Prospective Tissue Collection in Breast Cancer Patients Receiving Preoperative Systemic Therapy
Phase: NA
Conditions: Stage IIA Breast Cancer, Stage IIB Breast Cancer, Stage IIIA Breast Cancer, Stage IIIB Breast Cancer, Stage IIIC Breast Cancer, Stage IV Breast Cancer
Sponsor: Albert Einstein College of Medicine

Title: Chemotherapy Before Surgery and Tissue Sample 
Collection in Patients with Stage IIA-IIIC Breast Cancer
IRB ID:  13-02-079
NCT01897441
IRB Approval Date:  05/11/2021

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Title: Prospective tissue collection in breast cancer patients receiving preoperative systemic therapy
Coordinating Center:
Albert Einstein Cancer Center Clinical Trials Office (CTO)
Montefiore Medical Center-Weiler Division
1825 Eastchester Rd., 2South, Rm 60, Bronx, NY 10461
Project Coordinator: Karen Fehn, RN, Phone 718-405-8446, 718-Fax 405-8230 
Email: kfehn@montefiore.org
Principal Investigator:
Jesus Anampa-Mesias, MD
Montefiore Medical Center-Einstein Campus
  
Department of Medical Oncology, 2nd floor
1695 Eastchester Road, Bronx, New York 10461
Phone 718-405-8404; Email: janampa@montefiore.org
Co-principal Investigators: 
Della Makower, MD
Montefiore Medical Center-Einstein Campus
  
Department of Medical Oncology, 2nd floor
1695 Eastchester Road, Bronx, New York 10461
Phone 718-405-8404; Email: dmakower@montefiore.org
Sun Young Oh, MD
Montefiore Medical Center-Einstein Campus
  
Department of Medical Oncology, 2nd floor
1695 Eastchester Road, Bronx, New York 10461
Phone 718-405-8404; Email: suyoung@montefiore.org
Susan Fineberg, MD, 
Montefiore Medical Center- Moses Division
Department of Pathology
111 East 210th Street, Central 313,  Bronx, NY 10467
Phone 718-920-4976; Email: sfineber@montefiore.org
Kathleen Whitney, MD
Montefiore Medical Center-Einstein Campus
  
Department of Pathology
1825 Eastchester Road, Bronx, New York 10461
Phone 718.904.2947; Email: kwhitney@montefiore.org
Maja Oktay, MD, PhD
Montefiore Medical Center- Moses Division
Department of Pathology
111 East 210th Street, Central 313, Bronx, NY 10467
Phone 718-920-6091; Email: moktay@montefiore.org
                                                  
Cristina Montagna, PhD
                                                  
Albert Einstein College of Medicine
                                                
Department of Genetics and Pathology R401
                                                      
1301 Morris Park Ave., Bronx, NY 10461
                                                      
Phone: 718 678 1158; Email: cristina.montagna@einstein.yu.edu
Protocol statistician:                   Nan Xue, PhD
                                                      Albert Einstein College of Medicine
                                                      Division of Biostatistics
                                                      Phone: 718-430-2431; Email: xiaonan.xue@einstein.yu.edu
Summary of Protocol Changes :
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
Amendement 1 (Version 4.1 1/9/17) : (1) Cover page: removed Dr. Montgomery, Koenigsberg, 
McDaid and co-investigators; (2) Schema and section 4.1: eliminate pre-randomization, all patients receive 
sequential pacitaxel-AC (unless physician chooses AC-paclitaxel sequence), (3) Schema and section 4: 
trastuzumab (+/- pertuzumab) plus carboplatin/docetaxel or paclitaxel added as a treatment option for 
HER2-positive disease to reflect a new standard of care option, additional stratum added (stratum III) to 
include neaodjuvant endocrine therapy (4) Schema and section 5.1 : prior version allowed FNA or FNA 
followed by core biopsy, which was more made more explicitly clear in current protocol version and 
consent,  and reduced optional core biopsies from 5 to 3, (5) consolidated background section and study 
objectives, (6) statistical section and accrual goal modified, (6) deleted Appendix A (RCB staging) and B 
(AJCC staging), as these are considered part of standard care 
 
TABLE OF CONTENTS
SCHEMA....................................................................................................................................................4
1.
HYPOTHESIS & OBJECTIVES .....................................................................................................5
2.  BACKGROUND...................................................................................................................................5
3.
PATIENT SELECTION....................................................................................................................9
4.
MANAGEMENT PLAN..................................................................................................................10
5.
SPECIMEN COLLECTION AND BIOPSY PROCEDURES.....................................................12
6. 
CORRELATIVE STUDIES............................................................................................................14
7.
STUDY CALENDAR.......................................................................................................................17
8.
MEASUREMENT OF EFFECT.....................................................................................................18
9.
REGULATORY AND REPORTING REQUIREMENTS...........................................................18
10. DATA SAFETY AND MONITORING BOARDS .........................................................................19
11. STATISTICAL CONSIDERATIONS............................................................................................19
12. REFERENCES: .................................................................................................................................22
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SCHEMA
 
 
Breast Cancer

Eligible for neoadjuvant chemotherapy or endocrine treatment.

