A Phase 2 Study of Eribulin Followed by Doxorubicin and Cyclophosphamide as Preoperative Therapy for HER2-negative Inflammatory Breast Cancer
Phase: PHASE2
Conditions: Inflammatory Breast Cancer, Human Epidermal Growth Factor 2 Negative Carcinoma of Breast
Sponsor: Dana-Farber Cancer Institute



DF/HCC Protocol #: 15-292 
Protocol Version Date: April 23, 2021 
 
 
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Protocol Type / Version # / Version Date:     Amendment #8/Version #13/April 23, 2021 
 
 
1 
SCHEMA 
Cohort A: Preoperative eribulin followed by ddAC 
 

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Pre-treatment breast biopsy #1
Eribulin x 1 (cycle 1 day 1)
Breast biopsy #2: 1 wk after eribulin 
(cycle 1 day 8)
Eribulin  q 3 wks x  3 cycles
dd AC q 2 wks x 4 cycles
Mastectomy (harvest residual tumor) 
Radiation therapy + endocrine therapy 
 
Drugs:  
AC – doxorubicin + cyclophosphamide dd 
– dose-dense 
 
 
 
SCHEMA:  
Cohort B: Preoperative ddAC followed by eribulin 

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Pre-treatment breast biopsy #1
dd AC x 1 (cycle 1 day 1)
Breast biopsy #2: 1 wk after AC (cycle 1 
day 8)
dd AC q 2 wks x 3 cycles
Eribulin  q 3 wks x  4 cycles
Mastectomy (harvest residual tumor) 
Radiation therapy + endocrine therapy 
 
Drugs:  
AC – doxorubicin + cyclophosphamide dd 
– dose-dense 
 
 
 

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Imaging subset study
Cohort A:  DCE-MRI (N=5)
Pre-treatment breast DCE-
MRI
1 wk after eribulin (cycle 1,day 8) breast 
DCE-MRI
 
 
 
 
 
Imaging subset study
Cohort B:  DCE-MRI (N=5)
Pre-treatment breast 
DCE-MRI
1 wk after AC (cycle 1,day 8) breast 
DCE-MRI
TABLE OF CONTENTS 
 

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SCHEMA ........................................................................................................................................2
SCHEMA: .......................................................................................................................................3
1.1 
Study Design.........................................................................................................................7
1.2 
Primary Objectives for both Cohort A and Cohort B:..........................................................8
1.3 
Secondary Objectives for both Cohort A and Cohort B:......................................................9
2. BACKGROUND.........................................................................................................................9
2.1 
Inflammatory Breast Cancer.................................................................................................9
2.2 
IND Agent ..........................................................................................................................10
2.3 
Other Agent(s) ....................................................................................................................11
2.4 
Rationale.............................................................................................................................12
2.5 
Correlative Studies Background.........................................................................................13
3. PARTICIPANT SELECTION (FOR BOTH COHORTS A AND B)......................................14
3.1 
Eligibility Criteria...............................................................................................................14
3.2 
Exclusion Criteria ...............................................................................................................16
3.3 
Inclusion of Women and Minorities...................................................................................16
4. REGISTRATION PROCEDURES...........................................................................................17
4.1 
General Guidelines for DF/HCC and DF/PCC Institutions................................................17
4.2 
Registration Process for DF/HCC ......................................................................................17
5. TREATMENT PLAN ...............................................................................................................17
5.1 
Treatment Regimen.............................................................................................................17
5.2 
Pre-Treatment Criteria (for both Cohort A and B) .............................................................19
5.3 
Agent Administration .........................................................................................................20
5.4 
General Concomitant Medication and Supportive Care Guidelines...................................22
5.5 
Criteria for Taking a Participant Off Protocol Therapy......................................................24
5.6 
Duration of Follow-Up .......................................................................................................25
5.7 
Criteria for Taking a Participant Off Study ........................................................................26
6. DOSING DELAYS/DOSE MODIFICATIONS FOR COHORT A AND B............................26
6.1 
Eribulin ...............................................................................................................................26
6.2 
Doxorubicin/cyclophosphamide (AC)................................................................................29
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS....................................31
7.1 
Expected Toxicities.............................................................................................................31
7.2 
Adverse Event Characteristics............................................................................................32
7.3 
Expedited Adverse Event Reporting...................................................................................33
7.4 
Reporting Serious Adverse Events to 
........................................................................33
7.5 
Expedited Reporting to Hospital Risk Management ..........................................................34
7.6 
Routine Adverse Event Reporting......................................................................................34
8. PHARMACEUTICAL INFORMATION .................................................................................35
8.1 
Eribulin mesylate................................................................................................................35
8.2 
Doxorubicin ........................................................................................................................37
8.3 
Cyclophosphamide .............................................................................................................41
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES (COHORT A AND B)...........46
9.1 
Biomarker Studies ..............................................................................................................46
9.2 
Laboratory Correlative Studies...........................................................................................47
9.3 
Special Studies....................................................................................................................48

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9.4 
Imaging Sub-study..............................................................................................................49
10. STUDY CALENDAR.............................................................................................................52
11. MEASUREMENT OF EFFECT.............................................................................................58
11.1 
Clinical Evaluations ........................................................................................................58
11.2 
Pathological Response Parameters..................................................................................58
11.3 
Disease-Free Survival, Time to Treatment Failure, Overall Survival ............................58
12. DATA REPORTING / REGULATORY REQUIREMENTS ................................................59
12.1 
Data Reporting ................................................................................................................59
12.2 
Data Safety Monitoring...................................................................................................60
12.3 
Collaborative Research and Future Use of Data and Biospecimens...............................60
13. STATISTICAL CONSIDERATIONS....................................................................................61
13.1 
Study Design/Endpoints..................................................................................................61
13.2 
Sample Size, Accrual Rate and Study Duration..............................................................62
13.3 
Stratification Factors.......................................................................................................63
13.4 
Interim Monitoring Plan..................................................................................................63
13.5 
Analysis of Primary Endpoints .......................................................................................63
13.6 
Analysis of Secondary Endpoints ...................................................................................63
13.7 
Reporting and Exclusions ...............................................................................................64
14. PUBLICATION PLAN...........................................................................................................65
15. REFERENCES........................................................................................................................65
APPENDIX A: PERFORMANCE STATUS CRITERIA ............................................................68
APPENDIX B: PROCEDURES FOR RESEARCH CORE BIOPSY OF BREAST....................68
APPENDIX C: MASTECTOMY TISSUE COLLECTION PROTOCOL...................................71
APPENDIX D: BIOASSAY TEMPLATES .................................................................................71
OBJECTIVES 
 
1.1 
Study Design 
 
Cohort A: A total of 16 patients with newly diagnosed HER2-negative inflammatory breast 
cancer (IBC) without visceral metastasis will receive preoperative therapy with eribulin given 
day 1 and day 8 every 21 days for 4 cycles followed by doxorubicin and cyclophosphamide (AC) 
given every 14 days (dose-dense (dd)) for 4 cycles.  
 
•
A baseline research breast biopsy is obtained prior to beginning eribulin therapy. A 
second research breast biopsy is obtained 8 days after the first dose of eribulin.  
  
•
A cohort of patients (5) will participate in an imaging sub-study and undergo a research 
DCE-MRI of the breast after the baseline research breast biopsy. A second research 
DCE-MRI of the breast will be performed prior to the second research biopsy of the 
breast.  
 
Cohort B: A total of 16 patients with newly diagnosed HER2-negative inflammatory breast 
cancer (IBC) without visceral metastasis will receive preoperative therapy with doxorubicin and 

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cyclophosphamide (AC) given every 14 days (dose-dense (dd)) for 4 cycles followed by eribulin 
given day 1 and day 8 every 21 days for 4 cycles.  
 
•
A baseline research breast biopsy is obtained prior to beginning AC therapy. A second 
research breast biopsy is obtained 8 days after the first dose of AC.  
  
•
A cohort of patients (5) will participate in an imaging sub-study and undergo a research 
DCE-MRI of the breast after the baseline research breast biopsy. A second research 
DCE-MRI of the breast will be performed prior to the second research biopsy of the 
breast.  
 
Cohort A and B: 
 
3 to 5 weeks following the completion of preoperative study therapy, patients deemed surgically 
operable proceed to total mastectomy and axillary lymph node dissection, where residual cancer 
is obtained for correlative studies. 
 
•
Patients whose disease is not deemed surgically resectable following the completion of 
preoperative study therapy, may proceed to definitive radiation therapy given to the 
affected breast and regional lymph nodes beginning approximately 3 to 5 weeks after 
completion of preoperative therapy. 
•
Patients whose disease is rendered surgically resectable following radiation therapy may 
proceed to total mastectomy and axillary lymph node dissection approximately 4 to 8 
weeks following the completion of radiation therapy. Residual cancer will be obtained for 
correlative studies.  
•
Definitive radiation is given to the post-surgical chest wall and regional lymph nodes 
beginning approximately 3 to 6 weeks after surgery. 
 
•
Endocrine therapy with tamoxifen or an aromatase inhibitor, with or without ovarian 
suppression, will be given for 5-10 years if estrogen receptor (ER) and / or progesterone 
receptor (PR) positive. Endocrine therapy will begin either during or after completing 
radiation therapy as per standard of care practices.  
 
•
After completion of protocol therapy, participants in both cohorts will be followed every 
3 months for 1 year, then every 6 months for 4 years, then annually until death. 
 
 
1.2 
Primary Objectives for both Cohort A and Cohort B: 
 
• To determine pathologic complete response (pCR) rate after preoperative therapy for 
HER2-negative inflammatory breast cancer. 
 

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1.3 
Secondary Objectives for both Cohort A and Cohort B: 
 
•
To determine the efficacy of preoperative therapy for HER2-negative IBC defined as 
disease-free survival (DFS), time to treatment failure (TTF), and overall survival (OS). 
•
To assess the residual cancer burden (RCB) after preoperative therapy in HER2-negative 
inflammatory breast cancer. 
 
1.4        Correlative Objectives for both Cohort A and Cohort B: 
 
•
To correlate levels of expression of a panel of 20 pre-selected genes encoding for 
proteins involved with angiogenesis or epithelial-to-mesynchaml transition (EMT) with 
disease outcome (pCR, RCB, DFS, OS).  
 
•
To correlate changes in imaging (Ktrans ve, vp, and initial area under the curve (iAUC)) 
with genomic changes determined on core biopsies of the breast sampled at the same 
time points. 
 
 
2. BACKGROUND 
 
2.1 
Inflammatory Breast Cancer 
 
Inflammatory breast cancer (IBC) accounts for 2-5% of all invasive breast cancer. IBC is 
classified as a “cliniopathologic” diagnosis, whereby documentation of invasive breast 
carcinoma is established in the setting of unique clinical characteristics including a rapid onset of 
breast enlargement, pain, diffuse erythema and edema (peau d’orange) usually occurring within 
3-6 months.  The breast cancer often presents without a palpable mass, and dermal lymphatic 
involvement with cancer is demonstrated in approximately 75% of the cases.1 It is the effect of 
dermal lymphatic involvement, not infiltration of inflammatory cells that result in the clinical 
changes observed in IBC.  The median age at presentation is less than that seen in non-IBC, and 
there is a greater incidence of IBC among African American women. 2, 3 
 
The intrinsic biology of IBC is such that advanced disease is present at the time of diagnosis. 
Approximately 55-85% of patients present with metastasis to the axillary and / or supraclavicular 
lymph nodes, and distant metastatic disease is present at diagnosis in approximately 20-40% of 
women. 4  Even in the absence of metastatic disease at presentation, the likelihood of developing 
distant metastasis is extremely high, supporting a role for chemotherapy as the mainstay of 
treatment. Historically, surgery and radiation therapy alone resulted in a 54% relapse rate within 
18 months, translating into a median survival of 1.2 years. 5 The addition of chemotherapy to the 
initial treatment of IBC, i.e. trimodality therapy with neoadjuvant chemotherapy, followed by 
mastectomy and radiation therapy, improved the median overall survival to 3.8 years, translating 
into an approximately 50% 5-year overall survival. 1 
 

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The extent of locoregional disease precludes mastectomy as primary treatment for IBC, therefore 
preoperative chemotherapy has become the standard of care; however, the optimal chemotherapy 
regimen has yet to be determined. Anthracycline-containing regimens employed preoperatively 
have resulted in 40-45% 5-year overall survival, whereas the addition of taxanes to these 
regimens improved the pathologic complete response rate and overall survival. 6-8 The current 
chemotherapy regimens utilized as pre-operative treatment for IBC most commonly include 
anthracyclines and taxanes, yet the poor overall survival rates still necessitate ongoing 
investigation into improved pre-operative regimens with the goal of improving disease outcome.  
 
 
2.2 
IND Agent 
 
Eribulin 
 
Eribulin mesylate is a synthetic derivative of the natural product halichondrin B, a large 
polyether macrolide isolated from a marine sponge (Halichondria okadai). Halichondrin B 
exhibits anti-cancer activity through a microtubule-destabilizing anti-mitotic mechanism of 
action.9-11 Eribulin exerts its effects by binding to the plus end of microtubules, leading to tubulin 
sequestration into nonproductive aggregates, preventing tubulin polymerization and microtubule 
dynamics. Suppression of microtubule growth interferes with normal mitotic spindle formation, 
and blocks the prometaphase portion of mitosis,12 leading to irreversible cell cycle blockade at 
G2/M, disruption of mitotic spindles, and cell death via apoptosis after prolonged mitotic 
blockage.13 In vitro studies demonstrate that analogues of halichondrin B inhibit cell growth at 
nanomolar concentrations in a wide variety of cancer cell types, including breast, ovary, colon 
and melanoma.14 
 
The dosing regimen for eribulin selected based on results of Phase 1 studies is 1.4 mg/m2 
administered i.v. on Days 1 and 8 of a 3-week cycle. In an early Phase 2 trial, E7389-A001-201, 
the response rate (RR) in patients with breast cancer previously treated with anthracycline and 
taxanes was 11.5%.15  Based on these results, the effectiveness and safety of eribulin were 
further explored in Study E7389-A001-211, which included patients with locally advanced or 
metastatic breast cancer who had received between two to five prior chemotherapy regimens, 
including an anthracycline, taxane, and capecitabine, and who had progression on or within 6 
months of their last chemotherapy regimen. The response rate for eribulin was 9.3% in these 
heavily pretreated subjects.16 
 
In a Phase 3 trial, E7389-G000-305 (“EMBRACE”), eribulin (1.4 mg/m2 on Days 1 and 8 of a 
3week cycle) was compared with treatment of the physician’s choice (TPC; any cytotoxic or 
hormonal agent) in subjects heavily pre-treated for locally advanced or metastatic breast 
cancer.17  In the analysis of the primary endpoint of overall survival, patients receiving eribulin 
had a longer median overall survival of 13.1 months compared with 10.7 months for TPC (P = 
0.04, stratified log-rank test; hazard ratio = 0.81). Objective RR was 12% for eribulin mesylate 
and 5% for TPC (P = 0.005). Grade 3 or 4 eribulin-related adverse events included 

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asthenia/fatigue (7.6%), neutropenia (44%), and peripheral neuropathy (8.4%). Twelve percent 
of eribulin-treated subjects and 7% of TPC-treated subjects experienced treatment related serious 
AEs. Based on the results of this Phase 3 trial, eribulin was approved by the FDA in November 
2010 for metastatic breast cancer pre-treated with at least 2 prior regimens including 
anthracycline and taxane. 
 
The tolerability of eribulin and its ease of use suggest possible treatment advantages in 
comparison to other available agents for advanced disease (i.e. taxanes). Preclinical and clinical 
data suggest that eribulin mesylate may exhibit less neurotoxicity, myalgia/arthralgia, and 
hypersensitivity than other chemotherapeutic agents. The most common AEs with single-agent 
eribulin mesylate are asthenia/fatigue and neutropenia. In addition, eribulin requires minimal 
preparation, can be administered quickly, and does not require premedication to prevent 
hypersensitivity. 
 
Dosing justification 
The starting dosage of eribulin mesylate in this study, 1.4 mg/m2 administered as an i.v. infusion 
over approximately 2 to 5 minutes on Days 1 and 8 of a 3-week cycle, is a modification based on 
results of four phase 1 and four Phase 2 clinical studies of eribulin monotherapy. Four Phase 1 
dose-finding studies were conducted to evaluate escalating doses of eribulin mesylate and 
determine the maximum tolerated dose (MTD) of eribulin mesylate as: (1) a 1-hour i.v. infusion 
on Days 1, 8 and 15 of a 28-day cycle (E7389-A001-101); (2) a 1-hour i.v. infusion on Day 1 of 
a 21-day cycle (E7389-A001-102); (3) an i.v. bolus on Days 1, 8 and 15 of a 28-day cycle 
(National Cancer Institute [NCI] Study 5730); and (4) a bolus infusion on Days 1 and 8 of a 
21day cycle (E7389-J081-105). The MTD was determined to be 1.4 mg/m2 for the bolus 
injection and 1.0 to 2.0 mg/m2 for the infusion. 
 
