Clinical trial NCT01234532
Conditions: Estrogen Receptor-negative Breast Cancer HER2-negative Breast Cancer Progesterone Receptor-negative Breast Cancer Stage I Breast Cancer Stage II Breast Cancer Stage IIIA Breast Cancer Triple-negative Breast Cancer
Sponsor: University of Maryland, Baltimore
Start date: 2010-10-01; Completion date: 2017-05-01
Enrollment: 5 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase II trial is studying how well giving entinostat and anastrozole together works in treating postmenopausal women with triple-negative breast cancer that can be removed by surgery. Entinostat may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Estrogen can cause the growth of breast cancer cells. Hormone therapy using anastrozole may fight breast cancer by blocking the use of estrogen by the tumor cells. Giving entinostat together with anastrozole may be an effective treatment for breast cancer.

Detailed description:
PRIMARY OBJECTIVES:

I. To evaluate the safety and tolerability of entinostat in combination with anastrozole or tamoxifen. (Pilot) II. To determine the optimal dose of entinostat in combination with anastrozole or tamoxifen for phase II. (Pilot) III. To determine baseline and percentage change in proliferative index (Ki67) before and after treatment with entinostat and anastrozole/tamoxifen in triple negative breast cancer (TNBC). (Phase II) IV. To determine the estrogen receptor (ER) expression after treatment with entinostat and anastrozole/tamoxifen in TNBC. (Phase II)

SECONDARY OBJECTIVES:

I. To evaluate baseline and change in the expression levels of progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), cytokeratin 5/6 (CK5/6), and aromatase before and after treatment with entinostat and anastrozole/tamoxifen.

II. To assess baseline and change in tumor tissue histone H3 and H4 acetylation before and after treatment with entinostat and anastrozole/tamoxifen.

III. To assess the clinical and pathological response to preoperative combination of entinostat and anastrozole/tamoxifen in TNBC.

TERTIARY OBJECTIVES:

I. To correlate the levels of histone H3 and H4 acetylation in tumors with the changes in Ki67 and ER.

II. To evaluate baseline and change in gene methylation silencing and expression of candidate genes in tissues and in circulating DNA, including estrogen receptor (ER)-alpha, ER-beta, RAR-beta, cyclin D2, Twist, RASSF1A, and HIN-1.

III. To correlate entinostat trough concentrations with histone H3 and H4 acetylation in tumors as well as the change in Ki67 and ER.

IV. To evaluate baseline and change in the global gene expression profile before and after treatment with entinostat and anastrozole/tamoxifen.

OUTLINE: This is a multicenter, pilot study followed by a phase II study.

Patients receive entinostat orally (PO) once daily on days 1, 8, 15, 22, and 29 and anastrozole PO once daily on days 4-29. Patients then undergo mastectomy or lumpectomy.

Tumor tissue samples are collected at baseline or from original diagnosis, and during surgery for correlative studies by IHC and RT-PCR. Blood samples are also collected at baseline, on days 1 and 15, and during surgery for correlative studies.

After completion of study therapy, patients are followed up for 30 days.

Arms: arm 1: Neoadjuvant entinostat daily on days 1, 8, 15, 22, and 29 + anastrozole daily on days 4-29 followed by surgery ie either lumpectomy or mastectomy.

Correlative studies will be performed utilizing tissue and blood. A baseline tumor biopsy is done prior to study entry or archival tissue from diagnosis may be used and a representative tumor sample is submitted at time of surgery.

Bloods are drawn for correlative sciences on day 1 and 15 of treatment prior to entinostat dosing and 30 mins post and again on day of surgery.
Interventions: intervention 1: entinostat intervention 2: anastrozole intervention 3: diagnostic laboratory biomarker analysis intervention 4: therapeutic conventional surgery
Intervention descriptions: intervention 1: orally intervention 2: Given PO intervention 3: Correlative studies will be performed utilizing tissue and blood. A baseline tumor biopsy is done prior to study entry or archival tissue from diagnosis may be used and a representative tumor sample is submitted at time of surgery.

Bloods are drawn for correlative sciences on day 1 and 15 of treatment prior to entinostat dosing and 30 mins post and again on day of surgery. intervention 4: Lumpectomy or mastectomy will be performed follwoing day 29 of study therapy

Protocol documents:
Prot_SAP_000.pdf: 
Protocol #: GCC0927 
 
 
 
 
 
Version Date: 09/09/2014 
TITLE:  A PILOT AND PHASE II STUDY OF ENTINOSTAT AND 
ANASTROZOLE/TAMOXIFEN IN WOMEN WITH TRIPLE NEGATIVE BREAST 
CANCER TO EVALUATE BIOMARKERS AND SURROGATES FOR RESPONSE 
 
Principal Investigator: 
Katherine Tkaczuk, MD 
University of Maryland Greenebaum Cancer Center 
Telephone: (410) 328-2594 
Fax: (410) 328-6896 
E-mail address: ktkaczuk@umm.edu 
 
 
 
 
Co-Investigators: 
 
Saranya Chumsri, MD 
 
 
Mayo Clinic Florida 
 
 
4500 San Pablo Road 
 
 
Jacksonville, FL 32224 
Telephone: (904) 953-6822 
Fax: (904) 953-2315 
E-mail: Chumsri.saranya@mayo.edu  
 
 
 
Co-Investigators for Correlative Studies: 
 
Angela H. Brodie, Ph.D. 
University of Maryland GCC 
685 West Baltimore Street 
Baltimore, MD 21201 
Telephone: (410) 706-3137 
Fax: (410) 706-0032 
E-mail: abrodie@umaryland.edu   
Saraswati Sukumar, Ph.D 
The Sidney Kimmel Comprehensive Cancer Center at Johns 
Hopkins 
1650 Orleans Street, Rm 143 CRB 
Baltimore MD 21231-1000 
Telephone: (410) 614-2479 
Fax: (410) 614-4073 
Email: sukumsa@jhmi.edu 
 
Gauri J. Sabnis, PharmD, Ph.D. 
University of Maryland GCC 
685 West Baltimore Street 
Baltimore, MD 21201 
Telephone: (410) 706-4458 
Fax: (410) 706-0032 
E-mail: gsabn001@umaryland.edu 
 
Cristina Campassi 
University of Maryland 
Department of Radiology 
22 S. Greene Street 
Baltimore, MD 21201 
Telephone: (410) 328-3215 
Fax: (410) 328-5508 
E-mail: ccampassi@umm.edu  
 
Olga B. Ioffe, M.D. 
University of Maryland 
Department of Pathology 
22 S. Greene Street 
Baltimore, MD 21201 
Telephone: (410) 328-5525 
Fax: (410) 328-5508 
E-mail: oioffe@umm.edu  
Michelle Rudek, Pharm.D., Ph.D. 
The Sidney Kimmel Comprehensive Cancer Center at Johns 
Hopkins 
1650 Orleans Street  CRB1 Rm 184 
Baltimore, MD 21231-1000 
Telephone: 410-955-1129 
Fax: 410-502-0895 
E-mail: mrudek2@jhmi.edu  
 
 


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Statistician: 
Olga Goloubeva, PhD 
University of Maryland 
Medical School Teaching Facility, Suite 261 
10 South Pine Street 
Baltimore, Maryland 21201 
Telephone: (410) 706-8502 
Fax: (410) 706-8548 
E-mail: ogoloubeva@som.umaryland.edu 
 
Responsible Research Nurse: 
Nancy Tait 
University of Maryland Greenebaum Cancer Center 
22 S. Greene Street 
Baltimore, MD 21201 
Telephone: (410) 328-3546 
Fax: (410) 328-8616 
E-mail address: ntait@umm.edu 
 
Jane Lewis 
University of Maryland Greenebaum Cancer Center 
22 S. Greene Street 
Baltimore, MD 21201 
Telephone: (410) 328-7856 
Fax: (410) 328-8616 
E-mail address: jlewis@umm.edu 
Pharmacy Contact: 
Myounghee Lee 
Investigational Drug Services (IDS) 
University of Maryland Greenebaum Cancer Center 
22 S. Greene Street 
Baltimore, MD 21201 
Telephone: (410) 328-5468 
Fax: (410) 328-7326 
E-mail address: Mlee1@umm.edu 
 
 
 
 
Contract #  
  
NCI Supplied Agent: 
 
Commercial Agents: 
 
Entinostat or SNDX-275 (IND 61198; NSC 706995) 
 
Anastrozole (AstraZeneca) 
 
 
 
Protocol History: 
 
Version Date 
Description of Action 
10/29/09 
Original Submission to NCI 
1/8/10 
Follow Response 
10/28/10 
Amendment clarifying the shipping of the anastrozole, updating the research team, and defining 
the primary end points.  
1/19/11 
Version date 1/19/11. Face Sheet: Added Dr. Henry of U Mich as Investigator/participating site. 
Removed Dr. Kamboj from NorthShore, Dr. Kraft and MUSC as a participating site. Added 
California Cancer Consortium before listing of CCC sites. Adjusted investigator table as needed. 
Section 10.3 Study Days: deleted Methylation study profile on week 3, day 15.  
11/9/11 
Version date 11/9/11.  In response to RRA for Entinostat risk profile update. Section 7.1 updated 
with Entinostat CAEPR updated to Version 2.3, October 18, 2011. 
11/20/11 
- 
Disapproved 
Version date 11/20/11.  Face sheet: Title updated, added Northwestern, Indiana, and St. Joseph’s 
Medical PI and contact info, removed Loyola and Medical College of Wisconsin, updated Phase II 
Regulatory Manager and Responsible Data Manager.  Schema updated with addition of 
metastatic triple negative breast cancer and premenopausal women.  Section 1 Objectives 
updated with references to tamoxifen.  Section 2.4 updated with tamoxifen clinical experience.  
Section 2.5 rationale for using tamoxifen was added. Section 3.1 Inclusion Criteria updated with 
the number of unstained slides required and inclusion criteria for first and second cohort.  Section 
5.1 Agent Administration updated in response to addition of new cohorts.  Section 5.2.1 was 
updated to evaluate entinostat and anastrozole and entinostat and tamoxifen separately to 
determine the dose for phase II.  Section 5.4 updated to specify duration of therapy for each 
cohort.  Section 6.1 amended due to addition of tamoxifen.  Sections 7.2 and 8 were updated with 


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tamoxifen information.  Section 9 updated with instructions on obtaining a core specimen from the 
surgical sample after treatment for the first cohort and instructions on obtaining core biopsy 
specimens for the second cohort and timing clarifications were made to blood sample collections.  
Section 10 study calendar for first cohort updated with pregnancy test and tamoxifen information 
and study calendar for second cohort added and visit schedule was updated with these changes.  
Section 12 Statistical Considerations updated with changes for the new cohorts.  References 
updated.  Appendix C updated with tamoxifen.     
1/3/12 
Version date 1/3/12.  Face sheet: Title updated, added Northwestern, Indiana, and St. Joseph’s 
Medical PI and contact info, removed Loyola and Medical College of Wisconsin, updated Phase II 
Regulatory Manager and Responsible Data Manager.  Schema updated with addition of 
metastatic triple negative breast cancer and premenopausal women.  Section 1 Objectives 
updated with references to tamoxifen.  Section 2.4 updated with tamoxifen clinical experience.  
Section 2.5 rationale for using tamoxifen was added. Section 3.1 Inclusion Criteria updated with 
the number of unstained slides required  and inclusion criteria for first and second cohort.  Section 
4 removed old U of C registrar.  Section 5.1 Agent Administration updated in response to addition 
of new cohorts.  Section 5.2.1 was updated to evaluate entinostat and anastrozole and entinostat 
and tamoxifen separately to determine the dose for phase II.  Section 5.4 updated to specify 
duration of therapy for each cohort.  Section 6.1 amended due to addition of tamoxifen.  Section 
7.5.1 updated to as paper AdEERS forms are no longer accepted.   Sections 7.2 and 8 were 
updated with tamoxifen information.  Section 7.5.2 updated Cancer Clinical Trials Office contact 
information.  Section 9 updated with instructions on obtaining a core specimen from the surgical 
sample after treatment for the first cohort and instructions on obtaining core biopsy specimens for 
the second cohort and timing clarifications were made to blood sample collections.  Section 9.1.2 
updated with the requirement of ≥3 slides.  Section 10 study calendar for first cohort updated with 
pregnancy test and tamoxifen information and study calendar for second cohort added and visit 
schedule was updated with these changes.  Section 12 Statistical Considerations updated with 
changes for the new cohorts.  References updated.  Appendix C updated with tamoxifen.     
8/15/12 
Amendment submitted to clarify inclusion criteria, logistics regarding samples and shipping 
updated and non-participating sites removed from the face page of the protocol.  
4/15/14 
Change in the sponsorship of the trial, the coordinating center, registration process, agent 
ordering, and AE/SAE reporting. 
9/9/14 
PI change to Dr. Katherine Tkaczuk 
 
 


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SCHEMA 
A PILOT AND PHASE II STUDY OF ENTINOSTAT AND ANASTROZOLE/TAMOXIFEN IN WOMEN WITH TRIPLE 
NEGATIVE BREAST CANCER TO EVALUATE BIOMARKERS AND SURROGATES FOR RESPONSE 
Patient population:  
First Cohort: Operable stage I-II triple negative breast cancer (ER, PR, and HER2 negative) in pre- and 
postmenopausal women 
 
Second Cohort: Unresectable locally advanced or stage IV metastatic triple negative breast cancer in pre- and 
postmenopausal women with ≤ 2 previous systemic chemotherapy and accessible tumors for biopsies, excluding 
bone only metastasis 
Sample size: 12-41 patients 
Schema: 
 
* As per recommended dose for phase II 
* *For premenopausal patients, tamoxifen 20 mg oral daily will be used instead of anastrozole. 
***≥ 3 unstained slides of 5 micron thickness 
 
 
•Postmenopausal and 
premenopausal** 
•Stage I-II 
•Operable 
•ER/PR/HER2 –ve 
•Adequate tumor 
sample***
Entinostat * mg oral weekly 
Starting on day 1 
+ 
Starting on day 4 
Anastrozole 1 mg oral daily 
Or 
Tamoxifen 20 mg oral daily 
if premenopausal** 
 
For 15-29 days 
Surgery 
Within 6 days 
after the last 
dose of 
entinostat 
Tissue collection 
 
 
Tissue collection 
First cohort: Operable stage I-II 
•Postmenopausal and 
premenopausal** 
•Unresectable/stage IV 
•Accessible tumor for 
biopsy 
•ER/PR/HER2 –ve 
Biopsy #1 
Tissue collection 
Second cohort: Unresectable locally advanced and stage IV metastatic ≤ 10 patients 
Biopsy #2 
Tissue collection 
Continue 
until disease 
progression 
Restage q 6 
weeks 
 
Entinostat * mg oral 
weekly 
Starting on day 1 
+ 
Starting on day 4 
Anastrozole 1 mg 
oral daily 
 Or 
Tamoxifen 20 mg oral 
daily if 
premenopausal** 
For 15-29 days 


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   REGIMEN DESCRIPTION 
Agents 
Premedications/Precauti
ons 
Dose 
Route 
Schedule 
Length of 
Treatment 
Entinostat 
Taken with a meal after the 
first two or three bites 
*** mg 
oral 
weekly starting 
on day 1 
15-29 days 
(Preferably 22 days) 
Anastrozole 
- 
1 mg once daily 
oral 
everyday 
starting on day 4 
Tamoxifen 
- 
20 mg once daily 
oral 
everyday 
starting on day 4 
***Doses as appropriate for assigned dose level. 
 