Meet protocol eligibility criteria

Provide written informed consent
Stratum I:
HER2-Negative Tumor
Chemotherapy Indicated

Blood sample - pretreatment

Fine needle aspiration of tumor -  before treatment and 2-3 weeks after treatment begins

Core needle biopsy (optional) - before treatment and 2-3 weeks after treatment begins
Surgery After Completion of Neoadjuvant Chemotherapy or Endocrine Therapy
 Tissue collection and banking
Standard post protocol care
Stratum II:
HER2-Positive Tumor
Chemotherapy Indicated
Stratum III:
ER-Pos, HER2-Neg Tumor
Endocrine Therapy Indicated
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1.
HYPOTHESIS & OBJECTIVES
We hypothesize that the chemotherapy combinations/agents that are commonly used to treat breast cancer 
(including doxorubicin/cyclophosphamide, paclitaxel trastuzumab, pertuzumab, carboplatin/docetaxel) 
will have different biological outcomes on breast cancer cell fate; specifically cell death versus senescence. 
Accelerated senescence is a potent tumor suppressive response that halts the proliferation of cancer cells 
following treatment with cytotoxic chemotherapy. Our hypothesis predicts that persistent senescent cells 
and or / senescent reverted cells drive disease progression, and/or epithelial to mesenchymal transition 
(EMT). This is mediated via the pro-inflammatory activity of the senescence-associated secretory 
phenotype (SASP), a unique property of senescent cells and their resistant counterparts. Thus, the presence 
of these cells within a tumor mass likely contributes to disease progression, drug resistance and metastasis, 
ultimately leading to death. 
We propose to evaluate the effects of commonly used standard chemotherapy regimens on the 
propensity for senescence, and as a consequence EMT; and to create a biorepository for future studies. 
Objectives: 
(1) To evaluate the effects of preoperative neoadjuvant chemotherapy (+/- anti-HER2 therapy) on 
invasive Mena isoforms (ie Menainv, MenaCalc), TMEM score, and cancer stem cells
(2) To create a biospecimen repository for future studies derived from patients with breast cancer 
receiving standard neoadjuvant chemotherapy. 
2.  BACKGROUND
a) Preoperative Neoadjuvant Chemotherapy 
A metanalysis of 9 randomized trials including 3496 patients with operable breast cancer has 
shown comparable survival rates whether chemotherapy was given preoperatively (neoadjuvant) or 
postoperatively (adjuvant)1.  Patients with operable breast cancer typically undergo surgery first followed 
by adjuvant chemotherapy, but neoadjuvant chemotherapy may be indicated in certain clinical scenarios, 
including: (1) locally advanced inoperable breast cancer (eg, inflammatory disease, large primary tumor, 
bulky adenopathy) in which breast conservation is not an option without therapeutic downstaging induced 
by systemic therapy, (2) operable disease in which downstaging with systemic therapy may facilitate beast 
conservation when it would otherwise might not have been feasible.2-4 The goal of neoadjuvant 
chemotherapy is to induce a pathologic complete response (pCR) in the breast or breast and lymph nodes, 
which has been shown to be a short-term surrogate indicative of long-term cure. 2-4 In fact, the U.S. Food 
and Drug Administration has recently issued draft guidance recognized the value of pCR in the breast and 
lymph nodes after neoadjuvant chemotherapy as an acceptable endpoint in clinical trials supporting 
accelerated drug approval of a new agent/regimen. 5  
b) Type and Sequence of Preoperative Chemotherapy Regimens
 As previously stated, the goal of neoadjuvant chemotherapy is to induce a pCR. Approximately 
10% of patients with operable breast cancer achieve breast pCR  after 4 cycles of neoadjuvant AC 
(doxorubicin 60 mg/m2, cyclophosphamide 600 mg/m2) given every 3 weeks.6 A subsequent trial (B27) 
that compared preoperative sequential AC x 4 followed by docetaxel (100 mg/m2 every 3 weeks x 4) to 
AC alone x 4 demonstrated a significant improvement in pCR from 14% to 26%. 7  Other studies have 
shown that weekly paclitaxel therapy (80 mg/m2 weekly x 12) to be associated with a higher pCR rate in 
the breast and lymph  nodes compared with paclitaxel given every 3 weeks (225 mg/m2 x 4)  sequentially 
with an anthracycline-cyclophosphamide combination 8, including all  patients treated (28.2% 15.7%; P = 
.02), and patients with both ER-negative disease (48% vs. 23%) and ER-positive disease (22% vs. 11%).  
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The addition of HER2-directed therapy (ie, trastuzumab) to chemotherapy has been shown to further 
improve pCR rate.9 Sequential AC-taxane therapy (plus trastuzumab for HER2-positive disease) has 
therefore become an accepted standard of care for patients who are candidates for both postoperative 
adjuvant chemotherapy and preoperative neoadjuvant chemotherapy.
Evidence suggests that the paclitaxel-AC sequence of drug administration may be more effective 
than the standard sequence of AC-taxane therapy. Antiangiogenic therapy improves blood flow in tumors 
that exhibit disordered vascular beds, thereby improving drug delivery of cytotoxic agents.10 Taxanes have 
potent angiogenic effects11, and may also improve tumor blood flow by reducing tumor-associated vascular 
compression.12  Preoperative paclitaxel more effectively reduced interstitial fluid pressure (IFP) in primary 
breast cancers than AC in a study which included 54 patients with breast cancer treated with neoadjuvant 
AC or weekly paclitaxel.13 Weekly paclitaxel, when administered first, decreased the mean IFP by 36% (P 
= .02) and improved the tumor pO(2) by almost 100% (P = .003). In contrast, doxorubicin did not have a 
