Clinical trial NCT00244933
Conditions: Breast Cancer
Keywords: stage IV breast cancer recurrent breast cancer
Sponsor: Barbara Ann Karmanos Cancer Institute
Start date: 2004-02-01; Completion date: 2009-10-01
Enrollment: 19 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
RATIONALE: Drugs used in chemotherapy, such as gemcitabine hydrochloride and genistein, work in different ways to stop the growth of tumor cells, either by killing the cells or by stopping them from dividing. Giving gemcitabine hydrochloride together with genistein may kill more tumor cells.

PURPOSE: This phase II trial is studying how well giving gemcitabine hydrochloride together with genistein works in treating women with stage IV breast cancer.

Detailed description:
OBJECTIVES:

Primary

* Determine the objective response rate in patients with stage IV breast cancer treated with gemcitabine hydrochloride and genistein.

Secondary

* Determine the duration of response and survival of patients treated with this regimen.
* Determine the time to disease progression in patients treated with this regimen.
* Determine the quantitative and qualitative toxic effects of this regimen in these patients.
* Correlate plasma genistein levels with response in patients treated with this regimen.

OUTLINE: Patients receive oral genistein once daily on days -7 to 1. Patients also receive gemcitabine hydrochloride IV on days 1 and 8 and oral genistein once daily on days 1-21. Courses repeat every 21 days in the absence of disease progression or unacceptable toxicity.

PROJECTED ACCRUAL: A total of 36 patients will be accrued for this study.

Arms: arm 1: Gemcitabine IV-1000mg/m2: Days 1 \& 8 every 21 days Novasoy Orally-100 mg 2 times/day for 7 days; 2 times/day on Days 1-21 every 21 days.
Interventions: intervention 1: genistein intervention 2: gemcitabine intervention 3: Tumor biopsy
Intervention descriptions: intervention 1: Novasoy Orally-100 mg 2 times/day for 7 days; 2 times/day on Days 1-21 every 21 days. intervention 2: Gemcitabine IV-1000mg/m2: Days 1 \& 8 every 21 days intervention 3: Biopsy of tumor prior to dose of genistein (Novasoy)

Protocol documents:
Prot_SAP_000.pdf: 
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Karmanos Cancer Institute 
Wayne State University 
 
Phase II trial of gemcitabine and genistein in metastatic breast cancer patients, 
with biomarker assays 
 
 
PRINCIPAL INVESTIGATOR: 
Amy M. Weise, D.O. 
  
KARMANOS CANCER INSTITUTE 
WAYNE STATE UNIVERSITY 
4100 John R, 4-HWCRC 
Detroit, MI  48201 
 
 
 
 
 
OFFICE:  (313) 576-8952 
 
 
 
 
 
FAX:        (313) 576-8767 
 
 
 
 
 
PAGER:   (313) 745-5111 #0585 
 
 
 
 
 
E-MAIL: weise@karmanos.org   
 
 
 
 
 
 
CO-PRINCIPAL INVESTIGATOR: 
Fazlul Sarkar, Ph.D. 
(Correlative Research)  
 
HWCRC, Room 715 
 
 
 
 
 
KARMANOS CANCER INSTITUTE 
 
 
 
 
 
WAYNE STATE UNIVERSITY 
 
 
 
 
 
4100 John R, 7-HWCRC 
 
 
 
 
 
Detroit, MI  48201 
 
 
 
 
 
OFFICE:  (313) 576-8327 
 
 
 
 
 
E-MAIL:  sarkar@karmanos.org
 
CO-INVESTIGATORS: 
Lawrence Flaherty, MD 
Omer Kucuk, MD 
Michael Simon, MD, MPH 
Pat LoRusso, DO 
Elaina Gartner, MD 
Zeina Nahleh, MD 
 
DATA MANAGER: 
 
 
Matthew Gretkiewicz 
CLINICAL TRIALS OFFICE (CTO) 
 
 
 
 
 
KARMANOS CANCER INSTITUTE 
 
 
 
 
 
HARPER PROFESSIONAL BUILDING, Room 711 
 
 
 
 
 
4160 JOHN R 
 
 
 
 
 
DETROIT, MI 48201 
 
 
 
 
 
TEL:  (313) 576-9369 
 
 
 
 
 
PAGER:  (313) 745-5111 #9067 
 
 
 
 
 
FAX:  (313) 576-8368 
 
 
 
 
 
E-MAIL:  gretkiem@karmanos.org 
 
BIOSTATISTICIAN: 
 
 
Judith Abrams, Ph.D. 
 
 
 
 
 
KARMANOS CANCER INSTITUTE 
 
 
 
 
 
4160 JOHN R 
 
 
 
 
 
DETROIT, MI 48201 
 
 
 
 
 
OFFICE:  (313) 576-8651 
 
 
 
 
 
FAX:  (313) 576-8656 
 
 
 
 
 
E-MAIL:  abramsj@karmanos.org 
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TABLE OF CONTENTS 
 
 
 
 
 
 
 
PAGES 
 
PRECIS 
 
 
 
 
 
 
 
 
 
3 
1.0 
OBJECTIVES  
 
 
 
 
 
 
 
4 
 
2.0  
BACKGROUND 
 
 
 
 
 
 
 
4 
3.0 
DRUG INFORMATION 
 
 
 
 
 
 
10 
4.0 
ELIGIBILITY CRITERIA 
 
 
 
 
 
 
12 
5.0 
TREATMENT PLAN  
 
 
 
 
 
 
14 
6.0 
TREATMENT MODIFICATIONS FOR TOXICITY,  AND 
 
 
15 
CRITERIA FOR DISCONTINUATION FROM STUDY 
 
7.0 
CRITERIA FOR RESPONSE EVALUATION AND TOXICITY REPORTING 17 
 
8.0 
SCHEDULED EVALUATIONS ON STUDY 
 
 
 
 
18 
9.0 
CORRELATIVE STUDIES:  INSTRUCTIONS  
 
 
 
19 
10.0 
REGISTRATION PROCEDURE 
 
 
 
 
 
21 
11.0 
REPORTING OF ADVERSE EVENTS  
 
 
 