Eribulin mesylate 1.4 mg/m2 i.v. bolus was tested as the starting dose in four Phase 2 studies. In 
the first two Phase 2 studies, subjects initially received eribulin mesylate on Days 1, 8 and 15 in 
28-day cycles. Because the majority of subjects receiving the 28-day dosing schedule 
experienced dose delays, reductions, or omissions due to neutropenia on Day 15 of the cycle, an 
additional cohort of subjects who received eribulin mesylate 1.4mg/m2 on Days 1 and 8 in 21day 
cycles was added. This schedule appeared to be better tolerated than the 28-day schedule and 
demonstrated antitumor activity in all four Phase 2 studies. Thus, the optimal schedule of 
eribulin mesylate monotherapy based on the four studies appears to be 1.4 mg/m2 administered 
on Days 1 and 8 in 21-day (3-week) cycles. 
 
 
2.3 
Other Agent(s) 
 
Doxorubicin and cyclophosphamide (AC) – as per institutional standards 
 
Several studies confirmed the importance of anthracycline-based neoadjuvant (NAC) for IBC.6, 
18, 19 Bauer et al. compared the outcomes of 2 cohorts of patients with IBC: 28 patients treated 
between 1973 and 1988 received CMF with or without vincristine and prednisone (VP) and 10 

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patients treated after 1988 received FAC. 18 The median OS was significantly improved when 
anthracyclines were added, compared with CMF/VP (30 months vs. 18 months, respectively, p = 
.02), as was the 3-year OS (41% vs. 14%, respectively) and 3-year DFS (46% vs. 0%, 
respectively). The introduction of taxanes into the NAC armamentarium also resulted in 
improved outcomes. A retrospective review of 6 sequential clinical trials involving 240 patients 
with IBC treated at MD Anderson Cancer Center from 1973 and 2000, compared disease 
outcomes among patients treated with anthracycline-based NAC with or without paclitaxel (T).8 
The median PFS was improved with the addition of T (33 weeks with T vs. 26 weeks, p = .18), 
as was the median OS (52 weeks with T vs. 41 weeks, p = .11). Statistical significance was 
demonstrated in the ER negative sub-population. Horvath et al. also retrospectively compared 
their experience of anthracycline-based NAC versus docetaxel (D) with epirubicin (E) and found 
an improved 3-year OS with the taxane-based NAC; however, statistical significance was not 
achieved (75% with DE vs. 61%).20 Currently, the standard NAC for IBC includes an 
anthracycline-and taxane-based regimen, although the optimal combination or sequence of these 
agents has not been established.  
 
2.4 
Rationale 
 
Eribulin (E7389, Halaven) is a synthetic analog of Halichondrin B that suppresses microtubule 
growth, without effecting polymerization.21 Because of its unique mechanism of action, i.e., 
sequestration of tubulin into nonfunctional aggregates, eribulin has been found to be an effective 
agent against taxane resistant cancer. Two separate phase II studies of eribulin in the treatment 
breast cancer patients previously exposed to taxanes, anthracyclines, and capecitabine 
demonstrated significant response rates, prompting the EMBRACE trial which showed a survival 
advantage in highly pre-treated patients.19, 22, 23 These data strongly support the investigation of 
eribulin in IBC; a virulent and often chemo-resistant subtype of breast cancer. We hypothesize 
that the use of eribulin will result in a significant improvement in pCR compared with the 
historical outcome of 25%.8, 19  The improved pCR rate should translate into an improved 
disease-free survival and overall survival.  
Only 1/16 subjects enrolled in Cohort A achieved a pCR, therefore we did not proceed to the 
second stage of study enrollment. This prompted an updated assessment of the expected pCR 
from preoperative therapy for hormone receptor positive / HER2 negative IBC. More recent 
studies of preoperative therapy and its effect on subtypes of inflammatory breast cancer, has 
shown that the pCR rate of ER and / or PR positive, HER2 negative IBC is significantly less than 
10%.24 Our analysis of the DFCI IBC Registry (DF/HCC 11-035) demonstrated a pCR rate 
among the hormone receptor positive, HER2 negative subgroup to be 2%. This data supported 
the pCR rate seen in Cohort A, therefore we sought to expand the protocol to explore a drug 
sequencing question and link it with an exploration of impact on the correlative studies. The 
original protocol has been modified to include a Cohort B which changes the sequencing of the 
preoperative chemotherapy to ddAC followed by eribulin. The Correlative Studies will remain 
the same, which will allow a comparison of gene expression following eribulin administration 
versus AC chemotherapy exposure. An additional 16 patients will be enrolled in Cohort B.  
 

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2.5 
Correlative Studies Background 
 
Eribulin mesylate is a synthetic macrocytic ketone analogue of the marine sponge natural product 
halichondrin B, and an inhibitor of microtubule dynamics.  By inhibiting microtubular mechanics, 
eribulin exerts its primary effect by preventing cancer cells’ progression through the cell cycle, 
leading to apoptosis.  Importantly, eribulin has shown evidence of clinical efficacy against tumors 
that have progressed on other microtubule-targeted cytotoxics such as taxanes.  Recently, 
preclinical work has revealed two other potential mechanisms of action for eribulin.   
 
Eribulin induces a mesenchymal-to-epithelial transition 
 
Recently published data suggests that eribulin can induce a shift in the phenotype of triple-negative 
breast cancer (TNBC) cells from a mesenchymal state to a more epithelial state. 25 This 
“mesenchymal-to-epithelial transition” (MET) represents a shift in cellular phenotype from a 
motile, invasive, stem-like state to a less invasive epithelial one 26, 27.  In both mice and humans, 
evidence suggests that induction of MET might be associated with a reduction in tumor cell 
extravasation into the circulation and the formation of distant metastases 26, 28. 
 
In preclinical studies, TNBC cells treated with eribulin in vitro show a downregulation (at both the 
mRNA and protein levels) of mesenchymal markers including N-Cadherin, vimentin, Twist, Snail, 
ZEB1, and ZEB2 and a concomitant increase in the expression of classical epithelial markers 
including E-Cadherin and keratin-18.  Consistent with this, eribulin pre-treated cells showed a 
reduction in migratory and invasive capacities, and a relative ineptitude for colonizing distant 
organs and forming micrometastatic lesions 25.  Although speculative, this might relate to an 
eribulin-induced attenuation of Smad2/3 phosphorylation by TGF-beta 25.  Importantly, none of 
the described changes were observed when the same cells were treated with a cytotoxic with an 
alternate mechanism of action, 5-fluorouracil. 
 
2.5.2  Eribulin normalizes the vasculature of primary mammary tumors 
 
Preclinical work has also revealed that eribulin might have direct or indirect effects on mammary 
tumor vessels 29.  In two murine TNBC xenograft models, eribulin therapy was associated with an 
improvement of blood perfusion in the central region of tumors, an increase in vessel density, a 
reduction in vessel diameter, and a reduction in hypoxia.  Collectively, these changes are consistent 
with “vascular normalization” – a reversion of tumor vessels from an immature, abnormal 
phenotype to one more reminiscent of normal vessels 30.  Importantly this normalization was 
associated with an increased effect of concomitantly administered cytotoxics, and was not seen 
after treating tumors with capecitabine, a cytotoxic with a different mechanism of action.  
 
2.5.3  Proposed translational experiments 
 
Collectively, the above data provide intriguing insights into alternate and potentially unique 
mechanisms of action for eribulin. In the current study - A Phase 2 Study of Eribulin Followed by 
Doxorubicin and Cyclophosphamide as Preoperative Therapy for HER2-negative Inflammatory 

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Breast Cancer – we propose a series of correlative experiments seeking evidence of the 
abovementioned changes in response to eribulin treatment of human inflammatory breast cancer 
(IBC). An imaging sub-study will also be performed to evaluate the effects of eribulin on tumor 
vasculature. IBC represents an ideal model as it demonstrates both a high metastatic proclivity and 
robust angiogenesis. 
 
Although the preclinical data supports a specific effect of eribulin on gene expression and vascular 
normalization, these changes may be due to chemotherapy in general, therefore the same 
correlative analysis described above will be performed on biopsy samples and breast imaging after 
exposure to AC preoperative therapy.  
 
2.5.4 Additional Biomarker Analysis 
 
The study also requires mandatory blood samples for biomarker research, which may include, 
among other analyses, the assessment of circulating tumor DNA. There is increasing evidence 
that circulating DNA can be obtained from the blood specimens of cancer patients, which 
represents the DNA and mutational status of tumor cells37-42. The serial sampling gives the 
opportunity to evaluate changes in circulating biomarker levels over time that may allow further 
understanding of potential resistance mechanisms or of (early) indicators of recurrence. 
Additional candidate markers of response to treatments that emerge from other clinical or 
nonclinical studies may also be assessed in this study and can be assessed with different types of 
technologies. 
 
 
3. PARTICIPANT SELECTION (FOR BOTH COHORTS A AND B) 
 
3.1 
Eligibility Criteria 
  
Participants must have histologically confirmed invasive breast cancer. All histologic subtypes 
are eligible.  
 
Participants must NOT have HER2 positive status based on ASCO/CAP guidelines defined as: 
• IHC 3+ based on circumferential membrane staining that is complete, intense 
 
-AND/OR –  
 
• FISH positive based on one of the three following criteria: o Single-
probe average HER2 copy number ≥ 6.0 signals/cell; OR 
o
Dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥ 6.0 
signals/cell; OR 
o
Dual-probe HER2/CEP17 ratio ≥2.0 
 

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Age > 18 years. Because no dosing or adverse event data are currently available on the use of 
eribulin in participants <18 years of age, children are excluded from this study  
 
ECOG performance status ≤1 (Karnofsky ≥70%, see Appendix A) 
 
Participants must have normal organ and marrow function as defined below: 
 
− 
− 
− 
− 
leukocytes 
absolute neutrophil 
count platelets total 
bilirubin 
≥3,000/mcL 
≥1,500/mcL 
≥100,000/mcL 
within normal institutional limits 
− 
− 
AST(SGOT)/ALT(SGPT) 
creatinine 
OR 
≤2.5 × institutional upper limit of normal 
≤1.5 x institutional upper limit of normal 
− 
creatinine clearance 
≥60 mL/min/1.73 m2 for participants with creatinine 
levels above institutional normal. 
 
Patients must have the clinical diagnosis of inflammatory breast cancer.   
 
Patients must be without evidence of visceral or bone involvement with metastatic cancer on 
physical exam or any diagnostic study.  Extensive nodal involvement (distant or regional) is 
allowed.  
 
LVEF > 50% calculated by echocardiogram (ECHO) 
 
Patients may have bilateral breast cancer so long as one breast meets criteria for inflammatory 
breast cancer, and the breast with inflammatory breast cancer has never received prior 
therapy.  
 
The effects of eribulin on the developing human fetus are unknown.  For this reason and because 
other therapeutic agents used in this trial are known to be teratogenic, women of childbearing 
potential and men must agree to use adequate contraception (hormonal or barrier method of 
birth control; abstinence) prior to study entry and for the duration of study participation.  
Should a woman become pregnant or suspect she is pregnant while she or her partner is 
participating in this study, she should inform her treating physician immediately.  Men 
treated or enrolled on this protocol must also agree to use adequate contraception prior to the 
study, for the duration of study participation, and 4 months after completion of chemotherapy 
administration. 
 
Ability to understand and the willingness to sign a written informed consent document. 
 
 

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3.2 
Exclusion Criteria 
 
Participants who are receiving any other investigational agents. 
 
History of allergic reactions attributed to compounds of similar chemical or biologic composition 
to eribulin or other agents used in study. 
 
Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
 
Pregnant women are excluded from this study because eribulin is an agent with the potential for 
teratogenic or abortifacient effects.  Because there is an unknown but potential risk for 
adverse events in nursing infants secondary to treatment of the mother with eribulin, 
breastfeeding should be discontinued if the mother is treated with eribulin.  These potential 
risks may also apply to other agents used in this study. 
 
HIV-positive participants on combination antiretroviral therapy are ineligible because of the 
potential for pharmacokinetic interactions with eribulin.  In addition, these participants are at 
increased risk of lethal infections when treated with marrow-suppressive therapy.  
Appropriate studies will be undertaken in participants receiving combination antiretroviral 
therapy when indicated. 
 
 A baseline corrected QT interval of > 500 ms. Patients with left bundle branch block that is 
deemed not clinically significant may be enrolled with corrected QT > 500. 
 
Individuals with a history of a different malignancy are ineligible except for the following 
circumstances. Individuals with a history of other malignancies are eligible if they have been 
disease-free for at least 3 years and are deemed by the investigator to be at low risk for 
recurrence of that malignancy. Individuals with the following cancers are eligible if 
diagnosed and treated within the past 3 years: cervical cancer in situ, and basal cell or 
squamous cell carcinoma of the skin. 
 
Patients may not have received eribulin, paclitaxel, doxorubicin, or cyclophosphamide as 
antineoplastic therapy.   
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. Because breast 
cancer predominantly affects females, it is anticipated that male enrollment will be < 5% of the 
overall study population. 
 
 

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4. REGISTRATION PROCEDURES 
 
4.1 
General Guidelines for DF/HCC and DF/PCC Institutions 
 
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registrations must occur prior to the initiation of protocol therapy. Any participant not 
registered to the protocol before protocol therapy begins will be considered ineligible and 
registration will be denied. 
 
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist. 
 
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the Overall Principal Investigator (PI).  
 
4.2 
Registration Process for DF/HCC  
 
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.   
 
 
 
5. TREATMENT PLAN 
 
5.1 
Treatment Regimen 
 
Cohort A: Eribulin will be administered on day 1 and day 8, every 3 weeks, with 21 consecutive 
days defined as a treatment cycle. Eribulin will be administered for a total of 4 cycles. Treatment 
will be administered on an outpatient basis.  Reported adverse events and potential risks are 
described in Section 7.  Appropriate dose modifications are described in Section 6.   
 
Following the completion of eribulin, doxorubicin and cyclophosphamide (AC) will be 
administered on day 1, every 2 weeks, with 14 consecutive days defined as a treatment cycle. If 
AC is delayed due to toxicity and an investigator prefers drug administration every 3 weeks in 
order to maintain full dose, AC may be given on a 21 day cycle with approval of the Principal 
Investigator. AC will be administered for a total of 4 cycles. Treatment will be administered on 
an outpatient basis.  Reported adverse events and potential risks are described in Section 7.  
Appropriate dose modifications are described in Section 6.  No investigational or commercial 
agents or therapies other than those described below may be administered with the intent to treat 
the participant's malignancy. 
 
Cohort B: Doxorubicin and cyclophosphamide (AC) will be administered on day 1, every 2 
weeks, with 14 consecutive days defined as a treatment cycle. If AC is delayed due to toxicity 

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and an investigator prefers drug administration every 3 weeks in order to maintain full dose, AC 
may be given on a 21 day cycle with approval of the Principal Investigator. AC will be 
administered for a total of 4 cycles. Treatment will be administered on an outpatient basis.  
Reported adverse events and potential risks are described in Section 7.  Appropriate dose 
modifications are described in Section 6.  No investigational or commercial agents or therapies 
other than those described below may be administered with the intent to treat the participant's 
malignancy. 
 
Following the completion of AC, eribulin will be administered on day 1 and day 8, every 3 
weeks, with 21 consecutive days defined as a treatment cycle. Eribulin will be administered for a 
total of 4 cycles. Treatment will be administered on an outpatient basis.  Reported adverse events 
and potential risks are described in Section 7.  Appropriate dose modifications are described in 
Section 6. 
 
Treatment  
Agent 
Premedications; 
Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
Eribulin  
No routine 
premedication 
required 
1.4 mg/m2 – 
may be 
administered 
diluted in 100 
ml normal 
saline 
 
IV over 2-5 
minutes.  
 
Day 1 and 
day 8, 
every 21 
days x 4 
cycles 
Doxorubicin 
No routine 
premedication 
required 
60 mg/m2, 
IVP 
IVP over 3-5 
minutes; given 
prior to 
cyclophosphamide 
Day 1, 
every 14 
days x 4 
cycles 
Cyclophosphamide 
No routine 
premedication 
required 
600 mg/m2 
diluted 
in 
250500 
ml 
normal 
saline 
IV infusion 
administered per 
institutional 
guidelines; given 
after doxorubicin 
Day 1, 
every 14 
days x 4 
cycles 
21 days  
(3weeks) 
 
 
14 days 
(2weeks) 
•
Minor schedule changes owing to observed holidays, inclement weather, etc. are permitted. 
•
Patients may interrupt therapy for protocol-directed reasons (i.e. toxicity) or for personal 
preferences (holidays, vacations, etc).  Treatment should resume according to protocol 
guidelines.   
•
 will provide eribulin mesylate to DFCI Pharmacy for use in the study for dispensing free 
of cost to participants.  Patients and/or their insurance companies will be billed for the cost of 
doxorubicin and cyclophosphamide and their administration, as it is considered standard of 
care for inflammatory breast cancer.   
 

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5.2 
Pre-Treatment Criteria (for both Cohort A and B) 
 
Cycle 1-8, Day 1 
 

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20 
 
Physical examination will be performed.  
 
At every visit, vital signs and laboratory tests will be performed. Pretreatment criteria is required 
at day 1 of every cycle (including C1D1) and on day 8 when eribulin is being administered 
and will include: 
 
•
Absolute neutrophil count > 1,000/mcL. 
 