 
 
Dose Modification Schema for Postmenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Anastrozole 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
 
Dose Modification Schema for Premenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Tamoxifen 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
 


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TABLE OF CONTENTS 
SCHEMA ....................................................................................................................................................................... 4 
1. 
OBJECTIVES ....................................................................................................................................................... 8 
1.1. 
Primary Objectives ...................................................................................................................................... 8 
1.2. 
Secondary Objectives ................................................................................................................................. 8 
1.3. 
Exploratory Objectives ................................................................................................................................ 8 
2. 
BACKGROUND ................................................................................................................................................... 8 
2.1 
Epigenetics ................................................................................................................................................. 8 
2.2 
Histone Modification and Epigenetics ......................................................................................................... 9 
2.3 
Triple Negative Breast Cancer .................................................................................................................... 9 
2.4 
Entinostat Clinical Experience ..................................................................................................................... 9 
2.5 
Rationale ................................................................................................................................................... 10 
2.6 
Correlative Studies Background ................................................................................................................ 13 
3. 
PATIENT SELECTION ....................................................................................................................................... 14 
3.1 
Inclusion Criteria ....................................................................................................................................... 14 
3.2 
Exclusion Criteria ...................................................................................................................................... 16 
3.3 
Inclusion of Minorities ................................................................................................................................ 16 
4. 
REGISTRATION PROCEDURES ...................................................................................................................... 16 
4.1 
General Guidelines ................................................................................................................................... 16 
4.2 
Pretrial Screening...................................................................................................................................... 17 
5. 
TREATMENT PLAN ........................................................................................................................................... 17 
5.1 
Agent Administration ................................................................................................................................. 17 
5.2 
Study Plan ................................................................................................................................................. 19 
5.3 
General Concomitant Medication .............................................................................................................. 21 
5.4 
Duration of Therapy .................................................................................................................................. 21 
5.5 
Surgical Considerations for the First Cohort (Operable Stage I-II) ............................................................ 21 
5.6 
Postoperative Treatments ......................................................................................................................... 22 
5.7 
Duration of Follow Up ................................................................................................................................ 22 
5.8 
Criteria for Removal from Study ................................................................................................................ 22 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ..................................................................................................... 22 
6.1 
Recommended Dose Adjustments ............................................................................................................ 22 
6.2 
Supportive Care Guidelines ...................................................................................................................... 24 
7. 
ADVERSE EVENT REPORTING AND DATA SAFETY AND MONITORING PLAN ......................................... 24 
7.1 
Adverse Events (AEs) ............................................................................................................................... 24 
7.2  
Expedited reporting of severe adverse events .......................................................................................... 25 
7.3  
Expedited reporting by investigator to Syndax .......................................................................................... 25 
8. 
PHARMACEUTICAL INFORMATION ............................................................................................................... 25 
8.1  
Anastrozole ............................................................................................................................................... 25 
8.2 
Entinostat .................................................................................................................................................. 26 
8.3  
Tamoxifen ................................................................................................................................................. 27 
9. 
CORRELATIVE STUDIES ................................................................................................................................. 27 
9.1 
Tumor Tissue Samples ............................................................................................................................. 27 
9.2 
Blood Samples .......................................................................................................................................... 31 
10. 
STUDY CALENDAR AND VISIT SCHEDULE ................................................................................................... 34 
10.1 
Screening .................................................................................................................................................. 36 
10.2 
Registration ............................................................................................................................................... 36 
10.3 
Study Days ................................................................................................................................................ 36 
10.4 
End of Study (Treatment) / Withdrawal ..................................................................................................... 38 


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11. 
MEASUREMENT OF EFFECT ........................................................................................................................... 38 
11.1 
Pathologic Response Determination ......................................................................................................... 38 
11.2 
Clinical Response Determination .............................................................................................................. 39 
12. 
STATISTICAL CONSIDERATIONS ................................................................................................................... 39 
12.1 
Study Design/Endpoints ............................................................................................................................ 39 
12.2 
Sample Size/Accrual Rate ........................................................................................................................ 40 
12.3 
Analysis of Primary Endpoints .................................................................................................................. 41 
12.4 
Analysis of Secondary Endpoints .............................................................................................................. 41 
12.5 
Analysis of Exploratory Endpoints ............................................................................................................. 41 
APPENDIX A: ECOG PERFORMANCE STATUS SCALE .......................................................................................... 45 
APPENDIX B: AMERICAN JOINT COMMITTEE ON CANCER STAGING (AJCC) .................................................... 46 
APPENDIX C: STUDY DRUG DIARY ......................................................................................................................... 48 
 
 


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1. 
OBJECTIVES 
1.1. Primary Objectives 
Pilot Phase:  
1. 
To evaluate the safety and tolerability of entinostat in combination with anastrozole or tamoxifen. 
2. 
To determine the optimal dose of entinostat in combination with anastrozole or tamoxifen for phase 
II. 
 
Phase II: 
1. 
To determine baseline and percentage change in proliferative index (Ki67) before and after 
treatment with entinostat and anastrozole/tamoxifen in triple negative breast cancer (TNBC). 
2. 
To determine the estrogen receptor (ER) expression after treatment with entinostat and 
anastrozole/tamoxifen in TNBC. 
1.2. Secondary Objectives 
1. 
To evaluate baseline and change in the expression levels of progesterone receptor (PR), human 
epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), cytokeratin 
5/6 (CK5/6), and aromatase before and after treatment with entinostat and anastrozole/tamoxifen. 
2. 
To assess baseline and change in tumor tissue histone H3 and H4 acetylation before and after 
treatment with entinostat and anastrozole/tamoxifen. 
3. 
To assess the clinical and pathological response to preoperative combination of entinostat and 
anastrozole/tamoxifen in TNBC. 
1.3. Exploratory Objectives 
 
1. 
To correlate the levels of histone H3 and H4 acetylation in tumors with the changes in Ki67 and ER. 
2. 
To evaluate baseline and change in gene methylation silencing and expression of candidate genes 
in tissues and in circulating DNA, including estrogen receptor (ER)-alpha, ER-beta, RAR-beta, 
cyclin D2, Twist, RASSF1A, and HIN-1. 
3. 
To correlate entinostat trough concentrations with histone H3 and H4 acetylation in tumors as well 
as the change in Ki67 and ER. 
4. 
To evaluate baseline and change in the global gene expression profile before and after treatment 
with entinostat and anastrozole/tamoxifen. 
2. 
BACKGROUND 
In the era of postgenomic cancer biology, it is becoming increasingly evident that epigenetic modulation of gene 
expression plays a crucial role in tumorigenesis and tumor progression. Multiple epigenetic treatments have been 
validated to be successful strategies in cancer therapy. While the mortality rate in breast cancer has decreased in the 
past few decades, some subsets of breast cancer still have poor outlooks. With the advent of targeted therapy, 
particularly endocrine and anti-human epidermal growth factor receptor 2 (HER2) therapies, the outcome of breast 
cancer has changed dramatically. Nevertheless, approximately one fourth of breast cancers unfortunately lack 
expression of all three constructive cell surface receptors, namely estrogen receptor (ER), progesterone receptor 
(PR), and HER2, hence the term “triple negative” breast cancer (TNBC). Due to an absence of ER and/or PR, these 
tumors conventionally do not respond to endocrine therapy like tamoxifen and aromatase inhibitors, which have 
minimal toxicities and are generally well tolerated. For that reason, the treatment of these tumors depends solely on 
chemotherapy1. In our preliminary data, we found that entinostat, a histone deacetylase (HDAC) inhibitor, can induce 
re-expression of estrogen receptor and aromatase in TNBC cell lines and render them to be sensitive to aromatase 
inhibitor. With this promising data, we plan to evaluate the efficacy of this novel strategy in the neoadjuvant setting to 
target TNBC. 
2.1 Epigenetics 
 


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Epigenetics is a heritable process which refers to changes in gene expression caused by mechanisms other than 
alterations in the DNA sequence2. Epigenetic regulation plays a crucial role in normal development and allows for the 
transcriptional control of gene expression without changing the DNA sequence. Key machineries of epigenetic 
regulation include methylation of the promoter regions of the genes and posttranslational modification of histone, the 
main protein in the chromatin structure which serves as spools for DNA to wind3. In cancer cells, abnormal patterns of 
DNA methylation have been observed.  The abnormal patterns include both loss of methylation in the CpG-depleted 
regions where most CpG dinucleotides should be methylated and gains in methylation of CpG islands in gene 
promoter regions resulting in abnormal silencing of critical genes such as the tumor-suppressor genes4. 
 
2.2 Histone Modification and Epigenetics 
 
Chromatin is a complex structure that is comprised of DNA, RNA, and histones. Nucleosome is the fundamental 
repeating unit of chromatin which allows proper packaging of large genome in the nucleus. Each nucleosome 
composes of approximately 147 base pairs of DNA that wrapped around a histone octamer5. Posttranslational 
modifications of histones, mainly acetylation and methylation, determine the accessibility of the nucleosomal DNA to 
transcription factors and thereby the gene transcriptional activity. HDACs and histone acetyltransferases (HATs) are 
the key enzymes that are involved in posttranslational modifications of histones by selectively deacetylating or 
acetylating the ε-amino groups of lysine located near the amino termini of core histone proteins. Acetylation of the 
chromatin by HATs is associated with open chromatin and transcriptional activation, whereas deacetylation by 
HDACs correlates with close chromatin and gene silencing. The presence of the hypo-acetylated and 
hypermethylated histones H3 and H4 alone in the absence of the promoter hypermethylation can also silence certain 
genes with tumor-suppressor–like properties, such as p21WAF16. Eighteen human HDACs have been identified, 
which are generally subdivided into class I (HDACs 1, 2, 3, 8), class II (HDACs 4, 5, 6, 7, 9, 10), class III (sirtuins 1-7) 
and class IV (HDAC 11) based on sequence and functional homology. There is evidence that HDACs are associated 
with a wide range of tumors including neuroblastomas, melanomas, lymphomas, breast, prostate, lung, ovarian, 
bladder, and colon cancers. 
 
2.3 Triple Negative Breast Cancer 
 
Approximately 15-20% of all breast cancers do not express functional cell surface receptors like ER, PR, and HER2. 
Triple-negative tumors are typically associated with a higher histologic grade, increased mitotic count, pushing 
margins of invasion, and a stromal lymphocytic response. Patients with TNBC often have poorer prognosis compared 
to other breast cancer subtypes. It is commonly misconceived that TNBC is more sensitive to chemotherapy. 
Although higher pathologic complete response rate (pCR) was observed in TNBC (25–45%) compared to luminal 
breast cancers (6–7%), patients with TNBC had worse four-year distant disease free and overall survival7. It has 
been demonstrated in multiple studies that the loss of ER protein expression is a result of epigenetic changes in the 
tumors. Hypermethylation of the CpG islands within the ERα promoter has been observed in a significant fraction of 
breast cancers8. Moreover, HDAC1 has been reported to interact with and suppresses transcription activity of ERα. 
The interaction of HDAC1 with ERα is mediated by the activation function-2 (AF-2) domain and DNA-binding domain 
of ERα. Treatment of ER-negative breast cancer cells with TSA, an HDAC inhibitor, induced re-expression of ERα. 
These findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in 
association with a reduction in both ERα protein expression and transcriptional activity. Therefore, HDAC1 may be a 
potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers9. 
2.4 Entinostat Clinical Experience 
Entinostat is a histone deacetylase (HDAC) inhibitor that selectively inhibits HDACs 1, 2, and 3. By promoting 
hyperacetylation of nucleosomal histones, entinostat promotes transcriptional activation of certain set of genes which 
leads to inhibition of cell proliferation, induction of terminal differentiation, and apoptosis. Multiple studies both in vitro 
and in vivo demonstrated that entinostat can inhibit growth of multiple tumors including breast, lung, prostate 
pancreatic, renal cell, and glioblastoma10,11. Due to the selectivity of entinostat towards HDACs 1, 2, and 3, entinostat 
has a distinct safety and efficacy profiles compared to pan-HDAC inhibitors like vorinostat. Early clinical studies 
showed that entinostat has a remarkably long half-life which allows weekly or biweekly treatment dosing12. In phase I 
pharmacokinetic study, entinostat has a half-life ranging from 40 hours to 120 hours. The level of entinostat was still 
detectable at 168 hours after the dose of 6 mg/m2 to 12 mg/m2. Pharmacodynamic studies confirm an inhibition of 
HDAC by entinostat in phase I study participants at all dose levels (ranging between 2 and 12 mg/m2 and from once 
daily to every 2 weeks) tested to date by demonstrating increase in the histone hyperacetylation. Anti-cancer activity 
of entinostat has been observed in wide range of advanced solid tumors and hematologic malignancies across all 
range of tested doses.  For single agent, the MTD for solid malignancies is 4 mg/m² weekly 3 weeks on 1 week off, or 


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10 mg/m² every other week. In hematologic malignancies, the MTD is 6 mg/m² weekly 3 weeks on 1 week off. 
However, further analysis based on the body surface area and drug clearance showed that the fixed dosing is 
considered to be as accurate as dosing based on body surface area13. Entinostat is currently in phase 2 clinical 
development in multiple solid tumors and hematologic malignancies. 
 
In breast cancer, Syndax currently has an ongoing study of entinostat at the dose of 5 mg oral weekly in combination 
with exemestane, a steroidal aromatase inhibitor, 25 mg oral daily in the setting of endocrine resistant metastatic 
breast cancer. From personal communication with Syndax, this study demonstrates that this combination is safe with 
minimal toxicity. Based on these clinical experiences with entinostat, a dose of 5 mg given weekly has been selected 
as the regimen for this study in combination with anastrozole. Additional information on the chemistry, pharmacology, 
toxicology, preclinical findings, and clinical experience to date may be found in the Investigator’s Brochure. 
 
Although there is no prior clinical trial of entinostat in combination with tamoxifen, we do not expect to see significant 
increase in toxicity when compare between entinostat and tamoxifen vs. entinostat and aromatase inhibitor, 
particularly because these endocrine therapy agents are generally well tolerated drugs. In contrast to entinostat which 
specifically inhibits class I HDACs, vorinostat is a pan-histone deacetylase inhibitor that inhibits both class I and class 
II HDACs14. Due to the specificity of entinostat, the side effect profile is better with entinostat compared to vorinostat. 
Nevertheless, a prior phase II study of vorinostat in combination with tamoxifen in hormone receptor-positive 
metastatic breast cancer demonstrated that this combination is safe and tolerable15. In this trial, vorinostat was given 
at the dose of 400 mg daily for 21 out of 28 days and tamoxifen was given at the daily dose of 20 mg continuously. 
Most of the toxicities are grade 1 and 2. Grade 3 toxicities range from 2-16%. Most common toxicities include fatigue 
(16%) and neutropenia (16%) which are common toxicities of HDAC inhibitors rather than tamoxifen. There are only 
7% of patients who developed thrombosis related to tamoxifen. Taken this together, tamoxifen will be given at the 
dose of 20 mg daily in combination with entinostat 5 mg weekly. 
 
Since this trial was opened for accrual in 2009 under the National Cancer Institute’s Cancer Therapy Evaluation 
Program (NCI CTEP) IND 61198, there were a total of 7 women enrolled to date. However, due to funding and slow 
accrual considerations, NCI CTEP discontinued support for the trial earlier in 2013. Among these 7 patients, 4 
patients were treated with entinostat in combination with anastrozole and 3 patients were treated with entinostat and 
tamoxifen. There were 5 patients in the first cohort (operable stage I-III) and 2 patients in the second cohort 
(unresectable locally advanced and stage IV). With regards to response, there was one patient in the first cohort who 
achieved pathological complete response (pCR) after the treatment with entinostat and anastrozole prior to surgery. 
There was one more patient in the first cohort who had an increase in the expression of ER from 0% to 1%. She was 
treated with entinostat and tamoxifen. However, there was no reduction in Ki67 observed in this particular patient. 
Therefore, she did not meet our pre-specified criteria for response in this trial. 
With regards to toxicity, there was no dose limiting toxicity observed at the starting dose levels for both tamoxifen and 
anastrozole. Furthermore, there was no surgical delay due to toxicity from this preoperative treatment. The adverse 
events are as followed: 
 
Adverse Events 
Grade 
Number of patients 
Gastroesophageal reflux disease 
1-2 
4 
Nausea 
1-2 
3 
Fatigue 
1-2 
3 
Diarrhea 
1-2 
2 
Headache 
2 
1 
Hot flashes 
1 
1 
Myalgias 
3 
2 
White blood cell decreased 
1-2 
2 
Hypophosphatemia 
2 
1 
Neutrophil count decreased 
3* 
1 
*Entinostat was held and neutrophil count recovered to ≥ 1,000/mm3 within 3 days. 
2.5 Rationale 
In collaboration with Dr. Angela Brodie and Dr. Gauri Sabnis at the University of Maryland Greenebaum Cancer 
Center (UMGCC), we have found that entinostat can induce re-expression of ERα, androgen receptor, and 


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aromatase enzyme (CYP-19) in both in vitro16 and TNBC xenografts17, MDA-MB-231. This effect is dose dependent 
as shown below.  
 
 
 
 
As a single agent, entinostat is quite active in MDA-MB-231 with the half maximal inhibitory concentration (IC50) of 
84.2 nM. The combination of entinostat and letrozole demonstrated a marked synergy as shown in the figure below. 
 
 
 
By re-expressing aromatase and ERα, we hypothesized that entinostat can sensitize these TNBC cells to endocrine 
therapy. To test this hypothesis, we treated mice bearing MDA-MB-231 tumors with vehicle control, entinostat, 
letrozole, and the combination of letrozole and entinostat. In the combination group, entinostat was started 3 days 
prior to letrozole. Intriguingly, we found that the combination of letrozole and entinostat results in a significant and 
durable reduction in the tumor volume of MDA-MB-231 xenografts18 as illustrated in the figure below. In this xenograft 
model, starting entinostat 3 days prior to letrozole in order to induce expression of ER and aromatase is more 
effective than starting both of the treatment at the same time (data not shown). 
 
Control 
1 mg 
ERα 
β-actin 
SNDX-275 
2.5 mg 
5 mg
10 mg 
1 
1.2 
1.7 
2.9 
3.1 
1 
1.9 
2.4 
3.6 
5.1 
AR 
CYP-19 
1 
0.9 
2.5 
3.1 
4.2 


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Gnant et al  NEJM 2009;679 
In vitro study of anastrozole in combination with entinostat also demonstrated a comparable result with letrozole and 
entinostat in MDA-MB-231 as shown in the figure below. Therefore, we propose to conduct a pilot and phase II study 
of entinostat in combination with anastrozole in postmenopausal women with operable TNBC.  
 
