) 
                                          Address:  3170 UW Med. Fndtn. Centennial Bldg. 
                                                          1685 Highland Ave. 
                                                          Department of Pathology and Laboratory Medicine 
                                                          Madison, WI 53705 
                                                          e-mail address: smcgregor@wisc.edu 
 
 
 
Name:     Amy Fowler, MD, Ph.D. 
 
 
 
 
    1111 Highland Ave 
 
 
Address:  Department of Radiology, Room 6105 
 
 
 
 
    Madison, WI 53705 
 
 
 
 
    Telephone: (608) 262-9186 
 
 
 
          e-mail address: afowler@wisc.edu 
 
 
Study Coordinator:                    Name: 
Tamara Koehn 
Address: 600 Highland Avenue 
 
K4/667 Clinical Science Center 
 
Telephone: (608) 265-2999 
 
Fax: (608) 265-9657 
 
E-Mail: tmkoehn@medicine.wisc.edu 
 

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Statistician: 
                              Name:       Irene Ong 
 
                              Address:   1111 Highland Ave. 
                                                                         Department of Biostatistics and Medical                                           
Informatics 
                                                                         Madison, WI 53705 
                                                                         Telephone: (608) 265-8262 
                                                                         E-Mail: ong@biostat.wisc.edu  
 
 
 
NCI Supplied Agent: 
None 
 
Protocol Type / Version # / Version Date: 
Amendment / Version 5 /  8/23/2019 
 

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SCHEMA 
 
 
 
 
 
 
 
 
 
 
 
 
Locally
Advanced
HER2- BC
N=20
Br MRI, US
mammo
paclitaxel
80 mg/m2
C1D1
Biopsy
#1
Correlates
C1D2
paclitaxel
80 mg/m2
C1D8,15 &
C2-4
Br MRI, US
mammo
Pretreatment
FFPE slides for:
Mitotic index
Aneuploidy
CIN
Ki67
Mitotic spindle
Centrosome #
15ml blood
Standard
Of care
Surgery
Pathologic
Response
Genomic 
measurements of 
CIN
Posttreatment
Drug levels
Drug distribution
Mitotic index
Mitotic spindle
Centrosome #
Aneuploidy
CIN
Genomic
Measurements of
CIN
AC
C1D16 peak
(5 patients)
at 20 hours
Drug levels/distribution
Mitotic index
Aneuploidy
CIN
Genomics
Genomic
Measurements
of CIN
Biopsy
#2
N=5
 
Biopsy 
#3 
N≥5 
C4D16 20 hrs 
posttreatment 
 (≥5 patients)  
Drug levels/distribution 
Mitotic Index 
Mitotic Spindle 
Centrosome # 
Aneuploidy 
CIN 
Genomics 
C1D2 20 hrs 
N≥5 
Skin 
Biopsy 
N≥5 
20 ± 4hrs 
post C1D2 treatment 
Drug 
levels/distribution 
Mitotic Index 
Mitotic Spindle 
Centrosome # 
Aneuploidy 
CIN 
Genomics 

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TABLE OF CONTENTS 
 
 
Page 
SCHEMA ........................................................................................................................................v 
 
1. OBJECTIVES ..........................................................................................................................1 
1.1. Primary Objectives ...........................................................................................................1 
1.2. Secondary Objectives .......................................................................................................1 
 
2. BACKGROUND ......................................................................................................................1 
2.1 
Study Disease ...............................................................................................................1 
2.2 
Rationale and Correlative Studies .............................................................................2 
2.2.1 
Taxanes Effect on Mitosis ......................................................................................2 
2.2.2 The Mitotic Checkpoint ...........................................................................................2 
2.2.3 Biomarkers of Taxane Response .............................................................................3 
2.3 
Hypothesis ....................................................................................................................3 
 
3. PATIENT SELECTION..........................................................................................................3 
3.1 
Eligibility Criteria  ......................................................................................................3 
3.2 
Exclusion Criteria ........................................................................................................4 
3.3 
Inclusion of Minorities ................................................................................................5 
 
4. REGISTRATION PROCEDURES ........................................................................................5 
4.1  
General Guidelines.......................................................................................................5 
4.2  
Registration Process.....................................................................................................5 
 
5. TREATMENT PLAN ..............................................................................................................6 
5.1 
Administration .............................................................................................................6 
5.2 Timing of Surgery ..............................................................................................................6 
5.3 Data Acquisition and Management…………………………...………………………...6 
5.4 Sample Collection for Laboratory Analysis ....................................................................7 
5.5 Laboratory Analysis ..........................................................................................................8 
5.6 Future Studies……………………………………………………………………………9 
5.7 Duration of Follow Up .......................................................................................................9 
5.8 Criteria for Removal from Study .....................................................................................9 
 
6. DOSING DELAYS/DOSE MODIFICATIONS ..................................................................10 
 
7. PHARMACEUTICAL INFORMATION ............................................................................10 
7.1 
Adverse Events and Potential Risks .........................................................................10 
7.2 
Availability..................................................................................................................10 
 
8. ADVERSE EVENTS 
8.1 CTCAE term (AE description) and grade .....................................................................11 
8.2 Attribution of the AE .......................................................................................................11 
 
9.   OVERSIGHT AND MONITORING PLAN .......................................................................11 

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9.1 Monitoring and Reporting Guidelines ...........................................................................12 
9.2 Review and Oversight Requirements .............................................................................12 
9.2.1 Serious Adverse Event- Reported within 24 hours ................................................12 
9.2.2 Serious Adverse Event – Reported within 10 days ................................................13 
9.2.3 Study Progress Review ..........................................................................................13 
9.2.4 Expedited Reporting of Serious Adverse Events ...................................................14 
9.2.5 Expedited Reporting Table ....................................................................................15 
 
10. SAE REPORTING.................................................................................................................15 
 
11. STUDY CALENDAR ............................................................................................................17 
 
12. MEASUREMENT OF EFFECT ..........................................................................................17 
12.1 
Methods for Evaluation .............................................................................................17 
12.2 
Response Criteria- Pathologic ..................................................................................18 
12.3 
Response Criteria- Clinical Response ......................................................................18 
 
13. STATISTICAL CONSIDERATIONS .................................................................................18 
 
13.1 
Study Design/ Endpoints ...........................................................................................18 
 
13.2 
Sample Size/ Accrual Rate ........................................................................................18 
 
13.4 
Analysis of Secondary Endpoints .............................................................................19 
 
14. REFERENCES .......................................................................................................................20 
 

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1. OBJECTIVES 
 
1.1. 
Primary Objectives 
 
1.1.1. To test if cancers with high chromosomal instability (CIN) respond to paclitaxel 
better than low CIN cancers.  
 
1.2. 
Secondary Objectives 
 
1.2.1. To identify patient-specific differences in tumor levels and distribution of 
paclitaxel at 20 hours after first dose and patient-specific differences in peripheral 
non-tumor tissue (skin or plasma) paclitaxel levels 20 ± 4 hours after first dose. 
 