•
Platelets > 75,000/mcL. 
 
•
Total bilirubin ≤1.5 X institutional upper limit of normal. 
 
Alkaline phosphatase (ALP) ≤3 x institutional upper limit of normal.  
•
AST(SGOT)/ALT(SGPT) ≤ 3 X institutional ULN. 
 
•
Creatinine < 2.0 x ULN or creatinine clearance ≥50 mL/min. 
 
•
Non-hematologic toxicity CTCAE < Grade 3 or 4. 
 
 
5.3 
Agent Administration 
 
Eribulin 
 
•
Eribulin at a starting dose of 1.4 mg/m2 will be administered on an outpatient basis via IV 
infusion per institutional guidelines on Days 1 and 8 of each 21 day cycle, for a total of 4 
cycles.  
o Cohort B: First dose of eribulin will be administered 2 weeks after the last dose of 
AC is given (cycle 4, day 1).  
 
•
Eribulin dosing will be administered per institutional guidelines.  
 
 
Other agents 
 
Doxorubicin 
 
•
Doxorubicin and cyclophosphamide are administered concurrently (AC).  

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21 
 
o Cohort A: AC will be administered beginning 2 weeks after the last dose of eribulin 
is given (cycle 4, day 8).  
 
•
Doxorubicin 60 mg/m2 will be administered on an outpatient basis via IV. per 
institutional guidelines on Day 1 of each 14 day cycle, for a total of 4 cycles.  
 
 
Doxorubicin is administered immediately prior to the administration of 
cyclophosphamide.  
 
Cyclophosphamide 
 
•
Doxorubicin and cyclophosphamide are administered concurrently (AC). 
o  Cohort A: AC will be administered beginning 2 weeks after the last dose of 
eribulin is given (cycle 4, day 8).  
 
•
Cyclophosphamide 600 mg/m2 will be administered on an outpatient basis via IV 
infusion per institutional guidelines on Day 1 of each 14 day cycle, for a total of 4 cycles.  
 
 
•
Cyclophosphamide is administered immediately following the administration of 
doxorubicin.  
 
Other Modality(ies) or Procedures 
 
Surgery 
 
•
Primary breast surgery should be performed within 3 - 5 weeks after the last dose of 
chemotherapy (doxorubicin/cyclophosphamide for Cohort A, eribulin for Cohort B).  
Surgery must be performed at one of the participating institutions.  Prior to surgery, 
patients should be assessed for clinical response to preoperative treatment by MRI of the 
breasts.  Patients should undergo surgery with a total mastectomy; removal of the breast 
and level 1 and 2 axillary lymph node dissection.  Pathological specimens will be 
analyzed for tumor extent and grade, ER and PR status, HER2 expression, and other 
markers of tumor biology.  Given the high risk of local regional disease recurrence with 
IBC, reconstruction surgery is to be delayed for 6 – 12 months following the completion 
of radiation therapy.  
 
•
Patients whose disease is not surgically resectable following the completion of 
preoperative study therapy, may proceed to definitive radiation given to the affected 

DF/HCC Protocol #: 15-292 
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• 
22 
 
breast and regional lymph nodes beginning approximately 3 to 4 weeks after the last dose 
of preoperative chemotherapy. Patients whose disease is rendered surgically resectable 
following radiation therapy may proceed to total mastectomy and axillary lymph node 
dissection (as described above) approximately 4 to 8 weeks following the completion of 
radiation therapy.  
 
Radiation Therapy 
• Radiation therapy should be initiated within 3-6 weeks following surgery. Radiation should 
be delivered to the chest wall and regional lymph nodes. Treatment will be administered 
per institutional guidelines.Radiation can be administered at a local facility.  
Patients whose disease is not surgically resectable following the completion of 
preoperative study therapy may proceed to definitive radiation given to the affected 
breast and regional lymph nodes beginning approximately 3 to 4 weeks after completion 
of preoperative chemotherapy. 
Endocrine Therapy 
•
Appropriate postoperative adjuvant endocrine therapy should be administered in patients 
with ER and/or PR positive disease as defined by institutional guidelines.  
•
Either tamoxifen or an aromatase inhibitor, with or without ovarian suppression, should 
be administered as determined by the patient’s treating physician.  
•
Ovarian suppression should be offered to premenopausal patients who continue to have 
intact ovarian function following the completion of preoperative chemotherapy. Either 
tamoxifen or an aromatase inhibitor should be administered concurrently.  
•
This should be initiated 2-4 weeks following the completion of surgery and may be given 
concurrent with radiation therapy as per investigator discretion. Endocrine therapy should 
continue per institutional guidelines. 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
The weak inhibitory effect on cytochrome P450 enzymes exhibited by eribulin in vitro suggests a 
low risk of eribulin interaction with the pharmacokinetics of other drugs coadministered in usual 
clinic practice.  
 
All diagnostic, therapeutic, or surgical procedures relating to malignancy should be recorded in 
the electronic case report form (eCRF), including the date, indication, description of the 
procedure(s), and any clinical findings. All prior treatment or medication administered during the 
30 days preceding the first dose of study treatment and any concomitant therapy administered to 

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23 
 
the subject throughout the study until 30 days after the final dose of study treatment must be 
recorded on the Prior and Concomitant Therapy page of the eCRF. The generic name of the drug 
(or trade name for combination drugs) must be specified along with the duration of treatment and 
indication for use. If concomitant medication/therapy is administered for an AE, investigators 
will record that AE on the AE page of the eCRF. 
 
Any medication that is considered necessary for the subject’s welfare and that is not expected to 
interfere with the evaluation of study treatment may be given at the discretion of the investigator. 
Ancillary treatments will be given as medically indicated. 
 
Any changes in documented, permitted concomitant treatment already being taken at the start of 
the clinical study must be recorded on the eCRF, noting the type of medication, duration of use, 
and indication. 
 
• Antiemetics 

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Patients may be given antiemetics at the discretion of the treating physician.  
 
• Anticoagulants 
Anticoagulation with heparin, heparin derivatives, and/or warfarin may be given at the 
discretion of the treating physician. Patients receiving warfarin with therapeutic INR should 
have PT-INR checks according to standard institutional practice to maintain the target INR 
as determined by the treating investigator.  
 
• Growth Factors 
Patients may not receive erythropoietin while on study. The use of G-CSF should generally 
be administered to maintain neutrophil counts during eribulin administration as per ASCO 
guidelines. G-CSF should be routinely administered to maintain a 14 day cycle for 
doxorubicin/cyclophosphamide per institutional guidelines. The use of platelet growth 
factors are specifically prohibited on this protocol. Please consult the Principal Investigator 
with questions. Investigational growth factors are not permitted on this study.  
 
5.5 
Criteria for Taking a Participant Off Protocol Therapy 
 
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue for 
a total of 8 cycles of preoperative chemotherapy (Cohort A: eribulin for 4 cycles followed by 
doxorubicin/cyclophosphamide (AC) for 4 cycles; Cohort B: doxorubicin/cyclophosphamide 
(AC) for 4 cycles followed by eribulin for 4 cycles) or until one of the following criteria applies: 
 
•
Disease progression 
 
•
Intercurrent illness that prevents further administration of treatment 
 
•
Unacceptable adverse event(s) 
 
•
Participant demonstrates an inability or unwillingness to comply with the medication 
regimen and/or documentation requirements 
 
•
Participant decides to withdraw from the protocol therapy 
 
•
General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator 
 
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the case report form (CRF). Alternative care options will be discussed with the 
participant. 

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25 
 
 
A ODQ Treatment Ended/Off Study Form will be filled out when a participant is removed from 
protocol therapy. This form can be found on the ODQ website or obtained from the ODQ 
registration staff. 
 
In the event of unusual or life-threatening complications, treating investigators must immediately 
notify the Overall PI, Filipa Lynce M.D. at 617-632-3800.  
 
Preoperative chemotherapy 
 
Patients who discontinue chemotherapy due to toxicity or develop disease progression should not 
systematically be withdrawn from all study treatments. The following recommendations are 
given: 
 
Cohort A: 
•
Eribulin: patients who received at least 2 cycles of eribulin can proceed to 
doxorubicin/cyclophosphamide. Patient should then complete the treatment as per 
protocol. 
 
•
Doxorubicin / cyclophosphamide: in the case of discontinuation of 
doxorubicin/cyclophosphamide, patients should be assessed for eligibility for 
mastectomy, and if they are deemed surgical candidates, then they should proceed to 
mastectomy and continue on treatment as per protocol. 
 
Cohort B: 
•
Doxorubicin / cyclophosphamide (AC): patients who received at least 2 cycles of AC can 
proceed to eribulin. Patients should then complete the treatment as per protocol. 
 
•
Eribulin: in the case of discontinuation of eribulin, patients should be assessed for 
eligibility for mastectomy, and if they are deemed surgical candidates, then they should 
proceed to mastectomy and continue on treatment as per protocol. 
 
5.6 
Duration of Follow-Up 
 
After removal from protocol therapy, participants will be followed every 3 months for 1 year, 
then every 6 months for 4 years, then annually until death. Participants removed from protocol 
therapy for unacceptable adverse event(s) will be followed until resolution or stabilization of the 
adverse event. 
Patients who are no longer being followed at the treating institution will be contacted either 
directly or their current treating physician will be contacted to determine disease status 
until death.  
 

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26 
 
5.7 
Criteria for Taking a Participant Off Study 
 
Participants will be removed from study when any of the following criteria apply: 
•
Lost to follow-up 
•
Withdrawal of consent for data submission 
•
Death 
 
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF). 
 
A ODQ Treatment Ended/Off Study Form will be filled out when a participant comes off study. 
This form can be found on the ODQ website or obtained from the ODQ registration staff.  
 
 
6. DOSING DELAYS/DOSE MODIFICATIONS FOR COHORT A AND B 
 
Dose delays and modifications will be made as indicated in the following table(s). If any of the 
individual study drugs must be delayed for a day or more, all agents should be delayed for the 
same timeframe. Dose adjustments are to be made according to the highest grade toxicity or 
hematologic toxicity, whichever is more likely to result in a dose reduction. The descriptions and 
grading scales found in the revised NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0 will be utilized for dose delays and dose modifications. A copy of the 
CTCAE version 4.0 can be downloaded from the CTEP website  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
If possible, symptoms should be managed symptomatically. In the case of toxicity, appropriate 
medical treatment should be used (including anti-emetics, anti-diarrheals, etc.). 
 
All adverse events experienced by participants will be collected from the time of the first dose of 
study treatment, through the study and until the final study visit. Participants continuing to 
experience toxicity at the off study visit may be contacted for additional assessments until the 
toxicity has resolved or is deemed irreversible. 
 
6.1 
Eribulin 
 
The principle is to attempt to administer full doses of therapy on schedule. No more than 2 
dosereductions of eribulin mesylate chemotherapy should be made for toxicity. Growth factor 
support may be used instead of a dose reduction at the discretion of the investigator. If 
more than 2 dose-reductions would be required, eribulin should be discontinued.  
•
If eribulin is held for > 2 weeks for toxicity, eribulin should be discontinued.   

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27 
 
•
If eribulin is discontinued, the patient should proceed to doxorubicin/cyclophosphamide 
(AC) and proceed as outlined in the protocol (Cohort A) or proceed to surgery as 
outlined in the protocol (Cohort B). 
•
All dose modifications for eribulin are based on the dose level changes outlined below 
(Table 1). 
•
Instructions for management of toxicities related to eribulin are listed in Table 2. 
 
If the dose of eribulin cannot be administered as planned due to treatment-related toxicity, the 
dose should be delayed according to the following instructions: 
 
•
Day 1 of each cycle: If eribulin cannot be administered on day 1, the dose should be 
delayed until recovery to above mentioned values (criteria for eribulin administration). 
The day 1 dose will be rescheduled for when the criteria for eribulin administration are 
being met. The dose of eribulin may have to be reduced following a dose delay in 
accordance to the instructions for dose reduction (Table 2).  
•
Day 8 of each cycle: If eribulin cannot be administered on day 8, the dose should be 
delayed until recovery to above mentioned values (criteria for eribulin administration). 
The day 8 dose will be delayed for a maximum of 7 days and as follows: 
 
•
If the criteria for eribulin administration are met on, or before day 15 of a cycle, 
administer eribulin as recommended in Table 2; this dose is still called day 8. Eribulin 
administration on day 1of the next cycle must be no sooner than 14 days later. 
•
If the criteria for eribulin administration are NOT being met by day 15 of a cycle, omit 
the day 8 dose of eribulin for that cycle. Eribulin should be administered as 
recommended in Table 2 on day 1 of the next scheduled cycle assuming criteria to 
continue treatment are met within a maximum 2 week delay in therapy from originally 
planned day 8. 
Table 1: Eribulin mesylate dose modification levels 
Dose Level  
 
Eribulin mesylate Dose 
Starting Dose  
 
1.4 mg/m2 
First dose reduction 
1.1 mg/m2 
Second dose reduction 
0.7 mg/m2 
 
Table 2: Dose modifications for toxicity – Eribulin mesylate 
Toxicity 
Grade and Details 
Eribulin mesylate Dose 
Modifications 
Neutropenia: 
(ANC= absolute 
< 500 cells/mm3 lasting > 7 days in 
the previous cycle without use of 
growth factors 
Hold eribulin until recovery to grade 
<1 and reduce by 1 dose level. 
Prophylactic growth factor support should 
be instituted for subsequent cycles. 

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< 500 cells/mm3 lasting </= 7 days 
in the previous cycle without use of 
growth factors 
Hold eribulin until recovery to grade <1. 
Resume eribulin at the same dose with 
growth factor support. 
< 500 cells/mm3 lasting </= 7 days 
in the previous cycle despite use of 
growth factors 
 
Hold eribulin until recovery to grade <1, 
and reduce by 1 dose level. Continue 
ongoing prophylactic growth factor 
support for subsequent cycles. 
<1000 /mm3 with fever or infection 
without use of growth factors in the 
previous cycle 
Hold eribulin until recovery to grade <1. 
Resume eribulin at the same dose with 
growth factor support. 
neutrophil count) 
 
 
 
 
<1000 /mm3 with fever or infection 
despite use of growth factors 
Hold eribulin until recovery to grade <1, 
and reduce by 1 dose level, with 
prophylactic growth factor support. 
Toxicity 
Grade and Details 
Eribulin mesylate Dose 
Modifications 
 
<1000 /mm3 without fever or infection 
First occurrence: Hold eribulin until 
recovery to grade <1 then resume at the 
same dose with growth factor support. 
 
If uncomplicated neutropenia 
(<1000 /mm3 without fever or infection) 
occurs/recurs despite growth factor 
support, then hold eribulin until recovery 
to grade <1. 
Eribulin may be then be resumed at the 
same dose with growth factor support, or it 
may be reduced by one dose level, per 
investigator discretion. 
 
If a patient misses two consecutive 
Day 8 eribulin doses due to 
uncomplicated neutropenia, then hold 
eribulin until recovery to grade < 1 
and reduce by 1 dose level, and 
consider prophylactic growth factor 
support for subsequent cycles. 
Grade 3  
Hold eribulin until recovery to grade <1 
then reduce by 1 dose level. 
Platelets 
 
Grade 4 
Hold eribulin until recovery to grade <1 
then reduce by 1 dose level. 
Anemia 
Grade 4 
Hold eribulin until recovery to grade <1 
then reduce by 1 dose level. 
PRBC transfusions are permitted to treat 
anemia on protocol. 

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Grade 3, first occurrence 
Hold eribulin until recovery to grade <1. 
Maximize supportive care measures. If 
symptom recovery occurs within 1 week, 
then eribulin may be resumed at the same 
dose if deemed appropriate by the 
investigator, otherwise, reduce by 1 dose 
level. 
Grade 3, despite maximal supportive 
measures 
Hold eribulin until recovery to grade <1 
then reduce by 1 dose level. 
Non-hematologic 
toxicity 
 
 
Grade 4 
Hold eribulin until recovery to grade <1 
then reduce by 1 dose level. 
*Participants requiring a delay of >2 weeks should go off protocol therapy. 
**Participants requiring > two dose reductions should go off protocol therapy. 
Growth factor support may be used instead of a dose reduction at the discretion of the 
investigator, as outlined above.  
 