In premenopausal women, tamoxifen will be used instead of anastrozole. This is based on the fact that anastrozole 
with ovarian suppression may not adequately suppress estrogen production within 2-5 weeks after injection. It is 
commonly known that ovarian suppression with GnRH agonist initially produce surge of estrogen production, 
particularly within the first 2 weeks after injection19. This estrogenic surge can last up to 6 weeks which could have 
detrimental effect on tumor growth and our biologic endpoints, particularly the Ki67 expression.  Furthermore, the 
data from The Austrian Breast and Colorectal Cancer Study Group trial 12 (ABCSG12 trial) demonstrated a trend 
toward poor overall survival in premenopausal women receiving anastrozole in combination with goserelin compared 
to tamoxifen (figure below)20. This effect is more pronounced in overweight patients with BMI ≥ 25 kg/m2 which may 
be related to higher level of aromatase from subcutaneous tissue. Subset analysis of overweight patients 
demonstrated shorter disease free survival (DFS) and overall survival (OS) in patients taking anastrozole compared 
to tamoxifen (HR 0.625 95%CI 0.387-1.009 p = 0.052, and HR 0.336 95%CI 0.149-0.76, p = 0.006, respectively)21. 
This effect is believed to be secondary to an inadequate suppression of estrogen production with aromatase inhibitor 
and GnRH agonist in premenopausal women. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In our preclinical studies, it appears that tamoxifen also has similar synergistic effect with entinostat compared to 
other aromatase inhibitors in estrogen receptor-negative breast cancer cell lines in vitro (figures below). 
 


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2.6 Correlative Studies Background 
Ki67: Ki67 protein is a cellular marker for proliferation. Ki67 labeling has been reported to be a good prognostic 
marker and correlate well with the S phase fraction22 and mitotic index22,23. A change in the expression of Ki67 after 
short-term exposure to the investigational agent is frequently used to determine the efficacy, particularly in the 
neoadjuvant setting. For neoadjuvant chemotherapy, two previous studies24-26 showed a significant correlation 
between the reduction in Ki67 of more than 25% and a longer disease-free survival. However, subsequent studies26-
28 on the reproducibility of Ki67 measurements in the core needle biopsy suggested that a change in Ki67 score of at 
least 32-50% between two determinations is required to consider statistically significant and attributable to treatment 
effect for an individual patient. For neoadjuvant endocrine therapy, Dowsett et al. 29 reported that a reduction in Ki67 
could be observed in 2 weeks after neoadjuvant endocrine therapy and was largely maintained after 12 weeks of 
treatment in the majority of patients in the Immediate Preoperative “Arimidex” (anastrozole), Tamoxifen, or Arimidex 
Combined with Tamoxifen (IMPACT) trial. In this study, postmenopausal women with previously untreated ER-
positive breast cancer were randomized to receive a daily treatment of anastrozole, tamoxifen, or the combination of 
anastrozole and tamoxifen for 12 weeks before surgery. Biomarkers were measured in the core-cut biopsied 
specimens taken before and after 2 weeks of treatment as well as surgical specimens after 12 weeks of treatment. 
The geometric means of reduction in Ki67 at 2 and 12 weeks for anastrozole were 76% and 82% respectively. In their 
subsequent publication30, Ki67 expression after 2 weeks of treatment depending on the tertiles of tumor Ki67 
expression was more strongly associated with recurrence free survival (log-rank P = .008) than tumor Ki67 
expression at baseline (log-rank P = .07). However, there was no correlation made between the percentage change 
in the Ki67 expression and recurrence free survival. 
 
In our same preclinical study, there was a 63% reduction in Ki67 with the combination of entinostat and letrozole. 
There seems to be no significant change in Ki67 expression with single agent letrozole or entinostat. The figure below 
illustrates the Ki67 staining by immunohistochemistry in MDA-MB-231 (a TNBC cell line) mouse tumor xenografts 
treated with vehicle, letrozole, entinostat, or the combination of letrozole and entinostat. The percentage of Ki67-
positive cells is quantified by the ChromaVision ACIS system for immunohistochemistry image analysis which will 
also be used to analyze the specimens in this clinical trial. Therefore, we will use the cutoff point of 50% reduction in 
Ki67 to evaluate our primary endpoint. 


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EGFR and Cytokeratin 5/6: Recent data have identified five distinct subtypes of breast cancer based on gene 
expression profiling: luminal A, luminal B, HER2 overexpressing, normal breast-like, and basal-like31. These different 
subtypes of breast cancer are associated with different clinical course. Basal-like subtype comprises approximately 
20% of the breast cancer and has a poor prognosis32,33. Basal-like breast cancer is not entirely synonymous with 
TNBC since ER expression can be seen in 5%to 45% of basal-like cancers and up to 14% of basal-like cancers also 
express HER234. In contrast, TNBC are not necessarily basal-like cancers since a significant proportion of normal 
breast–like cancer subtype as defined by expression arrays would also lack hormone receptors and HER2. While the 
normal breast–like subtype is still poorly characterized, they are reported to have a better prognosis compared to 
basal-like cancers32,35. Nielsen et al.36 have reported that a panel of four antibodies (ER, HER1, HER2, and 
cytokeratin 5/6) can precisely identify basal-like tumors using standard immunohistochemical staining. Moreover, 
overexpression of EGFR was found to be associated with poor survival independent of tumor size and nodal status. 
 
Gene Expression Profile: Chromatin remodeling induced by different classes of HDAC inhibitors can result in 
significant changes in multiple gene expression. The recent development of high-throughput technologies provides a 
prevailing tool for a global analysis of gene expression. In the past, global gene expression profiling in the formalin-
fixed, paraffin-embedded (FFPE) tissue has been technically challenging since these technologies typically require 
substantial quantities of fresh or frozen tissue. Novel method like the cDNA-mediated Annealing, Selection, 
Extension, and Ligation (DASL) assay from Illumina has been developed to allow more comprehensive gene 
expression profiling of degraded RNA extracted from paraffin blocks using bead array format. The DASL assay has 
been shown to be highly reproducible and can be performed with as little as 50 ng of total RNA isolated from FFPE 
tissue that had been stored over 10 years37.  
 
Methylation Profile: In breast cancer, multiple genes are methylated and therefore silenced. Gene methylation is 
likely related to cancer progression. HDAC inhibitors may also alleviate gene repression that is mediated through 
promoter hypermethylation. It is presumed that HDAC inhibitors can increase expression of the genes that are not 
methylated but may not induce the expression of hypermethylated genes. Some of the genes that are often 
methylated in breast cancer include growth promoting hormone receptors such as ER-alpha, an important predictive 
factor of response to endocrine manipulations. Other genes that are often hypermethylated in breast cancer include 
cyclin D, Twist, RASSF1A, APC and HIN-1. Dr. Sukumar and other Johns Hopkins investigators have evaluated 
hypermethylation of a panel of seven genes using methylation-specific polymerase chain reaction (MSP) in a variety 
of breast tissues38-40. In invasive breast carcinomas, up to 100% of specimens contained at least one 
hypermethylated gene, 80% contained two, and 60% contained three or more methylated genes39. In 44 ductal 
carcinoma in situ (DCIS) specimens, 95% had at least one methylated gene. In contrast, the percentage of women 
with benign breast disease having at least one methylated gene was only 15%. Only one of 8 reduction 
mammoplasty specimens contained hypermethylated genes. More recently, Dr. Sukumar and colleagues have 
developed a novel method, quantitative multiplex methylation specific PCR (QM-MSP) to globally assess promoter 
hypermethylation for many genes simultaneously in small samples. QM-MSP is highly sensitive (1 in 104-105 copies 
of DNA) and linear over 5 orders of magnitude39. Therefore, it is also feasible to use the DNA sample obtained from 
the peripheral blood to perform this assay. 
3. 
PATIENT SELECTION 
 3.1 Inclusion Criteria 
Ki67 Expression 
Control = 39.7% 
Letrozole = 33.1% 
SNDX-275 = 35.3% 
Combination = 14.7% 


09/09/14 Version 10 
15 
 
3.1.1 
Female greater than or equal to 18 years. 
3.1.2 
Eastern Cooperative Oncology Group (ECOG) performance status <2 (see Appendix A). 
3.1.3 
Histologically confirmed adenocarcinoma of the breast. 
3.1.4 
Evidence of hormone insensitivity (ER and PR negative) of primary tumor tissue.  ER negative is 
define as ER 0 or < 1% staining by immunohistochemistry. PR negativity is defined as PR ≤ 10% 
staining by immunohistochemistry. 
3.1.5 
HER2 negative in the primary tumor tissue  as defined by: 
 
Immunohistochemistry (IHC) Grade 0 as defined by no staining observed or membrane staining that is 
incomplete and is faint/barely perceptible and within ≤ 10% of the invasive tumor cell 
 
IHC 1+ as defined by incomplete membrane staining that is faint/barely perceptible and within > 10% of the 
invasive tumor cell 
 
IHC Grade 2+ staining intensity by means of IHC analysis with no gene amplification below. 
 
No gene amplification on ISH based on  
o 
Single-probe average HER2 copy number < 4.0 signals/cell 
o 
Dual-probe HER2/CEP17 ratio < 2.0 with an average HER2 copy number < 4.0 signals/cell 
3.1.6 
Ability to understand and the willingness to sign a written informed consent document. 
3.1.7 
Patients must not have received any prior chemotherapy, radiation therapy, or endocrine therapy 
for their current breast cancer. Patients who received tamoxifen or raloxifene or another agent for 
prevention of breast cancer may be included as long as the patient has discontinued the treatment 
at least one month prior to baseline study biopsy. 
3.1.8 
Women of childbearing potential must have negative (serum or urine) pregnancy test within 7 days 
prior to registration. 
3.1.9 
Patients must have adequate tumor tissue sample prior to the enrolment available for correlative 
studies as defined below: 
 
Core needle biopsy or incisional biopsy samples that can provide ≥ 3 unstained sections of 5 micron thickness. 
Fine needle aspiration (FNA) sample alone is not sufficient except in the second cohort. 
 
Additional core needle biopsy needs to be performed in the patients who agree to participate in this study and do 
not have adequate tumor tissue sample. 
3.1.10 
Patients must have adequate organ and marrow function as defined below: 
 
Hemoglobin  
 
 
 
 
≥ 9.0 g/dL 
 
Leukocytes  
 
 
 
 
>2,500/mcL 
 
Absolute neutrophil count  
 
>1,100/mcL 
 
Platelets  
 
 
 
 
 
>100,000/mcL 
 
Total bilirubin 
 
 
 
 
within normal institutional limits 
 
AST(SGOT)/ALT(SGPT)  
 
<2.5 x institutional upper limit of normal 
 
Creatinine 
 
 
 
 
 
within normal institutional limits or creatinine clearance ≥ 60 mL/min/1.73 m2 
for patients with creatinine levels above institutional normal 
 
Additional Inclusion Criteria for the First cohort: 
 
3.1.11 
Unresected operable breast cancer that meets the following clinical stages (see Appendix B): 
 
T1b, T1c, or T2 
 
N0 or N1 
 
M0 (No distant metastasis) 
 
Additional Inclusion Criteria for the Second cohort: 
 
3.1.12 
Unresectable, inoperable, recurrent local-regional breast cancer or 
3.1.13 
Metastatic (stage IV) breast cancer 
3.1.14 
Patients must have measurable or evaluable disease (i.e. ascites or pleural/pericardial effusion). 
Patients with bone metastatic only will be excluded. 
3.1.15 
Patients must not have rapidly progressive disease, extensive visceral involvement, or any high risk 
characteristics that are not appropriate for this treatment as per investigator’s discretion. 
3.1.16 
Patients must receive at least one prior line of chemotherapy but not more 2 prior chemotherapy 
regimens for stage IV breast cancer. Prior chemotherapy in the adjuvant and /or neoadjuvant 
setting is permitted. However, patients must have finished chemotherapy at least 2 weeks prior to 
enrollment. 
3.1.17 
Patients must have an accessible tumor lesion from which a fine needle aspirate or preferably a 
core biopsy specimen can be obtained. Patients with FNA only samples are allowed in this cohort. 
Ascites or pleural/pericardial effusion alone is not sufficient. 


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3.1.18 
Patients must be willing to provide consents for 2 research biopsies. However, the pretreatment 
biopsy can be omitted in patients who have recent biopsy but have not been started on breast 
cancer treatment within 12 weeks prior to the registration and there is adequate tumor tissue 
sample as per section 3.1.9. 
 
3.2 Exclusion Criteria 
 
3.2.1 
Patients may not be receiving any other investigational agents. 
3.2.2 
Prior exposure to other HDAC inhibitors. However, prior valproic acid exposure is allowed providing 
≥ 30 days wash-out period. 
3.2.3 
History of allergic reactions or hypersensitivity to compounds of similar chemical or biologic 
composition to entinostat, benzamide, anastrozole, or tamoxifen. 
3.2.4 
Any medical condition which in the opinion of the investigator puts the patient at risk of potentially 
serious complications while on this therapy. 
Examples: HIV, unstable angina, uncontrolled heart failure or hypertension, uncontrolled 
hyperlipidemia, uncontrolled diabetes mellitus, uncontrolled systemic infection. 
3.2.5 
Previous or current systemic malignancy within the past 3 years other than breast cancer or 
adequately treated cervical carcinoma in situ or basal/squamous carcinoma of the skin. 
 
3.3 Inclusion of Minorities 
Women of all races and ethnic groups are eligible for this trial.  
 
 
4. 
REGISTRATION PROCEDURES 
4.1 General Guidelines 
Eligible patients will be entered on study at the University of Maryland Greenebaum Cancer Center by the Study 
Coordinator.  Following registration, patients should begin protocol treatment within 3 business days. All of the 
patients’ documents must be kept confidential in a secured area. 
To register a patient, the following documents should be completed by a research team member and sent to the 
Study Coordinator, Nancy Tait or Jane Lewis, via fax, (410) 328-1741, or e-mail, ntait@umm.edu or jlewis@umm.edu  
 
Copy of required laboratory tests 
 
Signed patient consent form 
 
HIPAA authorization form 
 
Other appropriate forms (e.g., Eligibility Screening Worksheet, Documentation of Pathology and 
Diagnosis, Registration Form)  
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
 Males 
 Total 
Hispanic or Latino 
10 
+ 0 
= 10 
Not Hispanic or Latino 
35 
+ 0 
= 35 
Ethnic Category: Total of all subjects 
45 
 
(A1) 
+  
 
0
 
(B1) 
=  
45 
(C1) 
Racial Category 
 
American Indian or Alaskan Native 
5 
+ 0 
= 5 
Asian 
5 
+ 0 
= 5 
Black or African American 
5 
+ 0 
= 5 
Native Hawaiian or other Pacific Islander 
5 
+ 0 
= 5 
White 
25 
+ 0 
= 25 
Racial Category: Total of all subjects 
45 
 
(A2) 
+ 0 
= 45 
 
(A1 = A2) 
 (B1 = B2) 
 (C1 = C2) 
 
Accrual Rate: 
2-3 
pts/month 
Total Expected Accrual:   
12 
Min 
45 
Max 


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To complete the registration process, the Coordinator will 
 
Assign the patient a study number 
 
Register the patient on the study 
4.2 Pretrial Screening 
Patients will be screened at the participating centers before entry to establish eligibility. The following assessments 
must be obtained within 3 weeks of trial entry: 
 
Medical history including all significant conditions. 
 
Concomitant therapy before entry documented to establish eligibility. 
 
ECOG performance status 
 
Physical examination including vital signs, height and weight 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, 
and location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, 
BUN, creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , 
alkaline phosphatase, LDH, uric acid, phosphorus, magnesium; and coagulation including PT with 
INR and PTT 
 
12-lead electrocardiogram (ECG) at baseline 
Following baseline breast imaging can be obtained within 6 weeks of trial entry: 
 
Baseline mammogram, breast ultrasound, or breast magnetic resonance imaging (MRI) as 
indicated 
5. 
TREATMENT PLAN 
5.1 Agent Administration 
Treatment will be administered on an outpatient basis.  Reported adverse events and potential risks for anastrozole 
and entinostat are described in Section 7.  Appropriate dose modifications for entinostat and anastrozole are 
described in Section 6.  No investigational or commercial agents or therapies other than entinostat, anastrozole, and 
tamoxifen may be administered with the intent to treat the patient's malignancy. 
In the first cohort: 
 
Treatment will be continued for a minimum of 15 days and a maximum of 29 days. If clinically acceptable, 
treatment should be preferably continued for 22 days prior to the definitive surgery. 
 
Anastrozole will be administered continuously starting on day 4 at a fixed dose of 1 mg oral once daily. 
 