1.2.2. To determine if paclitaxel levels are higher at 20h after the 3rd dose than after the 
first dose, and if levels are higher at 20h after the 12th dose than the 1st and 3rd 
dose. 
 
1.2.3. Compare pre-existing versus post-treatment antimitotic effects at 20h after the 1st 
dose, 20h after the 3rd dose, and 20h after the 12th dose. 
 
1.2.4. Correlate drug levels and distribution with biomarkers including mitotic index, 
aneuploidy, chromosomal instability, and Ki67.  
 
1.2.5. Correlate pathologic response and clinical response with biomarkers including 
mitotic index, aneuploidy, CIN and Ki67. 
 
1.2.6. To test if CIN increases in patient tumors in response to paclitaxel and to evaluate 
the feasibility of these measurements by genomic analysis. 
 
 
2. BACKGROUND 
 
2.1 
Study Disease 
 
Breast cancer is the most common cancer in women.  According to the 2005 
Surveillance Epidemiology and End Result (SEER) database, patients with 
locoregional spread have a 5-year survival rate of 83.6%1.  In this patient population, 
chemotherapy remains a cornerstone of treatment for breast cancer, and taxanes remain 
one of the most effective agents.  Several studies have demonstrated response rate to 
taxanes in the first line metastatic setting is 35-50%2.  Based on these results, taxanes 
have been incorporated into adjuvant breast cancer regimens.  For node positive cancer, 
addition of 4 cycles of paclitaxel 175mg/m2 q3 weeks after standard ACx4 (adriamycin 
and cyclophosphamide) improves survival3.  When delivered every two weeks with 
GCSF support, there is a better outcome and lower risk of neutropenic fever4.  Recently, 
investigators have suggested a potential benefit to sequential therapy with taxanes prior 
to AC.  Either sequence is tolerable but the dose intensity of chemotherapy is higher 
when taxanes are delivered first5.  Two studies of neoadjuvant taxane demonstrated an 

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increased rate of pathologic complete response (pCR) when taxane was given prior to 
an anthracycline-based chemotherapy rather than after6.   
Despite this success, a large number of patients do not respond to taxane therapy.  For 
example, the pCR rate for neoadjuvant paclitaxel does not exceed 35% even when 
combined with radiotherapy7.  Additionally, neuropathy and myelosuppression remain 
significant toxicities.  If there were a manner to predict the response to taxane therapy, 
patients with low likelihood of response could undergo alternative treatment and 
potentially avoid these toxicities.  Therefore, improved biomarkers of taxane response 
are needed for patient selection and personalization of optimal cancer therapy. 
 
2.2 Rationale for Correlative Studies  
 
2.2.1 Taxanes effect on mitosis.  Taxanes are microtubule stabilizers first identified as 
natural products from Taxus brevifolia.  Paclitaxel (Taxol®), the first member of 
this class, binds to β-tubulin and blocks microtubule depolymerization.  In the 
classic model, stabilized microtubules interfere with kinetochore-microtubule 
attachments, thereby activating the mitotic checkpoint, leading to mitotic delay and 
cell death.  Recent evidence suggests that paclitaxel can induce cell death through 
two distinct mechanisms, depending on the intracellular concentration of drug8,9.  
In one mechanism, long term mitotic delay leads to cell death directly from mitosis 
or after cells exit mitosis without division to form a single tetraploid cell.  Tumors 
with a strong mitotic checkpoint are expected to be sensitive to this mechanism, 
while tumors in which the mitotic checkpoint is impaired are expected to be 
resistant.  In the second mechanism, cells missegregate chromosomes as they exit 
mitosis, often generating more than two daughter cells.  This mechanism is 
predicted to cause cell death due to massive chromosome loss.   Aneuploid tumors 
are expected to be particularly sensitive to this mechanism.  However, these cell 
culture findings have not yet been verified in patient samples.  While patients are 
given a standard dose of paclitaxel based on previously established safety data from 
phase I trials, there is little available data on the concentration of paclitaxel within 
breast tumors following treatment.  Paclitaxel levels have been previously assessed 
following treatment in brain tumors10 but this is expected to differ significantly 
from extra-CNS tissue levels in part because of the blood-brain barrier.  Given the 
importance of intracellular concentration on the effect of paclitaxel, identification 
of the relative concentration in vivo would assist in development of in vitro breast 
cancer models using this medication.   
 
2.2.2    The Mitotic Checkpoint.  Mitosis is the critical event in the cell cycle when 
duplicated chromosomes are accurately segregated into daughter cells.  During 
mitosis, accurate segregation of sister pairs to daughter cells requires their 
attachment to opposite mitotic poles via microtubules.  This is achieved via 
assembly of a protein-rich structure at the centromere, known as the kinetochore.  
Because accurate segregation is crucial to maintaining genomic integrity, cells have 
evolved a ‘mitotic checkpoint’ which ensures that each chromosome sister pair is 
attached to the spindle, and bi-oriented (i.e. each member of a pair is attached to 
opposite pole).  If a single chromosome pair is unattached or is not bi-oriented, 
kinetochores generate a ‘wait signal,’ thereby activating the checkpoint.  This wait 

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signal requires a number of protein components including Mad1, Mad2, Bub1, 
BubR1, Bub3, and CENP-E. 
 
2.2.3 Biomarkers of taxane response.  Several investigators have sought to identify 
predictors of response to taxane therapy.  In an unbiased screen, gene expression 
arrays were performed from tumor samples prior to neoadjuvant chemotherapy 
with weekly paclitaxel followed by FAC (5-fluorouracil, doxorubicin and 
cyclophosphamide).  The gene that correlated best with pCR was Tau, which had 
previously been identified as a microtubule stabilizer11.  The authors sought to 
validate these findings through a retrospective analysis of 1,942 arrayed tissue 
specimens from NSABP B-28 (AC vs. AC followed by paclitaxel) and found that 
Tau expression by immunohistochemistry does not predict benefit from adding 
paclitaxel11.  Recent work by this group has sought to integrate expression data with 
genes responsible for paclitaxel resistance identified through an RNAi screen, but 
this is not yet validated12,13.   
 