significant effect as measured by ultrasound. These changes were independent of the tumor response to 
both drugs as evaluated by ultrasound, and the study was not powered to demonstrate a significant 
difference in clinical outcome based on the sequencing of drugs.  Recent studies in mouse models suggested 
that doxorubicin downregulates the surface programmed cell death 1 ligand 1 (PD-L1) in certain breast 
cancer cell types, which is a co-inhibitory molecule for cytotoxic T cells. Thus, it is hypothesized that this 
that might at least partially increased immunogenic apoptotic death.14 In contrast, in MDA-MB-468 breast 
cancer cell culture models, paclitaxel promotes PD-L1 mediated T cell apoptosis. These findings reveal a 
potential link between taxane chemotherapy and immunoresistance.15 Although it remains unknown 
whether the efficacy of anthracycline and paclitaxel combinations is affected by their sequence of 
administration, one phase III trial demonstrated a significantly higher pCR rate when the taxane followed 
by an anthracycline/cyclophosphamide-containing regimen was compared with the reverse sequence (20% 
vs. 15%, p=0.03), suggesting that the reverse sequence (taxane AC) may be somewhat more effective 
than the standard AC  taxane sequence. 8 In addition, a neoadjuvant randomized phase III trial (Neo-
tAnGo) in women with high-risk early breast cancer demonstrated a significant advantage in pCR (20% 
vs15%) with the sequence of paclitaxel+/-gemcitabine compared with the more conventional anthracycline  
first sequencing.16 Of note, the benefit was higher for the ER positive group. Moreover, a recent  
restrospective analysis  of the clinical outcome 
of the two sequences  in a large number of 
patients treated with neoadjuvant therapy 
reported that the sequence of  paclitaxel-
anthracycline compared to the antracycline-
paclitaxel is associated with lower risk of 
relapse.17 
Therefore, 
although 
either 
sequence of drug administration, (AC  
taxane sequence, or taxane AC sequence), 
are considered acceptable regimens for 
neoadjuvant chemotherapy treatment, there 
is tentative evidence that supports superior 
‘biological’ and clinical activity for taxane 
AC regimen.
c) TMEM as a measure of Cancer 
Cell 
Invasion 
and 
Interaction 
with 
Microenvironment
Highly invasive and motile cells are capable of 
metastasizing, 
non-proliferating 
and 
chemotherapy resistant. A multidisciplinary 
team of scientists from the Condeelis 
laboratory at Einstein and clinicians at 
 Figure 1: Migration of discohesive tumor cells leads to 
intravasation in mammary tumors. Tumor cell migration 
is believed to involve the tumor microenvironment-
initiated decreased expression of the epithelial isoform of 
Mena, Mena11a, and increased expression of MenaINV 
which promotes discohesion of the tumor and paracrine-
dependent tumor cell migration (bracket), as well as 
TMEM assembly leading to intravasation (box).
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Montefiore have shown that only a subset of tumor cells is capable of metastasizing and that these cells 
show reduced response to chemotherapy and express a distinct set of genes and their splice variants.18 Using 
multiphoton-based intra-vital imaging in mouse and rat mammary tumors the team identified a 
subpopulation of cancer cells that is discohesive and migratory (shown in bracket in Figure 1) and 
demonstrated that these discohesive, migratory tumor cells intravasate when associated with peri-vascular 
macrophages. 19-22. In addition, an in vivo invasion assay developed by Condeelis and Goswami has 
facilitated collection of invasive tumor cells from mammary tumors of rats, mice, and humans for 
expression profiling 23,24Many of the epigenetic changes in these cells are clustered in the motility pathways 
that control actin polymerization, directional cell movement toward EGF, and invadopodium formation 
25,26. The Mena family of actin binding proteins functions at the convergence of these motility pathways 
implying that Mena expression levels reflects the activity of motility pathways involved in the 
migratory/metastatic potential of cancer cells. Thus, Mena expression level is used as a summary marker 
for the motility pathways 24,26-28. Mena promotes actin polymerization by enhancing the activation of cofilin 
via increasing the sensitivity of the EGFR to EGF, interfering with the activity of inhibitory capping 
proteins and increasing actin filament elongation rates 26,29. This activity is essential for sustained directional 
cell movement in response to growth factors like EGF. Mena knockout mice crossbred with PyMT-
oncogene carrying mice develop dramatically less metastatic mammary tumors 30. Furthermore, the survival 
of these animals was prolonged due to the almost complete absence of hematogenous dissemination of 
tumor cells from the primary tumor 30. Breast carcinoma cells express three Mena isoforms: Menaclassic, 
MenaINV and Mena11a. Menaclassic contains only the constitutive exons, while the two splice variants 
MenaINV or Mena 11a contain the alternately-included exons INV or 11a, respectively. Mena is up regulated 
in rat, mouse and human mammary tumors 19. The Condeelis laboratory discovered that the observed 
increase of Mena expression in invasive and disseminating tumor cells reflects increased expression of both 
Menaclassic and MenaINV, and correlates with decreased Mena 11a expression relative to non-invasive, non-
intravasating tumor cells within the primary mammary tumor 19. They demonstrated that expression of 
MenaINV helps increase invasiveness of tumor cells in vitro and in vivo, and metastatic activity in mammary 