In patients who are not postmenopause as defined by any of the following criteria, tamoxifen will be administered 
continuously instead of anastrozole starting on day 4 at a fixed dose of 20 mg oral once daily. 
Postmenopausal as defined by any of the following criteria: 
 
Age ≥ 60 
 
Age < 60 with amenorrhea > 12 months and an intact uterus and an FSH and estradiol level within 
the postmenopausal range by institutional laboratory parameters. 
 
Age < 60 with previous hysterectomy must have FSH and estradiol level within the postmenopausal 
range by institutional laboratory parameters. 
 
Any age having undergone bilateral oophorectomy 
 
Entinostat will be administered at the assigned dose once a week (on day 1, 8, 15, 22, and 29) until the definitive 
surgery. Entinostat should be taken with a meal after the first two or three bites.  
o 
The initial starting dose for entinostat is 5 mg oral weekly. Dose de-escalation to 3 mg/week is permitted 
in the pilot phase if dose limiting toxicity is observed as defined in section 5.2.1.  
o 
Dose level for entinostat in phase II part of this study will be determined after the pilot phase is 
completed. 


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In the second cohort: 
1. 
Pretreatment biopsy (biopsy#1) will be performed within 3 weeks after registration. However, the pretreatment 
biopsy can be omitted in patients who have recent biopsy but have not been started on breast cancer treatment 
within 12 weeks prior to the registration and there is adequate tumor tissue sample as per section 3.1.9. 
 
Treatment will be started after pretreatment biopsy and will be continued until disease progression. The post-
treatment biopsy (biopsy#2) will be performed after a minimum of 15 days and a maximum of 29 days of 
treatment, but preferably close to 29 days. The post-treatment biopsy must take place within 6 days (preferably 
within 3 days) after the last dose of entinostat. After the post-treatment biopsy, treatment will be continued until 
disease progression regardless of the result of post-treatment biopsy. Restaging scans will be performed every 6 
weeks while on treatment. In the event that a patient cannot undergo the post-treatment biopsy for any reason, 
they will continue to be treated per study protocol. If a patient discontinues the study prior to 15 days, an attempt 
should be made to collect the biopsy when the decision is made to discontinue and prior to initiation of additional 
treatment, if possible. 
 
Anastrozole will be administered continuously starting on day 4 at a fixed dose of 1 mg oral once daily until 
disease progression. 
 
In patients who are not postmenopause as defined by any of the previously described criteria, tamoxifen will be 
administered continuously instead of anastrozole starting on day 4 at a fixed dose of 20 mg oral once daily until 
disease progression. 
 
Entinostat will be administered at the assigned dose once a week until disease progression. Entinostat should be 
taken with a meal after the first two or three bites.  
o 
The initial starting dose for entinostat is 5 mg oral weekly. Dose de-escalation to 3 mg/week is permitted 
if dose limiting toxicity is observed as defined in section 5.2.1.  
o 
Dose level for entinostat in phase II part of this study will be determined after the pilot phase is 
completed. 
The dose schemas are as shown below for both postmenopausal and premenopausal women:  


09/09/14 Version 10 
19 
 
 
Dose Modification Schema for Postmenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Anastrozole 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
or 
Dose Modification Schema for Premenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Tamoxifen 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
5.1.1 
Entinostat 
Entinostat is supplied as light brown (1 mg) or intense yellow (5 mg) coated tablets. Patients will self-administer 
entinostat as per assigned dose level shown in section 6.1. Tablets should be taken with a meal after the first two or 
three bites.  If the patient’s dose requires more than one tablet, the tablets should be taken one at a time. Unused 
tablets or empty bottles will be returned to the clinic for accountability purposes. Dose reductions for toxicity are 
summarized in section 6.2. The definitive surgery or post-treatment biopsy (biopsy#2) must take place within 6 days 
(preferably within 3 days) after the last dose of entinostat. 
 
Entinostat may cause fatigue or malaise; advise patient to exercise caution while driving a vehicle or operating 
machinery. Delay administration of H2-antagonists, antacids, or proton pump inhibitors, or other drugs that lower 
acidity for at least 2 hours after dosing entinostat.  
5.1.2 
Anastrozole 
Anastrozole will be taken by mouth once daily on a continuous basis starting on day 4 at the fixed dose of 1 mg per 
day. Anastrozole should be continued until the day of the surgery in the first cohort and until disease progression in 
the second cohort. 
5.1.3 
Tamoxifen 
In patients who are not postmenopause as defined in section 5.1 (premenopausal women), tamoxifen will be 
administered instead of anastrozole. Tamoxifen will be taken by mouth once daily on a continuous basis starting on 
day 4 at the fixed dose of 20 mg per day. Tamoxifen should be continued until the day of the surgery in the first 
cohort and until disease progression in the second cohort. 
 
5.2 Study Plan 
5.2.1 
Pilot Phase 
In the initial pilot phase, we will separately evaluate the safety of the combination of entinostat and anastrozole as 
well as entinostat and tamoxifen to find the optimal dose for the subsequent phase II part.  
The combination of entinostat and anastrozole will be administered in postmenopausal patients as defined in section 
5.1. Even though there is no previous clinical trial of entinostat in combination with anastrozole, we do not expect 
significant toxicity of this combination. An ongoing study of entinostat at the dose of 5 mg oral weekly with 
exemestane, a steroidal aromatase inhibitor, 25 mg oral daily in postmenopausal women with metastatic estrogen 
receptor-positive breast cancer progressing on a non-steroidal aromatase inhibitor demonstrates that so far this 
combination is safe with minimal toxicity (from personal communication with Syndax). Therefore, the first cohort of 3 


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postmenopausal patients will be treated with entinostat at the dose of 5 mg oral weekly with anastrozole 1 mg oral 
daily (dose level 1 as shown in the table below). If we do not observe any dose limiting toxicity (DLT), up to 3 
additional patients will be treated with dose level 1. If there is ≥ 1 patient experiencing DLT at dose level 1, the dose 
will be de-escalated to level -1. Up to 6 additional patients can be enrolled in the dose level -1.  
The dose modification schemas are as follow for both postmenopausal women: 
Dose Modification Schema for Postmenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Anastrozole 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
The combination of entinostat and tamoxifen will be administered in premenopausal patients as defined in section 
5.1. There is no previous study of tamoxifen in combination with entinostat. Based on previous phase II study of 
vorinostat and tamoxifen, we also do not expect significant toxicity of this combination. The first cohort of 3 
premenopausal patients will be treated with entinostat at the dose of 5 mg oral weekly with tamoxifen 20 mg oral daily 
(dose level 1 as shown in the table below). If we do not observe any DLT, up to 3 additional patients will be treated 
with dose level 1. If there is ≥ 1 patient experiencing DLT at dose level 1, the dose will be de-escalated to level -1. Up 
to 6 additional patients can be enrolled in the dose level -1. 
The dose modification schemas are as follow for both premenopausal women: 
Dose Modification Schema for Premenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Tamoxifen 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
Therefore, the total number of patients for the pilot phase of the study will be 6-12 for each treatment groups 
(entinostat and anastrozole as well as entinostat and tamoxifen) or the total of 12-24 patients. 
The phase II part will continue with the dose level in which there is ≤ 1 out of 6 patients experiencing a DLT. 
However, if there are ≥ 2 out of 6 patients experiencing DLT at dose level -1, the study will be terminated due to 
toxicity. 
The patients in the pilot phase will be included in the overall evaluation of response. 
 
Toxicity will be graded according to the active version of National Cancer Institute (NCI)-Common Terminology 
Criteria for Adverse Events (CTCAE) as per section 7.3. Patients in both cohorts (the first cohort-operable stage I-II 
and the second cohort-unresectable locally advance/metastatic) in the pilot phase will be used to evaluate for DLT. In 
the second cohort, only adverse events (AEs) that occur during the first 29 day will be considered for DLT. DLT is 
defined as one or more of the following treatment-related: 
 
1. 
Hematologic AEs  
a. 
Absolute neutrophil count (ANC) < 500/mm3 regardless of duration 
b. 
Failure of ANC to recover ≥ 1,000/mm3 within 14 days 
c. 
Grade ≥ 4 thrombocytopenia regardless of duration or grade 3 thrombocytopenia with significant 
bleeding. 
d. 
Failure of platelet  to recover ≥ 50,000/mm3 within 14 days 
e. 
Hemoglobin < 6.5 g/dL despite transfusion lasting > 7 days 
2. 
Grade ≥ 3 nonhematologic AEs (except for fatigue, diarrhea, and nausea/vomiting if manageable). 
3. 
Grade ≥ 3 diarrhea lasting > 2 days despite being treated with optimal medical therapy, or associated with 
fever or severe dehydration. 
4. 
Grade ≥ 3 fatigue lasting > 7 consecutive days. 


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5.2.2 
Phase II period 
After the pilot phase has been completed, the phase II period will be started with the dose level in which there is ≤ 1 
out of 6 patients experiencing any DLT. 
5.3 General Concomitant Medication  
All concomitant treatments and therapies, or medication administered during the 28 days preceding the screening 
study visit must be reported. The generic name of the drug (or trade name for combination drugs) must be specified 
along with the route of administration, dosage, frequency of dosage, and duration of treatment. Any medication which 
is considered necessary for the patient’s welfare, and which is not expected to interfere with evaluation of the study 
drug, may be given at the discretion of the investigator. Patients must notify the investigator of all concomitant 
medications taken through completion of the study.  
 
Throughout the study, investigators may prescribe any concomitant medications or treatments deemed necessary to 
provide adequate supportive care, such as potassium and phosphorus supplements and anti-emetics, with the 
exception of those listed below.  
 
The following concomitant medications are prohibited: 
 
Valproic acid, Zolinza™ (vorinostat), or any other HDAC inhibitor  
 
DNA methyltransferase inhibitors  
 
Other anti-cancer agents including Lupron or other approved luteinizing-hormone releasing 
hormone (LHRH) agonists such as goserelin or leuprolide  
 
Bisphosphonates initiated within 4 weeks prior to study start  
 
Other investigational agents  
 
Delay administration of H2-antagonists, antacids, or proton pump inhibitors, or other drugs that lower acidity 
for at least 2 hours after dosing entinostat.  
5.4 Duration of Therapy 
 
In the first cohort, treatment will be administered for a minimum of 15 days and up to 29 days prior to the 
definitive surgery. However if clinically acceptable, the preferred treatment duration is 22 days. The treatment 
should be continued until the time of the surgery (up to 29 days) or until one of the criteria in section 5.8 applies. 
o 
Anastrozole or tamoxifen treatment should be continued until the time of the surgery for a 
maximum of 29 days or until one of the criteria in section 5.8 applies.  
o 
Entinostat should be administered within 6 days prior to the definitive surgery. A minimum of 3 
doses (day 1, 8, and 15) are required to complete the study.  
 
In the second cohort, treatment including entinostat and anastrozole/tamoxifen will be continued until disease 
progression. Restaging scans will be performed every 6 weeks while on treatment. 
5.5 Surgical Considerations for the First Cohort (Operable Stage I-II) 
5.5.1 Marker Placement Prior to Initiation of Therapy 
A titanium marker or “clip” should be placed at the tumor site(s) prior to starting treatment in women who may be 
candidates for breast conservation surgery. This will identify the tumor location(s) for the surgical procedure in the 
event that there is a complete response to the preoperative treatment. 
5.5.2 Breast Surgery 
Following preoperative treatment, women will undergo breast conserving surgery or a mastectomy at the discretion of 
the treating surgeon. It is recommended that the definitive surgery take place within 6 days (preferably within 3 days) 
after the last dose of entinostat. Anastrozole should be continued until the day of the surgery. 
5.5.3 Axillary Staging 


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Pre-treatment 
 
Fine needle aspiration (FNA) or core biopsy of an axillary lymph node is permitted for any patient. 
 
A pre-neoadjuvant therapy sentinel lymph node biopsy (SLNB) for patients with clinically negative axillary 
lymph nodes is permitted. 
Post-treatment 
 
Post-neoadjuvant therapy axillary staging is required for all patients and will be performed according to the 
widely accepted American Society of Clinical Oncology (ASCO) guideline41. 
5.6 Postoperative Treatments 
5.6.1 Chemotherapy 
It is recommended that postoperative chemotherapy be administered. The adjuvant chemotherapy regimen is at the 
discretion of the treating physicians. 
5.6.2 Radiation Therapy 
Radiation therapy will be administered at the conclusion of all protocol treatment, when appropriate. 
5.7 Duration of Follow Up 
Patients will be followed for 30 days after the last dose of study medication or until death, whichever occurs first.  In 
patients who receive other primary treatment after entinostat and anastrozole administration and prior to surgery, we 
will collect a summary of the treatment given to correlate, if needed, with tissue samples from the surgical procedure 
– no additional data will be collected after that time.  Patients continuing to experience adverse events attributable to 
the study drug will be followed as needed until resolution or stabilization of the adverse events. Patients who either 
are found to be ineligible or refuse to start treatment after consenting will not be followed and their information will not 
be collected. 
5.8 Criteria for Removal from Study 
Patients will be removed from study when any of the criteria listed below applies.  The reason for study removal and 
the date the patient was removed must be documented in the case report form.  
 
Evidence of rapid disease progression during treatment at the discretion of the treating investigator. 
 
Non-compliance with the study protocol; including, but not limited to not attending the majority of scheduled 
visits. The Protocol Chair will determine when non-compliance should lead to removal from study.  
Note: The patients will still be included in the overall evaluation of response (intent-to-treat analysis). 
 
Unacceptable major toxicity or the dosing delay and/or delay in the surgery ≥ 2 weeks. 
Note: The patients will still be included in the overall evaluation of response (intent-to-treat analysis). 
 
Intercurrent illness or condition that would, in the judgment of the treating investigator, affect assessment of 
clinical status to a significant degree or require discontinuation of study treatment. 
 
At subject’s own request.  
Note: The reason for discontinuation from the study must be documented. The patients will be included in the overall 
evaluation of response (intent-to-treat analysis) if any protocol therapy was administered prior to withdrawal. 
 
Study is closed for any reason (e.g. new information shows that the patient’s welfare would be at risk if she 
continued study treatment). 
 
Subject withdraws consent for follow-up. 
 
Subject is lost to follow-up. 
 
Study is terminated for any reason. 
6. 
DOSING DELAYS/DOSE MODIFICATIONS 
6.1 Recommended Dose Adjustments  


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Although entinostat and anastrozole/tamoxifen have distinct toxicity profiles, they do share some adverse events 
such as fatigue and nausea. There is the theoretical possibility that one agent may potentiate the other and hence 
drug causality will not always be clear. In the event of uncertainty, dose reductions and/or delays will follow the most 
conservative approach, i.e., withholding both drugs until resolution of the event. 
 
6.1.1 
Non-Hematologic Toxicities of Entinostat 
 
The following rules will be followed for the management of adverse events that are definitely or probably due to 
entinostat alone. Grades are according to the active version of NCI CTCAE as per section 7.3. 
 
Toxicity 
Hold dose 
Treatment 
Recovery 
Dose reduction 
Grade ≥ 3 
Until recovery to grade 1 
or baseline 
Symptomatic remedies, 
start prophylaxis 
If recovered by next 
scheduled dose 
Resume at one dose level 
below the current dose 
 
 
 
If not recovered by next 
scheduled dose 
Skip this dosing; resume 
at one dose level below 
the current dose after 
recovery 
Recurrent 
Grade ≥ 3 
despite dose 
reduction 
Until recovery to grade 1 
or baseline 
Symptomatic remedies, 
start prophylaxis 
If recovered by next 
scheduled dose 
Resume at one dose level 
below the current dose 
 
 
 
If not recovered by next 
scheduled dose  
Skip this dosing; resume 
at one dose level below 
the current dose every 
other week after recovery 
Grade ≤ 2 
Until recovery to grade 1 
or baseline 
Symptomatic remedies, 
start prophylaxis 
If recovered by next 
scheduled dose 
Resume at the current 
dose level 
 
 
 
If not recovered by next 
scheduled dose 
Skip this dosing; resume 
at the current dose after 
recovery 
 
The dose modification schemas are as follow for both postmenopausal and premenopausal women: 
Dose Modification Schema for Postmenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Anastrozole 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
or 
Dose Modification Schema for Premenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Tamoxifen 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
 
6.1.2 
Hematologic Toxicities of Entinostat 
 
Myelosuppression is a common side effect of higher doses of entinostat. The guidelines in the table below will be 
followed for hematologic toxicities. 
 
ANC at planned dose 
 
PLT at planned dose 
Cycle Delivery 
≥ 1.0 x 109/L 
and 
≥ 80 x 109/L 
Resume at the current dose level 
< 1.0 x 109/L 
or 
< 80 x 109/L 
Delay until recovery to grade 1 or 


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baseline 
 
If the treatment is delayed for more than 2 weeks, the patient will be discontinued from treatment and followed until 
event resolution, and treated off-protocol at the investigator's discretion. 
 