6.2 
Doxorubicin/cyclophosphamide (AC) 
•
The principle is to attempt to administer full doses of therapy on schedule.  
•
If AC is delayed due to toxicity and an investigator prefers drug administration every 3 
weeks in order to maintain full dose, AC may be given on a 21 day cycle with approval 
of the Principal Investigator. 
•
No more than 1 dose-reduction of AC chemotherapy should be made for toxicity.  If 
more than 1 dose-reduction would be required, AC should be discontinued, and the 
patient should proceed to surgery as outlined in the protocol (Cohort A) or proceed to 
eribulin as outlined in the protocol (Cohort B).  
•
If AC is held for > 2 weeks for toxicity, AC should be discontinued. Patients may 
proceed to surgery as outlined in the protocol (Cohort A) or proceed to eribulin (Cohort 
B).  
•
All dose modifications for AC are based on the dose level changes outlined below (Table 
3). 
•
Instructions for management of toxicities related to AC are listed in Table 4. Table 3:  
doxorubicin/cyclophosphamide (AC) – dose modification levels 
Dose Level 
Doxorubicin dose 
Cyclophosphamide dose  
Starting dose 
60 mg/m2 
600 mg/m2 
First dose reduction 
45 mg/m2 
450 mg/m2 
 
Table 4:  Dose Modifications for toxicity - doxorubicin/cyclophosphamide (AC) 
Toxicity 
 
Modifications for AEs that 
occur during a cycle but 
RESOLVE PRIOR TO 
THE NEXT 
TREATMENT CYCLE a 
Modifications for AEs that 
REQUIRE A DELAY IN 
ADMINISTRATION OF 
THE TREATMENT 
CYCLEb 
HEMATOLOGICAL: 
 
 

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Neutrophils count decreased 
 
 
Grades 2, 3, 4  
Maintain dose 
Hold until ≥ 1000/mm3 If 
recovery takes: 
•
1 wk: maintain dose 
•
2 wks: ↓ one dose level 
Febrile neutropenia 
↓ one dose level or 
discontinue. 
 
HEMATOLOGICAL: 
 
 
Platelets 
 
 
Grades 2, 3 
 
 
 
 
 
Grade 4 
Maintain dose 
 
 
 
 
 
↓ one dose level 
Hold until ≥ 100,000/mm3. If 
recovery takes: 
•
1 wk: maintain dose 
•
2 wks: ↓ one dose level 
 
↓ one dose level or 
discontinue. 
GASTROINTESTINAL 
DISORDERS (if related to 
chemotherapy, despite medical 
management): 
 
 
Diarrhea 
 
 
Grade 2 
Grade 3 
Grade 4 
 
Maintain dose 
↓ one dose level discontinue 
↓ one dose level 
↓ one dose level  discontinue 
Mucositis oral (stomatitis) 
 
 
Grade 2 
Grade 3 
Grade 4 
 
Maintain dose ↓ 
one dose level 
discontinue 
↓ one dose level 
↓ one dose level discontinue 
Vomiting (despite antiemetics) 
 
 
Grade 2 
Grades 3, 4 
 
↓ one dose level (optional) 
↓ one dose level or 
discontinue 
↓ one dose level discontinue 
HEPATIC FUNCTION: 
 
 
Bilirubin or AST or ALP 
 
 increased 

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≥ 1000/mm3] and bilirubin [which must be ≤ the baseline grade]) on Day 1 of the next scheduled 
cycle of AC (ie, treatment can be given without delay). 
b Hold and check weekly. With exception of ANC and bilirubin, resume treatment when toxicity 
is ≤ grade 1. If toxicity has not resolved after 2 weeks of delay, discontinue 
doxorubicin/cyclophosphamide.  c Determination of "clinically significant" AEs is at the 
discretion of the investigator. 
 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Section 7.2) will determine whether the event requires expedited reporting in 
addition to routine reporting.  
 
7.1 
Expected Toxicities 
 
Most AEs on studies with eribulin are grade 1 or 2. Peripheral neuropathy was an important AE 
leading to discontinuation of therapy. The most frequently reported eribulin mesylate related 
AEs were asthenia/fatigue (65%), alopecia (60%), neutropenia (60%), nausea (44%), anemia 
(28%), pyrexia (23%), leucopenia (22%), anorexia (21%), constipation (19%), vomiting (18%), 
and peripheral neuropathy (only grade 3: 5.5%). Grade 4 neutropenia occurred in 32% of 
patients, and fever with neutropenia occurred in 5.5% of patients. The frequency of all other 
grade 3/4 AEs was less than 3%.31 
 
Grade 2 
 
 
 
 
 
 
Grade 3 
Grade 4 
↓ one dose level 
 
 
 
 
 
 
↓ one dose level 
Discontinue 
Hold until bilirubin returns 
to the baseline grade, and 
AST and alkaline 
phosphatase have returned 
to≤ grade 1. 
Then ↓ one dose level 
 
↓ one dose level 
Discontinue 
OTHER CLINICALLY 
SIGNIFICANT AEsc: 
 
 
Grade 3 
Grade 4 
↓ one dose level discontinue 
↓ one dose level 
Discontinue 
*Dose modifications must be based on AEs that occur during the cycle (column 2) and AEs 
present on the scheduled cycle Day 1 (column 3). 
**Dose modifications must be based on the AE requiring the greatest modification.  

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Adverse Events List(s) 
 
Refer to the Investigator’s Brochure for detailed eribulin mesylate information and FDA 
approved package insert for eribulin mesylate for more information. 
 
Adverse Event List(s) for eribulin mesylate 
 
AE of special interest of eribulin are: 
•
Hematological toxicity, 
•
Asthenia or fatigue, 
•
Alopecia, 
•
Nausea, 
•
Peripheral neuropathy. 
 
 
Adverse Event List(s) for Other Agent(s) 
 
Please refer to the FDA approved doxorubicin and cyclophosphamide package inserts for the 
comprehensive list of adverse events. 
 
AE of special interest for doxorubicin include: 
•
Hematological toxicity, 
•
Asthenia or fatigue, 
•
Alopecia, 
•
Nausea, 
•
Cardiac toxicity, 
•
Mucositis, 
•
Secondary leukemia.  
 
AE of special interest for cyclophosphamide include: 
•
Hematological toxicity, 
•
Asthenia or fatigue, 
•
Alopecia, 
•
Nausea, • Hemorrhagic cystitis. 
 
7.2 
Adverse Event Characteristics 
 
•
CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 

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33 
 
 
•
For expedited reporting purposes only:   
-
AEs for the agent(s) that are listed above should be reported only if the adverse event 
varies in nature, intensity or frequency from the expected toxicity information which 
is provided. 
-
Other AEs for the protocol that do not require expedited reporting are outlined in the 
next section (Expedited Adverse Event Reporting) under the sub-heading of 
ProtocolSpecific Expedited Adverse Event Reporting Exclusions. 
 
•
Attribution of the AE: 
-
Definite – The AE is clearly related to the study treatment. 
-
Probable – The AE is likely related to the study treatment. 
-
Possible – The AE may be related to the study treatment. 
-
Unlikely – The AE is doubtfully related to the study treatment. 
-
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting  
 
Investigators must report to the Overall PI any serious adverse event (SAE) that occurs after the 
initial dose of study treatment, during treatment, or within 30 days of the last dose of 
treatment on the local institutional SAE form.  
 
DF/HCC Expedited Reporting Guidelines 
 
Investigative sites within DF/HCC and DF/PCC will report SAEs directly to the DFCI 
Office for Human Research Studies (OHRS) per the DFCI IRB reporting policy. .  
 
 
7.4 
Reporting Serious Adverse Events to
 
 
Serious adverse events (SAEs), as defined below, where the Overall PI considers a relationship 
to the eribulin therapy to be at least a reasonable possibility, will be reported to 
 on a 
Medwatch 3500A form within one business day of the event. Serious adverse events (SAEs) are 
not related to eribulin therapy and non-serious AEs will be provided to 
 in the final study 
report and any interim reports provided.  
 
A serious adverse event (SAE) is any adverse event, occurring at any dose and regardless of 
causality that:  
•
Results in death 
•
Is life-threatening. Life-threatening means that the person was at immediate risk 
of death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form. 

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34 
 
•
Requires or prolongs inpatient hospitalization (i.e., the event required at least a 
24-hour hospitalization or prolonged a hospitalization beyond the expected length 
of stay). Hospitalization admissions and/or surgical operations scheduled to occur 
during the study period, but planned prior to study entry are not considered SAEs 
if the illness or disease existed before the person was enrolled in the trial, 
provided that it did not deteriorate in an unexpected manner during the trial (e.g., 
surgery performed earlier than planned). 
•
Results in persistent or significant disability/incapacity. Disability is defined as a 
substantial disruption of a person’s ability to conduct normal life functions. 
•
Is a congenital anomaly or birth defect; or 
•
Is an important medical event when, based upon appropriate medical judgment, it 
may jeopardize the participant and require medical or surgical intervention to 
prevent one of the outcomes listed above. Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home; blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
 
Events not considered to be serious adverse events are hospitalizations for: 
•
routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition, or for elective procedures 
•
elective or pre-planned treatment for a pre-existing condition that did not worsen 
•
emergency outpatient treatment for an event not fulfilling the serious criteria 
outlined above and not resulting in inpatient admission 
•
respite care 
 
The reports will be sent on MedWatch 3500A form to 
 at the number listed below: 
 
 
 safety fax number
 Email: 
 
 
 
7.5 
Expedited Reporting to Hospital Risk Management 
 
Participating investigators will report to their local Risk Management office any participant 
safety reports or sentinel events that require reporting according to institutional policy.  
 
7.6 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions to the Overall PI on the 
toxicity case report forms.  AEs reported through expedited processes (e.g., reported to the 
IRB, FDA, etc.) must also be reported in routine study data submissions. 
 
 

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8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational or other agents 
administered in this study can be found in Section 7.1. 
 
8.1 
Eribulin mesylate 
 
Refer to the Investigator’s Brochure for detailed agent information and eribulin mesylate  FDA 
approved package for more information. 
 
Description 
 
Chemical name: 11,15:18,21:24,28- Triepoxy-7,9-ethano-12,15-methano-
9H,15Hfuro[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-one,2-
[(2S)-3amino-2-hydroxypropyl]hexacosahydro-3-methoxy-26-methyl-20,27-
bis(methylene)(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,2
8R,29aS)-, methanesulfonate (salt) 
Empirical formula: C40H59NO11•CH4O3S Molecular 
weight: 826.0 (729.9 for free base). Structural formula:  
 
 
Other name: E7389, Halaven® 
Mode of action: Eribulin injection is a non-taxane microtubule dynamics inhibitor. 
Manufacturer:
 
Molecular weight: 826.0 (729.9 for free base). 
Distribution: Vd: 43-114 L/m2 
Protein binding: 49% to 65% Metabolism: Negligible Half-life, elimination: ~40 
hours 
Excretion: Feces (82%; predominantly as unchanged drug); urine (9%, primarily as 
unchanged drug) 
 

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Form 
 
Eribulin is clear, colorless, sterile solution for intravenous administration. Each vial 
contains 1mg of eribulin as a 0.5 mg/mL solution in ethanol: water (5:95). Eribulin is 
manufactured by 
 
 
Storage and Stability 
 
Store undiluted eribulin in the syringe for up to 4 hours at room temperature or for up to 
24 hours under refrigeration (40°F or/ 4°C). Store diluted solutions of eribulin for up to 4 
hours at room temperature or up to 24 hours under refrigeration. Discard unused portions 
of the vial.  
 
Compatibility 
 
Eribulin is compatible with saline (0.9% Sodium Chloride Injection). It is not compatible 
with solutions with dextrose. 
 
Handling 
 
Qualified personnel, familiar with procedures that minimize undue exposure to 
themselves and the environment, should undertake the preparation, handling, and safe 
disposal of the chemotherapeutic agent in a self-contained and protective environment.  
 
Availability 
 
Eribulin mesylate is a commercially available agent but will be supplied free-of-charge 
from Eisai. 
 
Preparation 
 
The amount of eribulin required will be withdrawn from the appropriate number of vials 
into a syringe. This may be injected directly as an i.v. infusion over approximately 2 to 5 
minutes or diluted in up to 100 mL 0.9% saline for i.v. infusion over approximately 2 to 
5 minutes. Do not dilute in or administer through an intravenous line containing solutions 
with dextrose. Do not administer in the same intravenous line concurrent with the other 
medicinal products. 
 
Administration 
 
It will be administered over approximately 2-5 minutes. Do not administer in the same 
intravenous line concurrent with the other medicinal products. 
 

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Ordering 
 
Eribulin will be provided by 
 and stored in the site research pharmacy. Under no 
circumstances will the investigator allow the study treatment(s) to be used other than as 
directed by this protocol. Clinical supplies will not be dispensed to any individual who is 
not enrolled in the study.  
 
Accountability 
 
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.) 
 
Destruction and Return 
 
At the end of the study, unused supplies of eribulin should be destroyed according to 
institutional policies. Destruction will be documented in the Drug Accountability Record 
Form. 
 
 
 
8.2 
Doxorubicin 
 
Refer to the FDA approved package description for more information. 
 
Description 
 
Doxorubicin is a cytotoxic anthracycline antibiotic isolated from cultures of 
Streptomyces peucetius var.caesius. Doxorubicin consists of a naphthacenequinone 
nucleus linked through a glycosidic bond at ring atom 7 to an amino sugar, daunosamine. 
Chemically, doxorubicin hydrochloride is (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-a-
Llyxo-hexopyranosyl)-oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-
methoxy5,12-naphthacenedione hydrochloride. The structural formula is as follows: 
 

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Doxorubicin binds to nucleic acids, presumably by specific intercalation of the planar 
anthracycline nucleus with the DNA double helix. The anthracycline ring is lipophilic, 
but the saturated end of the ring system contains abundant hydroxyl groups adjacent to 
the amino sugar, producing a hydrophilic center. The molecule is amphoteric, containing 
acidic functions in the ring phenolic groups and a basic function in the sugar amino 
group. It binds to cell membranes as well as plasma proteins. 
 
The cytotoxic effect of doxorubicin on malignant cells and its toxic effects on various 
organs are thought to be related to nucleotide base intercalation and cell membrane lipid 
binding activities of doxorubicin. Intercalation inhibits nucleotide replication and action 
of DNA and RNA polymerases. The interaction of doxorubicin with topoisomerase II to 
form DNA-cleavable complexes appears to be an important mechanism of doxorubicin 
cytocidal activity. 
 
Doxorubicin cellular membrane binding may affect a variety of cellular functions. 
Enzymatic electron reduction of doxorubicin by a variety of oxidases, reductases and 
dehydrogenases generates highly reactive species including the hydroxyl free radical 
OH•. Free radical formation has been implicated in doxorubicin cardiotoxicity by means 
of Cu (II) and Fe (III) reduction at the cellular level. 
 
 
 
Pharmacokinetics 
Pharmacokinetic studies, determined in patients with various types of tumors undergoing 
either single or multi-agent therapy have shown that doxorubicin follows a multiphasic 
disposition after intravenous injection. In four patients, doxorubicin has demonstrated 
dose-independent pharmacokinetics in the dose range of 30 to 70 mg/m2. 
 
Distribution 
The initial distribution half-life of approximately 5 minutes suggests rapid tissue uptake 
of doxorubicin, while its slow elimination from tissues is reflected by a terminal half-life 
of 20 to 48 hours. Steady-state distribution volume ranges from 809 to 1214 L/m2 and is 
indicative of extensive drug uptake into tissues. Binding of doxorubicin and its major 

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metabolite, doxorubicinol, to plasma proteins is about 74 to 76% and is independent of 
plasma concentration of doxorubicin up to 1.1 mcg/mL. 
 
Metabolism 
Enzymatic reduction at the 7 position and cleavage of the daunosamine sugar yields 
aglycones which are accompanied by free radical formation, the local production of 
which may contribute to the cardiotoxic activity of doxorubicin. Disposition of 
doxorubicinol (DOX-OL) in patients is formation rate limited, with the terminal half-life 
of DOX-OL being similar to doxorubicin. The relative exposure of DOX-OL, i.e., the 
ratio between the AUC of DOX-OL and the AUC of doxorubicin, compared to 
doxorubicin ranges between 0.4 and 0.6. 
 
Excretion 
Plasma clearance is in the range 324 to 809 mL/min/m2 and is predominately by 
metabolism and biliary excretion. Approximately 40% of the dose appears in the bile in 5 
days, while only 5 to 12% of the drug and its metabolites appear in the urine during the 
same time period. In urine, <3% of the dose was recovered as DOX-OL over 7 days. 
 
Form 
 
Commercially available from various manufacturers as lyophilized powder for 
reconstitution in 10-, 20-, 50-, 100-and 150-mg vials.  Also available as solution (2 
mg/ml) in 10-, 20-, 50- and 200-mg vials for injection.  Please refer to FDA-approved 
package insert for complete product information. 
 
Storage and Stability 
Intact vials of doxorubicin should be stored in the refrigerator.  Intact vials of powder for 
reconstitution should be stored at room temperature.  Reconstituted solutions are stable 
for 7 days at room temperature and 15 days under refrigeration when protected from 
light.  Commercially available solutions labeled as such are intended to be multi-dose 
vials. 
 
Compatibility 
 
Doxorubicin is compatible with saline (0.9% Sodium Chloride Injection). Doxorubicin 
should not be mixed with heparin or fluorouracil since it has been reported that these 
drugs are incompatible to the extent that a precipitate may form. Contact with alkaline 
solutions should be avoided since this can lead to hydrolysis of doxorubicin. Until 
specific compatibility data are available, it is not recommended that doxorubicin be 
mixed with other drugs. 
 
Handling 

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40 
 
 
Procedures for proper handling and disposal of anti-cancer drugs should be considered. 
Several guidelines on this subject have been published. There is no general agreement 
that all the procedures recommended in the guidelines are necessary or appropriate. 
However, given the toxic nature of this substance, the following protective 
recommendations are provided:  
Personnel should be trained in good technique for handling. 
 