tumors in vivo by increasing the sensitivity of the EGFR to EGF by 50 fold which increases paracrine 
signaling between macrophages and tumor cells (Fig. 1, bracket) 29,31. It is believed that Menaclassic co-
oligomerizes with MenaINV to form a Menaclassic/MenaINV hetero-tetramer so that they are co-localized and 
function together 19,26,32. On the other hand, Mena 11a is associated with epithelial growth 33 and epithelial 
integrity 34 in breast tumors. Thus cancer cells overexpressing Menaclassic/MenaINV hetero-tetramer may be 
essential for intravasation step of metastasis. 
Dr. Oktay  has identified the discohesive migratory, MenaINV expressing cell population shown in 
the bracket in Figure 1 in fine needle aspirate (FNA) samples from human invasive ductal carcinomas of 
the breast and shown that the level of MenaINV expression correlates with the number of macrophage 
assisted cancer cell intravasation sites called TMEM (“tumor microenvironment of metastasis”, box in 
Figure 1) 35. As mentioned above, TMEM intravsation sites have been identified in mouse by intravital 
imaging. They have been also identified in humans by triple immunostain using anti CD-31 antibodies for 
endothelial cells, anti-CD68 for macrophages and anti-Mena for cancer cells. Furthermore, TMEM score 
has been shown to correlate with the development of distant metastases in humans independent of other 
clinicopathologic features 36. Interestingly, the induction of cell death by cytotoxic agents such as 
doxorubicin is much lower in invasive migratory cell population than in the cells isolated from the whole 
tumor profiling 23,24, suggesting that Mena isoform expression and TMEM status can identify patients 
who are not good candidate for chemotherapy because they have high proportion of invasive, metastatic 
chemoresistant cells. Immunohistochemical (TMEM score) and cytological (Mena isoform expression) 
assays developed by Drs. Condeelis, Oktay and Goswami  can be easily incorporated into current clinical 
practice and used to identify patients with high risk of metastasis. 
d) Rationale for amdendment 1 (1/9/17): The protocol was modified to eliminate 
prerandomization for drug sequence (paclitaxel – AC vs. AC – paclitaxel) for patients with HER2-negative 
disease, as paclitaxel –AC sequence is an accepted standard, and to reflect changing standard of care for 
HER2-positive disease (TCH-P).  Based on preliminary data generated from the first 14 patients enrolled 
indicating that chemotherapy induces expression of invasive Mena isoforms (comparing pre-treatment 
FNA/biopsy with during treatment FNA/biopsy) and TMEM score (comparing pre-treatment biopsy and 
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post-treatment residual cancer), we now have focused the aims of this protocol on obtaining additional 
information regarding the effect of chemotherapy on invasive Mena isoforms, TMEM score, and cancer 
stem cells, and to clarify that patients who agree to only FNA (but decline core biopsy) are eligible.  
3.
PATIENT SELECTION
3.1
Eligibility Criteria
3.1.1    Patients must have histologically confirmed adenocarcinoma of the breast associated 
with the following clinical stage: IIA, IIB, IIIA, IIIB, or IIIC (see AJCC staging 
criteria, 7h Edition). Patients with stage IV disease are also eligible if there is an 
intention to perform breast surgery after neoadjuvant therapy is completed, or in 
patients participating in clinical trials where surgery after neoadjuvant therapy may be 
an option. 
3.1.2    ER, PR, and HER2/neu status documented by core needle biopsy of the primary tumor 
and/or regional lymph node must be known prior to beginning systemic therapy. 
3.1.3    Patients must have had a bilateral diagnostic mammogram within 6 months of 
registration, and may also have a targeted sonography of the breast and/or ipsilateral 
axilla and MRI if clinically indicated. 
3.1.4     Patients with clinically suspicious axillary lymph node involvement must have either 
aspiration cytology or biopsy prior to beginning therapy. 
3.1.5     It is strongly encouraged that all patients have metallic clips placed in the tumor prior 
to neoadjuvant therapy in order to facilitate evaluation for microscopic disease at the 
time of surgery; placement of clips is particularly encouraged for patients being 
considered for breast conserving surgery. 
3.1.6     No prior chemotherapy, irradiation, or definitive therapeutic surgery (eg, mastectomy 
or lumpectomy or axillary dissection) for this malignancy. Patients who have had a 
prior sentinel lymph node biopsy for this malignancy are eligible. 
3.1.7     Patients who received tamoxifen or another selective estrogen receptor modulator 
(SERM) for prevention or for other indications (e.g., osteoporosis, prior DCIS) are 
eligible. Tamoxifen therapy or other SERMs should be discontinued at least 1 week 
before the patient is enrolled on this study. 
3.1.8     Age >18 years.  Because breast carcinoma is a disease of adults that rarely occurs in 
children, children are excluded from this study. 
3.1.9     The patient is medically suitable candidate for preoperative chemotherapy and surgery 
in the judgment of the treating physicians.  
3.1.10 Ability to understand and the willingness to sign a written informed consent document, 
and to willing to provide blood samples before and during preoperative therapy. 
Patients are also asked but not required to have research biopsies performed before and 
after therapy.  
4.
MANAGEMENT PLAN
4.1
Treatment 
Note:..All drugs doses are based on actual body weight, and are regarded as standard of care. See 
section 5.2 for criteria for dose modification/delay. 
4.1.1 Paclitaxel alone (Stratum I) or plus Trastuzumab +/- Pertuzumab (Stratum II) 