Other investigators have evaluated cell-based predictors of response to neoadjuvant 
paclitaxel and radiotherapy7.  Surprisingly, pCR was not related to baseline Ki-67 
staining, a marker of cell cycle and proliferation.  However, response correlated 
well with a high fraction of mitotic cells per field at biopsy 24-72h after the first 
dose of paclitaxel.  Interpretation of these data is clouded because all studies include 
additional chemotherapy or radiotherapy prior to response assessment, and the 
analysis of mitotic index occurred over a large window of time after paclitaxel 
administration (24-72h).  In this study we would eliminate the variables of 
additional chemotherapy and variations based on time of analysis. 
 
     2.3        Hypothesis:  Our hypothesis is that paclitaxel levels increase chromosomal instability  
(CIN) in tumors and that this is lethal to tumors that have pre-existing CIN.   
 
3. PATIENT SELECTION 
 
3.1 
Eligibility Criteria 
 
3.1.1 Women with pathologically demonstrated breast cancer.  
 
3.1.2 Patients must be candidates for neoadjuvant paclitaxel chemotherapy by their 
treating oncologist.  No other investigational or commercial therapeutic agents 
may be given concurrently with the paclitaxel.   
 
 
3.1.3 Patients must not have metastatic disease on staging work-up with CBC and liver 
function studies. 
 
3.1.4 A formalin-fixed paraffin embedded tumor block (preferred) or unstained slides 
must be available from a prior biopsy of the primary tumor or lymph node.  A 
minimum of 8 slides must be available. 
 

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3.1.5 The primary tumor or lymph node must be readily biopsied by surgery or 
radiology teams. 
 
3.1.6 The primary tumor must be measurable by an imaging modality prior to 
treatment.  This imaging modality is to be repeated after completion of 4 cycles 
of paclitaxel and prior to surgery.  Such imaging modalities may include 
ultrasound, CT, mammography, or MRI.  MRI will be the preferred imaging 
modality if available because it has the highest accuracy and positive predictive 
value for predicting pathologic complete response14.All imaging will be 
performed per standard of care at the discretion of the treating physicians. 
 
3.1.7 Subjects may not have had prior systemic chemotherapy regimens administered 
for treatment of their current breast cancer.  However, studies (window studies, 
for example) that are deemed non-therapeutic, including those that utilize agents 
that are not FDA approved for the treatment of the patient’s current breast 
cancer, are permitted. 
 
3.1.8 Age >18 years.  Breast cancer is rare in women < 18 years old.  The safety of 
paclitaxel in pediatric population with breast cancer has not been established, 
therefore these patients are ineligible.   
 
3.1.9 Patients must have adequate organ and marrow function as determined by the 
treating oncologist. 
 
3.1.10  Patient must be willing to undergo additional biopsy of breast tumor or lymph 
node. 
 
3.1.11  Patient must have the ability and willingness to sign a written informed consent 
document. 
 
3.1.12 Women of childbearing potential (per UWCCC policy definition) must agree to 
use effective contraception as discussed with treating oncologist for the duration 
of the study. 
 
3.2  
Exclusion Criteria 
 
3.2.1 History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to paclitaxel including to other drugs formulated in 
Cremophor(R) EL (polyoxyethylated castor oil). 
 
3.2.2 Patients with known HIV due to concern that chemotherapy may cause further 
immunosuppression and potential infectious complications. 
 
3.2.3 Patients on non-aspirin anti-coagulation (Coumadin, heparins, or clopidogrel) or 
with documented bleeding disorders will be excluded due to risk of bleeding with 
biopsy.   

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3.2.4 Uncontrolled intercurrent illness including, but not limited to, ongoing or active 
severe infection, symptomatic congestive heart failure, unstable angina pectoris, 
cardiac arrhythmia, other malignancies requiring therapy or psychiatric 
illness/social situations that would limit compliance with study requirements. 
 
3.2.5 Pregnant women are excluded from this study because paclitaxel is a pregnancy 
category D drug and may cause deleterious effects to the fetus.  Because there is 
an unknown but potential risk for adverse events in nursing infants secondary to 
treatment of the mother with paclitaxel, breastfeeding should be discontinued if 
the mother is enrolled in the trial.   
 
3.3 
Inclusion of Minorities 
 
Women of all races and ethnic groups are eligible for this trial.  Men will be excluded 
from this study because they may have different tumor biology than women and may 
metabolize paclitaxel differently than women.   
 
4. REGISTRATION PROCEDURES 
 
4.1 
General Guidelines 
 
Eligible patients will be entered on study using the University of Wisconsin Carbone 
Cancer Center Oncore Database.  
 
In accordance with institutional policies approved by the U.S. Department of Health 
and Human Services, each subject must acknowledge consent for treatment as a 
human subject in this study.  Informed consent must be obtained prior to the initiation 
of protocol therapy. 
 
Following registration, patients should begin protocol treatment within 14 days.  
Issues that would cause treatment delays must be discussed with the Principal 
Investigator. If a patient does not receive protocol therapy following registration, the 
patient’s registration on the study may be canceled.  The Study Coordinator must be 
notified of cancellations as soon as possible. 
 
4.2 
Registration Process 
 
To register a patient, the following documents should be completed by the research 
nurse or data manager and maintained in the subject study chart: 
 
• Copy of required eligibility test results. 
• Signed patient consent form. 
• Signed HIPAA authorization form. 
 
The research coordinator at the site will then register the subject into the Oncore 
database prior to starting study treatment. Oncore will assign the unique subject 
number. 

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There will not be access to the Oncore database registration until documented IRB  
 
 
 
approval is obtained and entered into the Oncore database.   
 
5. TREATMENT PLAN 
 
5.1 Administration 
 
Treatment will be administered on an outpatient basis.  Reported adverse events and 
potential risks are described in Section 8.  No investigational or commercial agents or 
therapies other than those described below may be administered with the intent to 
treat the patient’s malignancy. 
 
Paclitaxel (Taxol®) 80 mg/m2 will be initiated as standard infusion on days 1, 8, 15 
of a 21-day cycle.   Patients will continue with paclitaxel 80 mg/m2 for cycles 2-4 
prior to surgery.  Dose modifications, delays in therapy, and administration of growth 
factors for all treatment will be left to the discretion of the treating physician.  
Modifications to the treatment regimen and reason for changes will be recorded for 
potential correlation with drug levels.   
 
5.2 Timing of surgery 
   
We anticipate that definitive breast surgery will be performed after completion of the 
final cycle of paclitaxel.  Patients will be allowed to receive additional neoadjuvant 
chemotherapy, such as AC, prior to surgery at the discretion of the treating physician, 
if deemed in the best interest of the patient.  This chemotherapy must be initiated after 
completion of paclitaxel and imaging of the tumor to assess response to paclitaxel.  
Surgery is anticipated to be performed 3-6 weeks after completion of chemotherapy.  
However, surgery may be delayed or performed earlier at the discretion of the treating 
physicians.  These modifications will not disqualify the patient from analysis in this 
protocol or result in removal of the patient from study.  The timing of definitive 
surgery relative to treatment will be recorded. 
 