6.1.3 
Toxicities likely from both drugs (diarrhea, anorexia, fatigue, and nausea) 
 
In the event that one of the above toxicities occurs, patients should be treated symptomatically and prophylactic 
treatment should be considered for subsequent administration of entinostat. In case grade 3 or 4 toxicity is observed 
both drugs should be held until recovery to grade 1 or baseline.  
 
If dosing is to be delayed by more than 2 weeks the patient will go off study. 
6.1.4 
 Other Adverse Events 
For any other NCI-CTCAE Grade 3 or 4 adverse events or any clinically significant, lower-grade adverse event, 
treatment with entinostat and anastrozole/tamoxifen should be interrupted for a maximum of 14 days until the patient 
recovers completely or the adverse event reverts to NCI-CTCAE Grade 1 or to baseline grade. If recurrence of 
adverse event after drug holiday / interruptions is observed a dose reduction to dose level -1 is recommended. Dose 
reduction should only be implemented when all supportive care measures have been exhausted without an 
improvement of patient status. In all cases where the subject is withdrawn due to unusual or unusually severe 
adverse event considered related to entinostat, the investigator must report the withdrawal as an SAE. 
6.2 Supportive Care Guidelines 
 
6.2.1 
Dehydration/dysgeusia 
 
In other studies, the use of popsicles and Gatorade has been found to be useful by some investigators in maintaining 
adequate hydration in the setting of dysgeusia.   
 
6.2.2 
Diarrhea 
 
Diarrhea should be treated promptly with appropriate supportive care, including loperamide.  Instruct patients to begin 
taking loperamide at the first signs of:  1) poorly formed or loose stool; 2) occurrence of more bowel movements than 
usual in one day; or, 3) unusually high volume of stool.  Loperamide should be taken in the following manner:  4 mg 
at first onset of diarrhea, then 2 mg after each unformed stool.  Daily dose should not exceed 16 mg/day.  Advise 
patients to drink plenty of clear fluids to help prevent dehydration caused by diarrhea.  Avoid loperamide if there is the 
presence of blood or mucus in the stool or if diarrhea is accompanied by fever.  If grade 3 or 4 diarrhea recurs despite 
treatment, discontinue further treatment with entinostat. 
 
6.2.3 
Additional Information 
 
When possible, symptoms should be managed symptomatically.  In case of toxicity, appropriate medical treatment 
should be used (including anti-emetics for nausea/vomiting, anti-diarrheals for diarrhea, as above, etc.). 
 
7. 
ADVERSE EVENT REPORTING AND DATA SAFETY AND MONITORING PLAN 
 
7.1 
Adverse Events (AEs) 
An Adverse Event is any untoward occurrence reflecting a change from baseline state after signing informed consent 
and up to 30 days from last administration of the study medication.  A Serious Adverse Event (SAE) is any AE that is 
1) Fatal, 2) Life Threatening, 3) Requires or prolongs hospital stay, 4) Results in a persistent or significant disability or 
incapacity. 5) Results in a congenital anomaly or birth defect or 6) Is considered by the Investigator as an important 
medical event. 
7.1.1 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 4.0 (Available through http://ctep.cancer.gov) will be utilized for AE reporting. AEs 
of any grade will be documented in the case report form. 


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7.1.2  
AEs can be “Expected” if described in the Informed Consent or “Unexpected”. The attribution of the 
AE is Definite if the AE is clearly related to study treatment; Probable if it is likely related to the study treatment; 
Possible if it is equally likely related to study treatment or to some other circumstance, and Unlikely if most probably 
some other event was responsible for the adverse event, and Unrelated if the Adverse Event is entirely explained by 
circumstances not caused by the study medication. 
7.1.3  
Data Safety Monitoring. The UMGCC Data Safety Monitoring / Quality Assurance Committee will 
review the study. The specific functions of the DSMB /QAC are contained in the UMGCC Clinical Investigators 
Handbook, available through http://www.umgcc.org/research/clinical_research.htm which is modeled closely after 
NCI policies and procedures for example, for adverse event reporting. (See http://www.ctep.nci.nih.gov.)  
7.2  Expedited reporting of severe adverse events 
The University of Maryland IRB has specific guidelines for expedited adverse event reporting. These policies can be 
found in the IRB’s policies and procedures manual which can be accessed using the following URL: 
http://www.hrpo.umaryland.edu/default.asp. The UM policy requires that any harm experienced by a subject or other 
individual, which in the opinion of the investigator is unexpected and probably related, be expeditiously reported to 
the IRB within 5 business days. 
For those events that require a change to the informed consent, the data entry staff who input these data will notify 
the appropriate research coordinator and regulatory coordinator of this change. 
7.3  
Expedited reporting by investigator to Syndax 
Serious adverse events (SAE) are defined above.  The investigator must inform Syndax in writing using a SAE form 
or MEDWATCH 3500A form of any SAE within 24 hours of being aware of the event.  The date of awareness should 
be noted on the report.  The written report must be completed and supplied to Syndax by facsimile within 24 hours/1 
business day at the latest on the following working day.  The initial report must be as complete as possible, including 
details of the current illness and (serious) adverse event, and an assessment of the causal relationship between the 
event and the investigational product(s).  Information not available at the time of the initial report (e.g., an end date for 
the adverse event or laboratory values received after the report) must be documented on a follow-up report.  A final 
report to document resolution of the SAE is required.  If this is a multicenter trial, please participating study sites must 
report SAEs to Syndax as described and within 24 hours of awareness.  Participating sites should also report SAEs 
to the primary study site. 
All SAEs will be reported to Syndax Drug Safety as soon as possible, but no later than fifteen calendar days from first 
knowledge of the event.  A copy will be sent to the FDA (if applicable) by the investigative site:  
Syndax Pharmaceuticals, Inc. 
Drug Safety/ Regulatory  
Attn: Miranda Rees 
460 Totten Pond Road, Suite 650 
Waltham, MA 02451 
mrees@syndax.com 
Main: 781-419-1400 
Direct: 781-419-1417 
 
CC: jhasapidis@syndax.com 
 
8. 
PHARMACEUTICAL INFORMATION 
8.1  Anastrozole 
8.1.1 
Description 
Anastrozole or Arimidex® is a non-steroidal aromatase inhibitor. It is chemically described as 1,3-
Benzenediacetonitrile, a, a, a’, a’-tetramethyl 5-(1H-1,2,3-triazol-1-ylmethyl) and the molecular formula C17H19N5, with 
a molecular weight of 293.4.  
8.1.2 
Formulation 


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The tablets are white, biconvex, film-coated containing 1 mg of anastrozole. The tablets are impressed on one side 
with a logo consisting of a letter “A” (upper case) with an arrowhead attached to the foot of the extended right leg of 
the “A” and on the reverse with the tablet strength marking “Adx1”. 
8.1.3 
Availability and Storage 
The commercial version of anastrozole will be supplied by AstraZeneca Pharmaceuticals. 
These tablets are supplied in bottles of 30 tablets of 1 mg anastrozole tablet (NDC 0310-0201-30). Anastrozole 
should be stored at controlled room temperature, 20-25oC (68-77oF). 
Additional information regarding the formulation as well as storage and handling instructions can be found in the 
approved package insert.  
8.1.4 
Drug Accountability 
Agent Inventory Records – The investigator, or a responsible party designated by the investigator, must maintain a 
careful record of the inventory and disposition of anastrozole.  
 
8.1.5 
Agent Ordering 
Anastrozole may be requested by the Principal Investigator (or their authorized designees).  Anastrozole will be 
supplied by AstraZeneca Pharmaceuticals.   
 
Agent may be requested by contacting Emmanuel Semmes, Investigational Drugs Clinical Specialist Pharmaceutical 
Services (telephone number 773-834-8749) (fax number 773-834-7461). The contact person at AstraZeneca 
Pharmaceuticals is Ms. Karen Bartels, ISS Manager (telephone number 302-886-1507). 
 
8.2 
Entinostat 
8.2.1 
Description 
 
Entinostat 
is 
a 
synthetic 
small 
molecule 
bearing 
the 
chemical 
name 
N-[[4-[[(2-
aminophenyl)amino]carbonyl]phenyl]methyl]-, 3- pyridinylmethyl ester and the molecular formula C21H20N4O3, with a 
molecular weight of 376.41. SNDX-275 or MS-275 or Entinostat is classified as an anti-neoplastic agent, specifically 
functioning as an inhibitor of histone deacetylases by promoting hyperacetylation of nucleosomal histones, allowing 
transcriptional activation of a distinct set of genes that leads to the inhibition of cell proliferation, induction of terminal 
differentiation, and/or apoptosis. 
8.2.2 
Formulation 
Entinostat is orally bioavailable and is supplied as light brown or yellow coated tablets containing 1 mg and 5 mg of 
active ingredient, respectively. Each tablet contains mannitol, carboxymethylstarch sodium, hydroxypropyl cellulose, 
potassium bicarbonate, and magnesium stearate as inactive ingredients. The tablet coat contains hydroxypropyl-
methyl cellulose, talc, titanium dioxide, and ferric oxide pigment as a coloring agent. 
8.2.3 
Availability and Storage 
Entinostat is an investigational agent supplied by Syndax Pharmaceuticals, Inc. 
 
Entinostat is supplied as 1 mg (light brown, in bottles of 40), or 5 mg (intense yellow, in bottles of 40) film-coated 
tablets (round-biconvex). Each tablet also contains mannitol, carboxymethyl starch sodium, hydroxypropyl cellulose, 
potassium bicarbonate, and magnesium stearate, hydroxypropyl methylcellulose, talc, titanium dioxide, iron oxide 
pigment (yellow) and iron oxide pigment (red). 


09/09/14 Version 10 
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Entinostat tablets may be shipped and stored at room temperature (15-30oC), and should be protected from light. 
Avoid temperature extremes. The pharmacist will dispense the investigational material to the patient at appropriate 
intervals throughout the study in childproof containers. Shelf life stability studies of the intact bottles are on-going. 
8.2.4 
Drug Accountability 
Agent Inventory Records – The investigator, or a responsible party designated by the investigator, must maintain a 
careful record of the inventory and disposition of entinostat. 
 
 
8.2.5 
Agent Ordering  
 
Agent may be requested by contacting Jeannette Hasapidis , VP of Clinical Operations (office phone 781-419-1404, 
mobile phone 781-724-6909, and email jhasapidis@syndax.com). 
8.3  Tamoxifen 
8.3.1 
Description 
 
Tamoxifen or Nolvadex® is a nonsteroidal antiestrogen. It is the trans-isomer of a triphenylethylene derivative. The 
chemical 
name 
is 
(Z)2-[4-(1,2-diphenyl-1-butenyl) 
phenoxy]-N, 
N-dimethylethanamine 
2-hydroxy-1,2,3- 
propanetricarboxylate (1:1) with a molecular weight of 563.62. 
8.3.2 
Formulation 
The tablets are round, biconvex, uncoated, white tablet identified with NOLVADEX 604 debossed on one side and a 
cameo debossed on the other side. Each tablet contains 20 mg equivalent of tamoxifen. 
8.3.3 
Availability and Storage 
Tamoxifen will be prescribed by the treating physician and covered by the patients insurance. 
 
These tablets should be stored at controlled room temperature 20-25°C (68-77°F). Dispense in a well closed, light-
resistant container. 
Additional information regarding the formulation as well as storage and handling instructions can be found in the 
approved package insert.  
9. 
CORRELATIVE STUDIES 
Both blood samples and tissue samples should be collected from both pilot and phase II subjects.   
9.1 Tumor Tissue Samples 
The submission of pathology materials from BOTH the original diagnostic procedure and the definitive surgery or 
biopsies are required for ALL patients.  
9.1.1 
Collections 
In the first cohort: 
Tumor tissue samples from the following time points will be obtained: 
1. 
Prior to starting treatment or tissue sample at the original diagnostic procedure. (T0) 
2. 
At the time of definitive surgery (i.e., mastectomy or lumpectomy). A separate tumor biopsy is not needed on 
the day of surgery; tissue will be taken from the surgical specimen. (T1) 


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Note: If a subject receives additional preoperative treatment after discontinuing study treatment and prior to surgery, 
every attempt should be made to collect a tissue sample for the study prior to initiation of the new treatment; in these 
cases, tissue will still be collected at the time of definitive surgery, as above. 
Every effort should be made to provide a core specimen from the surgical sample after treatment. The site pathologist 
should remove a core samples during the dissection of the surgical specimen (using a 5-mm skin punch biopsy 
device). The samples should be processed preferably within 1 hour, but may be processed up to 2 hours of removal 
from the patients. This core specimen should be fixed in formalin and processed separately as per the American 
Society of Clinical Oncology/College of American Pathologists Guideline Recommendations published in Journal of 
Clinical Oncology 201042. 
In the second cohort: 
Approximately six core biopsy specimens will be obtained with each biopsy. Alternative to core needle biopsy, fine 
needle aspirate (FNA) is allowed in this cohort of patients. Cytology cell blocks should be made from FNA specimens 
using routine institutional cytology protocol.  Biopsy should be obtained from soft tissue metastasis such as breast, 
liver, skin, lung, or intra-abdominal implants. Bone biopsy, paracentesis (ascites), and thoracocentesis (pleural 
effusion) specimens are not adequate.  
Tumor tissue samples from the following time points will be obtained: 
1. 
Pretreatment biopsy (biopsy#1) will be performed within 3 weeks after registration. However, the 
pretreatment biopsy can be omitted in patients who have recent biopsy but have not been started on breast 
cancer treatment within 12 weeks prior to the registration and there is adequate tumor tissue sample as per 
section 3.1.9. (T0-Second cohort) 
2. 
Post-treatment biopsy (biopsy#2) will be performed after a minimum of 15 days and a maximum of 29 days 
of treatment, but preferably close to 29 days. The post-treatment biopsy must take place within 6 days 
(preferably within 3 days) after the last dose of entinostat. If a patient discontinues the study prior to 15 days, 
an attempt should be made to collect the biopsy when the decision is made to discontinue and prior to 
initiation of additional treatment, if possible. 
9.1.2 
Paraffin Embedded Tissue Submission 
 
All samples must be submitted within ONE month after the patient’s definitive surgery or biopsy. All materials must 
be labeled with the institutional surgical pathology number, the study number, the patient’s study number, the cohort 
number (first or second), the treatment group (tamoxifen or anastrozole), and the designated specimen type (T0 or 
T1). The samples should not contain patients’ identifying information. 
Please be sure to use a method of shipping that is secure and traceable. Extreme heat precautions should be taken 
when necessary. Please use papers to pad the specimen shipments. Do not use bubble wrap. Shipping should be 
arranged only on Monday-Thursday by overnight service to assure receipt. Do not ship specimens on Friday or 
Saturday. Please notify all of the contact persons listed below via email and/or telephone PRIOR to shipping.  
A copy of the tissue tracking form should be included in the shipping package. Please note that if multiple samples 
are sent, only one copy of the form needs to be included.  
The specimens should be shipped as directed below:  
Specimens 
Purpose 
Shipping Address and Contact Information 
≥3 unstained 
slides* 
ER, PR, HER2, EGFR, CK5/6, 
Aromatase, histone H3, H4 
acetylation, and Ki67 expression by 
immunohistochemistry 
NOTE: A copy of the pathology 
report from the treating and/or 
referring institution and a copy of the 
operative report should be included 
Kimberly C. Tuttle, HT (ASCP) 
Pathology Biorepository and Research Core 
University of Maryland Medical Center 
22 S. Greene Street, Rm NBW58 
Baltimore, MD 21201 
Phone: (410) 328-5524 
Fax: (410) 328-5508 
Email: ktuttle@umm.edu  


09/09/14 Version 10 
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in this package. 
Tumor/lymph 
node block 
or 
≥3 unstained 
slides∞ 
Gene expression profiling and 
Methylation profiling 
Saraswati Sukumar, Ph.D. 
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 
1650 Orleans Street, Rm 143 CRB 
Baltimore MD 21231-1000 
Phone: (410) 614-2479 
Fax: (410) 614-4073 
Email: sukumsa@jhmi.edu 
 
*Five-micron (5 µM) thickness mounted on charged slides (do not oven bake slides) from both original diagnostic 
tumor blocks AND residual primary tumor or a positive lymph node blocks should be submitted as described above. 
 
∞The original diagnostic tumor block and at least one representative block of residual primary tumor or a positive 
lymph node containing the maximum amount of tumor are required. Although it is preferred that tissue blocks are 
submitted, for institutions that do not allow submission of the tissue blocks, please submit ≥3 unstained sections of 5 
µM thickness mounted on charged slides. 
9.1.3 
Methods 
9.1.3.1 Immunohistochemistry 
 
Immunohistochemistry (IHC) will be used to determine the pre-treatment and post-treatment expression of ER, PR, 
HER2, and Ki67. The level of expression of these proteins will be quantified by ChromaVision ACIS Assisted 
Quantitative Image Analysis. These studies will be performed by immunohistochemistry technicians in the pathology 
department at the University of Maryland which is certified by both Clinical Laboratory Improvement Amendments 
(CLIA) and College of American Pathologists (CAP). The specimens will be de-identified with a unique patient’s study 
number and stored in the tumor bank facility at University of Maryland. Hematoxylin and eosin stained sections will be 
examined by a pathologist to confirm the tissue source. Five-micron sections of formalin-fixed, paraffin-embedded 
tissue will be tested for the presence of immunohistochemically detectable ER, PR, HER2, and Ki67 expression. After 
60 minutes of heat induced epitope retrieval in citrate buffer, strepavidin biotin detection systems using Ventana 
reagents and instruments will be used. We will use ER, PR, HER2, Ki67, EGFR, and CK5/6 antibodies that have 
been validated and are currently in clinical use at University of Maryland pathology department as listed below.  
 