Paclitaxel (Stratum I or II ): 80 mg/m2 IV infusion over 1 hour weekly x 12 consecutive 
weeks. Paclitaxel premedication: administer dexamethasone 10 mg IV prior to the first 
paclitaxel dose. If no reaction after the first dose administer dexamethasone 4 mg IV (or 
lower doses if well tolerated without reaction), diphenhydramine 25-50 mg IV, and an H2 
blocker (cimetidine 300 mg, ranitidine 20 mg, or an equivalent) approximately 30-60 
minutes prior to each paclitaxel dose. Proceed with each weekly dose of paclitaxel (+/- 
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trastuzumab +/- pertuzumab) if:

Absolute neutrophil count > 1000/uL and platelets > 50,000/uL

No grade 3-4 toxicity, or prohibitive grade 2 toxicity

No evidence of clinical cardiac dysfunction

Trastuzumab (+/- pertuzumab) plus paclitaxel (Stratum II- HER2-positive disease 
only): Trastuzumab 8 mg/kg IV loading dose over 90 minutes, then 6 mg/kg every 3 weeks 
(after paclitaxel when given concurrently) for a total of 17 doses (51 weeks) To be 
administered after paclitaxel.  Pertuzumab (840 mg IV loading dose cycle 1, then 420 mg 
every 3 weeks for 6 cycles prior to surgery) may also be used at the discretion of the treating 
physician.

TCH+P or TCH - docetaxel/carboplatin, trastuzumab +/-  pertuzumab (Stratum II: 
Docetaxel 75 mg/m2 and carboplatin AUC 6 (Calvert formula) every 3 weeks for 6 cycles, 
plus ,trastuzumab (8 mg/kg loading dose cycle 1, then 6 mg/kg every 3 weeks) for up to 17 
doses before and after surgery (51 weeks), and pertuzumab (840 mg IV loading dose cycle 
1, then 420 mg every 3 weeks for 6 cycles prior to surgery). Patients to receive standard 
antiemetic premedication, plus dexamethasone 8 mg PO BID x 3 days beginning 1 day 
prior to each cycle. Pertuzumab may be omitted at discretion of treating physician.  Repeat 
every 3 weeks if:

Absolute neutrophil count > 1000/uL and platelets > 50,000/uL

No grade 3-4 toxicity, or prohibitive grade 2 toxicity

No evidence of clinical cardiac dysfunction

Granulocyte colony stimulating factor (G-CSF; filgrastim): G-CSF should not be used 
during paclitaxel unless: (1) treatment with paclitaxel is delayed due to neutropenia, (2) the 
patient has a serious or life-threatening documented or suspected infection associated with 
neutropenia (absolute neutrophil count [ANC]< 1000/mm3), or (3) or another complication 
that in the judgment of the treating physician could derive potential benefit from G-CSF. 
The drug should be used at a dose/schedule specified in the package insert. Pegfilgrastim 
should not be used during paclitaxel therapy. Pegfilgrastim (6 mg) should be given after 
each AC dose if given on an every 2 week schedule, or after TCH+/-P at discretion of 
treating physicians. 
4.1.2
Doxorubicin and Cyclophosphamide (AC) – Stratum I or II

Doxorubicin 60 mg/m2 IV over 5-10 minutes, Cyclophosphamide 600 mg/m2 IV 
infusion over 30-60 minutes. To be given 3very 2 weeks x 4 cycles. When given 
after paclitaxel, the first cycle should be initiated 3 weeks after the last paclitaxel 
and trastuzumab dose, or 2 weeks after the last paclitaxel dose when used without 
trastuzumab.  For patients in stratum II, AC may be omitted at discretion of treating 
physician depending upon clinical response to therapy, toxicity, or other factors, 
in accordance with standard clinical practice. 