5.3  Data Acquisition and Management 
Data elements will be extracted from subject medical records after informed consent 
and HIPAA authorization have been obtained. Data elements to be collected and 
maintained by the UWCCC OnCore database system and/or the researcher records 
include the following: 
 
• Subject name and MRN (recorded in OnCore for subject registration purposes 
ONLY) 
• Automated subject ID number (generated by OnCore) 
• Verification of inclusion and exclusion criteria 
• Demographic information of age, weight, height, race and ethnicity, history of prior 
cancer, family history of breast or ovarian cancer, menopausal status 
• Mammographic breast density, specific mammographic or ultrasound abnormality, 
pathologic diagnosis including cancer type with estrogen, progesterone and HER-

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2/neu status or particular benign diagnosis, tumor size, grade and stage, lymph node 
status, Ki67 score 
• BRCA1/2 mutation status, if known 
• Neoadjuvant therapy regimen received 
                   • Response to neoadjuvant therapy 
 
In order to maintain subject privacy, all research samples released from the BioBank 
will be coded with a unique identifier that can be linked to an individual only by a 
key maintained in the secure Oncore database. A link between the unique sample 
code to the MRN and subject name will be retained for the purposes of associating 
pathology data and subject outcomes with the functional effects of the subject-derived 
epithelial and stromal cells.  All personal identifiers, including subject name, date of 
birth, address, and date of study clinic visit will be removed from researcher records 
to preserve individual confidentiality.  This information will be kept by the principal 
investigator in a locked computer file and in the secure Oncore database. Coded data 
elements lacking identifiers will be stored separately on UWSMPH Department of 
Medicine workstations and researcher laptops. Study data elements will be shared 
exclusively with the investigators on this protocol; the key to the subject codes will 
not be shared. 
 
5.4 Sample Collection for Laboratory Analysis 
 
5.4.1 15 ml Blood will be drawn prior to chemotherapy to provide a sample of non-
tumorous control cells for genomic, histological, and paclitaxel concentration 
analysis.  This blood will be drawn into purple-top EDTA tubes and delivered to 
the University of Wisconsin 3P Lab for analysis.  
 
5.4.2 Archived tissue from a prior biopsy of the primary tumor (or lymph node) will be 
obtained and used for biomarker and genomic analysis.  One FFPE block, or 10 
unstained slides will be obtained from archived tissue. 
 
5.4.3 Tumor core biopsies will be obtained with goal timing as close as possible to 20 
hours following initiation of the 1st,  3rd, and 12th paclitaxel infusions. The biopsy 
after the 1st dose of paclitaxel is required; the biopsy after the 3rd and 12th dose of 
paclitaxel (C1D16  and C4D16, respectively )  are optional. Patients who have a 
biopsy after the 3rd dose of paclitaxel are not required to get a biopsy after the 12th 
dose, and similarly, patients who do not have the biopsy after the 3rd dose may 
still choose to undergo the biopsy after the 12th dose. . To achieve this, the UW 
Breast investigators will arrange to deliver the dose of chemotherapy on a Mon-
Thursday between 12 and 1 PM, seeing an enrolled patient in urgent clinic if 
needed.  Patients will have an appointment for breast biopsy at 8AM the following 
day so a breast biopsy will be able to be performed within 18-22h window, 
although we will actually attempt to perform this as close as possible to 20h post 
the start of paclitaxel administration.  The breast biopsy may be performed with or 
without image guidance.  The biopsy may be performed either by a coinvestigator 
or an interventional radiologist credentialed in breast biopsy. Time from initiation 
of paclitaxel to biopsy will be recorded. A total of 4 biopsy cores will be taken at 

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each biopsy time-point. Tissue (2 cores) will be flash frozen in liquid nitrogen and 
stored at -80°C or liquid nitrogen in the 3P laboratory until paclitaxel level 
measurements are performed. Additional samples (2 cores) will be fixed in 
formalin for analysis of biomarkers.  
 
For subjects consenting to the optional tumor biopsies after the 3rd and 12th 
paclitaxel doses, the investigator will make the final determination as to which 
subjects will undergo the biopsy. The investigator may use the following criteria 
to make this determination: tumor size, tumor location and treatment received.  
 
An optional skin biopsy (3-4 mm) will be obtained from consenting subjects as 
close as possible to 20 ± 4 hours following initiation of the 1st paclitaxel infusion. 
The skin biopsy may be performed by an appropriately credentialed medical 
provider on the protocol. Time from initiation of paclitaxel to skin biopsy will be 
recorded. Tissue will be flash frozen in liquid nitrogen and stored at -80°C or 
liquid nitrogen in the 3P laboratory until paclitaxel level measurements are 
performed.  
 
 
5.4.4 If excess residual tumor is present at time of surgery, a sample will be obtained 
for biomarker and genomic analysis.  One FFPE block, or 10 unstained slides, 
will be requested after pathologic review is complete for this analysis. 
 
5.5       Laboratory analysis 
 
5.5.1 Both tumor tissue and plasma samples taken at 20 hours will be assessed for 
paclitaxel levels by the 3P lab as previously described9. Tumor paclitaxel 
distribution will be measured by MALDI-TOF by the Li lab.  A blood sample (15 
mL drawn into purple-top EDTA tubes) will be drawn as close to the 20 hour 
timepoint as possible.  Time of blood draw and tissue biopsies will be recorded 
for analysis purposes. 
 
5.5.2 All tissue samples, including archival pretreatment biopsy (see 3.1.4) will be 
assessed for feasibility analysis of biomarkers including: 
 
5.5.2.1   Markers of proliferation:  Using H&E stained slides, the number of mitotic 
cells and interphase nuclei will be quantified.  Typically, mitotic index is 
measured as the ratio of mitotic cells over total cells.  Additional stains will be 
used to characterize the fraction and spindle morphology of mitotic cells.  For 
example, tissue will be analyzed by immunofluorescence of cells stained with 
DAPI, phospho-H3, alpha-tubulin, and Mad2.  Ki67 stain will be performed as 
a marker of proliferation. 
 
5.5.2.2    Aneuploidy:  To assay for aneuploidy in a parsimonious manner, we will 
use multicentromeric single-color fluorescence in-situ hybridization (FISH).  
For example, using probes specific for centromeres of chromosomes 1, 5 and 
19, we expect to see 6 signals per diploid cell.  Aneuploidy is determined from 

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fixed specimens at pretreatment, ~20h post 1st, 3rd, and 12th treatments, and at 
the time of surgery if there is sufficient residual tumor. 
 