Pregnant staff should be excluded from working with this drug. 
 
 Personnel handling doxorubicin should wear protective clothing: goggles, gowns 
and disposable gloves and masks. 
  
A designated area should be defined and the work surface should be protected by 
disposable, plastic-backed, absorbent paper. 
  
All items used for administration or cleaning, including gloves, should be placed in 
high-risk waste-disposal bags for high-temperature incineration. 
  
Spillage or leakage should be treated with dilute sodium hypochlorite (1% 
available chlorine) solution, preferably by soaking, and then water. 
  
All cleaning materials should be disposed of as indicated previously. 
  
In case of skin contact thoroughly wash the affected area with soap and water or 
sodium bicarbonate solution. However, do not abrade the skin by using a scrub 
brush. 
  
In case of contact with the eye(s), hold back the eyelid(s) and flush the affected 
eye(s) with copious amounts of water for at least 15 minutes. Then seek medical 
evaluation by a physician. 
  
Always wash hands after removing gloves. 
 
Availability 
 
Commercially available from various manufacturers. 
 
Preparation 
Reconstitute the vials with 5, 10, 25, 50, or 75 ml, respectively, of sodium chloride for 
injection, USP. 

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Administration 
 
Administer doxorubicin intravenously, either peripherally as a bolus injection or through 
a central venous line.  Avoid extravasation, since severe local tissue necrosis may result. 
Administration time is per institutional standards.  
 
Ordering 
 
Doxorubicin will be ordered by pharmacy as institutional policies.  
 
Accountability 
 
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.) 
 
Destruction and Return 
 
Unused supplies of doxorubicin should be destroyed according to institutional policies.  
 
8.3 
Cyclophosphamide 
 
Refer to the FDA approved package description for more information. 
 
Description 
 
Cyclophosphamide (INN, trade names 
Endoxan,  Cytoxan,  Neosar,  Procytox,  Revimmune), also known as cytophosphane, is a 
nitrogen mustard alkylating agent  from the oxazaphosphorine group. An alkylating agent 
adds an alkyl group to DNA. It attaches the alkyl group to the guanine base of DNA, at 
the number 7 nitrogen atom of the imidazole ring. This interferes with DNA replication 
by forming intrastrand and interstrand DNA crosslinks. 
 
Cyclophosphamide for Injection, USP is a sterile white powder containing 
cyclophosphamide monohydrate. Cyclophosphamide is a synthetic antineoplastic drug 
chemically related to the nitrogen mustards. Cyclophosphamide is a white crystalline 
powder with the molecular formula C7H15CI2N2O2P•H2O and a molecular weight of 
279.1. The chemical name for cyclophosphamide is 2-
[bis(2chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide monohydrate. 

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42 
 
Cyclophosphamide is soluble in water, saline, or ethanol and has the following structural 
formula: 
 
 
 
Pharmacokinetics 
Cyclophosphamide is administered as an inactive prodrug that must undergo activation to 
form the active metabolite 4 hydroxy cyclophosphamide through phase I metabolism by 
cytochrome P450 (CYP) enzymes 2B6, 2C8, 2C9, 2C19, 3A4 and 3A5. Detoxification is 
primarily through glutathione S transferases (GSTA1, GSTP1) and alcohol 
dehydrogenase (ALDH1, ALDH3). Concomitant therapy with inducers of 
cyclophosphamide metabolizing enzymes (e.g. CYP 2B6, 2C9, 3A4) enhances the 
enzyme expression and may potentially increase the formation of metabolites responsible 
for cytotoxicity. In contrast, the inhibitors could interfere with cyclophosphamide 
activation and may alter the effectiveness of cyclophosphamide treatment. 
The decline in CP plasma levels following an i.v. dose is biexponential with terminal 
half-life averaging 7 hours (1.8 to 12.4) for adults, and 4 hours (2.4 to 6.5) for children; 
daily administration of approximately 50 mg/Kg bid or qid (i.v. infusion) to children 
significantly decreased both plasma half life and urinary excretion of CP. With daily 
exposure or repeated high-dose administration (i.v.) of cyclophosphamide to adult 
patients, the half-life of CP decreased without an increase in urinary excretion, 
suggesting that the drug induces its own metabolism. After an i.v. dose, the NBP [4-
(nitrobenzyl)pyridine] plasma alkylating activity peaks 2 hours after administration, and 
declines with a half-life of 7.7 hours. Phosphoramide mustard in 3 patients, receiving 60-
75 mg/Kg cyclophosphamide, peaked 2 to 3 hours after the administration of CP at levels 
10 to 20% of the unchanged drug, and declined slowly with levels still detectable at 24 
hours. Even with doses as high as 80 mg/Kg, the plasma half-life of CP does not 
increase. 
The t½ and AUC of cyclophosphamide after a 5-day continuous infusion schedule of 
300-400 mg/m2/day, were similar to the t½ and AUC of a 1500 mg/m2 i.v. bolus. The 
AUC of the alkylating activity after 5-day i.v. infusion, however, was three times higher 
than the AUC of alkylating activity after 1500 mg/m2 i.v. bolus administration of 
cyclophosphamide.  
After CP administration to man and laboratory animals, significant differences in the 
pharmacokinetic parameters of the active metabolite 4-hydroxycyclophosphamide in 

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43 
 
both man and animals were found. In man, the active metabolite in blood was found at 
only low but longer lasting concentrations compared to the high and relatively short time 
concentration in blood of mice and rats, after a comparable dose. 
Distribution 
A mean apparent volume of distribution of cyclophosphamide was 0.56 L/Kg in adults 
and 0.67 L/Kg in children. Tissue Distribution of CP after i.v. administration to cancer 
patients indicated that both unchanged parent drug and metabolites in small quantities 
penetrate the blood brain barrier; brain tissue concentrations being similar to those in 
blood. Biopsies, performed 2 hours after CP infusion, indicated approximately 30% more 
radioactivity in lymph nodes compared to muscle, adipose tissue or skin, but relative 
proportions of unchanged drug metabolites were not established. 
Protein Binding: 12 to 14% of unchanged cyclophosphamide is protein-bound; the 
alkylating metabolites, however, are more extensively bound, namely 67% of the total 
plasma alkylating activity, and in another study, 39% of phosphamide mustard was 
protein-bound. 
Metabolism 
While chemically not reactive, the primary metabolites 4-hydroxycyclophosphamide and 
aldophosphamide are cytotoxic in vitro, and may represent transport forms of the 
alkylating moiety, phosphoramide mustard. The two primary metabolites can be further 
oxidized into the major urinary metabolites 5-ketocyclophosphamide and 
carboxyphosphamide. Nor-nitrogen mustard, a decomposition product of  
carboxyphosphamide, is an active alkylating agent with cytotoxicity in vivo and in vitro, 
however, little antitumour activity could be demonstrated; yet, it may play a role in the 
hematopoietic and other toxicities of cyclophosphamide. Another metabolite formed 
from aldophosphamide is acrolein, which has been identified as the most urotoxic 
species. 
 
Excretion 
In man, a generally higher proportion of the administered CP is excreted as metabolites 
in urine. Urinary recovery of radioactivity after intravenously administered 
14Ccyclophosphamide to patients ranged from 59 to 82% after 4 days, while not more 
than 20% of i.v. cyclophosphamide was excreted unchanged in urine at any dose level. 
Renal clearance estimates of between 5.3 and 11 mL/min indicate substantial renal 
tubular reabsorption. 
Form 
Commercially available as powder for injection in 100-mg, 200-mg, 500-mg, 1-g, and 2g 
vials.  Please refer to the FDA-approved package insert for complete product 
information. 

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44 
 
 
Storage and Stability 
Store intact vials of powder at room temperature (15° to 30°C). Reconstitute lyophilized 
cyclophosphamide is chemically and physically stable for 24 hours at room temperature 
or for 6 days in the refrigerator (2° to 8°C).  It does not contain any antimicrobial 
preservative, and care must therefore be taken to ensure the sterility of prepared solution. 
 
Compatibility 
 
It is prudent to monitor, among others, the following drugs if administered concurrent 
with cyclophosphamide: Colchicine, Probenecid, Sulfinpyrazone, Chlorambucil, 
Mercaptopurine. PROCYTOX should not be reconstituted with benzyl alcohol-preserved 
diluent solution such as bacteriostatic sodium chloride when used in children or infants, 
due to toxicity concerns in this age group (i.e., gasping syndrome in infants). Further, 
PROCYTOX should not be reconstituted or diluted with benzyl alcohol-containing 
diluents, as benzyl alcohol may catalyse the decomposition of cyclophosphamide. 
Therefore, it is recommended to reconstitute PROCYTOX with isotonic, sterile, Sodium 
Chloride Injection USP. 
 
Handling 
 
Preparation of PROCYTOX must take place in a Pharmacy or, in facilities where there is 
not a Pharmacy, in a Class II Type B or better, externally-vented biological safety 
cabinet. The biological safety cabinet should have airflow monitoring devices and should 
be certified at least annually. Only luer-lock connections should be used in the 
preparation of PROCYTOX. 
 
Procedures for proper handling and disposal of anti-cancer drugs should be considered. 
Several guidelines on this subject have been published. There is no general agreement 
that all the procedures recommended in the guidelines are necessary or appropriate. 
However, given the toxic nature of this substance, the following protective 
recommendations are provided:  
 
Personnel should be trained in good technique for handling. 
 
Pregnant staff should be excluded from working with this drug. 
 
 Personnel handling doxorubicin should wear protective clothing: goggles, gowns 
and disposable gloves and masks. 
  

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45 
 
A designated area should be defined and the work surface should be protected by 
disposable, plastic-backed, absorbent paper. 
  
All items used for administration or cleaning, including gloves, should be placed in 
high-risk waste-disposal bags for high-temperature incineration. 
  
Spillage or leakage should be treated with dilute sodium hypochlorite (1% 
available chlorine) solution, preferably by soaking, and then water. 
  
All cleaning materials should be disposed of as indicated previously. 
  
In case of skin contact thoroughly wash the affected area with soap and water or 
sodium bicarbonate solution. However, do not abrade the skin by using a scrub 
brush. 
  
In case of contact with the eye(s), hold back the eyelid(s) and flush the affected 
eye(s) with copious amounts of water for at least 15 minutes. Then seek medical 
evaluation by a physician. 
  
Always wash hands after removing gloves. 
 
Availability 
 
Commercially available from various manufacturers. 
 
Preparation 
Reconstitute 100-mg, 200-mg, 500-mg, 1-g, and 2-g vials with 5,10, 25, 50, or 100 ml of 
sterile water for injection respectively, for a final concentration of 20 mg/ml.  Vigorous 
shaking an/or gentle warming may be necessary.  Bacteriostatic water (paraben-preserved 
only) may also be used for reconstitution. 0.9% sodium chloride or 5% dextrose may also 
be used for reconstitution.   
 
Administration 
Intravenous administration preferably should be conducted as an infusion. To reduce the 
likelihood of adverse reactions that appear to be administration rate-dependent (e.g., 
facial swelling, headache, nasal congestion, scalp burning), cyclophosphamide should be 
injected or infused very slowly. Duration of the infusion also should be appropriate for 
the volume and type of carrier fluid to be infused. 
 
Ordering 
 

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46 
 
Cyclophosphamide will be ordered by pharmacy as institutional policies.  
 
Accountability 
 
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.) 
 
Destruction and Return 
 
Disposal of cyclophosphamide-contaminated clothing, gloves, utensils, broken glass etc. 
must be considered as hazardous waste. It must be deposited into a 4 mil thick 
polypropylene hospital trash bag (properly labeled), or be otherwise segregated and 
incinerated at above 1000 C. Chemical inactivation should, if possible, be avoided, since 
it is often ineffective and may produce byproducts that are more mutagenic than the 
parent drug. Unused supplies of cyclophosphamide should be destroyed according to 
institutional policies.  
 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES (COHORT A AND B) 
 
9.1 
Biomarker Studies 
 
The study also requires mandatory plasma/serum samples for biomarker research, which may 
include, among other analyses, the assessment of circulating tumor DNA. Additional candidate 
markers of response to treatments that emerge from other clinical or nonclinical studies may also 
be assessed in this study and can be assessed with different types of technologies. 
 
Handling of Samples: 
 
Research blood collection is mandatory for all patients for DNA isolation and cell-free 
circulating DNA analysis. The samples will be banked in the DF/HCC Core and Blood Tissue 
Bank in order to extract germline and cell-free DNA to be used as normal DNA reference for 
tumor tissue-based studies and for future research purposes. These specimens will become the 
property of the DF/HCC. 
 
The following research blood samples are required: 
•
Two 10 mL lavender top (EDTA Fisher #366643) tubes of whole blood at baseline (or at 
any time after registration but before initiating study therapy). 

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47 
 
•
Two 10 mL Streck blood collection tubes will be collected at baseline, prior to the first 
dose of AC (Cycle 5 Day 1) for Cohort A and prior to the first dose of Eribulin (Cycle 5 
Day 1) for Cohort B, the presurgery visit, and the post-surgical visit (8 tubes total). 
 
All samples should be de-identified and labeled with the Participant initials, Participant Study ID 
number and date of collection and time point (e.g., “Baseline” or “Presurgery”). Processing 
instructions for whole blood and circulating cfDNA are provided in the table below. 
 
Sample Type 
Processing Instructions 
Lavender Top 
Tubes: Whole 
Blood for 
genomic 
analysis 
Collect 20 cc of whole blood by standard venous phlebotomy into the 
EDTA (purple top) tubes provided. 
Tube should be labeled with patient study number, patient initials, 
and date /time of collection. 
After collection, invert the tubes 10 times to ensure adequate mixing 
and anticoagulation. DO NOT centrifuge this tube. Store tube at 
room temperature until ready for shipment. 
Ship in the ambient temperature (cold pack) compartment of the 
shipping kit. 
Do not freeze. 
Streck Tubes: 
Circulating cfDNA 
Collect 20 mL of whole blood into 2 x 10 ml Streck BCT tubes 
Invert tubes 10 times. 
Streck tubes should be kept at 6°C (42.8°F) to an ambient 
temperature of 37°C (98°F) until shipping. 
Streck tubes should be kept at 6°C (42.8°F) to an ambient 
temperature of 37°C (98°F) during shipping. 
Do not freeze specimens. 
 
DO NOT FREEZE OR REFRIGERATE STRECK TUBES. Streck tubes must be stored at room 
temperature to preserve the sample. 
 
Please email the DFCI Core Lab with the sample information the prior to the scheduled blood 
draw at: 
  
 
 
9.2 
Laboratory Correlative Studies 
 
Reversion of EMT phenotype (MET) and Normalization of the tumor vessel phenotype 
 
Collection and handling of specimens is the same for both cohorts. 
 
Collection of Specimen(s) 
 

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48 
 
A core needle breast tumor biopsy (14 gauge) will be acquired from all participants prior 
to starting protocol therapy, and on cycle 1 day 8 of treatment. Any residual tumor 
present in the mastectomy specimen will also be obtained. Research breast biopsies 
(baseline and cycle 1 day 8) are mandatory. 
 
Handling of Specimens(s) 
 
For research breast biopsies, 6-8 cores will be collected per timepoint (2 FFPE, 4 OCT). 
Two cores will be immediately formalin fixed before being embedded in paraffin blocks 
and stored.  Four cores will be embedded in OCT and then immediately frozen on dry ice 
and stored at -80°C.   
 
For any mastectomy tissue, the tumor section size should be about 20 mg. Only residual 
tumor measuring > 1 cm found within the mastectomy specimen on gross examination 
will be collected. Half of the specimen will be frozen on dry ice and stored at -80°C and 
half will be immediately formalin-fixed before being embedded in paraffin blocks and 
stored. Tumor tissue frozen in OCT may be stored at -80°C indefinitely. 
 
Coded laboratory specimens will be stored in the Tumor Bank of the DFCI. These 
specimens will become the property of DFCI.  Patients will be informed that their 
specimens may be used for research by investigators at DF/HCC. 
 
 
9.3 
Special Studies 
 
Reversion of EMT phenotype (MET) and Normalization of the tumor vessel phenotype 
 
Outcome Measure 
 
Preclinical data suggests that eribulin induces two specific biological effects in breast 
cancers: Reversion of EMT phenotype (MET) 25, 32 and normalization of the tumor vessel 
phenotype 29, 33.  Exploratory studies will be performed during this trial to determine if 
similar changes are seen after eribulin treatment of human inflammatory breast cancer. 
Identical exploratory studies will be performed after exposure to AC to determine if the 
biological effects are specific to eribulin, or due to chemotherapy in general.  
 
Method of Assessment 
 
•
RNA extracted from biopsy specimens with >50% tumor cellularity 
•
Reverse transcription to cDNA using standard protocols 
 
•
Expression of 10 EMT-related genes determined in each sample by RT-qPCR 
•
TGFB1, TGFB2, CDH1, KRT18, CDH2, VIM, TWIST1, SNAI2, ZEB1, ZEB2  

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49 
 
•
Primers designed in-house (PrimerBlast) 
•
2 housekeeping genes included 
 
•
Expression of angiogenesis-related genes determined in each sample by RT-qPCR 
•
15 genes selected based on pre-clinical data. Each has a defined role in modulating 
vessel phenotype or EC-PVC interactions 
•
VEGFA, VEGFR1, VEGFR2, VEGFR3, PGF, DLL4, JAG1, NOTCH4, EFNB2, 
EPHA2, EPHB1, WNT5A, WNT11, BPM4, CA9. 
 