 
22 
12.0 
DATA AND SAFETY MONITORING  
 
 
 
 
23 
13.0 
STATISTICAL CONSIDERATIONS 
 
 
 
 
 
23 
14.0 
BIBLIOGRAPHY 
 
 
 
 
 
 
 
25 
15.0 
APPENDICES:  
 
 
 
 
 
 
 
29 
 
 
APPENDIX I:  STUDY SCHEMA 
 
 
 
 
29 
 
 
 
APPENDIX II:  STUDY CALENDAR  
 
 
 
30 
 
 
APPENDIX III:  SWOG PERFORMANCE STATUS 
 
 
31 
 
 
APPENDIX IV:  NCI-CTEP COMMON TOXICITY CRITERIA 
32 
 
 
 
 
 
 
 
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Phase II trial of gemcitabine and genistein in metastatic breast cancer patients, with 
biomarker assays 
 
 
PRECIS 
 
 
 
Metastatic breast cancer remains an incurable disease.  Single agent conventional chemotherapy such as 
gemcitabine yields response rates in the 20-25 % response rates, with progression free survival durations of ~6 
months.  There is public and scientific interest in soy isoflavones such as genistein as a breast cancer prevention 
or treatment agent.  Recently, genistein has been shown to induce breast cancer growth inhibition and apoptosis 
in preclinical studies, in part by inhibiting Akt and NF-κB signal pathways.  When soy isoflavones are 
administered to humans at a well tolerated dose, NF-κB signaling is inhibited in human peripheral lymphocytes, 
demonstrating bioactivity at these doses.  Moreover, combinations of gemcitabine and genistein have 
demonstrated at least additive inhibitory effects in vitro in carcinoma cells.  Although there is significant lay and 
scientific interest in genistein’s effects in human breast cancer, there are no peer-reviewed published data 
regarding the in vivo effects of genistein in patients with breast cancer.  Thus, the major rationale is that the 
addition of genistein may increase the effectiveness without added toxicity.  Therefore, we propose to combine 
gemcitabine 1,000 mg/m2 IV days 1 and 8, with genistein 100mg PO BID days 1-21, with cycles q21 days, for 
first or second line treatment of patients with metastatic breast cancer.  Prior to cycle 1, day 1, genistein will be 
administered alone for seven days for the collection of baseline plasma levels, and for the collection of 
accessible tumor tissue for molecular biomarker studies.  Along with standard dose modification guidelines for 
toxicity, prospective safety monitoring and stopping rules are incorporated into the study design insure the 
safety of this protocol.  The primary objective is to estimate the objective response rate.  Secondary clinical 
objectives are to obtain data on the quality of the responses, overall survival, and the toxicity and tolerance to 
this combination.  Secondary translational objectives are to assess plasma genistein levels and explore 
associations with responses, and to explore the effect of genistein alone on tumor biomarkers in order to better 
understand its mechanisms. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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1.0 
OBJECTIVES 
 
1.1 
Primary Objective 
 
1.1.1 
To estimate the objective response rate in patients with metastatic breast cancer treated with the 
combination of gemcitabine and genistein as first or second line therapy. 
 
1.2 
Secondary Objectives
 
1.2.1 
To obtain data on duration of response, time to disease progression, and duration of survival. 
 
1.2.2 
To estimate the quantitative and qualitative toxicities of this regimen. 
 
1.2.3 
To assay the plasma genistein levels and explore associations with responses. 
 
1.2.4 
Explore the in vivo effects of genistein in human breast cancer tissue biomarkers (Ki67, 
TUNEL, phosphorylated-Akt, and NF-κB), and by cDNA microarray analysis. 
 
 
2.0 
BACKGROUND
 
2.1 
Metastatic Breast Adenocarcinoma 
 
Approximately 190,000 new cases of breast cancer are diagnosed each year in the USA (1).  Although 
early stage disease is potentially curable through a multimodality approach involving surgery, 
chemotherapy and radiation, metastatic breast cancer remains a largely incurable disease accounting for 
41,000 deaths each year (1, 2). For the vast majority of these patients, metastatic breast cancer is an 
incurable disease with a median survival of only 2 to 3 years after diagnosis (2). Long-term remissions 
with combination chemotherapy have been reported to occur in only 2-3% of patients (3).  Historically, 
the main objective of treatment in the metastatic setting was the improvement in the quality of life. 
Recently, several trials have demonstrated a prolongation in median survival of women with metastatic 
breast cancer with effective systemic therapy (4, 5).  These trials demonstrate the importance of 
introducing novel active agents or combinations of agents that could potentially improve survival as 
well as palliate symptoms.  
 
2.2 
Gemcitabine
 
Gemcitabine (2,2’-difluorodeoxcytidine, dFdC) is a nucleoside analog initially synthesized as a potential 
anti-viral drug and had excellent activity against both RNA and DNA viruses using cell culture assays.  
Despite early encouraging in vitro results, the in vivo therapeutic index was disappointingly low and the 
drug was not developed further.  Concurrent evaluation of the cytotoxic activity of the compound 
demonstrated that gemcitabine was a potent and specific deoxycytidine analog with an acceptable in 
vivo therapeutic index.  The mechanism of action and metabolism of gemcitabine have been well 
characterized. Gemcitabine inhibits the synthesis of DNA by at least two mechanisms.  Gemcitabine is 
phosphorylated by deoxycytidine kinase to dFdC-5'-monophosphate (dFdCMP).  Difluorodeoxyuridine 
is a product of gemcitabine deaminations and is inactive.  dFdCMP is further metabolized to dFdC5'-
diphosphate (dFdCDP) and dFdC-5'triphosphate (dFdCTP), which when incorporated into DNA, results 
in masked chain termination.  In comparison to Ara-C incorporation into DNA, dFdCTP is less readily 
excised from DNA by DNA exonuclease.  Thus, dFdCTP accumulates intracellularly to a greater degree 
than Ara-C-CTP, which may account, at least in part, for its different spectrum of pre-clinical and 
clinical activity.  In addition, dFdCDP inhibits ribonucleotide reductase resulting in reduced formation 
of deoxyribonucleotides (6).  Other intracellular effects of gemcitabine include stimulation of 
deoxycytidine kinase and inhibition of deoxycytidine monophosphate deaminase (7). 
 