Immunostaining 
Antibody 
ER 
SP1 rabbit monoclonal Ventana prediluted anti-ER antibody 
PR 
1E2 rabbit monoclonal Ventana prediluted anti-PR antibody 
HER-2 
4B5 rabbit monoclonal Ventana prediluted anti-HER-2/neu antibody 
Ki67 
30-9 rabbit monoclonal Ventana prediluted anti-Ki67 antibody 
EGFR 
3C6  mouse monoclonal Ventana prediluted anti-EGFR antibody 
CK5/6 
D5&16B4 mouse monoclonal Ventana prediluted anti-CK5/6 antibody 
Aromatase 
Mouse monoclonal antibody (Novartis #677)43  
 
Scoring systems: 
 
 
ER and PR: Will be scored as the percentage of positive staining tumor nuclei on the examined slide. Five 
percent immunoreactivity is considered a positive result. This is based on comparison of our results to that 
of clinically validated ER and PR assays, determined by several large clinical studies. 
 
HER-2/neu: Will be scored as followed - score 0: no membrane staining is observed.  1+: weak, incomplete 
staining in any proportion of the invasive tumor cells.  2+: complete membrane staining in less than or equal 
to 30% of the invasive tumor cells.  3+: strong complete membrane staining in more than 30% of the 
invasive tumor cells. 


09/09/14 Version 10 
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 
Ki-67: Will be assessed by counting 500 to 1000 cells, and will be reported as percent positive cells based 
on nuclear expression using ChromaVision ACIS system to quantify.  
 
EGFR: Will be assessed by counting 500 to 1000 cells, and will be reported as percent positive cells based 
on membrane expression using ChromaVision ACIS system to quantify.  
 
CK5/6: Will be assessed by counting 500 to 1000 cells, and will be reported as percent positive cells based 
on cytoplasmic expression using ChromaVision ACIS system to quantify. 
 
Aromatase: Will be assessed by counting 500 to 1000 cells, and will be reported as percent positive cells 
based on cytoplasmic expression using ChromaVision ACIS system to quantify. 
 
Various other cancer specimens (expression in > 30% of the tumor cells) will be used as positive controls. Buffer will 
replace primary antibody for negative controls. All of the immunohistochemical staining will be quantitatively analyzed 
by ChromaVision ACIS system for immunohistochemistry image analysis. Quantitation of both staining intensity as 
well as assessment of numbers of positive stained cells will be determined by the agreement of a consensus of 
pathologists and researchers. These studies will be carried out under the direction of Dr. Olga Ioffe in the Department 
of Pathology at University of Maryland. 
 
9.1.3.2 Gene Expression Profiling 
 
Additional biopsy for fresh tissue sample is not required in this study. We will use the cDNA-mediated Annealing, 
Selection, Extension, and Ligation (DASL) assay from Illumina which has been developed to allow more 
comprehensive gene expression profiling of degraded RNA extracted from paraffin blocks using bead array format as 
previously described37. Illumina’s Bead Array Technology is based on 3-micron silica beads that self assemble in 
microwells on either of two substrates: fiber optic bundles or planar silica slides. When randomly assembled on one 
of these two substrates, the beads have a uniform spacing of ~5.7 microns. Each bead is covered with hundreds of 
thousands of copies of a specific oligonucleotide that act as the capture sequences in one of Illumina’s assays. 
BeadArray technology is utilized in Illumina's iScan System for a broad range of DNA and RNA analysis applications. 
 
All promising findings of gene expression changes will also be validated by the quantitative RT-PCR methods. 
Change in expression of genes as a result of treatment will be determined by IHC or quantitative RT-PCR. Although 
IHC can be easily accomplished on a single slide, it may not be sensitive to small changes in expression and 
expression measurements are semi-quantitative. Quantitative (real-time) RT-PCR can provide better quantification of 
expression of gene product, and recent studies have shown that this approach can be applied to RNA isolated from 
paraffin-embedded tissues, when small sequences are amplified. The overall goal of these studies is to identify 
candidate markers for response and molecular profiles that might be relevant to an understanding of drug 
mechanisms. Although statistical methods will provide some indication of significance of particular molecular 
changes, validation will ultimately require additional testing in independent samples. 
 
9.1.3.3 Methylation of Relevant Genes 
 
Both gene expression profiling and Methylation of relevant genes will be performed under the direction of Dr. 
Saraswati Sukumar, the co-director of breast cancer program at the Sidney Kimmel Comprehensive Cancer Center at 
Johns Hopkins. 
 
Methylation of relevant genes (ERalpha, APC-1, RARbeta, cyclin D2, Twist, RASSF1A, and HIN-1) will be evaluated 
by Quantitative Multiplex Methylation-Specific Polymerase Chain Reaction (QM-MSP) as previously described39,44. 
This analysis provides a quantitative assessment of methylation in each gene as well as units of cumulative 
methylation of the gene panel.  The prediction is that methylation in specific genes will reduce as a result of treatment 
and will result in re-expression of the genes. Re-expression of the silenced genes will be tested by Q-R-PCR in RNA 
extracted from the same samples.   
 
9.1.3.4 Tumor Histone H3 and H4 Acetylation 
 
Analysis for tumor histone H3 and H4 acetylation will be performed at University of Maryland Pathology Department 
under direction of Dr. Olga Ioffe. The histone acetylation will be assessed in formalin-fixed, paraffin-embedded 
section by immunohistochemical staining using the method that was previously described in a phase II trial of 
vorinostat in recurrent glioblastoma multiforme45. However, we will use ChromaVision ACIS system for 
immunohistochemistry image analysis to quantify the expression of tumor histone H3 and H4 acetylation. The 
polyclonal antibodies that will be used are listed below: 
 
Acetylated histone H3, lysine 9 (1:25 dilution; Cell Signaling technology catalog# 9671L) 
 
Acetylated histone H4, lysine 8 (1:100 dilution; Cell Signaling technology catalog# 2594S) 


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9.2 Blood Samples 
 
9.2.1 Collections 
Blood samples from the following time points will be obtained in all groups of patients including both 
tamoxifen/anastrozole and the first/second cohort. Blood samples should be collected in the following venipuncture 
tubes: 
1. 
For pharmacokinetic study (PK): One 6 ml, sodium heparin coated venipuncture tubes (BD vacutainer® 
green top tube).  
2. 
For Methylation profile (MP): Two 8.5 ml, Red/Gray tiger top serum separator tubes (SST) 
For patients in the pilot phase and stage 1 of the phase II portion (the initial 12 patients):  
 
Day of Rx 
Label 
Time Points 
Purposes 
1 
B0 
Prior to starting treatment 
PK and MP 
1 
B1 
30 minutes after the first dose of entinostat 
PK 
15 
B2 
Day 15 (±1 day) after the first treatment BEFORE entinostat 
PK 
15 
B3 
Day 15 (±1 day)  after the first treatment 30 minutes AFTER entinostat 
PK 
Surgery/Biopsy 
B4 
At the time of definitive surgery (i.e., mastectomy or lumpectomy) or the 
post-treatment biopsy* 
MP 
 
For patients in the stage 2 of the phase II portion (20 patients): 
Day of Rx 
Label 
Time Points 
Purposes 
1 
B5 
Prior to starting treatment 
PK and MP 
15 
B6 
Day 15 (±1 day) after the first treatment BEFORE entinostat 
PK 
Surgery/Biopsy 
B7 
At the time of definitive surgery (i.e., mastectomy or lumpectomy) or the 
post-treatment biopsy* 
MP 
*Blood sample at the time of definitive surgery or post-treatment biopsy can be omitted if definitive surgery or post-
treatment biopsy occurs within 3 days after day 15 blood collection. 
9.2.2 Blood Sample Processing and Storage 
All samples should be labeled with the study number, patient’s study number, the date and time of collection, the 
cohort number (first or second), the treatment group (tamoxifen or anastrozole), and designated specimen type in 
section 8.2.1 (i.e. B0/B1/B2/B3). The samples should not contain patients’ identifying information. 
9.2.2.1 Pharmacokinetic Samples 
Blood will be collected for entinostat pharmacokinetics in a sodium heparin vacutainer. Samples will be obtained at 
baseline on Day 1 and pre-dose Day 15. In the initial dose finding portion of the study, patients will also have 2 
additional samples obtained at 0.5 hr (historical time to Cmax) on Day 1 and Day 15. Each sample will require 5 mL of 
patient blood.  Samples will be placed on ice immediately and centrifuged within 30 minutes of collection at 1,000 
x g for 10 minutes in a refrigerated centrifuge. Plasma will be stored at –20°C until analysis. Entinostat concentrations 
will be determined using a validated LC/MS/MS method that will be developed by the Analytical Pharmacology Core 
Laboratory at the Sidney Kimmel Comprehensive Cancer Center (SKCCC) at Johns Hopkins 46.  
 
9.2.2.2 Methylation Profile 
 
The samples should be processed within two hours of drawing the blood. 
 
 
Draw blood into two 8.5 ml SST red/grey vacutainer tubes (BD vacutainer® catalog# 367988) and invert 
gently 5 times to initiate clotting. 
 
Allow blood to clot for a minimum of 30 minutes, but not longer than 2 hours. 
 
Centrifuge 15-20 minutes at 3,000 rpm (1,100-2,000 x g) at room temperature or 4oC. 


09/09/14 Version 10 
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 
Aliquot the serum (top layer) into 6 cryovials (~1 ml each aliquots).  There should be approximately 3 ml 
of serum per vacutainer tube.  All of the serum should be stored.  
 
Serum will be stored at -70oC or colder until batch shipment or analysis. 
 
9.2.3 Blood Sample Submission 
Instructions for the collection and shipping of samples are included below. All materials must be labeled with the 
study number, the patient’s study number, patient initials, date of collection, the cohort number (first or second), the 
treatment group (tamoxifen or anastrozole), and the designated specimen type (i.e. B0, B1, or B2). The samples 
should not contain patients’ identifying information. Please be sure to use a method of shipping that is secure and 
traceable. Extreme heat precautions should be taken when necessary. Shipping should be arranged only on Monday-
Wednesday by overnight service to assure receipt. Do not ship specimens on Thursday, Friday, Saturday, or 
Sunday (specimens drawn on Friday should be sent the following Monday). Please notify the contact person listed 
below for each type of specimens via email and/or telephone PRIOR to shipping.  
A copy of the tissue tracking form and/or the blood and serum tracking form should be included in the shipping 
package. Please note that if multiple samples are sent, only one copy of the form needs to be included. 
9.2.3.1 Pharmacokinetic Samples 
All specimens should be properly labeled as described above. NOTE: In the event that baseline specimens are 
available but the patient is not successfully registered to the protocol, do not submit the patient's specimens. Blood 
samples for pharmacokinetic study should be processed and stored until batch shipment at the end of the study. 
Samples should be shipped on dry ice and overnighte shipmentsshould occur on Monday through Wednesday 
except when the following day is a holiday.  A fax or call should be place to the Analytical Pharmacology Core 
Laboratory prior to shipment providing the shipment tracking information. 
 
Address shipments and any question regarding specimen processing to: 
 
Analytical Pharmacology Core Laboratory  
Attn: Entinostat/Anastrozole Study Samples 
1650 Orleans Street CRB1, Rm 184 
Baltimore, MD 21231-1000 
Phone: 410-955-1129 
Phone with voicemail: 410-502-7192 
Fax: 410-502-0895 
 
9.2.3.2 Methylation Profile 
 
All specimens should be properly labeled as described above. Blood samples for Methylation profile should be 
processed and stored until batch shipment at the end of the study. Samples should be shipped in dry ice overnight on 
Monday through Wednesday except when the following day is a holiday. Please contact Dr. Saraswati Sukumar 
prior to the shipment and the shipment tracking information should be provided. 
 
Address shipments and any question regarding specimen processing to: 
Saraswati Sukumar, Ph.D. 
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 
1650 Orleans Street, Rm 143 CRB 
Baltimore MD 21231-1000 
Phone: (410) 614-2479 
Fax: (410) 614-4073 
Email: sukumsa@jhmi.edu 
 
9.2.4 Methods 
9.2.4.1 Pharmacokinetic Study 
Pharmacokinetic analysis will be performed by Michelle A. Rudek, Pharm.D, Ph.D. at the Analytical Pharmacology 
Core Laboratory at SKCCC at Johns Hopkins.  Patients in the pilot phase as well as stage 1 of the phase II 


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component of the study will have plasma samples collected four times to assess entinostat pharmacokinetics.  Steady 
state trough concentrations (Css,min) will be the pretreatment concentration on Day 15.  These pharmacokinetic 
parameters will be used to explore the relationship between entinostat exposure with pharmacodynamic (PD) 
endpoints such toxicity and histone acetylation in the tumors. Levels of entinostat will be determined using a validated 
LC/MS/MS method by Dr. Michelle Rudek at the Analytical Pharmacology Core Laboratory at Johns Hopkins46. The 
pharmacokinetic schedule has been summarized below. 
 
For patients in the pilot phase and stage 1 of the phase II portion (the initial 12 patients):  
1. 
Day 1 
Pretreatment 
B0  
2. 
Day 1 
0.5 hour after entinostat (Cmax) 
 
B1 
3. 
Day 15  
Pretreatment (Css,min) 
 
B2 
4. 
Day 15 
0.5 hour after entinostat (Cmax) 
 
B3 
 
For patients in the stage 2 of the phase II portion (20 patients): 
1. 
Day 1  
Pretreatment 
B5  
 
2. 
Day 15  
Pretreatment (Css,min) 
B6 
 
9.2.4.2 Methylation Profiling 
 
DNA Methylation studies will be performed at the Breast Cancer Research Laboratories at Johns Hopkins under the 
direction of Dr. Sara Sukumar. We expect that over 70% of patients will have circulating DNA at baseline. QM-MSP 
studies will be done on DNA extracted from serum obtained from each subject. Primers and probes for QM-MSP 
analysis have been designed to specifically amplify hypermethylated promoter sequences of Cyclin D2, RAR-beta, 
APC-1, Twist, RASSF1A, HIN-1, and ER-alpha. PCR will be performed in separate wells for each primer/probe set. 
Fluorogenic PCR amplification will be carried out using a 7,900 Sequence detector (Perkin-Elmer Applied 
Biosystems) as previously described. 


09/09/14 Version 10 
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10. STUDY CALENDAR AND VISIT SCHEDULE 
Baseline evaluations are to be conducted within 3 weeks prior to start of protocol therapy.   
For the first cohort: 
Procedure 
Pre-
study 
Week 1 
Week 2 
Week 3 
Week 4 
Week 5 
Surgery a 
Off 
treatment 
D1 
D4-7 
D8 
D9-14 
D15 
D16-21 
D22 
D23-28 
D29 
D30-32 
 
 
Minimum treatment 
Optional 
 
 
Treatment: 
Entinostat 
 
A 
 
A 
 
A 
 
A 
 
A 
 
 
 
Anastrozole/Tamoxifen 
 
 
B 
B 
B 
B 
B 
B 
B 
B 
B 
 
 
Clinical assessment: 
Informed consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Current medications 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Physical exam 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Vital signs 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Performance status 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Adverse event evaluation 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Tumor measurement b 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Laboratory test: 
CBC w/diff, plts 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Chemistries c 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
PT/INR, PTT 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
Electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test g 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Correlative studies (Research procedures): 
Pharmacokinetic study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Pilot phase, stage 1 
 
Xd 
 
 
 
Xd 
 
 
 
 
 
 
 
 Stage 2 
 
Xe 
 
 
 
Xe 
 
 
 
 
 
 
 
Methylation profile 
 
Xf 
 
 
 
 
 
 
 
 
 
Xf 
 
Tumor sample 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
A: Entinostat: as per appropriate dose level PO weekly on day 1, 8, 15, 22 and 29 ± 2 days. Preferably 22 days. 
B: Anastrozole for postmenopausal women: 1 mg PO daily starts on day 4 and continue until the day of definitive 
surgery (lumpectomy or mastectomy). 
 