Premedication: Administer antiemetic therapy in accordance with NCCI guidelines.

Pegfilgrastim 6 mg SC on day 2 of each cycle

Repeat every 2 weeks x 4 cycles if: (a) absolute neutrophil count > 1500/uL, platelets 
> 100,000/uL, (b) recovered from non-hematologic toxicity to grade 1 or less (except 
alopecia)
4.13
Neoadjuvant Endocrine Therapy – Stratum III
Patients with ER-positive, HER2-negative disease may receive neoadjuvant endocrine 
therapy with an aromatase inhibitor (anastrozole 1 mg po daily, letrozole 2.5 mg daily, or 
exemestane 25 mg po daily) for 4-6 months prior to surgery (or longer if clinically indicated). 
4.1.4
Chemotherapy Dose Modifications (StrataI & II)

Proceed with each dose of chemotherapy if:

Absolute neutrophil count > 1000/uL and platelets > 50,000/uL (> 100,000/uL for AC)
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
No persistent grade 3-4 toxicity, or prohibitive grade 2 toxicity

No evidence of clinical cardiac dysfunction

Patients who experience the toxicities outlined below should have the chemotherapy dose 
modified according to the criteria outlined in the table below. 
Toxicity
Paclitaxel or AC
Grade 3 or 4 febrile neutropenia (fever > 38.5C 
and ANC < 1,000/mm3):
Reduce 1 dose level
Grade 3 or 4 non-hematologic toxicity 
Reduce 1 dose level
Grade 2 non-hematologic toxicity
Reduce 1 dose level if poorly tolerated and 
attributable to therapy
Dose Reduction
Paclitaxel 
Dose
AC Dose
Docetaxel 
Dose
Carboplatin 
Dose
Initial Dose
80 mg /m2
A-60 mg/m2; C-
600 mg/m2
75 mg/m2
AUC6
First Reduction
Reduce 25% - 
60 mg/m2
Reduce 25% 
(A45, C450 
mg/m2)
60 mg/m2
AUC4.5
Second Reduction
Reduce 50% - 
45 mg/m2
Reduce 50% 
(A30, C300 
mg/m2)
45 mg/m2
AUC3
 
4.1.5
Surgery 
Patients will be reassessed for surgery following completion of chemotherapy or endocrine 
therapy.  Primary surgery may consist of a modified radical mastectomy, radical 
mastectomy, segmental mastectomy or lumpectomy with an axillary lymph node 
dissection, or sentinel lymph node biopsy without further axillary dissection if the sentinel 
node biopsy is negative if deemed medically appropriate. The goal of surgery is to 
completely debulk the tumor and achieve tumor free margins. The local pathologist should 
carefully evaluate the specimen for microscopic residual disease, including searching for 
the prior biopsy tract and/or metallic clips placed preoperatively. The surgery should be 
performed about 4 weeks (range of 2-6 weeks) after the last chemotherapy dose  unless it 
needs to be delayed because of persistent adverse events or other medical problems. 
4.1.5
Post-Protocol Therapy 
The following portions of the treatment are recommended after surgery in accordance with 
the standard of care: 

Adjuvant chest wall/breast irradiation if standard indications met (eg, if tumor > 5 cm, 
inflammatory carcinoma, 4 or more positive axillary nodes, or 1-3 positive nodes with 
extranodal extension or large metastasis)

Adjuvant hormonal therapy for a minimum of 5 years (for patients with ER and/or PR-
positive disease)

Postoperative trastuzumab (for up to 51 weeks, or up to 17 doses of trastuzumab given 
before and after surgery (if HER2-positive). 
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5.
SPECIMEN COLLECTION AND BIOPSY PROCEDURES 
5.1. Tumor Fine Needle Aspiration +/- Core Biopsies 
Consented patients will undergo serial tumor sampling  and blood specimen collection at:

Baseline  (before treatment initiation)

2-3 weeks after the first chemotherapy dose
o
Stratum I: 2 weeks after the first chemotherapy dose, (ie, just before the 3rd planned 
paclitaxel dose) 
o
Stratum II: 2 weeks after first chemotherapy dose (ie, just before the 3rd planned paclitaxel 
dose if paclitaxel dose) or 3 weeks after first chemotherapy dose (if carboplatin/docetaxel 
used