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Of particular importance is the fact that the therapeutic effects of gemcitabine at the maximum tolerated 
dose level are dependent on the administration schedule. The drug was injected intraperitoneally to mice 
in various schedules at equitoxic maximum tolerated dose levels, resulting in a reversible weight loss 
that varied between 5% and 15%. Generally, it was found that treatment with 120 mg/kg gemcitabine, 
injected four times at 3-day intervals, was more effective than the schedules of daily (five times 2.5 to 
3.5 mg/kg) or weekly (two times 240 mg/kg) injections (8).  Gemcitabine, but not cytosine arabinoside 
(Ara-C), had a broad spectrum of anti-tumor activity against seven different types of murine solid 
tumors. The activity of gemcitabine was also schedule dependent (9).  Additional experiments were 
performed on normal mice bearing the colon 26-10 murine colon carcinoma. The effect of a continuous 
intravenous infusion system was investigated by giving two injections of 15 mg/kg gemcitabine over 24 
hours at a 7-day interval. Interestingly, the efficacy of treatment increased dramatically with this 
infusion schedule, producing complete remissions in most tumors (8). 
 
Other investigators have shown that the 3-day interval schedule also was active in human pancreas and 
lung carcinoma xenografts (8).  Gemcitabine was tested against 12 human carcinoma xenografts. When 
given on an every 3 day x 4 schedule, the following percent inhibitions (at maximally tolerated doses 
(MTD); MTD/2) in tumor growth were seen: MX-1 mammary (93%; 80%), CX-1 colon (92%; 82%), 
HC-1 colon (96%; 92%), GC3 colon (98%; 94%), VRC5 colon (99%; 100%), LX-1 lung (76%; 61%), 
CALU-6 lung (75%; 38%), NCI-H460 lung (45%; 46%), HS766T pancreatic (73%; not tested), PaCa-2 
pancreatic (69%; 40%), PANC-1 pancreatic (70%; 60%), and BxPC-3 pancreatic (9%; 19%). In 
contrast, only the LX-1 lung carcinoma xenograft was responsive to Ara-C treatment, which inhibited 
tumor growth by a marginal 62%. Thus, like its activity against murine solid tumors, gemcitabine has 
excellent anti-tumor activity against a broad spectrum of human solid tumors (9).   
 
Following the demonstration of broad-spectrum cytotoxic activity in pre-clinical models, Phase I human 
studies for gemcitabine began in September 1987 (10).  Doses ranging from 10 to 1,000 mg/m2 were 
administered over 30 minutes weekly times 3 weeks every 4 weeks. The maximum-tolerated dose was 
790 mg/m2. The dose-limiting toxicity was myelosuppression, with thrombocytopenia and anemia 
quantitatively more important than granulocytopenia. Non-hematologic toxicity was minimal. The 
maximum dFdC plasma concentration, reached after 15 minutes of infusion, was proportional to the 
total dose administered. Elimination, due mainly to deamination, was rapid (terminal half-life (t1/2), 8.0 
minutes) and dose independent. The deamination product 2', 2’-difluorodeoxyuridine (dFdU) was 
eliminated with biphasic kinetics characterized by a long terminal phase (t1/2, 14 hours); it was the sole 
metabolite detected in urine. The plasma and cellular pharmacology of gemcitabine was studied during a 
Phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC 
dose, which ranged between 53 and 1,000 mg/m2 per 30 min. The cellular pharmacokinetics of an active 
metabolite, dFdC 5'-triphosphate (dFdCTP) was determined in mononuclear cells of 22 patients by 
anion-exchange high-pressure liquid chromatography. The rate of dFdCTP accumulation and the peak 
cellular concentration were highest at a dose rate of 350 mg/m2 per 30 min, during which steady-state 
dFdC levels of 15-20 µM were in plasma.  A comparison of patients infused with 800 mg/m2 over 60 
min. with those receiving the same dose over 30 min. demonstrated that the dFdC steady-state 
concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were 
similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that 
observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by 
mononuclear cells incubated in vitro was maximal at 10-15 µM dFdC. These studies suggest that the 
ability of mononuclear cells to use dFdC for triphosphate formation is saturable (11). 
 
A Phase I study utilizing twice-weekly injections was conducted in 50 eligible and evaluable patients.  
Twenty-nine patients received drug by 30-minute infusion at doses of 5-90 mg/m2, and 22 patients by 5-
minute bolus at 30-150 mg/m2. The primary dose limiting toxicities were marrow suppression and flu-
like symptomatology. Thrombocytopenia was dose limiting at 75 mg/m2 on the infusion schedule and 
150 mg/m2 on the 5-minute schedule. Flu-like symptoms with fever rigors and malaise occurred the day 
of injection in many patients (12).  In another Phase I study, patients received gemcitabine at 
therapeutically active doses (> 875 mg/m2/week x 3 every 28 days) (13).  Except for one patient, all 
were given gemcitabine doses exceeding 1,000 mg/m2 (1 patient at 875, 3 at 1,095 and 11 at 1,370 
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mg/m2) for a total of 50 delivered courses. Dose-limiting hematologic toxicity was found at 1,370 
mg/m2/wk as underscored by the higher number of toxic treatment delays requiring subsequent dose 
attenuation in 6 of 11 patients. Toxicity was mild and easily managed. WHO grade 2-3 toxicity included 
leukopenia (53%), thrombocytopenia (20%), anemia (53%), AST/ALT rises (27%), emesis (40%) and 
fever (grade 2 only) (60%).  An integrated database of toxicity was also analyzed according to starting 
dose (800, 1000 or 1250 mg/m2) in a subset of 665 chemo-naive patients to see whether an increased 
dose resulted in increased toxicity. In general, only small, clinically insignificant differences in toxicity 
were seen between the three dose groups. Although neutropenia appeared to increase as starting dose 
increased (grade 3 or 4, 19.4%, 23.2%, 28.3%, respectively), this was not associated with an increased 
incidence of infection. In some cases, toxicity decreased with increasing dose but this may have been 
because of imbalances between the patient groups. These findings indicate that not only is gemcitabine 
well tolerated, but also the use of higher doses may be possible (13) 
 