Timing of Assessment 
 
•
Prior to starting protocol therapy and on Cycle 1, day 8 
•
Residual tumor harvested from mastectomy specimen. 
 
Method of Data Recording 
 
All RT-qPCR data (raw data) will be stored in an electronic database within the 
Department of Cancer Biology, Dana-Farber Cancer Institute.  Data will be de-identified 
to clinical details or patient demographics.  The database will also be copied and 
maintained on a second storage drive also held within the Department of Cancer Biology. 
 
9.3.6    Timing of Data Recording 
 
•
RT-qPCR for both cohorts will be conducted at the completion of the study, and data 
stored immediately as described. 
 
9.4 
Imaging Sub-study 
 
9.4.1 
Eribulin and vessel normalization 
 
•
This analysis will be performed on a sub-set of the patient population (N=5 from 
Cohort A; N=5 from Cohort B) 
 
 
9.4.2 
Outcome Measure 
 
Pre-clinical data demonstrated vascular remodeling among MX-1 and MDA-MB-231 
human breast cancer xenograft models 5 to 6 days after a single exposure to eribulin. 29 
The Ktrans values from tumor rim and core regions following treatment with eribulin 
resulted in a significant increase of Ktrans in tumor cores by day 6. The iAUC histogram 
analysis of the MX-1 xenograft model treated with eribulin demonstrated a rightward 
shift of the curve resulting in a convergence of the curves for tumor and rim between 
days 6 and 10. These results suggest that eribulin treatment induces tumor vascular 
remodeling which may contribute to its efficacy in human cancers. Exploratory studies 

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50 
 
will be performed during this trial to determine if similar changes are seen after eribulin 
treatment of human inflammatory breast cancer. Identical studies will be performed after 
exposure to AC to determine if these vascular effects are specific to eribulin, or due to 
chemotherapy in general.  
 
9.4.3 
Method of Assessment 
 
•
Patients will undergo a baseline DCE-MRI of the affected breast, and then receive their first 
therapeutic dose of eribulin (Cohort A) or AC (Cohort B).  Approximately 6-8 days later, 
DCE-MRI of the affected breast is repeated on the same scanner (Siemens 3T scanner). 
 
•
Changes in Ktrans, ve and vp determination of the tumor core and rim will be analyzed, as will 
changes in the iAUC computed pre-treatment and 6-8 days post-eribulin (Cohort A) or AC 
(Cohort B) dosing. 
 
9.4.4 
Timing of Assessment 
 
•
Imaging must be done after the research breast biopsy scheduled at baseline (prior to starting 
protocol therapy) and before cycle 1, day 8 research biopsy (after one dose of protocol 
therapy).  
 
9.4.5 
Method of Data Recording 
 
•
DCE MRI Acquisition. 
 
DCE MRI will be performed at the Dana-Farber Cancer Institute using a Siemens 3T 
scanner with a breast coil. Dynamic scan will be acquired prior to and after contrast 
agent gadopentetate dimeglumine, Gd- DTPA, is injected intravenously via a power 
injector. 
•
Quantify Ktrans, Normalized vessel area. 
 
The semiquantitative parameter, iAUC, and the quantitative physiological 
parameters, 
Ktrans, ve and vp, will be calculated for all tumor voxels from the DCE-MRI data.34  
 
•
Compare peripheral rim of tumor and central core of tumor. 
Regions-of-Interest (ROIs) will be manually drawn on the Ktrans map to select either 
the core or the rim of the lesion. The ROIs will be copied from the Ktrans map to the 
ve, vp and iAUC maps. Mean and histogram analysis in these ROIs will be compared 
between lesion rim and core.  
 
9.4.6 
Timing of Data Recording 

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•
Data recording of each DCE-MRI scan will be performed shortly after acquisition.  
 
 
 
 
 
 
 
 
 
 
 

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52 
 
10. STUDY CALENDAR 
 
Baseline evaluations are to be conducted within 4 weeks (28 days) prior to start of protocol therapy unless otherwise noted.  Baseline 
imaging may be performed up to 35 days prior to the start of protocol therapy. .   
 
Assessments must be performed prior to administration of any study agent. Study assessments and agents should be administered 
within + 2 days of the protocol-specified date, unless otherwise noted. 
 
Cohort A: 
 
Screening  
(< -28 
days prior 
to 
treatment) 
Pre-operative therapy 
Pre-surgical 
evaluation 
 
Surgeryk 
Post-surgical 
evaluation 
Follow-up Visitsm 
 
 
Cycle 1-4, 
eribulin day 
1 
Cycle 1-4, 
eribulin day 8 
Cycle 5-8, 
AC day 1 
 
 
 
 
Eribulin mesylate 
 
A 
A 
 
 
 
 
 
Doxorubicin / 
cyclophosphamid 
e (AC) 
 
 
 
A 
 
 
 
 
Concurrent meds 
X 
X 
 
X 
X 
 
X 
 
Physical exama, 
X 
X 
 
X 
X 
 
X 
 
Clinical tumor 
(breast/lymph 
nodes) 
assessmenta 
X 
X 
 
X 
X 
 
 
 

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53 
 
Vital signsb 
X 
X 
X 
X 
X 
 
X 
 
Performance 
status 
X 
X 
 
X 
X 
 
X 
 
CBC w/diff, plts 
Xc 
X 
X 
X 
X 
 
X 
 
Serum chemistryd 
Xc 
X 
X 
X 
X 
 
X 
 
Magnesium 
 
Xe 
 
 
 
 
 
 
EKG (12-lead) 
X 
 
 
 
 
 
 
 
Adverse event 
evaluation 
 
X 
 
X 
X 
 
X 
 
Pregnancy Testf 
X 
 
 
 
 
 
 
 
Mammogram/ 
ultrasound 
X 
 
 
 
Xh 
 
 
Xi 
PET/CT 
X 
 
 
 
 
 
 
 
CT 
chest, 
abdomen, pelvis 
X 
 
 
 
 
 
 
 
Clinical Breast 
MRI 
X 
 
 
 
Xh 
 
 
 
Cardiac Echog 
X 
 
 
 
 
 
 
 
Mastectomyj 
 
 
 
 
 
X 
 
 
Radiation 
therapyk 
 
 
 
 
 
 
 
X 
Endocrine 
therapyk 
 
 
 
 
 
 
 
X 

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54 
 
Research breast 
tissue collectionl 
X 
 
X 
 
 
X 
 
 
DCE-MRIn 
X 
 
X 
 
 
 
 
 
Research blood 
collectiono 
 
X 
 
X 
X 
 
X 
 
 
A: Eribulin mesylate – 1.4mg/m2 IV day 1, 8 every 21 days x 4 cycles. One cycle = 21 days.  
B: AC – doxorubicin 60mg/m2 IV and cyclophosphamide 600mg/m2 IV day 1 every 14 days x 4 cycles. One cycle = 14 days. Begin AC 2 weeks 
(14 days +/2 days) after cycle 4, day 8 of eribulin.  
a:     Physical examination performed on Day 1 of every cycle  (+/- 2 days). Physical examination must be performed         by a 
medically qualified individual such as a licensed physician, physician’s assistant, or advanced registered nurse practitioner. b.     
Vital signs should include: BP, HR, respiratory rate, body temperature and weight.  Height performed at baseline only. c:     If 
screening labs done < 7 days of C1D1, they do not need to be repeated 
d:     Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total protein, SGOT [AST], 
SGPT [ALT],   sodium. 
e:    Magnesium only required at C1D1 f: 
Serum or urine pregnancy test (women of childbearing 
potential). g:     ECHO is preferred but MUGA will be accepted.  h:    Breast imaging must be done 1-
2 weeks after completing preoperative treatment, prior to surgery. i:     Breast imaging per standard 
practice recommendations. j:     Surgery to occur 3 to 5 weeks following completion of AC. 
k:     Radiation therapy to begin approximately 3 to 6 weeks following surgery. Endocrine therapy (for ER and/or PR positive disease) per 
institutional standards may begin concurrent with or after completion of radiation therapy per physician preference.  
l:     Breast biopsy performed at baseline and prior to cycle 1, day 8. Research biopsies can be done the same day as treatment. Baseline biopsy can 
be done any time prior to initiating treatment, cycle 1, day 1. Cycle 1 day 8 biopsies can be done within 1 day prior to therapy.  Tissue is also to 
be collected at the time of surgery. See section 9.2 for tissue collection guidelines. 
m:   Every 3 months (+/- 1 month) x 1 year, then every 6 months (+/- 1 month) x 4 years, then annually.  Patients can be evaluated at any time as 
deemed clinically necessary by the investigator. 
n:    DCE-MRI performed in 5 patients. Imaging performed at baseline and prior to cycle 1, day 8. Baseline imaging can be performed any time 
prior to initiating treatment (cycle 1, day 1) but imaging must be performed after the research breast biopsy at baseline and the second DCEMRI 
must be performed prior to the cycle 1, day 8 research biopsy.  DCE-MRI of the affected breast is repeated on the same scanner (Siemens 3T 
scanner). There must be between 5-8 days between both DCE-MRI scans. 
o:    Research blood samples collected at screening OR C1D1, C5D1, pre-surgical evaluation and post-surgical evaluation. See Section 9.1 for details. 
 
 

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55 
 
 
 
Cohort B: 
 
Screening  
(< -28 days 
prior to 
treatment) 
Pre-operative therapy 
Pre-surgical 
evaluation 
 
Surgeryk 
Post-surgical 
evaluation 
Follow-up 
Visitsm 
 
 
Cycle 1-4, 
AC day 1 
Cycle 1 day 8 
Cycle 5-8, 
eribulin day 1 
Cycle 5-8, 
eribulin day 8 
 
 
 
 
Eribulin mesylate 
 
 
 
B 
B 
 
 
 
 
Doxorubicin / 
cyclophosphamide 
(AC) 
 
B 
 
 
 
 
 
 
 
Concurrent meds 
X 
X 
 
X 
 
X 
 
X 
 
Physical exama, 
X 
X 
 
X 
 
X 
 
X 
 
Clinical tumor 
(breast/lymph 
nodes) assessmenta 
X 
X 
 
X 
 
X 
 
 
 
Vital signsb 
X 
X 
 
X 
X 
X 
 
X 
 
Performance status 
X 
X 
 
X 
 
X 
 
X 
 
CBC w/diff, plts 
Xc 
X 
 
X 
X 
X 
 
X 
 
Serum chemistryd 
Xc 
X 
 
X 
X 
X 
 
X 
 
Magnesium 
 
 
 
Xe 
 
 
 
 
 
EKG (12-lead) 
X 
 
 
 
 
 
 
 
 
Adverse event 
evaluation 
 
X 
 
X 
 
X 
 
X 
 

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56 
 
B-HCGf 
X 
 
 
 
 
 
 
 
 
Mammogram/ 
ultrasound 
X 
 
 
 
 
Xh 
 
 
Xi 
PET/CT 
X 
 
 
 
 
 
 
 
 
CT 
chest, 
abdomen, pelvis 
X 
 
 
 
 
 
 
 
 
Clinical Breast 
MRI 
X 
 
 
 
 
Xh 
 
 
 
Cardiac Echog 
X 
 
 
 
 
 
 
 
 
Mastectomyj 
 
 
 
 
 
 
X 
 
 
Radiation therapyk 
 
 
 
 
 
 
 
 
X 
Endocrine 
therapyk 
 
 
 
 
 
 
 
 
X 
Research breast 
tissue collectionl 
X 
 
X 
 
 
 
X 
 
 
DCE-MRIn 
X 
 
X 
 
 
 
 
 
 
Research blood 
collectiono 
 
X 
 
 
X 
X 
 
X 
 

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57 
 
 
A: Eribulin mesylate – 1.4mg/m2 IV day 1, 8 every 21 days x 4 cycles. One cycle = 21 days.  
B: AC – doxorubicin 60mg/m2 IV and cyclophosphamide 600mg/m2 IV day 1 every 14 days x 4 cycles. One cycle = 14 days. Begin AC 2 weeks (14 days +/2 
days) after cycle 4, day 8 of eribulin.  
a:     Physical examination performed on Day 1 of every cycle  (+/- 2 days). Physical examination must be performed         by a 
medically qualified individual such as a licensed physician, physician’s assistant, or advanced registered nurse practitioner. b.     
Vital signs should include: BP, HR, respiratory rate, body temperature and weight.  Height performed at baseline only. 
c:     If screening labs done < 7 days of C1D1, they do not need to be repeated 
d:     Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total protein, SGOT [AST], SGPT [ALT],  
sodium. 
e:    Magnesium only required at C5D1 (prior to first dose of eribulin). 
f: 
Serum or urine pregnancy test (women of childbearing 
potential). g:     ECHO is preferred but MUGA will be accepted.  
h:    Breast imaging must be done 1-2 weeks after completing preoperative treatment, prior to surgery. 
i:     Breast imaging per standard practice recommendations. j:     
Surgery to occur 3 to 5 weeks following completion of AC. 
k:     Radiation therapy to begin approximately 3 to 6 weeks following surgery. Endocrine therapy (for ER and/or PR positive disease) per institutional standards 
may begin concurrent with or after completion of radiation therapy per physician preference.  
l:     Breast biopsy performed at baseline and prior to cycle 1, day 8. Research biopsies can be done the same day as treatment. Baseline biopsy can be done any 
time prior to initiating treatment, cycle 1, day 1. Cycle 1 day 8 biopsies can be done within 1 day prior to therapy.  Tissue is also to be collected at the time of 
surgery. See section 9.2 for tissue collection guidelines. 
m:   Every 3 months (+/- 1 month) x 1 year, then every 6 months (+/- 1 month) x 4 years, then annually.  Patients can be evaluated at any time as deemed clinically 
necessary by the investigator. 
n:    DCE-MRI performed in 5 patients. Imaging performed at baseline and prior to cycle 1, day 8. Baseline imaging can be performed any time prior to initiating 
treatment (cycle 1, day 1) but imaging must be performed after the research breast biopsy at baseline and the second DCE-MRI must be performed prior to the 
cycle 1, day 8 research biopsy.  DCE-MRI of the affected breast is repeated on the same scanner (Siemens 3T scanner). There must be between 5-8 days 
between both DCE-MRI scans. 
o:    Research blood samples collected at screening OR C1D1, C5D1, pre-surgical evaluation and post-surgical evaluation. See Section 9.1 for details. 
 

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58 
 
 
11. MEASUREMENT OF EFFECT 
 
11.1 
Clinical Evaluations 
 
For the purposes of this study, participants should be reevaluated during preoperative therapy 
every 1 cycle by physical examination. Photographs of the affected breast may be obtained. If 
extensive nodal involvement is present, patients should be evaluated with scans prior to cycle 5 
day 1 (first dose of AC for Cohort A or first dose of eribulin for Cohort B) and prior to surgery.  
 
Tumor response will be evaluated based upon resolution of edema, erythema and any density 
palpable within the effected breast. Breast imaging and clinical exam will determine final criteria for 
surgical respectability.   
 
In the case of skin changes, documentation by color photography is recommended. 
 
 
11.2 
Pathological Response Parameters 
 
Pathologic Complete Response (pCR) 
 
 
Complete pathologic disease response (pCR) is defined as absence of invasive carcinoma 
within the breast and axillary lymph nodes following preoperative therapy. Participants 
whose disease is not surgically resectable following preoperative treatment are 
considered as not having pCR. 
 
Residual cancer burden (RCB)  
 
Residual cancer burden (RCB) after preoperative therapy will be determined, as defined 
by Symmans et al.35  
 
11.3 
Disease-Free Survival, Time to Treatment Failure, Overall Survival 
 
•
Overall Survival: Overall Survival (OS) will be defined two ways: among patients 
who undergo surgery, as time from surgery until death from any cause; and among 
all patients, as the time from treatment initiation until death from any cause. 
Censoring will use the date last known alive. 
 
Disease-Free Survival: Disease-Free Survival (DFS) will be defined among patients who 
undergo surgery, as time from surgery until occurrence of one of the events below.  
 
•
Ipsilateral local-regional invasive breast cancer recurrence (i.e., an invasive breast 
cancer in the axilla, regional lymph nodes, chest wall and / or skin of the ipsilateral 
breast, or skin of the contralateral breast)  

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•
Distant recurrence (i.e., evidence of breast cancer in any anatomic site other than 
local-regional disease described above) that has either been histologically 
confirmed or clinically diagnosed as recurrent invasive breast cancer.  
 
•
Contralateral invasive breast cancer. 
 
•
Death attributable to any cause including breast cancer, non-breast cancer, or 
unknown cause (but cause of death should be specified if at all possible). 
 
•
Second primary cancers other than breast.  
 
Time to Treatment Failure: Time to treatment failure (TTF) will be defined among all 
patients from time of treatment initiation until occurrence of one of the events below or 
occurrence of progressive disease during preoperative therapy or treatment of disease that 
is not surgically resectable.    
 