At the time of definitive surgery (residual tumor/ tumor bed - section 7.0).
Biospecimens will be collected and the study conducted in accordance with the recommendations of 
the NCI-BIG Breast Cancer Intergroup37,38. 
Procedures for collection and processing specimens will be stipulated in detail in a Standard Operation 
Procedure Manual developed specifically for this protocol. A recent study evaluated the aliquots of fresh 
primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNA later at 
room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline 
and 40 minutes thereafter.39 Although sample preservation in RNAlater was associated with improved RNA 
yield and quality, whereas cold ischemia increased RNA fragmentation as measured by the 3'/5' expression 
ratio of control genes, expression levels of single genes and multigene signatures that are of diagnostic 
relevance in breast cancer were mostly unaffected by sample preservation method, or prolonged cold 
ischemic duration. This indicates that for  
The following guidelines apply to sampling and specimen processing:

All patients will have fine needle biopsy (FNA) procedure), sonographically guided if necessary. 
Patients may elect to have: (1) FNA only, (2) FNA plus core biopsies

The procedure may be billed to the insurance company if performed to confirm/establish a 
diagnosis or biomarkers (e.g., ER, PR, HER2) at baseline, but not in other circumstances (e.g. 
during treatment biopsy). 

Five FNA passes (all patients) followed  3 core biopsies (if patient agrees) will be obtained with 
25 and 12 gauge needles respectively. The specimens should be processed a follows:
o
FNA: immediately placed in cytology solution
o
1st core biopsy (if done): 10% formalin (to confirm histologic presence of tumor) 
o
2rd core biopsy (if done): RNA Later (for gene expression studies in future). RNAlater® 
samples should be shipped on a cold pack. For more information on RNAlater, see 
http://www.ambion.com/techlib/tn/114/6.html 
o
3rd : Snap frozen 
5.2 Blood Specimens

Draw peripheral blood using vacutainer into two Acid Citrate Dextrose (ACD tubes (yellow 
top, draw volume 6 ml/tube), then gently invert tube 8-10 times at baseline (before treatment).

Label tube with patient ID number 

Place specimen on cool pack deliver specimen and form to Einstein Biorepository on same 
day. 

Samples will be processed in the Einstein biorepository for storage of peripheral blood 
mononuclear cells (PBMC’s) and plasma. 
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6. 
CORRELATIVE STUDIES
6.1 TMEM 
TMEM stain is a triple immunostain for predicting metastatic risk in which 3 antibodies are applied 
sequentially, and developed separately with different chromogens on a Bond Max Autostainer as described 
previously 36. TMEM is defined as the direct apposition of an endothelial cell, a perivascular macrophage, 
and a Mena expressing tumor cell. The antibodies used are CD31 (endothelial cell), CD68 (macrophage) 
and pan Mena (intravasation competent tumor cell) 36.  A pan-Mena mouse monoclonal (A351F7D9) will 
be used to identify Mena positive tumor cells. The assessment of TMEM scores will be performed using 
Adobe Photoshop on 10 contiguous 400x digital images of the most representative areas of the tumor. The 
total TMEM for each image will be tabulated, and the scores from all ten images will be summed to give a 
final TMEM density for each patient sample, expressed as the number of TMEM per 10 400x fields (total 
magnification). Two pathologists will score TMEM, including Dr. Oktay. TMEM score will be obtained 
on pre-treatment core biopsies and the post-treatment excision.
6.2 qRT-PCR for Mena isoform expression and Flow Cytometry for Cancer Stem Cells
PCR analyses will be performed using SyBR Green kit and ABI 7300 sequence detector. PCR primers used 
for the detection of Menaclassic, MenaINV and Mena 11a isoforms have been described by us previously 19. 
These primers do not recognize other Ena/VASP family members. Also, macrophages do not express Mena 
40. Therefore, PCR detects Mena levels in tumor cells only. PCR analysis will be performed on FNA 
samples obtained before treatment and on FNA samples obtained from surgical resections of chemoresistant 
tumors. The percentage of CD44high/CD24low breast cancer stem cells (BCSCs) by flow cytometry will be 
quantified. 
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7.
STUDY CALENDAR
Parameter
Specimen Collection Time Points
Correlative studies
Research Blood 
Samples 
o
Pretreatment 
 

Biospecimen banking
Research Tumor 
Samples
o
Pretreatment 
o
2-3 
weeks 
after 
first 
chemotherapy dose (section 
5.1) 

TMEM 
(from 
pretreatment 
diagnostic core biopsy, and week 2-
3 biopsy if done)

qPCR Mena isoform (FNA)

Cancer stem cells (FNA)
Tissue 
Collection 
at 
Surgery
 
o
At time of surgical procedure 

AJCC Y stage (routine pathlogy)

RCB Score (study pathologist)

TMEM, qPCR Mena isoform, and 
cancer stem cells in residual tumor if 
present 
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8.
MEASUREMENT OF EFFECT
Pathological Response
The pathologic response to treatment will be assessed by in all patients as follows:

 “y staging” denoting prior neoadjuvant chemotherapy using AJCC Y version 7.0, as 
described in the pathology synoptic report

 Residual Cancer Burden" will be assessed by one of the study pathologists as 
described 
(http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3). 41 
9.
REGULATORY AND REPORTING REQUIREMENTS
9.1 Registration and Data Reporting 

All patients must be screened for eligibility and meet all eligibility criteria before being 
offered participation in the trial

Patients who meet eligibility criteria for stratum A  may be consented for participation if 
the eligibility criteria are met.