2.2 
Gemcitabine In Breast Cancer 
 
Table 1.  Phase II trials of single agent gemcitabine in advanced breast cancer 
 
Author
Year
N
Resp. 
Eval. 
Pts.
% prior 
chemo
% prior 
chemo. 
for met. 
disease
No. prior 
chemo. 
treatments 
for met. 
disease 
% with 
prior 
anthra.
mg/m
2 
dose and 
schedule mg/m
2/week
Response 
rate
% grade 4 
neutropenia % FN (3)
Carmichael 
1995 44 
40 
65% 
43% 
0 - 1 
39% 
800 q wk x 
3, q 4 wks
600 
25% 
7% 
2% 
Brodowicz(1) 
1998 24 
 
100% 
 
2 - 3 
100%
1250 q wk 
x 3, q 4 
wks 
937.5 
12% 
 
 
Possinger 
1999 42 
42 
24% 
0% 
0 
 
1000 q wk 
x 3, q 4 
wks 
750 
14% 
5% 
0% 
Spielman(2) 
2001 47 
41 
100% 100% 
1 - 2 
100%
1200 q wk 
x 3, q 4 
wks 
900 
29% 
4% 
0% 
Blackstein 
2002 39 
35 
49% 
0% 
0 
 
1200 q wk 
x 3, q 4 
wks 
900 
37% 
0% 
0% 
Proposed trial 
 
 
 
 
 
0-1 
 
1200 q wk 
x 2, q 3 
weeks 
800 
 
 
 
       ____________________________________ 
1. 
Abstract 
2. 
All patients had a prior response to the anthracycline for metastatic disease 
3. 
Febrile neutropenia 
 
Five Phase II studies have evaluated the activity of gemcitabine in metastatic breast cancer (Table 1). In 
the first study, Carmichael et al. reported 44 patients with advanced disease were given a dose of 800 
mg/m2 on days 1, 8, and 15 every 28 days (14).  Twenty-six patients had received prior chemotherapy 
either in the adjuvant (7 patients) or metastatic setting (19 patients). The mean administered dose of 
gemcitabine was 725 mg/m2/injection, or 543 mg/m2/week. The response rate was 25%.  In the second 
study Brodowicz preliminarily reported a response rate of only 12%, but all patients had prior 
chemotherapy and gemcitabine was third or fourth line chemotherapy for metastatic disease (15).  In the 
fourth study, only patients with previous metastatic disease responsive to anthracycline-based 
chemotherapy were enrolled (16).  Forty-seven patients were treated with gemcitabine at a dose of 1,200 
mg/m2 on days 1,8 and 15 every 28 days, with a delivered dose of 754 mg/m2/week.   The median time 
to progression of the responders and the median survival of all the patients were 8.1 and 18.6 months, 
respectively. The response rate was 29% of which 11% were complete responses.  In the fifth study, 
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Blackstein reported 39 patients with advanced breast cancer of whom 21 had received prior adjuvant 
chemotherapy were treated with gemcitabine 1,200 mg/m2 on days 1, 8 and 15 every 28 days (17), with 
a delivered dose of 1,053 mg/m2/week.   The response rate in this study was 37% of which 4 were 
complete responses.  In comparison, the response rates in anthracycline-resistant disease reported with 
paclitaxel (18) and docetaxel (19) were 6-29% and 30-41%, respectively. In conclusion gemcitabine 
administered at a dose of 800-1,250 mg/m2 on days 1 and 8 every 21 days, or days 1,8, and 15 every 28 
days is tolerable and active in metastatic breast cancer.  Thus, gemcitabine appears to be a very 
reasonable single agent for patients with metastatic breast disease.   
 
The main toxicity of gemcitabine is myelosuppression.  However, in all these studies, in which 
gemcitabine is administered at an intended dose of 600 – 900 mg/m2/wk, the incidence of grade 4 
neutropenia was < 7%, with rare instances of febrile neutropenia (Table 1).   Therefore, it is reasonable 
to propose gemcitabine at a dose of 1,000 mg/m2 on days 1 and 8 every 21 days, which would yield a 
dose of 800 mg/m2/wk, which is well within the dose range of achieving clinical responses dosing that 
produces a very low incidence of neutropenia or febrile neutropenia.   
 
 
2.3 
 Aberrant Akt and NF-κB signaling in human breast cancer 
 
2.3.1   Akt pathway in breast cancer 
 
Akt is an oncogenic serine-threonine kinase that is significantly activated by the estrogen receptor (ER) 
and breast cancer related receptor tyrosine kinases (RTKs) such as EGFR, Her-2, insulin receptor (IR) 
and the insulin like growth factor-1 receptor (IGF-1R).  We and others have shown that Akt1 is 
overexpressed or activated in the majority of breast cancer cell lines and tumors (20, 21).  Roth et al. has 
reported that Akt1 and Akt3 is overexpressed in most breast cancer cell lines and in the majority of 
primary breast cancer samples, especially in ER negative tumors (20).  Thirty-eight percent of breast 
cancers have elevated Akt1 kinase activity or expression of phosphorylated Akt (pAkt) by 
immunohistochemistry (IHC) (21).  Ahmad et al. has reported that Akt1 levels are elevated in a panel of 
human breast cancer cell lines, and that MCF-7 cells stably overexpressing WT-Akt1 exhibited 
increased anchorage independent growth in response to serum, IGF-1, or estrogen (22).  Heregulin and 
amplified Her-2 may both activate Akt in human breast cancer cell lines (23, 24).  Recent in vitro data 
also indicate that Akt may augment ERα signaling by specific phosphorylation of ERα at Ser167.  
Stable overexpression of Akt1 in MCF-7 cells confers ligand independent activation and increased 
transactivation by ERα, and decreases growth inhibition and apoptosis in response to tamoxifen.  Bcl-2 
is also significantly induced in these cells (25).   A recent multivariate analysis of a cohort of tamoxifen 
and/or goserelin treated breast cancer showed that expression of pAkt in tumors was an independent 
predictor of distant metastatic relapse (26).  These data together suggest that Akt plays a mechanistic or 
predictive role in tamoxifen resistance, and that inhibition of Akt may be therapeutic in breast cancer. 
 