5.5.2.3 Sequencing analysis: For patients who have multiple biopsies, assessments 
of fixed specimens (tumor, skin, blood) at pretreatment, ~20h post 1st, 3rd , and 
12th  treatments, and, at the time of surgery if there is sufficient residual tumor, 
will be performed using techniques such as SNP-CHIP and sequencing.  
Genome wide analysis will provide alternative measures for aneuploidy and 
CIN and will provide information about genetic alterations in paclitaxel-
responsive and paclitaxel unresponsive tumors. 
 
5.6      Future Studies 
 
Additional blood and tissue specimens remaining will be stored for future studies 
related to cancer research. All future projects using the banked data or samples from 
this study will be submitted to the Institutional Review Board (IRB) for review and 
approval or exemption.  Distribution of banked samples will be limited to researchers 
within UW-Madison.  Request for samples would be directed to the study PI (Dr. 
Burkard) who will determine if the banked samples would be suitable for use in that 
study.  Prior to distribution, scientific and IRB approval of the research study would 
need to be provided to the Dr. Burkard.   
 
After study-related testing is complete, the blood and tissue samples from subjects 
will be stored in the Burkard Lab. These samples will be kept until they are exhausted 
or for a maximum of 10 years, whichever comes first. Subjects will be permitted to 
withdraw their samples from the study or the bank. They may withdraw their samples 
by informing the study PI. 
To protect the confidentiality of subjects in this study, the blood and tissue samples 
will be coded. The key will be saved in computer files that are protected via 
passwords and access rights. The key will include: subject name, medical record 
number, procedure dates or pathology accession number (biopsy and surgery), and 
the sample identification codes (one per subject per sample). For subjects who are 
deemed eligible and are registered to the trial their subject ID number will also be 
listed on the key. The rest of the research data will also be saved in secure computer 
files or in the OnCore database which is only accessible via username, password, and 
access rights. 
 
After subjects sign the informed consent form, the blood and tissue samples will be 
obtained by the research staff and coded prior to the samples being provided to the 
Burkard lab. The Burkard lab is only accessible to individuals involved with the 
research completed in that lab. 
 
5.7       Duration of Follow Up 
 
Patients will be followed until time of surgery at which time all tissue and plasma 
sample collection will have been collected and their response to neoadjuvant 
chemotherapy will be assessed. 

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Subjects may receive additional systemic therapy (following paclitaxel) prior to 
planned surgery, at the discretion of the treating physician.  Adverse events that occur 
during this period, up until surgery, will be recorded. 
 
5.8       Criteria for Removal from Study 
 
Patients will be removed from the study if they withdraw consent.  If the core biopsy 
on day 2 is not able to be obtained or the patient is not able to receive the 1st complete 
dose of paclitaxel, they will be removed from the study.  If they are not able to 
complete the study due to any reason after day 2, their initial serum and tissue will 
still be analyzed.   
 
6. DOSING DELAYS/DOSE MODIFICATIONS  
 
Dosing delays and dose modifications will be at the discretion of the treating physician.  
Such adjustments will be recorded for potential correlation to biomarkers. 
 
7. PHARMACEUTICAL INFORMATION 
 
7.1 Adverse events and potential risks (Micromedex®).  The adverse events and potential 
risks associated with paclitaxel (Taxol®) include:  
 
7.1.1 Common  
• Dermatologic: Alopecia (55% to 96%) 
• Hematologic: Anemia, any grade (47% to 96%), leucopenia (90%), 
neutropenia, any grade (78% to 100%), thrombocytopenia, any grade (4% to 
68%) 
• Gastrointestinal: Diarrhea (16% to 90%), inflammatory disease of mucous 
membrane (5% to 45%), nausea and vomiting, any grade (4% to 88%) 
• Immunologic: Immune hypersensitivity reaction, any grade (2% to 45%) 
• Musculoskeletal: Arthralgia, myalgia 
• Neurologic: Peripheral neuropathy, any grade (42% to 79%) 
 
7.1.1 Serious 
• Cardiovascular: Atrial fibrillation (rare), Cardiac dysrhythmia (less than 
1%), Cardiotoxicity, Congestive heart failure, Myocardial infarction (rare), 
Supraventricular tachycardia (rare) 
• Gastrointestinal: Gastrointestinal perforation, Nausea and vomiting, Grade 3 
or greater (10% to 29%) 
• Hematologic: Anemia, Grade 3 or greater (2% to 34%), Deep venous 
thrombosis, Febrile neutropenia (2% to 55%), Neutropenia, Grade 4 (14% to 
81%), Thrombocytopenia, Grade 3 or greater (1% to 17% 
• Immunologic: Anaphylaxis, Immune hypersensitivity reaction, Grade 3 or 
greater (up to 4%), Opportunistic infection (up to 76%), Sepsis 
• Neurologic: Grand mal seizure (less than 1%), Peripheral neuropathy, Grade 

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3 or greater (up to 10%), Seizure 
• Respiratory: Pulmonary embolism 
 
7.2 Availability 
 
Paclitaxel (Taxol®) is an FDA approved medication for treatment of curable breast 
cancer and is available by prescription at pharmacies throughout the United States. 
 
 
 
8 ADVERSE EVENTS  
8.1 
Only adverse events related to the research biopsies and research only blood draws 
will be collected. CTCAE term (AE description) and grade:  The descriptions and 
grading scales found in the revised NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 4.03 will be utilized for AE assessment.  A copy of the 
CTCAE version 4.03 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov). 
 
8.2 
Attribution of the AE: 
Definite 
 
The AE is clearly related to the study procedures. 
Probable 
 
The AE is likely related to the study procedures. 
Possible 
 
The AE may be related to the study procedures. 
Unlikely 
 
The AE is doubtfully related to the study procedures. 
            Unrelated 
      The AE is clearly NOT related to the study procedures. 
 
8.3 
Expected Adverse Events resulting from the administration of paclitaxel, any other 
systemic therapy and surgical intervention will not be reported. Unanticipated serious 
adverse events will be reported as outlined in sections 9 and 10.  
 