•
Ipsilateral local-regional invasive breast cancer recurrence (i.e., an invasive breast 
cancer in the axilla, regional lymph nodes, chest wall and / or skin of the ipsilateral 
breast, or skin of the contralateral breast) . 
 
•
Distant recurrence (i.e., evidence of breast cancer in any anatomic site other than 
local-regional disease described above) that has either been histologically 
confirmed or clinically diagnosed as recurrent invasive breast cancer.  
 
•
Contralateral invasive breast cancer. 
 
•
Death attributable to any cause including breast cancer, non-breast cancer, or 
unknown cause (but cause of death should be specified if at all possible). 
 
•
Second primary cancers other than breast. 
 
For DFS and TTF, patients without an event will be censored at the date of last disease 
assessment. 
 
 
12. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 (Adverse 
Events: List and Reporting Requirements). 
 
12.1 
Data Reporting 
 

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60 
 
Method 
 
The ODQ will collect, manage, and perform quality checks on the data for this study. 
 
Responsibility for Data Submission 
 
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the ODQ according to the schedule set by the ODQ. 
 
12.2 
Data Safety Monitoring 
 
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of clinical specialists with 
experience in oncology and who have no direct relationship with the study. Information that 
raises any questions about participant safety will be addressed with the Overall PI and study team. 
 
The DSMC will review each protocol up to four times a year or more often if required to review 
toxicity and accrual data. Information to be provided to the committee may include: up-to-date 
participant accrual; current dose level information; DLT information; all grade 2 or higher 
unexpected adverse events that have been reported; summary of all deaths occurring with 30 days 
of intervention for Phase I or II protocols; for gene therapy protocols, summary of all deaths 
while being treated and during active follow-up; any response information; audit results, and a 
summary provided by the study team. Other information (e.g. scans, laboratory values) will be 
provided upon request.  
 
12.3 
Collaborative Research and Future Use of Data and Biospecimens 
 
Tissue, blood, bodily fluids, and other materials derived from these will be collected in this study 
to analyze genes, DNA, RNA, proteins and cells for the study’s correlative endpoints and potential 
future research, utilizing new types of biomarker testing as it becomes available.  
 
These samples and any data generated as a part of these clinical trials may be used for future 
research studies and may be provided to collaborating investigators both within and outside of the 
DF/HCC for either correlative endpoints or secondary use. Samples and data may be shared with 
outside non-profit academic investigators, as well as with for-profit pharmaceutical investigators 
or commercial entities, with whom we collaborate. When samples or data are sent to collaborators 
and when any research is performed on them, all information will be identified with a code, and 
will not contain any PHI, such as name, birthday, or MRNs.  
 
In order to allow the greatest amount of research to be performed on the specimens and 
information generated as a part of this trial, researchers in this study may share results of genetic 
sequencing with other scientists. De-identified specimen or genetic data may be placed into one of 
more publicly-accessible scientific databases, such as the National Institutes of Health’s Database 

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61 
 
for Genotypes and Phenotypes (dbGaP). The results from the correlative research on this study 
will be shared with these public databases. Through such databases, researchers from around the 
world will have access to de-identified samples or data for future research. More detailed 
information, beyond the public database, may only be accessed by scientists at other research 
centers who have received special permission to review de-identified data. 
 
 
13. STATISTICAL CONSIDERATIONS 
 
The primary objective of this phase II trial is to determine pathologic complete response (pCR) 
rate after preoperative therapy with either 4 cycles of eribulin mesylate (E) followed by 4 cycles 
of doxorubicin/cyclophosphamide (AC) or after 4 cycles of AC followed by 4 cycles of eribulin in 
HER2 negative inflammatory breast cancer, as well as to assess the residual cancer burden (RCB) 
after preoperative therapy.  
 
In the original design participants in Cohort A were to be enrolled using a single-arm, two-stage 
design. Cohort A did not meet the criteria to proceed to the second stage, with 1/16 patients achieving 
pCRs (0.0625; 90% CI 0.0032 to 0.2638). Cohort B are enrolled using a single-arm, two-stage 
design, with revised design criteria. 
 
The original study (Cohort A) was designed based upon published literature suggesting a 25% 
pCR rate following anthracycline/taxane preoperative therapy in ER- IBC.8 . More recent 
literature in IBC specifically and generally across all early breast cancer, indicate pCR rates are 
lower in ER+ than ER- disease (especially ER+/HER2-), which were the majority of patients 
enrolled in Cohort A. We have subsequently estimated that the pCR rate for ER+/HER2- IBC is 
significantly lower than 25%, approximately 2% based upon an analysis of DFCI IBC registry 
data (DF/HCC 11-035). For this reason, we modified the null and alternative hypotheses for 
Cohort B, as well as the α error, and plan enrollment of 16 patients, as was done for the first 
stage of Cohort A.  
 
In both cohorts, following surgery, radiation therapy and endocrine therapy are given according to 
standard of care. Participants will be followed post-therapy for recurrence and survival. 
Efficacy of protocol therapy, defined as disease-free survival (DFS), time to treatment failure (TTF), 
and overall survival (OS), will also be assessed. 
 
For correlative studies, research biopsies will be obtained prior to start of preoperative therapy, and 
prior to day 8 of cycle 1 of preoperative therapy. A sample of any residual disease within the 
breast at time of surgery will be obtained. 
 
13.1 
Study Design/Endpoints 
 
13.1.1 Primary Endpoint. 
 

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62 
 
The primary endpoint is pCR, as defined in Section 11.2.  
 
13.1.2 Design 
 
The study (Cohort A) was planned using Simon minimax two-stage design. We propose 
that if the proportion of participants experiencing pCR is ≤ 0.10 then the preoperative 
EAC regimen is considered minimally effective, versus an alternative hypothesis that the 
EAC regimen is worthy of further study if proportion pCR ≥ 0.30. After testing the 
regimen on 16 participants in the first stage, the study will be terminated if ≤ 1 have pCR, 
If the preoperative EAC regimen is actually not effective, there is an α=0.095 probability 
of concluding that it is (target α=0.10); if the EAC regimen is actually effective, there is 
an 0.097 probability of concluding that it is not (target β=0.10).  
 
For Cohort B, the design will use a two-stage design. We propose that if the proportion of 
participants experiencing pCR is ≤ 0.02 then the preoperative ACE regimen is considered 
minimally effective, versus an alternative hypothesis that the ACE regimen is worthy of 
further study if proportion pCR ≥ 0.23. After testing the regimen on 12 participants, the if 
no patient has pCR then enrollment will cease; whereas if ≥1 patient has pCR then 
enrollment will proceed to 16 patients. The regimen is considered promising if ≥ 2 of 16 
have pCR. If the regimen is actually not worthy of further study, there is an α=0.038 
probability of concluding that it is (target α=0.05); if the regimen is actually effective, 
there is an 0.098 probability of concluding it is (target β=0.10). If 2 of 16 patients have 
pCR then the two-sided 90% CI unadjusted for the two-stage design is (0.0226 to 
0.3438). 
 
13.1.3 Primary Analysis 
The number and proportion of participants achieving pCR following preoperative 
treatment will be summarized with 2-sided confidence interval (CI), separately for each 
cohort. For Cohort A, both 80% and 90% CI will be reported; for Cohort B, 90% CI will 
be reported. The pCR rates will also be summarized according to hormone receptor status 
(ER and/or PR positive, or triple-negative).  
 
13.2 
Sample Size, Accrual Rate and Study Duration 
 
If the trial continues to the second stage, then 25 patients will be enrolled (Cohort A); otherwise 
16 patients will be enrolled in the first stage. In Cohort B, 16 patients will be enrolled. If a patient 
does not initiate protocol therapy, she will be replaced. Accrual is expected to be 10-12 patients 
per year for 2 years. Given the historic data of median OS among IBC patients with ER positive 
(3.2 years) or triple negative (2.0 years), the study duration to assess OS is anticipated to last for 
at least 5 years. 36, 37  
 

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63 
 
 
Accrual Targets 
Sex/Gender 
Ethnic Category 
Females 
 
Males 
 
Total 
Hispanic or Latino 
1 
+
 
=
 
Not Hispanic or Latino 
24 
+
 
=
 
Ethnic Category: Total of all subjects 
 
25 
+
 
0 
=
 
25 
Racial Category 
 
American Indian or Alaskan Native 
 
+
 
=
 
Asian 
1 
+
 
=
 
Black or African American 
1 
+
 
=
 
Native Hawaiian or other Pacific 
Islander 
0 
+
 
=
 
White 
23 
+
 
=
 
Racial Category: Total of all subjects 
 
25 
+
 
0 
=
 
25 
 
 
 
13.3 
Stratification Factors 
 
N/A 
 
13.4 
Interim Monitoring Plan 
 
See Section 12.2 
 
13.5 
Analysis of Primary Endpoints 
 
See Section 13.1 
 
13.6 
Analysis of Secondary Endpoints 
 
Therapeutic Efficacy Objectives 
Time-to-event distributions for DFS, TTF and OS will be summarized using the method 
of Kaplan-Meier and two-sided 90% CI for the medians will be summarized (endpoints 
defined in Section 11.3), separately by cohort. Exploratory analyses will summarize 
postsurgery DFS and post-surgery OS distributions according to pCR and/or according to 

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64 
 
RCB and estimate hazards ratios with 90% CI using Cox proportional hazards models, 
for the two cohorts combined.  
 
Residual Cancer Burden following preoperative treatment is defined according to Symmans 
et al35.  The number and proportion of participants achieving RCB-0, I, II, III will be 
summarized with two-sided 90% exact binomial CIs. 
 
Correlative Objectives  
 
Assess the changes in gene expression following administration of 1 cycle of eribulin, as 
seen in preclinical models: 
 
The paired expression levels of each gene at the two time points will be summarized 
graphically and descriptively. Wilcoxon signed rank test will be used to determine if there 
are any significant changes in the expression level of each gene between Biopsy 1 and 
Biopsy 2, and secondarily between and Biopsy 1 and Mastectomy among the subgroup 
who do not achieve pCR (i.e., have residual invasive tumor in breast or lymph nodes at 
mastectomy).  If 25 patients are enrolled, and assuming 80% (n=20) have adequate RNA 
and expression data in paired biopsy samples, there is 80% power to detect a 0.6 SD 
change using a signed rank test (two-sided =0.10).  
 
Exploratory analysis will be performed to determine if there is any correlation between the 
changes in imaging (ktrans, ve, vp and iAUC) and genomic changes determined on core 
biopsies of the breast sampled at the same time points. 
 
Assess the association of changes in gene expression with pCR and RCB at mastectomy: 
Exploratory analyses will summarize gene expression changes separately according to pCR 
and RCB status. The analyses will be descriptive.  
 
The association of correlative biomarkers and clinical outcomes will be exploratory and 
hypothesis-generating and will not adjust for multiple comparisons in any statistical 
inferences. 
  
In additional exploratory analyses, the changes will be dichotomized at the median, and post-
surgery DFS and OS summarized according to degree of change using KM method. 
 
13.7 
Reporting and Exclusions 
 
Evaluation of Toxicity 
 
The frequencies of adverse events (AE) while on study treatments will be summarized and 
tabulated according to treatment phase (preoperative vs. post-surgical) and at any time. 

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65 
 
Enrolled patients who received at least one dose of study treatment will be included in the 
analyses. 
 
Evaluation of the Primary Efficacy Endpoint 
 
All participants who initiate the preoperative treatment phase will be considered as evaluable 
for the preoperative treatment endpoints of pCR and RCB. Patients who do not initiate 
preoperative treatment would also be excluded from analyses of time-to-event endpoints. 
 
 
14. PUBLICATION PLAN 
 
The data will be collected and analyzed by Dr. Filipa Lynce, Dr. Meredith Regan, ScD from the 
Department of Biostatistics and Computational Biology, and key co-investigators. The results will 
be shared with
 in advance of publication. It is anticipated that results will be 
made public within 12 months of the end of data collection. Initial release may be an abstract that 
meets the requirements of the International Committee of Medical Journal Editors. The primary 
endpoint of the study will not be reported until the study is closed to accrual and there is adequate 
follow-up time to estimate the response rate. A report will also be published in a peer-reviewed 
journal. A full report of the outcomes will be made public no later than three (3) years after the end 
of data collection. 
 
15. REFERENCES   
 
1.
Kim T, Lau J, Erban J. Lack of uniform diagnostic criteria for inflammatory breast cancer 
limits interpretation of treatment outcomes: a systematic review. Clin Breast Cancer. 2006;7: 
386-395. 
2.
Woodward WA, Cristofanilli M. Inflammatory breast cancer. Semin Radiat Oncol. 2009;19: 
256-265. 
3.
Anderson WF, Chu KC, Chang S. Inflammatory breast carcinoma and noninflammatory 
locally advanced breast carcinoma: distinct clinicopathologic entities? J Clin Oncol. 2003;21: 
2254-2259. 
4.
Walshe JM, Swain SM. Clinical aspects of inflammatory breast cancer. Breast Dis. 2005;22: 
35-44. 
5.
Zucali R, Uslenghi C, Kenda R, Bonadonna G. Natural history and survival of inoperable 
breast cancer treated with radiotherapy and radiotherapy followed by radical mastectomy. 
Cancer. 1976;37: 1422-1431. 
6.
Ueno NT, Buzdar AU, Singletary SE, et al. Combined-modality treatment of inflammatory 
breast carcinoma: twenty years of experience at M. D. Anderson Cancer Center. Cancer Chemother 
Pharmacol. 1997;40: 321-329. 
7.
Baldini E, Gardin G, Evagelista G, Prochilo T, Collecchi P, Lionetto R. Long-term results of 
combined-modality therapy for inflammatory breast carcinoma. Clin Breast Cancer. 2004;5: 
358363. 

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8.
Cristofanilli M, Gonzalez-Angulo AM, Buzdar AU, Kau SW, Frye DK, Hortobagyi GN. 
Paclitaxel improves the prognosis in estrogen receptor negative inflammatory breast cancer: the M. 
D. Anderson Cancer Center experience. Clin Breast Cancer. 2004;4: 415-419. 
9.
Hirata Y, Uemura D. Halichondrins: antitumor plyether macrolides from a marine sponge. 
Pure Appl Chem. 1986: 701-710. 
10.
Bai RL, Paull KD, Herald CL, Malspeis L, Pettit GR, Hamel E. Halichondrin B and 
homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of 
tubulin-based mechanism of action by analysis of differential cytotoxicity data. The Journal of 
biological chemistry. 1991;266: 15882-15889. 
11.
Hirata Y, Uemura D. Halichondrins: antitumor plyether macrolides from a marine sponge. 
Pure Appl Chem. 1986;58: 701-710. 
12.
Jordan MA, Kamath K, Manna T, et al. The primary antimitotic mechanism of action of the 
synthetic halichondrin E7389 is suppression of microtubule growth. Molecular cancer therapeutics. 
2005;4: 1086-1095. 
13.
Kuznetsov G, Towle MJ, Cheng H, et al. Induction of morphological and biochemical 
apoptosis following prolonged mitotic blockage by halichondrin B macrocyclic ketone analog 
E7389. Cancer research. 2004;64: 5760-5766. 
14.
Towle MJ, Salvato KA, Budrow J, et al. In vitro and in vivo anticancer activities of synthetic 
macrocyclic ketone analogues of halichondrin B. Cancer research. 2001;61: 1013-1021. 
15.
Vahdat LT, Pruitt B, Fabian CJ, et al. Phase II study of eribulin mesylate, a halichondrin B 
analog, in patients with metastatic breast cancer previously treated with an anthracycline and a 
taxane. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 
2009;27: 2954-2961. 
16.
Cortes J, Vahdat L, Blum JL, et al. Phase II study of the halichondrin B analog eribulin 
mesylate in patients with locally advanced or metastatic breast cancer previously treated with an 
anthracycline, a taxane, and capecitabine. Journal of clinical oncology : official journal of the 
American Society of Clinical Oncology. 2010;28: 3922-3928. 
17.
Cortes J, O'Shaughnessy J, Loesch D, et al. Eribulin monotherapy versus treatment of 
physician's choice in patients with metastatic breast cancer (EMBRACE): a phase 3 open-label 
randomised study. Lancet. 2011;377: 914-923. 
18.
Bauer RL, Busch E, Levine E, Edge SB. Therapy for inflammatory breast cancer: impact of 
doxorubicin-based therapy. Ann Surg Oncol. 1995;2: 288-294. 
19.
Ellis GK, Barlow WE, Gralow JR, et al. Phase III comparison of standard doxorubicin and 
cyclophosphamide versus weekly doxorubicin and daily oral cyclophosphamide plus granulocyte 
colony-stimulating factor as neoadjuvant therapy for inflammatory and locally advanced breast 
cancer: SWOG 0012. J Clin Oncol. 2011;29: 1014-1021. 
20.
Horvath Z, Torday L, Hitre E, et al. Inflammatory breast cancer-comparing the effectivity of 
preoperative docetaxel-epirubicine protocol to conventional antracycline-containing chemotherapy 
to achieve clinical benefit and complete pathological response. Pathol Oncol Res. 2011;17: 541-550. 
21.
Vahdat LT, Pruitt B, Fabian CJ, et al. Phase II study of eribulin mesylate, a halichondrin B 
analog, in patients with metastatic breast cancer previously treated with an anthracycline and a 
taxane. J Clin Oncol. 2009;27: 2954-2961. 