Tamoxifen for premenopausal women: 20 mg po daily starts on day 4 and continue until the day of definitive 
surgery (lumpectomy or mastectomy). 
 
a: 
Within 6 days (preferably within 3 days) after the last dose of entinostat. 
b: 
Tumor measurement: clinical tumor size, character, mobility, and location of the breast mass. 
c: 
Chemistries:  sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose, total bilirubin, calcium, total 
protein, albumin, AST (SGOT), ALT (SGPT) , alkaline phosphatase, LDH, uric acid, phosphorus, and magnesium 
d:  Day 1:   
 
Prior to starting treatment  
 
 
 
 
 
30 minutes after the first dose of entinostat 
 
Day 15 (±1 day): BEFORE entinostat 
 
 
 
 
 
30 minutes AFTER entinostat 
e:  Day 1:   
 
Prior to starting treatment  
 
Day 15 (±1 day): BEFORE entinostat 
f: 
Day 1:   
 
Prior to starting treatment 
 
The day of definitive surgery (lumpectomy or mastectomy) 
g: 
Serum or urine pregnancy test should be performed within 7 days prior to registration in women of childbearing 
potential 
NOTE: Visit Windows ± 2 days 


09/09/14 Version 10 
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For the second cohort: 
Procedure 
Pre-
study 
Week 1 
Week 2 
Week 3 
Week 4 
Week 5 
Continue 
until 
disease 
progression 
h 
Off 
treatment 
D1 
D4-7 
D8 
D9-
14 
D15 
D16-
21 
D22 
D23-28 
D29 
D30-32 
Treatment: 
Entinostat 
 
A 
 
A 
 
A 
 
A 
 
A 
 
 
 
Anastrozole/Tamoxifen 
 
 
B 
B 
B 
B 
B 
B 
B 
B 
B 
 
 
Clinical assessment: 
Informed consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Current medications 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Physical exam 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Vital signs 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Performance status 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Adverse event evaluation 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Tumor measurement for 
palpable or visible tumors 
b 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
CBC w/diff, plts 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Chemistries c 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
PT/INR, PTT 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test g 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Correlative studies (Research procedures): 
Pharmacokinetic study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Pilot phase, stage 1 
 
Xd 
 
 
 
Xd 
 
 
 
 
 
 
 
 Stage 2 
 
Xe 
 
 
 
Xe 
 
 
 
 
 
 
 
Methylation profile 
 
Xf 
 
 
 
Xf 
 
 
Biopsies 
X 
 
 
 
 
Xa 
 
 
 
A: Entinostat: as per appropriate dose level PO weekly until disease progression. 
B: Anastrozole: 1 mg PO daily starts on day 4 and continue until disease progression. 
 
Tamoxifen for premenopausal women: 20 mg po daily starts on day 4 and continue until disease progression. 
 
a: 
The post-treatment biopsy (biopsy#2) will be performed after a minimum of 15 days and a maximum of 29 days 
of treatment, but preferably close to 29 days. The post-treatment biopsy must take place within 6 days 
(preferably within 3 days) after the last dose of entinostat. 
b: 
Tumor measurement for palpable or visible tumor: clinical tumor size, character, mobility, and location of the 
breast mass. Restaging scans will be performed every 6 weeks while on treatment. 
c: 
Chemistries:  sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose, total bilirubin, calcium, total 
protein, albumin, AST (SGOT), ALT (SGPT) , alkaline phosphatase, LDH, uric acid, phosphorus, and magnesium 
d:  Day 1:   
 
Prior to starting treatment  
 
 
 
 
 
30 minutes after the first dose of entinostat 
 
Day 15 (±1 day): BEFORE entinostat 
 
 
 
 
 
30 minutes AFTER entinostat 
e:  Day 1:   
 
Prior to starting treatment  
 
Day 15 (±1 day): BEFORE entinostat 
f: 
Day 1:   
 
Prior to starting treatment 
 
The day of post-treatment biopsy 
g: 
Serum or urine pregnancy test should be performed within 7 days prior to registration in women of childbearing 
potential 


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h: 
After the post-treatment biopsy, treatment will be continued until disease progression. Restaging scans will be 
performed every 6 weeks while on treatment. Follow up visits after the post-treatment biopsy will be every 4 
weeks or at physician’s discretion. 
NOTE: Visit Windows ± 2 days 
 
VISIT SCHEDULE 
The study consists of a maximum 21-day (3 weeks) screening phase prior to registration, start of study drug 
administration within 3 business days of registration. The follow-up phase will last 30 days after the last dose of study 
medication or until death, whichever occurs first. 
10.1 
Screening  
Each potential patient will be screened before the start of the study to determine her eligibility for participation. These 
tests are to be conducted within 3 weeks prior to study registration. The following procedures will be performed:  
 
 
Informed consent (signed and dated by patient) 
 
Medical history and demographics  
 
Inclusion/Exclusion Criteria 
 
Concomitant medications and treatments will be recorded from 1 month prior to the start of study 
treatment.  
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass. The patient will be examined by one of the treating investigators or his/her 
designee at each specified time point and after the final dose of treatment. 
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate (HCO3− 
or CO2), BUN, creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT 
(SGPT) , alkaline phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Coagulation study: PT with INR and PTT 
 
12-lead electrocardiogram (ECG)  
 
Collection of tumor sample: Parrafin blocks and/or unstained slides 
10.2 
Registration  
Registration will take place once the consented patient has completed the necessary screening procedures and is 
deemed eligible for study entry by the investigator or designee. Each registered patient will be assigned a unique 
identification number. Treatment should begin within 3 business days after registration. Please see registration 
process in more detail in section 4.2. 
10.3 
Study Days  
The period called “Study Days” begins when the patient receives the initial dose of entinostat (Week 1 Day 1). 
Assessments are scheduled once a week with a visit window of ± 2 day until the time of definitive surgery 
(lumpectomy or mastectomy) or the post-treatment biopsy. However, this may be repeated more often, as clinically 
indicated. A maximum 2-week delay for resolution of study drug-related toxicities is allowed. A patient should be 
discontinued from the study if a greater than 2-week delay occurs. 
Week 1 Day 1 
On Week 1 Day 1 only, the required assessment may be omitted if an acceptable screening assessment was 
performed within 3 days prior to Week 1 Day 1. Week 1 Day 1 is dictated by the day the patient receives the first 
dose of entinostat.  
 
 
Assessment of Concomitant Treatments including medication use 


09/09/14 Version 10 
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 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Study Drug Administration, and Drug Accountability  
 
Methylation profile study 
 
Pharmacokinetic study:  
o 
Pilot phase and stage 1 patients 
 
Prior to the first dose of entinostat 
 
30 minutes after the first dose of entinostat 
o 
Stage 2 patients 
 
Prior to the first dose of entinostat 
 
Administration of entinostat as per appropriate dose level orally 
 
Week 3 Day 15 
 
Day 15 after the first dose of entinostat. 
 
 
Assessment of Concomitant Treatments including medication use 
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Study Drug Administration, and Drug Accountability  
 
Pharmacokinetic study:  
o 
Pilot phase and stage 1 patients 
 
Prior to the day 15 dose of entinostat 
 
30 minutes after the day 15 dose of entinostat 
o 
Stage 2 patients 
 
Prior to the day 15 dose of entinostat 
 
Administration of entinostat as per appropriate dose level orally 
 
Day 8, 22, and 29 after the first dose of entinostat 
 
 
Assessment of Concomitant Treatments including medication use 
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Study Drug Administration, and Drug Accountability  
 
Administration of entinostat as per appropriate dose level orally 
 
For the first cohort: 
 
Surgery day 
 
The definitive surgery (i.e. lumpectomy or mastectomy) should be performed within 6 days (preferably within 3 days) 
after the last dose of entinostat. 
 
 
Assessment of Concomitant Treatments including medication use 
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 


09/09/14 Version 10 
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 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Coagulation study: PT with INR and PTT 
 
Collection of tumor sample: Parrafin blocks and/or unstained slides 
 
Methylation profile study 
 
For the second cohort: 
 
The post-treatment biopsy (biopsy#2) day 
 
The post-treatment biopsy (biopsy#2) will be performed after a minimum of 15 days and a maximum of 29 days of 
treatment, but preferably close to 29 days. The post-treatment biopsy must take place within 6 days (preferably within 
3 days) after the last dose of entinostat. 
 
 
Assessment of Concomitant Treatments including medication use 
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Clinical tumor assessment by a breast examination including clinical tumor size, character, mobility, and 
location of the breast mass.  
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
 
Coagulation study: PT with INR and PTT 
 
Collection of tumor sample: Parrafin blocks and/or unstained slides 
 
Methylation profile study 
 
After the post-treatment biopsy, treatment will be continued until disease progression.  
 
Restaging scans will be performed every 6 weeks while on treatment.  
 
Follow up visits after the post-treatment biopsy will be every 4 weeks or at physician’s discretion. 
10.4 
End of Study (Treatment) / Withdrawal  
All patients must continue to be observed for 30 days after the last dose of study medication or until death; whichever 
occurs first. At the end of the study treatment, the following procedures should be performed within 4 weeks.  
 
 
Assessment of Concomitant Treatments including medication use 
 
Physical examination including examination of major body systems, height, body weight, ECOG 
performance status, and vital signs (temperature, blood pressure, heart rate, respiratory rate) 
 
Laboratory studies: CBC with diff; chemistries including sodium, potassium, chloride, bicarbonate, BUN, 
creatinine, glucose, total bilirubin, calcium, total protein, albumin, AST (SGOT), ALT (SGPT) , alkaline 
phosphatase, LDH, uric acid, phosphorus, and magnesium 
11. MEASUREMENT OF EFFECT 
For the purposes of this study, patients should be reevaluated weekly until the definitive surgery.   
 
11.1 Pathologic Response Determination 
 
Pathological response in the breast, and when appropriate, in the axilla will be assessed. 
 
 
11.1.1  Primary Endpoint Definitions for Ki67 and ER Expression 
 
For the primary endpoints, pathological response for Ki67 and ER expression will be assessed in the primary breast 
tumor. As per the updated guideline from the American Society of Clinical Oncology (ASCO) and Colleges of 


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American Pathologists (CAP) published in June 2010, the recommended ER positivity is at least 1% positive tumor 
nuclei in the tumor samples.  
 
 
Ki67: 
≥ 50% reduction in Ki67 after treatment with entinostat and anastrozole. 
 
ER: 
≥ 1% staining of ER is considered a positive result. 
 
11.1.2 
Pathologic Response 
 
 
Pathologic Complete Response (pCR): No viable invasive cancer in pathologic specimen, as determined by 
histological examination. We will report whether in situ disease only was present in the specimen versus no 
disease. We will also report for each woman whether she had a pCR in the breast only, in the lymph nodes 
only, or in both.  
 
Nodal Disease: We will report the number of women who had positive lymph node prior to starting study 
drugs, or at the time of surgery: 0, 1-3, 4-10, or >10 lymph nodes. 
 
11.2 Clinical Response Determination 
 
Clinical response in the breast will be evaluated, and when appropriate, in the axilla, infraclavicular, supraclavicular 
regions, and skin. At the time of study enrollment, patients will have a breast examination. Physical examination 
findings will be documented including clinical tumor size, character, mobility, and location of the breast mass. In 
addition, T4 features must be described. The patient will be examined by one of the treating investigators or his/her 
designee at each specified time point and at the time of the surgery. 
 
11.2.1 Endpoint Definitions for Clinical Response (UICC criteria) 
 
 
Complete response (cCR) in the breast on physical exam will be defined as the absence of any palpable 
abnormality: i.e., no skin or breast thickening, mass, or associated skin or nipple changes. CR will be 
recorded separately for the breast and axilla. 
 
Partial response (cPR) in the breast will be defined as a 50% or greater decrease in the product of 
biperpendicular diameters as measured with a ruler, compared with the prechemotherapy measurement. 
 
Stable disease (cSD) in the breast will be a defined as palpable disease which does not fit the definition of 
PR or PD. 
 
Progressive disease (cPD) will be defined as an increase in the product of biperpendicular diameters of 25% 
or greater compared to the original measurement. 
 
Inevaluable disease (cID) is defined as breast cancer that is not palpable in two dimensions. 
 
12. STATISTICAL CONSIDERATIONS 
 
12.1 
Study Design/Endpoints 
 
This is a Pilot and Phase II, single-arm, open-labeled, multi-center study of entinostat in combination with 
anastrozole/tamoxifen in women with triple negative breast cancer. The main objective of this study is to assess the 
safety and efficacy of the combination of entinostat and anastrozole/tamoxifen.  The primary endpoints, that we will 
use to evaluate the efficacy of the entinostat and anastrozole/tamoxifen, are the reduction in proliferative index (Ki67) 
and upregulation of ER after treatment with entinostat and anastrozole/tamoxifen therapy. We define patient’s 
response as at least 50% reduction in the Ki67 index AND greater than 1% ER-positivity OR pathologic complete 
response in the post-treatment surgical specimens. For the evaluation of these biological endpoints, the analysis will 
include ALL of the patients in both cohorts (the first cohort-operable stage I-II and the second cohort-unresectable 
locally advance/metastatic) as well as both anastrozole and tamoxifen groups.  
 
In the pilot phase, the classic cohort-of-3-study design will be used. The primary endpoint of this part of the study is to 
determine the recommended dose for the phase II part as well as to evaluate the safety of both combinations. The 
dose levels are defined below for both pre- and postmenopausal groups. For safety, each combination (entinostat 
and anastrozole as well as entinostat and tamoxifen) will be evaluated separately. For both groups (anastrozole and 
tamoxifen), the first cohort of 3 patients will be treated starting at dose level 1 (d1). If ≤ 1 patient experiences DLT, an 
additional 3 patients will be treated at this dose level (d1). If ≥ 2 patients in the initial cohort of 6 patients suffer DLT, 
then up to 6 additional patients will be enrolled at the dose level -1. The phase II part will continue with the highest 
dose level with < 2 out of 6 patients experiencing a DLT. Therefore, we will need 6-12 patients for each combination 


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in this part of the study. The 6 patients from the pilot phase of each combination that are treated with the 
recommended dose level for the phase II will be included in the primary endpoint analysis. 
 
The dose modification schemas are as follow for both postmenopausal and premenopausal women: 
 
 
 
 
 
 
 
 
or 
 
 
 
In the phase II part, the Simon’s two-stage design will be used for the study to ensure that the number of patients 
exposed to this new treatment is minimized. The analysis for this part of the study will include ALL of the patients in 
both cohorts (the first cohort-operable stage I-II and the second cohort-unresectable locally advance/metastatic) as 
well as both anastrozole and tamoxifen groups. The total sample size for the phase II part of the study is 32 with 12 
and 20 patients in the first and second stage respectively. The 6 patients in the pilot phase of each combination that 
are treated with the recommended dose level for phase II will be included in the first stage of the phase II part of the 
study. The study will be terminated, if there is no response among the first 12 patients. If ≥ 1 response in these 12 
patients is observed, then 20 more patients will be accrued. The combination will be considered promising, if ≥ 4 out 
of 32 patients have a response. This design yields 87% power. The probability of early stopping and declaring that 
this combination has no sufficient activity is 0.54, if the true success rate is 5% and 0.07 if the true response rate is 
20%. If the true proportion of patients with Ki67 reduction combined with ER up-regulation is 0.20, the probability of 
concluding that the drug has sufficient activity is 0.87 and 0.07 if the true proportion is 0.05. To allow about 10% 
inevaluability, the phase II trial will accrue 35 eligible patients.  Statistical analysis will be done using S-plus (TIBCO, 
version 8.0). All descriptive statistics will be reported. The multivariate analysis of variance or its non-parametric 
alternative will be applied to compare biomarkers’ expression levels. The 90% confidence intervals will be 
constructed for the observed proportions. All statistical tests will be two-sided, and done at the 0.05 level of 
significance. 
 
12.2 
Sample Size/Accrual Rate 
For the pilot phase, the study will require 12-24 patients to complete, allowing for 3-6 patients per dose level and 2 
predefined dose levels for 2 combination treatments (entinostat and anastrozole as well as entinostat and tamoxifen). 
In the phase II period, the study will require 12-35 patients to complete. However, we will include the 12 patients in 
the pilot phase that are treated with the recommended dose level of each combination for phase II in the primary 
endpoint analysis. Therefore, the total sample size for this study is 12-41 patients. At an anticipated accrual rate of 
15-20 patients/year, we anticipate that the phase II part will need to accrue patients for anywhere from 7 to 33 
months. 
NOTE: The maximum total sample size will most likely remain 41 patients. The only situation that this trial will require 
additional 6 patients is that there are ≥ 2 patients in the first cohort of 6 patients suffer DLT in dose level 1 in both 
combination treatments. As of November 2011, there were 3 patients enrolled in the pilot phase of this study. There is 
no DLT observed in this first 3 patients treated with entinostat and anastrozole. Furthermore, a phase II study of 
Dose Modification Schema for Postmenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Anastrozole 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
1 
Dose Modification Schema for Premenopausal Women 
Dose 
Level 
Dose 
Entinostat (mg) 
Tamoxifen 
(mg) daily 
-1 
3 mg weekly (on day 1, 8, 15, 22, and 29) 
20 
1 
5 mg weekly (on day 1, 8, 15, 22, and 29) 
20 


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entinostat and AIs including anastrozole was recently completed which demonstrated that this combination is safe 
and tolerable with the majority of AEs being only grade 1 or 247. Taken this together, it is highly unlikely that there will 
be ≥ 2 patients in the first cohort of 6 patients suffer DLT in dose level 1 in entinostat and anastrozole group. 
 