 
2.3.2 
NF-κB pathway in breast cancer  
 
The nuclear factor kappa B (NF-κB) transcription factor is often activated in tumor cells. Growth factors 
such as the epidermal growth factor (EGF), cytokines and mitogens activate the phosphatidyinositol 3-
kinase, protein kinase C and subsequently NF-κB (27).  Activation of NF-κB by Akt may explain the 
pro-transforming effects of Akt.  When the pathway is unstimulated NF-κB is sequestered in the cytosol 
of cells by the inhibitory I-κB protein.  NF-κB activity is induced by Akt via activation of a kinase, IKK, 
a direct Akt substrate, which then phosophorylates I-κB at two conserved serines in the N-terminal 
domain, leading to its ubiquitin mediated degradation (28, 29).  This releases NF-κB to translocate into 
the nucleus where it transactivates promoters of genes involved with cell cycle progression, such as 
cyclin D1 (30) or survival function such as cellular inhibitors of apoptosis (CIAPs) (31).   
 
NF-κB is constitutively elevated in human breast cancer cell lines compared to untransformed breast 
cells, and its inhibition causes apoptosis (32, 33).  Her-2 activation of NF-κB also occurs through Akt–
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IKK mediated degradation of IKB (34).  NF-κB expression is also elevated in the majority of human 
breast cancer tissues (33, 35).   Furthermore NF-κB has been shown to be low in the cytosol but elevated 
in the nucleus of breast cancer cells indicating that NF-κB is not only overexpressed but also is 
functional (35).  The role of NF-κB in anti-apoptosis (36, 37), invasion (38), cell cycle regulation (39) 
and growth of cells has been clearly demonstrated in breast cancer.  In breast cancer cells NF-κB has 
been shown to upregulate secretion of urokinase-type plasminogen activator (38) and transcription of 
matrix metalloproteinase (40) both of which are promote cell invasion and metastasis.  NF-κB has also 
been shown to disturb the cell cycle regulation in breast cancer cells through increased cyclin D1 
transcription and retinoblastoma phosphorylation (39).  Hence, NF-κB is a molecular target in breast and 
other cancers, in its own right, as numerous drugs are being developed which antagonize NF-κB activity 
(41). 
 
NF-κB also appears to have a role in chemoresistance.  Inhibition of NF-κB via adenoviral delivery of I-
ĸB (42), or inhibition of proteasome-mediated degradation of I-ĸB increases apoptosis and 
chemosensitizes cancer cell lines (43).  Introduction of the I-ĸB vector into breast cancer cell lines 
sensitizes these cells to the effects of paclitaxel (44).  In a preclinical model, non-small cell lung cancer 
cells treated with a plasmid expressing I-ĸB resulted in increased apoptosis with gemcitabine as 
compared to the cells not treated with the vector (45).  On the other hand, introduction of the NF-κB 
gene into cell lines lacking the NF-κB overexpression resulted in resistance to gemcitabine (46).  Thus, 
inhibition of NF-κB activity may sensitize cancer cells to gemcitabine chemotherapy.  
 
 
2.4  
Genistein  
 
Flavonoids compromise the most common group of plant polyphenols.  More than 5,000 different 
flavonoids have been described and classified based on chemical structure into six subgroups (47).  
Genistein (4,5,7-trihydroxyisoflavone) is a naturally occurring flavonoid of the isoflavone subgroup 
present in soybeans (47).  Genistein containing nutritional supplements have become popular in the 
public domain, with claims for potential breast cancer prevention or treatment, such that patients will 
seek administration of these without prospective vigorous clinical trial data.  However, there is 
accumulating preclinical data that indicate that genistein has potential for the treatment or prevention of 
breast cancer, and suggest that careful clinical studies are warranted. 
 
Genistein has been shown to elicit molecular changes that result in inhibition of cell growth and 
induction of apoptosis in different cancer cell lines. In the squamous cell cancer cell lines, genistein can 
induce cell cycle arrest through upregulation of p21waf1 and downregulation of cyclin B1 (48).  
Genistein also induces apoptosis via upregulation of bax and downregulation of Bcl-2 gene expression 
(48).  Furthermore, it downregulates the expression of matrix metalloproteinase 2 and 9 (48).  In 
testicular cancer cell lines, genistein can induce apoptosis through activation of caspase-3 protease (49).  
In colon cancer cell lines, genistein can induce apoptosis through topo-isomerase II-mediated DNA 
breakage (50).  In the breast cancer cell lines, genistein has been shown to block cells at the G2M phase 
(51).  Genistein also can induce apoptosis through modulation of ERK phosphorylation and c-fos 
expression in breast cancer cell lines (52).   
 
Recent evidence indicates that genistein is also a potent inhibitor of Akt and NF-κB pathways.  Our 
laboratory has shown that genistein may facilitate apoptosis in cancer cells by inhibiting several critical 
intracellular survival pathways, including the inhibition of the PI3-kinase – Akt dependent signaling and 
NF-κB activation (48, 53).  Genistein has also been shown to inhibit NF-κB activation in prostate cancer 
(53) and head and neck squamous cell cancer cell lines (48). 
 