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Protocol Version Date: December 18, 2018 
 
 
67 
 
22.
Cortes J, Vahdat L, Blum JL, et al. Phase II study of the halichondrin B analog eribulin 
mesylate in patients with locally advanced or metastatic breast cancer previously treated with an 
anthracycline, a taxane, and capecitabine. J Clin Oncol. 2010;28: 3922-3928. 
23.
Cortes J, O'Shaughnessy J, Loesch D, et al. Eribulin monotherapy versus treatment of 
physician's choice in patients with metastatic breast cancer (EMBRACE): a phase 3 open-label 
randomised study. Lancet. 2011;377: 914-923. 
24.
Masuda H, Brewer TM, Liu DD, et al. Long-term treatment efficacy in primary 
inflammatory breast cancer by hormonal receptor- and HER2-defined subtypes. Ann Oncol. 
2014;25: 384-391. 
25.
Yoshida T, Ozawa Y, Kimura T, et al. Eribulin mesilate suppresses experimental metastasis 
of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to 
mesenchymal-epithelial transition (MET) states. Br J Cancer. 2014;110: 1497-1505. 
26.
Yang J, Mani SA, Donaher JL, et al. Twist, a master regulator of morphogenesis, plays an 
essential role in tumor metastasis. Cell. 2004;117: 927-939. 
27.
Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with 
properties of stem cells. Cell. 2008;133: 704-715. 
28.
Yu M, Bardia A, Wittner BS, et al. Circulating breast tumor cells exhibit dynamic changes in 
epithelial and mesenchymal composition. Science. 2013;339: 580-584. 
29.
Funahashi Y, Okamoto K, Adachi Y, et al. Eribulin mesylate reduces tumor 
microenvironment abnormality by vascular remodeling in preclinical human breast cancer models. 
Cancer Sci. 2014;105: 1334-1342. 
30.
Goel S, Duda DG, Xu L, et al. Normalization of the vasculature for treatment of cancer and 
other diseases. Physiol Rev. 2011;91: 1071-1121. 
31.
Twelves C, Cortes J, Vahdat LT, Wanders J, Akerele C, Kaufman PA. Phase III trials of 
eribulin mesylate (E7389) in extensively pretreated patients with locally recurrent or metastatic 
breast cancer. Clin Breast Cancer. 2010;10: 160-163. 
32.
Dezso Z, Oestreicher J, Weaver A, et al. Gene expression profiling reveals epithelial 
mesenchymal transition (EMT) genes can selectively differentiate eribulin sensitive breast cancer 
cells. PLoS One. 2014;9: e106131. 
33.
Agoulnik SI, Kawano S, Taylor N, et al. Eribulin mesylate exerts specific gene expression 
changes in pericytes and shortens pericyte-driven capillary network in vitro. Vasc Cell. 2014;6: 
3. 
34.
Tofts PS, Brix G, Buckley DL, et al. Estimating kinetic parameters from dynamic 
contrastenhanced T(1)-weighted MRI of a diffusable tracer: standardized quantities and symbols. J 
Magn Reson Imaging. 1999;10: 223-232. 
35.
Symmans WF, Peintinger F, Hatzis C, et al. Measurement of residual breast cancer burden to 
predict survival after neoadjuvant chemotherapy. J Clin Oncol. 2007;25: 4414-4422. 
36.
Overmoyer B, H. G, LE. W, et al. Clinical outcomes of triple negative inflammatory breast 
cancer treated with contemporary anthracycline and taxane preoperative therapy support further 
investigation of therapeutic targets. 2014 San Antonio Breast Cancer Symptosium, 2014. 
37.
Li J, Gonzalez-Angulo AM, Allen PK, et al. Triple-negative subtype predicts poor overall 
survival and high locoregional relapse in inflammatory breast cancer. Oncologist. 2011;16: 1675-
1683. 

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APPENDIX A: PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
100 
Normal, no complaints, no evidence 
of disease. 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
1 
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
70 
Cares for self, unable to carry on 
normal activity or to do active work. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
2 
In bed <50% of the time.  
Ambulatory and capable of all self-
care, but unable to carry out any 
work activities.  Up and about 
more than 50% of waking hours. 
50 
Requires considerable assistance and 
frequent medical care. 
40 
Disabled, requires special care and 
assistance. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
10 
Moribund, 
fatal 
processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 
  
 
 
APPENDIX B: PROCEDURES FOR RESEARCH CORE BIOPSY OF BREAST  
 
Site: DFCI 
Biopsy Tissue Collection Protocol: I. 
Supplies: 

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69 
 
•
Tissue-Tek Cryomold 
•
Standard size 25x20x5mm (#4557) 
•
Fisher Scientific Cat# NC9511236 
 
OR 
•
Intermediate size 15x15x5mm (#4566) 
•
Fisher Scientific Cat# NC9542860 
•
Tissue-Tek O.C.T. Compound, 4oz (#4583): Fisher Scientific Cat# NC9638938 
•
Small specimen bag: Fisher Cat# 01-002-37 (or equivalent) 
•
Forceps: Fisher Cat# NC9832137 (or equivalent) 
•
Dry Ice and Cooler 
•
Cryoware Pen: Fisher Cat# 13-382-88 
•
Protocol® 10% Zinc Formalin Cat# 032-065 
 
II.   Procedure: 
•
Prepare cryomolds ahead of time: Squeeze small amount of OCT into cyromolds* – enough for 
there to be a thin layer covering the bottom let settle on a flat surface for a minute or two. It is 
crucial for future sectioning that these core biopsies are placed on a very flat OCT surface, so the 
best way to make sure these mold are flat is to freeze the bottom layer on a -80 freezer shelf.  
•
Bring all supplies needed to the biopsy as the tissue needs to be frozen or fixed in formalin 
immediately.  
•
You should ideally collect between 5 and 8 separate cores.Priority for core collection: 
1.
Frozen in OCT 
2.
Frozen in OCT 
3.
Formalin (for FFPE) 
4.
Formalin (for FFPE) 
5.
And any additional cores: Frozen in OCT 
•
For frozen samples: As soon as the specimen is ready, use disposable forceps to gently 
place only 1 core into each cryomold. Gently pick up the tissue with the forceps from one 
end of the core biopsy, being careful not to crush the tissue and immediately lay out the 
fresh biopsy tissue onto the center of the mold. Be sure to lay the tissue as flat to the 
mold, and as straight as possible. Keep molds on dry ice at all times. 
•
Once the specimen is in the cryomold, cover with more OCT making sure the tissue is 
entirely submerged.  
•
Immediately place cryomold with OCT and tissue onto dry-ice making sure the cryomold 
is level and will not tip over.  
•
The OCT will freeze into a solid white block within 5-10 minutes 
•
Once the blocks have completely frozen they can be put into a specimen bag and sealed. 
More than one block can be put into a bag. 
•
The bag should be labeled with: 
•
Patient name 
•
DFCI Study #:  
•
DFCI MRN # 
•
Date of biopsy 
•
Time Point (Biopsy #1, Biopsy #2, or Surgery) 
•
Number of blocks in the bag 

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•
For formalin fixed samples: As soon as the specimen is ready, use disposable forceps to 
gently place only 1 core into each formalin jar.  
•
The formalin containers should be labeled as bags above. 
•
All frozen research tissue should then be brought to the DF/HCC Core Blood and Tissue 
Bank on the day of collection. Samples in formalin should be brought to the BWH 
Specialized Histopathology Core. Cryomolds should be kept on dry ice at all times. 
 
*If time permits, the cryomolds can be placed onto the dry-ice once a thin layer of OCT has been put it but 
before the tissue is put in. Once this is frozen or begins to freeze, the tissue can be placed on top of the 
now frozen OCT and then covered with more liquid OCT and then placed back onto the dry ice to freeze 
completely.  
 
 

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APPENDIX C: MASTECTOMY TISSUE COLLECTION PROTOCOL 
I.  Procedure: 
•
Keep tissue on dry ice in insulated container as it is transported to the Institutional Tissue 
Bank. 
•
Place tumor into sterile petri dish in a cryostat at -20°C.  Using a sterile scalpel blade, cut 
two to four adjacent slices from the tumor which are representative of the tumor and which 
are grossly free of stroma/fat and necrosis.    There is no limitation on size of tissue 
obtained so long as the residual tumor within the mastectomy specimen is > 1 cm. One to 
two sections are placed immediately into formalin until it is collected by the study 
coordinator to be processed into paraffin embedded blocks. 
•
The other one to two pieces of tumor are placed on a chuck, layered with OCT, and allowed 
to freeze.  One block is faced off and multiple 5 μm thick sections are placed on a charged 
and labeled glass slide.  The slide is stained with hemotoxylin and eosin (H+E), coversliped, 
and then evaluated for tumor percentage (T), stroma/fat/blood percentages (S), and 
necrosis percentage (N).  The tumor percentage and stroma/fat/blood percentage should be 
based on area occupied by nuclei and not by that of cytoplasm. These results are written on 
the slide, recorded in the database, and the slides are stored in the Tissue Bank. 
*If time permits, the cryomolds can be placed onto the dry-ice once a thin layer of OCT has been put it but 
before the tissue is put in. Once this is frozen or begins to freeze, the tissue can be placed on top of the 
now frozen OCT and then covered with more liquid OCT and then placed back onto the dry ice to freeze 
completely.  
 
 
APPENDIX D: BIOASSAY TEMPLATES 
 
RNA extraction from frozen tumor specimens 
 
Frozen sections (20 micron thickness) will be cut from tissue cores embedded in OCT.  One slide from 
each specimen will be stained according to standard protocols with Haematoxylin and Eosin.  Only 
samples with at least 50% tumor cellularity will be included in the analysis. 
 
Total RNA will be extracted from samples according to standard in-house protocols.  RNA 
concentration and purity will be measured using the NanoDrop 1000 apparatus (ThermoScientific).  
RNA will then be stored at -80C until required for use. 
 
Reverse transcription  
 
RNA will be reverse transcribed using the iScript Reverse Transcription Supermix (Bio-Rad) according 
to manufacturer’s instructions.  A total of between 200ng and 1000ng of RNA will be reverse 
transcribed, depending upon the amount of total RNA available from each sample.  cDNA will be 
stored at -20C until utilized.  

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72 
 
 
Primer Design 
 
A single primer pair will be designed for RT-qPCR to each of the genes described in the protocol.  
Primers will be designed using the Primer-BLAST online tool (National Centre for Biotechnology 
Information) with attention paid to GC content (50-60%), melting temperature (50-65 degrees C), 
avoidance of secondary structure targeting, and minimization of 3’- and 5’-primer complementarity to 
avoid primer-dimer formation.  Primers will be ordered through the Dana-Farber Cancer Institute’s 
Molecular Biology Core Facility.  
 
RT-qPCR assays 
 
RT-qPCR will be performed in 96-well optical plates using an QT-qPCR instrument held in the 
Department of Cancer Biology, Dana-Farber Cancer Institute (Applied Biosystems).  Each RT-qPCR 
assay will be performed in triplicate, in addition to triplicate assays for two housekeeping genes.   
 
RT-qPCR will be set up in 20uL reactions per well as follows: 
1. SYBR qPCR Mastermix (BioRad): 10uL 
2. 3’-primer (5 pmol/uL): 0.3uL 
3. 5’-primer (5 pmol/uL): 0.3uL 
4. Nuclease free water: 7.4uL 
5. cDNA template: 2uL 
 
the RT-qPCR cycling conditions will be as follows: 
1. 50°C 2 min, 1 cycle 
2. 95°C 10 min, 1 cycle 
3. 95 °C 15 s -> 60 °C 30 s -> 72 °C 30 s, 40 cycles 
4. 72°C 10 min, 1 cycle 
 
 

DANA-FARBER CANCER INSTITUTE 
Nursing Protocol Education Sheet 
Protocol Number: 
15-292 
Protocol Name: 
  A Phase 2 Study of Eribulin Followed by Doxorubicin and Cyclophosphamide as 
Preoperative Therapy for HER2-negative Inflammatory Breast Cancer 
DFCI Site PI: 
Filipa Lynce MD 
DFCI Research Nurse: 
Margaret Haldoupis, Elizabeth Kasparian, Kathleen Roche, , Janet LaPointe, Victoria Brock; 
Christine Wong, Jaclyn Colucci  
Page the DFCI research nurse or DFCI site PI if there are any questions/concerns about the protocol.   
Please also refer to ONC 15: Oncology Nursing Protocol Education Policy 
*** Remember to check the ALERT PAGE*** 
              SPECIAL NURSING CONSIDERATIONS UNIQUE TO THIS PROTOCOL 
Study Agent 
 
Eribulin is a synthetic derivative of the natural product halichondrin B, which is isolated from a marine sponge. 
Halichondrin B exhibits anti-cancer activity through a microtubule-destabilizing anti-mitotic mechanism of action. 
Eribulin exerts its effects by binding to microtubules which causes interference in the normal cell cycle. This 
interference leads to cell death via apoptosis. Doxorubicin is a cytotoxic Anthracycline antibiotic. 
Cyclophosphamide is a nitrogen mustard alkylating agent. 
 
A cycle is defined as 21 days, cycles 1-4 of Eribulin, then every 14 days during cycles 5-8 of AC (Section 5.1) 
Dose Calc. 
•
Eribulin is dosed in mg/m2-Section 5.1.  
Doxorubicin is dosed in 
mg/m2 
•
Cyclophosphamide is dosed in mg/m2 
For All 3 Study Drugs: Body weight will be recorded on each treatment day and dosing BSA will be calculated per 
Institutional Guidelines.  (Section 5.3). 
Study Drug  
Administration 
Agent Administration Guidelines are found in Section  5.3 
•
Eribulin IV administered over approximately 2 to 5 minutes on Days 1 and 8 of every 21 day cycle for a total of 
4 cycles. (Section 5.3) 
•
Doxorubicin and Cyclophosphamide are administered concurrently (AC) beginning 2 weeks after the last 
dose of Eribulin is given (cycle 4, day 8).  
•
Doxorubicin 60 mg/m2 will be administered on an outpatient basis via I.V. push over approximately 3 to 5 
minutes on Day 1 of each 14 day cycle, for a total of 4 cycles.  
•
Doxorubicin is a vesicant, and is administered per Institutional Standard.  
•
Doxorubicin is administered immediately prior to the administration of Cyclophosphamide.  
•
Cyclophosphamide 600 mg/m2 will be administered on an outpatient basis via I.V. infusion over 
approximately 30 minutes on Day 1 of each 14 day cycle, for a total of 4 cycles.  
•
Cyclophosphamide is diluted in 250-500 ml normal saline, and its administration is per institutional 
standard/package insert. 
•
Cyclophosphamide is administered immediately following the administration of doxorubicin.  
•
If AC is delayed due to toxicity, drug administration every 3 weeks on a 21 day cycle may be given with the 
approval of the Principal Investigator. ( Section 6.2) 
•
Endocrine Therapy should be administered in Participants with EER/PR positive disease as defined by 
Institutional guidelines.   
Dose  
Mods & Toxicity 
Criteria to Treat, Dose Modifications/Dosing Delay for Toxicity are outlined in Section 5.6 
•
This protocol uses NCI CTCAE criteria, version 4.0 
•
Criteria to Treat is found in Section 5.2 
•
Expected toxicities are outlined in Section 7.1 
•
Dose modifications and delays for all drugs are outlined in Section 6.1 tables 1 and 2 for Eribulin. 
•
See Section 6.2, and tables 3 and 4 for Doxorubicin and Cyclophosphamide 
•
Maximum amount of dose reductions for Eribulin is 2. (Section 6.1)If Eribulin is held for greater than 2 weeks 
for toxicity, it should be discontinued. (Section 6.1) 
•
Management of toxicities related to Eribulin are listed in Table 2. 

•
No more than 1 dose-reduction of AC chemotherapy should be made for toxicity. (Section 6.2)  
If AC is 
held for >2 weeks for toxicity, Participants should be discontinued. (Section 6.1)  
Management of toxicities 
related to AC are listed in Table 4.  
Protocol #15-292: Drafted 10/9/15 MOD Approved 10/9/15 MOD; R: 12/19/2016 (amd. # 4) MML; R: 2/5/2017 (amd. #5) 
MML 
 
Con 
Meds 
Concomitant Therapy Guidelines are in Section 5.4 
•
Permitted meds are in Section 5.4 
•
Platelet growth factors are specifically prohibited. (Section 5.4) 
•
No Erythropoietin allowed while on study. (Section 5.4) 
•
GCSF is allowed during Doxorubicin / Cyclophosphamide cycles 5-8 
Req  
Data  
Study Calendar and Assessment Required data are outlined in Section 10.0 
Tips 
All study drugs require documentation of exact administration time. 
 
 
 

Protocol #15-292: Drafted 10/9/15 MOD Approved 10/9/15 MOD; R: 12/19/2016 (amd. # 4) MML; R: 2/5/2017 (amd. #5) 
MML