9 OVERSIGHT AND MONITORING PLAN 
The UWCCC Data and Safety Monitoring Committee (DSMC) is responsible for 
the regular review and monitoring of all ongoing clinical research in the 
UWCCC.  A summary of DSMC activities are as follows: 
 
• Reviews all clinical trials conducted at the UWCCC for subject safety, protocol 
compliance, and data integrity. 
• Reviews all Serious Adverse Events (SAE) requiring expedited reporting, as 
defined in the protocol, for all clinical trials conducted at the UWCCC, and 
studies conducted at external sites for which UWCCC acts as an oversight body. 
• Reviews all reports generated through the UWCCC DSMS elements (Internal 
Audits, Quality Assurance Reviews, Response Reviews, Compliance Reviews, 
and Protocol Summary Reports) described in Section 9.1 of this document. 
• Notifies the protocol Principal Investigator of DSMC decisions and, if applicable, 
any requirements for corrective action related to data or safety issues. 
• Notifies the CRC of DSMC decisions and any correspondence from the DSMC to 
the protocol Principal Investigator. 

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• Works in conjunction with the UW Health Sciences IRB in the review of relevant 
safety information as well as protocol deviations, non-compliance, and 
unanticipated problems reported by the UWCCC research staff.  
• Ensures that notification is of SAEs requiring expedited reporting is provided to 
external sites participating in multi-institutional clinical trials coordinated by the 
UWCCC. 
 
 
9.1 
      Monitoring and Reporting Guidelines 
 
UWCCC quality assurance and monitoring activities are determined by study sponsorship 
and risk level of the protocol as determined by the PRMC.  All protocols (including 
Intervention Trials, Non-Intervention Trials, Behavioral and Nutritional Studies, and trials 
conducted under a Training Grant) are evaluated by the PRMC at the time of committee 
review.  UWCC monitoring requirements for trials without an acceptable external DSMB are 
as follows: 
      Intermediate Monitoring 
Protocols subject to intermediate monitoring generally include UW Institutional 
Phase I/II and Phase II Trials. These protocols undergo review of subject safety at 
regularly scheduled DOWG meetings where the results of each subject’s treatment 
are discussed and the discussion is documented in the DOWG meeting minutes. The 
discussion includes the number of subjects enrolled, significant toxicities, dose 
adjustments, and responses observed. Protocol Summary Reports are submitted on a 
semi-annual basis by the study team for review by the DSMC. 
 
9.2 
Review and Oversight Requirements    
 
9.2.1 Serious Adverse Event – Reported Within 24 Hours 
Serious Adverse Events requiring reporting within 24 hours (as 
described in the protocol) must also be reported to the Data and Safety 
Monitoring Committee (DSMC) Chair via an email to 
saenotify@uwcarbone.wisc.edu within one business day.  The OnCore 
SAE Details Report must be submitted along with other report 
materials as appropriate (NCI AdEERS form or FDA Medwatch Form 
#3500 and/or any other documentation available at that time of initial 
reporting). The DSMC Chair will review the information and 
determine if immediate action is required. Within 5 calendar days, all 
available subsequent SAE documentation must be submitted 
electronically along with a 24 hour follow-up SAE Details Report and 
a completed UWCCC SAE Routing Form to 
saenotify@uwcarbone.wisc.edu. All information is entered and tracked 
in the UWCCC database. 

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The Principal Investigator notifies all investigators involved with the 
study at the UWCCC, the IRB, the sponsor, and the funding agency 
and provides documentation of these notifications to the DSMC. 
If the SAE occurs on a clinical trial in which the UW PI serves as the 
sponsor-investigator, the PI reviews the event to determine whether the 
SAE requires reporting to the FDA and other participating 
investigators.   
For a multiple-institutional clinical trial the PI is responsible for 
ensuring SAEs are reported to the FDA as well as to all participating 
investigators. 
 
See Section 10 for detailed instructions on SAE reporting. 
 
9.2.2 Serious Adverse Event – Reported within 10 Days 
Serious Adverse Events requiring reporting within 10 calendar days 
(as described in the protocol) must also be reported to the Data and 
Safety Monitoring Committee (DSMC) Chair via an email to 
saenotify@uwcarbone.wisc.edu.  The OnCore SAE Details Report 
must be submitted along with other report materials as appropriate 
(NCI AdEERS form or FDA Medwatch Form #3500 and/or any other 
documentation available at that time of initial reporting). The DSMC 
Chair will review the information and determine if further action is 
required. All information is entered and tracked in the UWCCC 
database. 
The Principal Investigator notifies all investigators involved with the 
study at the UWCCC, the IRB, the sponsor, and the funding agency 
and provides documentation of these notifications to the DSMC. 
If the SAE occurs on a clinical trial in which the UW PI serves as the 
sponsor-investigator, the PI reviews the event to determine whether the 
SAE requires reporting to the FDA and other participating 
investigators.   
For a multiple-institutional clinical trial the PI is responsible for 
ensuring SAEs are reported to the FDA as well as to all participating 
investigators. 
 
See Section 10 for detailed instructions on SAE reporting. 
 
9.2.3 Study Progress Review 
Protocol Summary Reports (PSR) are required to be submitted to the 
DSMC in the timeframe determined by the risk level of the study 
(quarterly; semi-annually; or annually). The PSR provides a 
cumulative report of SAEs, as well as instances of non-compliance, 

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protocol deviations, and unanticipated problems, toxicities and 
responses that have occurred on the protocol in the timeframe 
specified.  PSRs for those protocols scheduled for review are reviewed 
at each DSMC meeting. 
Protocol Summary Reports enable DSMC committee members to 
assess whether significant benefits or risks are occurring that would 
warrant study suspension or closure. This information is evaluated by 
the DSMC in conjunction with other reports of quality assurance 
activities (e.g., reports from Internal Audits, Quality Assurance 
Reviews, etc.) occurring since the prior review of the protocol by the 
DSMC.  Additionally, the DSMC requires the study team to submit 
external DSMB or DSMC reports, external monitoring findings for 
industry-sponsored studies, and any other pertinent study-related 
information.  
In the event that there is significant risk warranting study suspension 
or closure, the DSMC will notify the PI of the DSMC findings and 
ensure the appropriate action is taken for the protocol (e.g., suspension 
or closure).  The DSMC ensures that the PI reports any temporary or 
permanent suspension of a clinical trial to the sponsor (e.g., NCI 
Program Director, Industry Sponsor Medical Monitor, Cooperative 
Group Study Chair, etc.) and other appropriate agencies. DSMC 
findings and requirements for follow-up action are submitted to the 
CRC. 
 
9.2.4 Expedited Reporting of Serious Adverse Events 
 
Depending on the nature, severity, and attribution of the serious 
adverse event an SAE report will be phoned in, submitted in writing, 
or both according to Table 9.2.6 below. Serious adverse events must 
also be reported to the UW IRB according to their reporting 
guidelines. 
 
Determine the reporting timeframe for the SAE in question by using 
the following table.   
 