Several clinical studies have been done with soy products or supplements.  We have shown that 
Novasoy, which contains ~40% genistein, inhibits NF-κB activation in humans without toxicity.  In a 
clinical study involving six healthy male volunteers, 50 mg of genistein administered twice daily for 
three weeks resulted in inhibition of NF-κB activation by TNF-α in peripheral lymphocytes.  The 
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volunteers experienced no side effects (54).  Moreover, a phase II clinical study at Wayne State 
University has been completed in which 41 patients with prostate cancer received Novasoy at 100 mg 
BID for six months.  Overall a decrease in PSA was noted, without any side effects (Hussein et al., 
manuscript submitted) (55).  Recently, a limited, preliminary phase I clinical study with 
pharmacokinetics was reported in male volunteers with a soy supplement similar in content to Novasoy.  
Fifteen healthy volunteers were given single doses at 1, 2, 4, 8 or 16 mg/kg, with three patients at each 
level.   At 1 and 2 mg/kg doses levels (equivalent to 70 and 140mg for a 70kg man), no clinical or 
laboratory toxicity was observed.   At 4, 8 and 16 mg/kg (~280, 560, and 1120 mg doses, respectively), 
there were 7 cases of grade 1 - 2 adverse events, including 3 episodes of hypophosphatemia judged to be 
“clinically insignificant,” one pedal edema, one loss of appetite, one abdominal tenderness, and one 
leukopenia (grade 2 at 16 mg/kg).  Mean peak plasma total genistein levels in three men receiving 
8mg/kg (~560 mg) was ~ 20 µM, with a pseudo t1/2 of ~9.2 h, and the major route of elimination through 
the urine (56).  Thus, safe, biologically active does of genistein may prevent or treat breast cancer by not 
only inhibiting Akt and its downstream effector, NF-κB, but by increasing the efficacy of chemotherapy 
such as genistein.  Moreover, monitoring NF-κB activity in peripheral blood mononuclear cells 
(PBMNC) may serve as an surrogate marker of genistein activity in clinical trials.   
 
 
2.5 Rationale of the study 
 
2.5.1 Clinical Rationale 
 
Systemic chemotherapy can palliate symptoms as well as prolong survival of patients with metastatic 
breast cancer (4, 5).    Introduction of novel agents in this setting is important in order to further improve 
the clinical outcome of patients.  Gemcitabine at a dose of 800-1200 mg/m2 has significant activity 
comparable to the taxanes as second line agent in breast cancer (14, 17, 57).  The toxicity of gemcitabine 
is mainly myelosuppression.  Dose limiting myelosuppression occurs in about 10% of patients treated 
with gemcitabine (16).  However, in the Phase II trials in metastatic breast cancer no treatment related 
mortality was observed (16).  Genistein has garnered increasing scientific interest as a breast 
chemoprevention agent.  Genistein may inhibit several molecular targets that are relevant to human 
breast cancer, and may modulate sensitivity to gemcitabine.  These include Akt and NF-κB, which are 
overexpressed and/or activated in the majority of breast cancer cell lines and primary tumors (39, 58).  
Overexpression of the NF-κB in cancer cell lines results in resistance to gemcitabine (45).  Inhibition of 
NF-κB can sensitize cells to the effects of chemotherapeutic agents (44), including gemcitabine (46).  
Genistein is an inhibitor of the NF-κB pathway as demonstrated in healthy volunteers (54) as well as in 
cancer cell lines (48, 59).  Genistein at doses proposed in this study has no side effects (Hussein et al., 
manuscript submitted) (55).  Thus, there is increasing evidence that genistein may have 
chemoprevention, or chemotherapeutic applications, especially in combination with other cytotoxic 
chemotherapies such as gemcitabine.  Further clinical studies with genistein are clearly indicated.   We 
hypothesize that combining genistein and gemcitabine in patients with metastatic breast cancer could 
improve the therapeutic effectiveness of gemcitabine through decreasing chemo-resistance via 
modulation of the Akt and NF-κB pathways without added toxicity.  
 
 
 
 
 
2.5.2 Translational Rationale 
 
The two general translational aims are to: 1.  Determine if higher plasma levels of genistein are 
associated with clinical responses to the combined treatment; and 2.  Explore effects of genistein alone 
on in vivo tumor biology.   Although there have been numerous recent reports of the in vitro effects of 
genistein on human cancer cell lines (reviewed in Section 2.5), there are no studies that have 
corroborated or even explored the in vivo effects in human tumor specimens.  Therefore, in patients who 
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have easily accessible tumor and are willing to undergo biopsies before and after seven days of 
genistein, we will perform studies to determine if genistein inhibits cell cycle, induces apoptosis, and 
inhibits Akt and NF-κB activation as would be predicted from preclinical studies.  Further, we will 
explore the effects on other potential biomarkers through cDNA microarray analysis when sufficient 
cDNA can be prepared from paired specimens.  This will provide novel insights and a better 
understanding on the mechanisms of genistein effects on malignant breast cancer cells. 
 
 
3.0  
DRUG INFORMATION
 
3.1 Gemcitabine (Gemzar)
 
Chemistry: Gemcitabine HCl is 2-deoxy-2, 2-difluorocytidine monohydrochloride (beta isomer). 
 
Clinical Pharmacology: Gemcitabine pharmacokinetics are linear and are described by a 2-
compartment model.  Population pharmacokinetic analyses of combined single-and multiple-dose 
studies showed that the volume of distribution of gemcitabine was significantly influenced by duration 
of infusion and gender.  Clearance was affected by age and gender.  Differences in either clearance of 
volume of distribution based on patient characteristics or the duration of infusion; result in changes in 
half-life and plasma concentrations. 
Gemcitabine half-life for short infusions ranged from 32-94 minutes, and the value for long infusions 
varied from 245-638 minutes, depending on age and gender, reflecting a greatly increased volume of 
distribution with longer infusions.  The lower clearance in women and the elderly result in higher 
concentrations of gemcitabine for any given dose.  The volume of distribution was increased with 
infusion length.  Volume of distribution of gemcitabine was 50 L/m2 following infusions lasting <70 
minutes, indicating that gemcitabine, after short infusions, is not extensively distributed into tissues.  
For long infusions, the volume of distribution rose to 370 L/m2, reflecting slow equilibration of 
gemcitabine within the tissue compartment.  The maximum plasma concentrations of dFdU (inactive 
metabolite) were achieved up to 30 minutes after discontinuation of infusions, and the metabolite is 
excreted in urine without undergoing further biotransformation.  The metabolite did not accumulate with 
weekly dosing, but its elimination is dependent on renal excretion, and could accumulate with decreased 
renal function.  The effects of significant renal or hepatic insufficiency on the disposition of gemcitabine 
have not been assessed. 
 