Patients who meet the eligibility criteria for stratum B or C (HER2-negative disease, 
candidate for preoperative chemotherapy) must be pre-randomized first with the Cancer 
Clinical Trials Office and receive a stratum assignment to stratum B or C. After pre-
randomization, patients must be offered the trial and consented. 

Data and specimen collection will be collected using study specific case report form. 
9.2 Adverse Event Reporting

Adverse events will be reported in accordance with guidelines established by the Institutional 
Review 
Board 
(.http://www.einstein.yu.edu/docs/administration/institutional-review-
board/policies/AE-internal.pdf). All of the treatment given as a component of this protocol is 
consistent with standard of care. Only the biopsies are considered research-related procedures 
and require adverse event reporting, including: 

DEATH: The following must be reported: (a) deaths that occur while subject is on study 
treatment, or (b) deaths that occur within thirty 30 days of conclusion of study intervention.  
All deaths, whether or not anticipated or related to the protocol, must be reported to the 
designated IRB within 48 hours of the PI’s knowledge of the death. Within 10 business 
days of informing the designated IRB, the Principal Investigator is required to submit a 
completed Adverse Event Report to the designated IRB.

SERIOUS EVENT: A serious event is defined as (a) an adverse event, whether or not 
anticipated, that has the potential to cause significant impairment of health or death; or (b) an 
adverse event that prompts the Principal Investigator to suspend the research protocol, 
(temporarily or permanently) even if the risk is explicitly identified in the Consent Form; or (c) 
events that occur with a frequency or degree of severity greater than anticipated. (Examples of 
Serious Events are: disability, inpatient hospitalization, prolongation of hospitalization, 
congenital anomaly, laboratory anomalies critical to safety evaluations, and serious adverse 
drug reactions.) The following must be reported: (a) serious events that occur while subject is 
on study treatment, or (b) serious events that occur within thirty (30) days of conclusion of 
study intervention All reportable serious events must be reported to the designated IRB 
within 48 hours by phone, e-mail, or fax. Within 10 business days of initial notification to 
the designated IRB the Principal Investigator is required to submit a completed Adverse 
Event Report. 

NON-SERIOUS UNANTICIPATED EVENT: A reportable non-serious unanticipated event 
is an adverse event that is not explicitly identified in the consent form and/or in the 
investigator's brochure, does not pose a serious impairment to a subject's health, and is not life-
threatening. Non-Serious Unanticipated Events must be reported to the designated IRB 
within 30 days via submission of a completed Adverse Event Report. 
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10. DATA SAFETY AND MONITORING BOARDS
All trials initiated by the Montefiore Medical Center are subject to oversight by the Albert Einstein 
Cancer Center Data Safety Monitoring Board (DSMB).  This board meets once a month with any 
additional meetings scheduled when needed.  The responsibilities are as follows:

Familiarize themselves with the research protocol (s)

Review interim analyses of outcome data and cumulative toxicity data summaries to 
determine whether the trial should continue as originally designed, should be changed, or 
should be terminated based on these data.  

The Albert Einstein Cancer Center DSMB reviews trial performance information such as 
accrual information.  

The Albert Einstein Cancer Center DSMB also determines whether and to whom 
outcome results should be released prior to the reporting of study results.

All adverse events are reviewed by the committee with assurances that these have been in 
fact sent for review to all pertinent IRBs.

Review of reports of related studies to determine whether the monitored study needs to be 
changed or terminated.

Review major proposed modifications to the study prior to their implementation (e.g., 
termination, dropping an arm based on toxicity results or other reported trial outcomes, 
increasing target sample size).
Following each Albert Einstein Cancer Center DSMB meeting, provide the study leadership with 
written information concerning findings for the trial as a whole related to cumulative toxicities 
observed and any relevant recommendations related to continuing, changing, or terminating the 
trial.  The study leadership will provide information on cumulative toxicities and relevant 
recommendations to the local principal investigators to be shared with their IRB’s.
11. STATISTICAL CONSIDERATIONS
The primary aim of the study is to examine the difference in change of the outcome variables (Mena 
isoforms, cancer stem cells, and TMEM score) before treatment and 2-3 weeks after chemotherapy 
treatment begins. Two-sampled t-test will be used for this comparison. Appropriate transformation may be 
used to improved normality of the outcome variable. With 46 patients in stratum I and II  (92 total), our 
study will have 80% power to detect a difference in change of 0.60SD of each outcome variable in each 
stratum, a moderate to large effect size42..As an exploratory analysis, we will also conduct multivariable 
linear regression model to examine the treatment difference in change in outcome variables while adjusting 
for other clinical and patient characteristics. We will also perform an exploratory analysis in 20 patients in 
stratum III. 
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