 
 
 
 
 
 
 
 
 
 
 

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9.2.5 
Expedited Reporting Table 
 
Reporting Requirements for Serious Adverse Events 
NOTE: Investigators MUST immediately report to the UWCCC DSMC and any other parties outlined in the 
protocol ANY Serious Adverse Events, that are related to the research procedure (post paclitaxel biopsy) (21 
CFR 312.64). 
An adverse event is considered serious if it results in ANY of the following outcomes: 
1) Death. 
2) A life-threatening adverse event. 
3) An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization 
for ≥ 24 hours. 
4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
5) A congenital anomaly/birth defect. 
6) Important Medical Events (IME) that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they may 
jeopardize the patient or subject and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria* MUST be immediately reported to the 
UWCCC within the timeframes detailed in the table below: 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 
Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
hospitalization  ≥ 
24 hrs 
10 Calendar Days 
24-Hour; 5 
Calendar Days 
Not resulting in 
Hospitalization ≥ 
24 hrs 
Not required 
10 Calendar Days 
* [research team: Only adverse events related to the research procedure (post paclitaxel biopsy) 
require expedited reporting.  ] 
 
Expedited AE reporting timelines are defined as: 
• 
24-Hour; 5 Calendar Days – The AE must initially be reported within 24 hours of learning of the 
AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report. 
• 
10 Calendar Days – A complete expedited report on the AE must be submitted within 10 
calendar days of learning of the AE 
 
 
10 SAE Reporting 
 
10.1  SAE Requiring [24] Hour Reporting Occurs at UWCCC: 
 
             A.  Report to the UWCCC: 
 
Reference the SAE SOP (Standard Operating Procedure) and the SAE 
Reporting Workflow for DOTs on the UWCCC website 
(http://www.uwccc.wisc.edu) for specific instructions on how and what 
to report to the UWCCC for [24] hour initial and follow-up reports.  A 
follow-up report is required to be submitted within 5 calendar days 
of the initial [24] hour report.   

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For this protocol, the following UWCCC entities are required to be 
notified: 
 
a) saenotify@uwcarbone.wisc.edu 
b) Study PI: Mark Burkard, MD, PhD 
c) Disease Oriented Team Manager: Tammy Koehn 
d) Any other appropriate parties listed on the SAE Routing Form (for 
follow-up reports only) 
 
B.  Report to the IRB: 
 
       
 
Consult the UW-IRB website for reporting guidelines.   
      
10.2 
SAE Requiring [10] Day Reporting Occurs at UWCCC:  
 
A.  Report to the UWCCC: 
 
Reference the SAE SOP and the SAE Reporting Workflow for DOTs 
on the UWCCC website (http://www.uwccc.wisc.edu) for specific 
instructions on how and what to report to the UWCCC for [10] day 
reports.   
 
                                    For this protocol, the following entities are required to be notified: 
 
a) saenotify@uwcarbone.wisc.edu 
b) Any appropriate parties listed on SAE Routing Form 
 
B.  Report to the IRB: 
 
       
 
Consult the UW-IRB website for reporting guidelines. 
  
C.  Report to the FDA: 
 
                                    The treating physician and/or PI may report unexpected serious adverse  
                                    events to the FDA using the voluntary reporting form (FDA Form 3500)  
                                    at their discretion. 
 
 
 
 
 
 
 
 
 

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11 STUDY CALENDAR 
 
Test/Assessment 
Prior to 
Initiation of 
Paclitaxel  
Cycle 
1, day 
2 
Cycle 1 
Day 162 
 
 
 
 
Cycle 
2-4, 
day 1 
After 
completion 
of 
paclitaxel2  
Surgery  
History and Physical 
x 
 
 
 
x 
 
x 
ECOG PS 
x 
 
 
 
x 
 
 
Pregnancy Test 
Will be 
confirmed if 
ran by 
treating 
physician 
 
 
 
 
 
 
AST, ALT, CBC 
Per 
oncologist 
 
 
 
x 
 
 
Blood sample for genomics and 
paclitaxel levels 
x 
 
 
 
 
 
 
Tumor/serum Paclitaxel Level 
and Distribution 
 
x 
x 
 
 
x 
 
Optional skin biopsy3 
 
x 
 
 
 
 
 
Toxicity Evaluation1 
x 
 
 
 
x 
 
 
Archived FFPE sample 
x* 
 
 
 
 
 
x 
Fresh Biopsy or tumor sampling 
for analysis—4 cores (CIN, 
mitotic index, etc.)2 
 
x 
x 
 
 
x 
 
Pathologic Evaluation for 
Response 
 
 
 
 
 
 
x 
Imaging Evaluation per 12.1 
x 
 
 
 
 
x 
 
* Slides obtained from prior biopsy of tumor may be obtained at any point after subject signs 
consent. 
1. Only toxicities related to research only biopsies and blood draws will be collected and 
recorded. 
2.C1D16 and C4D16  are optional and collections are only completed if patient consents. 
3. Skin biopsy is optional and collection is only completed if patient consents.  
 
12 MEASUREMENT OF EFFECT 
 
12.1 Methods for Evaluation  
 
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.  Follow-up evaluations should be performed prior to surgery. 
The same method of assessment and the same technique (MRI, ultrasound, CT, 
mammography, or clinical exam) should be used to characterize each identified and 

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reported lesion at baseline and prior to surgery. MRI will be preferentially used for 
evaluation when medically reasonable.  Imaging will be performed upon 
completion of paclitaxel chemotherapy, prior to any additional chemotherapy. 
 
12.2 
Response Criteria- Pathologic  
 
For the purposes of this study, patients should be evaluated for a pathologic response 
once at time of surgery.  A pathologic complete response (pCR) will be defined as the 
absence of residual invasive cancer within both the breast and lymph nodes.  In 
addition, pathologic evidence of significant tumor cell reduction will be noted. 
 
12.3 
Response Criteria- Clinical Response 
 
The clinical response will be determined similar to RECIST 1.1 criteria,15 modified to 
allow measurements of breast tumors by MRI, ultrasound, or mammogram as 
previously described16 
 
13. STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
 
We will treat CIN as a continuous variable by 6-chromosome FISH as the average % 
of cells that deviate from the modal number for each of the 6 chromosome FISH 
signals.  Response to paclitaxel will also be considered as a continuous variable 
where response is determined by the % decrease in linear measurement of tumor size 
on the greatest dimension per RECIST-like criteria (as per 12.3).  We will correlate 
Taxol response with CIN.    Using alpha=0.05, 1-beta=80% and power our sample to 
distinguish the correlation coefficient of r = 0.6 from r=0 (null hypothesis). Thus, we 
estimate the study will require 19 patients.   We anticipate dropout or other technical 
issue will affect data interpretation of at least one patient so we will enroll 20 
subjects.  Results will be interpreted in the context of other studies that evaluate the 
correlation of taxane sensitivity with CIN measured by FISH. 
 