Human Toxicity:  Hematologic:  Myelosuppression is the dose-limiting toxicity with gemcitabine, but 
<1% of patients discontinued therapy for anemia, leukopenia, or thrombocytopenia.  Red blood cell 
transfusions were required by 19% of patients.  The incidence of sepsis was less than 1%.  Petechiae or 
mild blood loss (hemorrhage), from any cause, were reported in 16% of patients; less than 1% of 
patients required platelet transfusions.  Patients should be monitored for myelosuppression during 
gemcitabine therapy and dosage modified or suspended according to the degree of hematologic therapy 
(see Section 8.0).  Gastrointestinal:  Nausea and vomiting were commonly reported (69%) but were 
usually mild to moderate.  Severe nausea and vomiting (WHO grade 3/4) occurred in <15% of patients.  
Diarrhea was reported by 19% of patients, and stomatitis by 11% of patients.   Hepatic:   gemcitabine 
was associated with transient elevations of serum transaminases in approximately two-thirds of patients, 
but there was no evidence of increasing hepatic toxicity with either longer duration of exposure to 
gemcitabine or with greater total cumulative dose.  Renal:  Mild proteinuria and hematuria were 
commonly reported.  Clinical findings consistent with the hemolytic-uremic syndrome (HUS) were 
reported in 6/24 patients (0.25%) receiving gemcitabine in clinical trials.  Four patients developed HUS 
on gemcitabine therapy, two immediately post-therapy.  Renal failure may not be reversible, even with 
discontinuation of therapy, and dialysis may be required.  Fever:  The overall incidence of fever was 
41%.  This is in contrast to the incidence of infection (16%) and indicates that gemcitabine may cause 
fever in the absence of clinical infection.  Fever was frequently associated with other flu-like symptoms 
and was usually mild and clinically manageable.  Rash: Rash was reported in 30% of patients.  The rash 
was typically a macular or finely granular maculopapular pruritic eruption of mild-to-moderate severity, 
involving the trunk and extremities.  Pruritus was reported for 13% of patients.  Pulmonary:  Dyspnea 
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was reported in 23% of patients, severe dyspnea in 3%.  Dyspnea may be due to underlying disease, 
such as lung cancer (40% of study population) or pulmonary manifestations of other malignancies.  
Dyspnea was occasionally accompanied by bronchospasm (<2% of patients).  Rare reports of 
parenchymal lung toxicity consistent with drug-induced pneumonitis have been associated with the use 
of gemcitabine.  Edema:  Edema (13%), peripheral edema (20%), and generalized edema (<1%) were 
reported.  Less than 1% of patients discontinued due to edema.  Flu-like Symptoms:   "Flu syndrome" 
was reported for 19% of patients.  Individual symptoms of fever, asthenia, anorexia, headache, cough, 
chills, and myalgia, were commonly reported.  Fever and asthenia were also reported frequently as 
isolated symptoms.  Insomnia, rhinitis, sweating, and malaise were reported infrequently.  Less than 1% 
of patients discontinued due to flu-like symptoms.  Infection:  Infections were reported for 16% of 
patients.  Sepsis was rarely reported (<1%).  Alopecia:  Hair loss, usually minimal, was reported by 15% 
of patients.  Neurotoxicity:  There was a 10% incidence of mild parasthesias and a <1% rate of severe 
parasthesias.  Extravasation:  Injection-site-related events were reported for 4% of patients.  There were 
no reports of injection-site necrosis.  Gemcitabine is not a vesicant.  Allergic:   Bronchospasm was 
reported for less than 2% of patients.  Anaphylactoid reaction has been reported rarely.  Gemcitabine 
should not be administered to patients with a known hypersensitivity to this drug.  Cardiovascular:  
Two percent of patients discontinued therapy with gemcitabine due to cardiovascular events such as 
myocardial infarction, cerebrovascular accident, arrhythmia, and hypertension.  Many of these patients 
had a prior history of cardiovascular disease. 
 
Pharmaceutical Data: Gemcitabine is supplied as a lyophilized powder in sterile vials containing 200 
mg or 1 g of gemcitabine as the hydrochloride salt (expressed as the free base), mannitol, and sodium 
acetate.  Drug vials will be reconstituted with normal saline added to the vial to make a solution ideally 
containing 10 mg/mL or less.  The concentration for 200 mg and 1 g vials should be nor greater than 40 
mg/mL.  An appropriate amount of drug will be prepared with normal saline and administered as a 
continuous infusion for 30 minutes.  Once the drug has been reconstituted, it should be stored at room 
temperature and used within 24 hours. 
 
Storage and Stability: Store at controlled room temperature (20-250C), should be handled and 
disposed of in a manner consistent with other anti-cancer drugs. 
 
Route of Administration: Intravenous infusion over 30 minutes. 
 
Supplier: Commercially available from Eli Lilly and Company, Indianapolis, Indiana, 46285. 
 
3.2  Genistein (Soy Isoflavones) 
 
 
Novasoy ® tablets containing 50 mg soy isoflavones will be obtained at no charge from Archer 
Daniels Midland (ADM) Company (Decatur, IL). Novasoy tablet is a well-defined soy 
concentrate tablet manufactured to contain 50 mg soy isoflavones per tablet. The contents have 
been analyzed by HPLC and contain soy isoflavones genistein, daidzein and glycitein at a ratio 
of 1.1:1:0.1, thus each 50 mg tablet provides roughly 25 mg of genistein. The isoflavones are 
present in both conjugated (40%) and unconjugated (60%) forms. In our previous studies we 
have measured serum genistein and daidzein levels and found them to significant increase after 
one week and remain stable for the remaining period upon continuous dosing without change in 
serum concentrations. The half-life of genistein after a single oral dose is approximately 3-4 
hours. We have shown in our previous studies that Novasoy taken 1 tablet (50 mg) daily is 
sufficient to have a biological effect with significant reduction in oxidative stress markers. Soy 
isoflavones are generally considered safe and are available in nutrition stores and pharmacies 
without prescription. No adverse health effects have been attributed to use of soy isoflavone 
supplements.  
 
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Chemistry:  Genistein is supplied as a major component of an isoflavone extract of soy and is classified 
as a nutritional product.  Genistein’s chemical structure is 4,5,7-trihydroxyisoflavone. 
  
Administration:    Patients will be instructed to take their capsules twice daily with breakfast and  
dinner. They may also take the study capsules at any time during the day if they forget to take them with 
the designated meals. Patients will return their remaining study capsules. A capsule count will be made 
and the number of remaining capsules will be placed in patient’s study folder by the study coordinator. 
Capsule containers (partial and empty) need to be returned for accountability. 
 