12.3 
Analysis of Primary Endpoints 
Pilot Phase 
 
To address the safety of both regimens, a maximum width 90% confidence interval for any grade 3 or higher toxicity 
will be approximately 30%. For 35 patients in this study, if the true unknown probability of a rare toxicity is 10%, the 
probability of observing 1 or more toxicities is 97%, and if the true toxicity rate is 5% then the probability of observing 
one or more rare toxicities is 83%. 
 
In the first cohort, because the study participants are awaiting a curative surgery, greater than 10% delay in surgery 
(more than 2 weeks) is unacceptable.  Thus, if ≥ 2 delays are observed in the first ten participants, the dose will be 
de-escalated to level -1.  If ≥ 2 additional delays are observed after dose modification, consideration will be made to 
further change in dose or schedule of study medication.   
 
Phase II 
 
Regarding the ER and Ki67 analysis, we will also include the evaluable patients from the pilot phase for this analysis. 
For the evaluation of these biological endpoints, the analysis will include ALL of the patients in both cohorts (the first 
cohort-operable stage I-II and the second cohort-unresectable locally advance/metastatic) as well as both 
anastrozole and tamoxifen groups. When the sample size is 41, a single-group repeated measures analysis of 
variance with a 0.05 significance level will have adequate above 80% power to detect a difference in average 
expression levels of ER and Ki67 between the baseline and after treatment values when an effect size is 0.1. With 
effect size of 0.125 and higher, power increases to 90%.  
 
The 95% confidence intervals will be constructed for the observed proportions. Exploratory data analysis and 
appropriate graphs will be used to decide whether data transformation (e.g. log or square-root) is necessary to assure 
an approximate normality.  All descriptive statistics will be reported for ER and Ki67 expression. We will use general 
linear model approach and/or its non parametric alternative, the Wilcoxon test, to assess whether there is any 
evidence of changes due to treatment. All statistical tests will be two-sided done at the 0.05 level of significance. 
Statistical analysis will be done using S-plus (TIBCO version 8.0). 
 
12.4 
Analysis of Secondary Endpoints 
Baseline and after treatment PR, HER2, EGFR, CK5/6, aromastase, tissue histone H3 and H4 acetylation will be 
treated as continuous variables. We may also use the multivariate analysis of variance to compare correlated 
biomarkers’ expression. Correlation between biomarkers will be estimated and tested. The repeated measures model 
approach will be also used. Categorical outcome data (e.g., number of proteins expressed) will be recorded, 
proportions will be estimated and compared using the Fisher’s exact test.  
 
Clinical and pathological response rate with the corresponding 90% or 95% CI will be estimated for all eligible 
patients. The possible association between response rate and dose level will also be assessed.    
 
 
12.5 
Analysis of Exploratory Endpoints 
12.5.1 
To correlate the levels of histone H3 and H4 acetylation with the changes in Ki67 and ER, the appropriate to 
the data coefficient of correlation will be calculated. With 35 eligible subjects on the study we will have above 
80% power to test the null hypothesis of some positive association against the research hypothesis of strong 
positive correlation, for instance, rho of 0.30 versus rho of at least 0.70. 
12.5.2 
To evaluate gene methylation profile, the outcomes of interest are gene methylation silencing and 
expression of candidate genes in tissue and in circulating DNA (ER-alpha, ER-beta, RAR-beta, cyclin D2, 
Twist, RASSF1A, and HIN-1) which are categorical and continuous respectively.  Measurements of the 
baseline and change in gene methylation profile will be measured.  The methylation silencing data for each 
gene will be tabulated and proportions will be estimated and compared using Fisher’s exact test.  For gene 
expression, exploratory plots, means and 95% confidence intervals will be calculated.  We will use t-tests 
and signed-rank tests to determine whether or not the data shows evidence of change from baseline. 


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12.5.3 
For the pharmacokinetic study, summary statistics (mean, standard deviation, median) and graphical 
techniques (boxplots, scatter-plots, histograms) will be used to characterize the behavior of 
pharmacodynamic (PD) and pharmacokinetic (PK) measurements. Regarding the exploratory endpoints of 
correlation between entinostat exposure and PD endpoints (i.e. acetylation of histone H3 and H4), A Mann-
Whitney U test and logistic regression will be used to evaluate the association between entinostat exposure 
and PD endpoints as well as the change in Ki67 and ER expression as a categorical variable (i.e. response 
or no response). The entinostat exposure parameters explored will be plasma concentrations.   
12.5.4 
For the global gene expression analysis, we will have tissue samples from between 12-41 women enrolled 
in this study.  Each of the tissue samples will be preprocessed using DASL assay from Illumina as described 
above.  The resulting CEL files will be imported and stored on the Gene Traffic database in the Biostatistics 
Core (Core 4).  Gene Traffic software will be used for quality control and identification of any experimental 
artifacts, including spatial patterns on chips.  GCRMA will be applied to the CEL file results using the “Affy” 
library within the Bioconductor suite using R statistical software.48 The GCRMA method summarizes probe 
set information to obtain one gene expression measurement per probe set on the array.  Significance 
analysis of microarrays (SAM) will then be applied to the gene expression data to find genes which are 
differentially expressed between before and after treatment tissues.49  The SAM statistic is similar to 
performing a two sample t-test for each gene under consideration, but it includes an adjustment to stabilize 
the coefficient of variation of the SAM statistic as a function of the standard deviation of the gene expression 
(across genes).  As part of the SAM approach, a permutation method will be used to determine a cutoff for 
significance.  Specifically, the permutation method takes the original data (the G x 6 matrix of expressions, 
where G is the number of genes) and permutes the data within each row of the expression matrix.  The SAM 
analysis is then repeated on the permuted data.  The distribution of the SAM statistics from the permuted 
analysis is called the “null” distribution, meaning that it is the distribution of SAM statistics we would expect 
to see if there were no relationship between gene expression and treatment (or, treatment modality).  We 
then compare the distribution of the SAM statistics based on original data to the null distribution and choose 
a cutoff such that our false-discovery rate is 5%.  That is, we choose a cutoff so that we would only expect 
that about 5% of the genes we claim to be differentially expressed would not be.   
 


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APPENDIX A: ECOG Performance Status Scale 
 
Grade 
Descriptions 
0 
Normal activity.  Fully active, able to carry on all pre-disease performance without restriction. 
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). 
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. 
3 
In bed >50% of the time.  Capable of only limited self-care, confined to bed or chair more than 50% 
of waking hours. 
4 
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  Totally confined to bed or 
chair. 
5 
Dead. 
 
 


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APPENDIX B: American Joint Committee on Cancer Staging (AJCC) 
 
T – Primary Tumor 
TX 
Primary tumor cannot be assessed 
T0 
No evidence of primary tumor 
Tis 
Carcinoma in situ 
Tis (DCIS) 
Ductal carcinoma in situ 
Tis (LCIS) 
Lobular carcinoma in situ 
Tis (Paget) 
Paget’s disease of the nipple with no tumor 
  Note:  Paget’s disease associated with a tumor is classified according to the size of the tumor. 
T1 
Tumor ≤ 2 cm in greatest dimension 
T1mic 
Microinvasion ≤ 0.1 cm in greatest dimension 
T1a 
Tumor > 0.1 cm but not > 0.5 cm in greatest dimension 
T1b 
Tumor > 0.5 cm but not > 1 cm in greatest dimension 
T1c 
Tumor > 1 cm but not > 2 cm in greatest dimension 
T2 
Tumor > 2 cm but not > 5 cm in greatest dimension 
T3 
Tumor > 5 cm in greatest dimension 
T4 
Tumor of any size with direct extension to 
(a) chest wall or 
(b) skin, only as described below 
T4a 
Extension to chest wall, not including pectoralis muscle 
T4b 
Edema (including peau d’orange” or ulceration of the skin of the breast, or satellite skin nodules 
confined to the same breast 
T4c 
Both T4a and T4b 
T4d 
Inflammatory carcinoma 
 
N – Regional lymph nodes 
NX 
Regional lymph nodes cannot be assessed (e.g., previously removed) 
N0 
No regional lymph node metastasis 
N1 
Metastasis in movable ipsilateral axillary lymph node(s) 
N2 
Metastases in ipsilateral axillary lymph nodes fixed or matted, or in clinically apparent ipsilateral 
internal mammary nodes in the absence of clinically evident axillary lymph node metastasis 
N2a 
Metastasis in ipsilateral axillary lumph nodes fixed to one another (matted) or to other structures 
N2b 
Metastasis only in clinically apparent ipsilateral internal mammary nodes and in the absence of 
clinically evident axillary lymph node metastasis 
N3 
Metastasis in ipsilateral infraclavicular lymph node(s), or in clinically apparent ipsilateral internal 
mammary lymph node(s) and in the presence of clinically evident axillary lymph node metastasis; 
or metastasis in ipsilateral supraclavicular lymph node(s) with or without axillary or internal 
mammary lymph node involvement 
N3a 
Metastasis in ipsilateral infraclavicular lymph node(s) and axillary lymph node(s) 
N3b 
Metastasis in ipsilateral internal mammary lymph node(s) and axillary lymph node(s) 
N3c 
Metastasis in ipsilateral supraclavicular lymph node(s) 
 


09/09/14 Version 10 
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APPENDIX B (Continued) 
 
pN – Regional lymph nodes 
pNX 
Regional lymph nodes cannot be assessed  (e.g., previously removed or not removed for 
pathologic study) 
pN0 
No regional lymph node metastasis histologically, no additional examination for isolated tumor cells 
pN0(i-) 
No regional lymph node metastasis histologically, negative IHC 
pN0(i+) 
No regional lymph node metastasis histologically, positive IHC, no IHC cluster > 0.2 mm 
pN0(mol-) 
No regional lymph node metastasis histologically, negative molecular findings (RT-PCR) 
pN0(mol+) 
No regional lymph node metastasis histologically, positive molecular findings (RT-PCR) 
pN1mi 
Micrometastasis (> 0.2 mm, none > 2.0 mm) 
pN1 
Mestastasis in one to three axillary lymph nodes and/or in internal mammary nodes with 
microscopic disease detected by sentinel lymph node dissection but not clinically apparent 
pN1a 
Mestastasis in one to three axillary lymph nodes 
pN1b 
Metastasis in internal mammary nodes with microscopic disease detected by sentinel lymph node 
dissection but not clinically apparent 
pN1c 
Mestastasis in one to three axillary lymph nodes and in internal mammary lymph nodes with 
microscopic disease detected by sentinel lymph node dissection but not clinically apparent 
pN2 
Metastasis in four to nine axillary lymph nodes, or in clinically apparent internal mammary lymph 
nodes in the absence of axillary lymph node metastasis 
pN2a 
Metastasis in four to nine axillary lymph nodes (at least one tumor deposit > 2.0 mm) 
pN2b 
Metastasis in clinically apparent internal mammary lymph nodes in the absence of axillary lymph 
node metastasis 
pN3 
Metastasis in 10 or more axillary lymph nodes, or in infraclavicular lymph nodes, or in clinically 
apparent ipsilateral internal mammary lymph nodes in the presence of one or more positive axillary 
lymph nodes; or in more than three axillary lymph nodes with clinically negative microscopic 
metastasis in internal mammary lymph nodes; or in ipsilateral supraclavicular lymph nodes 
pN3a 
Metastasis in 10 or more axillary lymph nodes (at least one tumor deposit > 2.0 mm), or metastasis 
to the infraclavicular lymph nodes 
pN3b 
Metastasis in clinically apparent ipsilateral internal mammary lymph nodes in the presence of one 
or more positive axillary lymph nodes; or in more than three axillary lymph nodes and in internal 
mammary lymph nodes with microscopic disease detected by sentinel lymph node dissection but 
not clinically apparent 
pN3c 
Metastasis in ipsilateral supraclavicular lymph nodes 
 
 
M – Distant metastasis 
MX 
Distant metastasis cannot be assessed 
M0 
No distant metastasis 
M1 
Distant metastasis 
 


09/09/14 Version 10 
48 
 
APPENDIX C: Study Drug Diary 
 
Study Drug Diary: Entinostat 
 
Please complete this diary every time you take your entinostat.  You should also use this diary to record any side 
effects that you experience and medications that you take other than your study drugs (entinostat and anastrozole or 
tamoxifen).  Please be sure to bring this diary with you to your next doctor’s visit. 
 
You will take entinostat once a week until you have your surgery (i.e. lumpectomy or mastectomy). Entinostat is a 
tablet either in light brown (1 mg) or intense yellow (5 mg). Entinostat should be taken with a meal after the first two or 
three bites.  
 
You are currently taking: 
 
 
3 mg of entinostat weekly 
 
 
5 mg of entinostat weekly 
 
Please contact your study doctor or nurse with any new complaints or symptoms that you have after starting to take 
entinostat.  If you have severe symptoms, your dose of entinostat may be changed or you may be told to stop taking 
entinostat.  Please do not make any changes in your entinostat dose without speaking with your study doctor or 
nurse.   
 
Dose 
# 
Due Date 
Due Time 
Date Taken 
Time Taken 
# of Pills 
Taken 
Comments 
(Side effects, complaints, other 
medications) 
1 
 
 
 
 
 
 
2 
 
 
 
 
 
 
3 
 
 
 
 
 
 
4 
 
 
 
 
 
 
5 
 
 
 
 
 
 
 
Note:  For any time that you did not take your entinostat (Example:  You forgot to take it), write the reason that you 
did not take it in the “Comments” field. 
 
 
Completed by: 
 
 
 
 
 
 
 
Date:    
 
 
 
 
Signature of Patient 
 
 
Reviewed by: 
 
 
 
 
 
 
 
Date:    
 
 
 
 
Signature of Study Staff 
 


09/09/14 Version 10 
49 
 
APPENDIX C (Continued) 
 
Study Drug Diary: Anastrozole or Tamoxifen 
 
Please complete this diary every time you take your anastrozole or tamoxifen.  You should also use this diary to 
record any side effects that you experience and medications that you take other than your entinostat and 
anastrozole/tamoxifen.  Please be sure to bring this diary with you to your next doctor’s visit. 
 
You will take one tablet of anastrozole (1 mg) or tamoxifen (20 mg) by mouth once a day started on day 4 after the 
first dose of entinostat. Anastrozole and tamoxifen are white tablets that can be taken with or without food. You 
should take anastrozole or tamoxifen at around the same time every day.  
 
Please contact your study doctor or nurse with any new complaints or symptoms that you have after starting to take 
anastrozole or tamoxifen.  If you have severe symptoms, your dose of anastrozole or tamoxifen may be changed or 
you may be told to stop taking anastrozole or tamoxifen.  Please do not make any changes in your anastrozole or 
tamoxifen dose without speaking with your study doctor or nurse.   
 
Dose 
# 
Due Date 
Due Time 
Date Taken 
Time Taken 
# of Pills 
Taken 
Comments 
(Side effects, complaints, other 
medications) 
1 
 
 
 
 
 
 
2 
 
 
 
 
 
 
3 
 
 
 
 
 
 
4 
 
 
 
 
 
 
5 
 
 
 
 
 
 
6 
 
 
 
 
 
 
7 
 
 
 
 
 
 
8 
 
 
 
 
 
 
9 
 
 
 
 
 
 
10 
 
 
 
 
 
 
11 
 
 
 
 
 
 
12 
 
 
 
 
 
 
13 
 
 
 
 
 
 
14 
 
 
 
 
 
 
15 
 
 
 
 
 
 
16 
 
 
 
 
 
 
17 
 
 
 
 
 
 
18 
 
 
 
 
 
 
19 
 
 
 
 
 
 
20 
 
 
 
 
 
 


09/09/14 Version 10 
50 
 
APPENDIX C (Continued) 
 
Study Drug Diary: Anastrozole or Tamoxifen 
 
Dose 
# 
Due Date 
Due Time 
Date Taken 
Time Taken 
# of Pills 
Taken 
Comments 
(Side effects, complaints, other 
medications) 
21 
 
 
 
 
 
 
22 
 
 
 
 
 
 
23 
 
 
 
 
 
 
24 
 
 
 
 
 
 
25 
 
 
 
 
 
 
26 
 
 
 
 
 
 
27 
 
 
 
 
 
 
28 
 
 
 
 
 
 
29 
 
 
 
 
 
 
30 
 
 
 
 
 
 
31 
 
 
 
 
 
 
32 
 
 
 
 
 
 
 
Note:  For any time that you did not take your anastrozole or tamoxifen (Example:  You forgot to take it), write the 
reason that you did not take it in the “Comments” field. 
 
 
Completed by: 
 
 
 
 
 
 
 
Date:    
 
 
 
 
Signature of Patient 
 
 
Reviewed by: 
 
 
 
 
 
 
 
Date:    
 
 
 
 
Signature of Study Staff