 
13.2 
Sample Size/Accrual Rate 
 
The planned sample size is 20 patients, with accrual expected ~10 per year.  The 
study is expected to accrue all patients within 2-3 years.  The study will be terminated 
at the time when all patients have completed operative management and data 
collection using patient identifiers is complete.  Subjects who are deemed unevaluable 
due to inadequate tissue or any other reason will be replaced. 

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13.4 Analysis of Secondary Endpoints 
 
 
 
Secondary endpoints such as mitotic index, aneuploidy, and paclitaxel levels will be 
analyzed by fluorescence in situ hybridization, immunofluorescence, 
immunohistochemistry, and liquid chromatography as previously described16-18..  
More specifically for each secondary endpoint:  
 
13.4.1 To identify patient-specific differences in tumor/non-tumor levels and tissue 
distribution of paclitaxel at 20 hours after first dose. 
Tissue paclitaxel levels are measured by high-performance liquid chromatography 
(HPLC)of tumor and plasma samples and comparing variability between patients 
with descriptive statistics. Paclitaxel tissue distribution is measured by matrix-
assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-
TOF MS) to compare variability in distribution patterns between patients with 
descriptive statistics.  
 
13.4.2 To determine if paclitaxel levels are higher at 20h after the 3rd dose than after the 
first dose, and if levels are higher at 20h after the 12th dose than the 1st and 3rd 
dose.  
This is measured by high-performance liquid chromatography of tumor and 
plasma samples and comparing difference at two time points within the same 
patient with paired statistics.  
 
13.4.3 Compare pre-existing versus post-treatment antimitotic effects at 20h after the 1st 
dose and 20h after the 3rd and 12th dose. 
This is measured by tissue analysis of tumor samples as described previously 16 
with phospho-histone H3 and stains for spindle morphology to quantify mitotic 
index and mitotic characteristics at two time points using paired statistical 
analysis. 
 
13.4.4 Correlate drug levels and tissue distribution with biomarkers including mitotic 
index, aneuploidy, chromosomal instability, and Ki67.  
 
13.4.5 Correlate pathologic response and clinical response with biomarkers including 
mitotic index, aneuploidy, CIN and Ki67. 
 
Measurements of aneuploidy, CIN, and Ki67 will be performed as previously 
described 18.  These correlations will be considered preliminary and results will be 
interpreted in the context of other studies being performed in metastatic breast 
cancer. 
 
13.4.6 To test if CIN increases in patient tumors in response to paclitaxel and to evaluate 
the feasibility of these measurements by genomic analysis. 
 
Multiple genomic analyses have been proposed to measure CIN and aneuploidy in 
tumors.  These include whole genome sequencing with high depth, SNP-array 
analysis, Comparative Genomic Hybridization and others.  However, this is 

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rapidly changing field with new technologies and analytic methods emerging 
weekly.  We anticipate performing high-depth whole genome sequencing from 
three independent core samples per biopsy as a pilot analysis.  Based on our 
experience with initial samples, and on available technologies and analytic 
methods available at the time of analysis, we may select another genomic 
sequencing and/or hybridization methods to optimally determine the changes in 
CIN.  Fluorescence-in-situ hybridization will be used as a companion method as 
described18. 
 
REFERENCES: 
 
1. 
Surveillance Epidemiology and End Results (SEER). Available from: 
http://seer.cancer.gov/. 
2. 
Gradishar WJ, Krasnojon D, Cheporov S, et al. Significantly longer progression-free 
survival with nab-paclitaxel compared with docetaxel as first-line therapy for metastatic breast 
cancer. J Clin Oncol. 2009;27(22):3611-3619. 
3. 
Henderson IC, Berry DA, Demetri GD, et al. Improved outcomes from adding sequential 
Paclitaxel but not from escalating Doxorubicin dose in an adjuvant chemotherapy regimen for 
patients with node-positive primary breast cancer. J Clin Oncol. 2003;21(6):976-983. 
4. 
Citron ML, Berry DA, Cirrincione C, et al. Randomized trial of dose-dense versus 
conventionally scheduled and sequential versus concurrent combination chemotherapy as 
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Intergroup Trial C9741/Cancer and Leukemia Group B Trial 9741. J Clin Oncol. 
2003;21(8):1431-1439. 
5. 
Puhalla S, Mrozek E, Young D, et al. Randomized phase II adjuvant trial of dose-dense 
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Wildiers H, Forceville K, Paridaens R, Joensuu H. Taxanes and anthracyclines in early 
breast cancer: which first? Lancet Oncol;11(3):219-220. 
7. 
Chakravarthy AB, Kelley MC, McLaren B, et al. Neoadjuvant concurrent paclitaxel and 
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8. 
Brito DA, Rieder CL. The ability to survive mitosis in the presence of microtubule 
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cells. Cell Motil Cytoskeleton. 2009;66(8):437-447. 
9. 
Yang Z, Kenny AE, Brito DA, Rieder CL. Cells satisfy the mitotic checkpoint in Taxol, 
and do so faster in concentrations that stabilize syntelic attachments. J Cell Biol. 
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10. 
Fine RL, Chen J, Balmaceda C, et al. Randomized study of paclitaxel and tamoxifen 
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Clin Cancer Res. 2006;12(19):5770-5776. 
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Pusztai L, Jeong JH, Gong Y, et al. Evaluation of microtubule-associated protein-Tau 
expression as a prognostic and predictive marker in the NSABP-B 28 randomized clinical trial. J 
Clin Oncol. 2009;27(26):4287-4292. 
12. 
Juul N, Szallasi Z, Eklund AC, et al. Assessment of an RNA interference screen-derived 
mitotic and ceramide pathway metagene as a predictor of response to neoadjuvant paclitaxel for 
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13. 
Swanton C, Marani M, Pardo O, et al. Regulators of mitotic arrest and ceramide 
metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. 
Cancer Cell. 2007;11(6):498-512. 
14. 
Croshaw R, Shapiro-Wright H, Svensson E, Erb K, Julian T. Accuracy of clinical 
examination, digital mammogram, ultrasound, and MRI in determining postneoadjuvant 
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15. 
Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid 
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Zasadil LM, Andersen KA, Yeum D, et al. Cytotoxicity of paclitaxel in breast cancer is 
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Choudhary A, Zachek B, Lera RF, et al. Identification of selective lead compounds for 
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Denu RA, Zasadil LM, Kanugh C, Laffin J, Weaver BA, Burkard ME. Centrosome 
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