 
Human toxicity: Genistein did not have any side effects in previous studies; therefore,  we 
do 
not 
expect toxicity in this study.  
 
Storage: At room temperature. 
 
Supplier:  Archer Daniels Midland, Decatur, IL 
 
 
4.0 
ELIGIBILITY CRITERIA  
 
 
 
4.1 
Inclusion Criteria
 
a) 
Patients must have histological or cytological diagnosis of breast cancer.    
 
b) 
Patients must have clinical and/or radiological evidence of metastatic disease (stage IV). 
 
c) 
Patients must have measurable disease by RECIST criteria.  Prior radiation permitted as long as 
at least one measurable disease site is outside the radiation field 
 
d) 
Patients may have had any prior cytotoxic chemotherapy treatments for metastatic disease 
excluding gemcitabine.  Any prior adjuvant therapy is permitted (except for gemcitabine).  
Patients must have failed within 24 months of completion of adjuvant (or neoadjuvant) taxane-
based therapy or after a taxane therapy for metastatic disease.  
 
e) 
Patients with prior hormone therapy must have documented disease progression on the hormone 
therapy. 
 
f) 
Patients must not have received any prior gemcitabine.   
 
g) 
Patients must have performance status of 0-2 on SWOG scale (see Appendix III). 
 
h) 
Patients must have adequate bone marrow function:  absolute neutrophil count >1,500/cmm, 
platelet count >100,000/cmm, and hemoglobin > 10g/l. 
 
i) 
Adequate liver function:  bilirubin < 3.0 mg/dL; transaminases (AST/ALT) < 2.5 times upper 
limit of institutional normal. 
 
j) 
Adequate renal function:  creatinine < 1.5 mg/dL. 
 
k) 
Patients must be informed of the investigational nature of this study and must give written 
informed consent prior to the receiving of treatment per this protocol. 
 
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Women with child bearing potential must practice effective birth control while receiving 
treatment. 
 
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m) 
Prior chemotherapy, biologic, investigational , or major surgical therapy must be completed at 
least 3 weeks prior to initiation of protocol therapy. 
 
n) 
Prior radiation therapy is allowed, however, patients must have recovered from the acute effects 
of the treatment, and have completed radiation at least 4 weeks prior to starting protocol 
therapy. 
 
o) 
Patients may have hormone receptor positive or negative breast cancer.  Any prior hormonal 
therapy, either adjuvant or for metastatic disease is permitted, as long as patients are off all 
hormone therapy for at least 2 weeks prior to starting protocol therapy. 
 
p) 
All soy supplements (including all soy based pills, liquids, concentrates) must be discontinued 
for at least 1 week.   Dietary soy that may be normally part of a meal (e.g., tofu) may be 
ingested once per week.  A daily multivitamin pill is permitted.   
 
q) 
Patients must discontinue all other nutritional supplements, herbal agents, high doses of 
antioxidants (e.g., vitamin C, D, or E) including any that may interact with, antagonize, or alter 
or imitate the potential effects of either gemcitabine or genistein.   
 
4.2 
Exclusion Criteria 
 
a) 
History of active central nervous system (CNS) metastases.  Patients with previously treated 
CNS metastases are permitted if > 3 months prior to enrollment, clinically stable without 
steroids or antiseizure medications. 
 
b) 
Serious concomitant systemic disorders incompatible with the study (at the discretion of the 
investigator). 
 
c) 
History of other malignancy, except for cervical carcinoma, or basal or squamous skin cancers.  
For these cancers, patient must have been treated with a curative intent be in complete in 
remission. 
 
d) 
Unresolved bacterial infection requiring treatment with antibiotics. 
 
e)  
Pregnant or lactating women may not participate in the study.  
 
j) 
Patients infected with HIV-1 virus because of the undetermined effect of this chemotherapy 
regimen in these patients, and the potential for interaction with anti-HIV medications. 
 
 
 
 
 
 
 
5.0 
TREATMENT PLAN  
 
 
5.1 
Dosage Selection and Administration Procedures
  
TABLE 2.  The Starting Dose Levels 
 
 
Cycle and Day 
Gemcitabine (I.V.) 
Novasoy (P.O.)1
Dose 
 
1000 mg/m2
100 mg 
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Cycle 1,  
Days -7 to -1 
----------------------- 
BID x 7 days2
Schedule 
Cycles 1 to N, 
Days 1-21 
Days 1 and 8  
Q 21 days 
BID2  
Days 1-21 
Q21 days 
 
 
 
 
1Genistein (Novasoy) should be taken with breakfast and dinner. 
2Patients should be told not to take genistein A.M. dose on cycle 1, days –7 and –1, and on 
cycle 2, day 1, until plasma for genistein level is collected in clinic. 
 
The body surface area will be calculated according to actual height and weight at the beginning of 
treatment and this will be used to calculate the dose of gemcitabine.  Doses can be recalculated if weight 
declines by >10%.  Treatment may be administered on an outpatient basis. 
 
Gemcitabine 1,000 mg/m2 intravenously administered over 30 minutes on days 1 and 8 of each 21-day 
cycle.  Please refer to Table 2 for the initial dose level. 
 
 
5.2 
Duration of Therapy 
 
Patients with metastatic cancer will continue until disease progression and/or undue or intolerable 
toxicity. 
 
 
5.3 
Concomitant Therapy 
 
No other chemotherapy, immunotherapy, hormonal therapy (excluding anticoagulants, appetite 
stimulants and replacement steroids), nutritional or herbal supplements (except for a multi-vitamin) 
radiation therapy, or experimental medications will be permitted while the patients are on study.  To 
prevent nausea and vomiting, the prophylactic use of anti-emetics including dexamethasone, is allowed.  
Any disease progression requiring other forms of specific anti-tumor therapy will be a cause for early 
discontinuation from this study.  Bisphosphonates are permitted.  Patients should receive full supportive 
care as necessary.  Patients may receive hematopoietic growth factors as clinically indicated. 
 
 
 
 
 
 
 
 
 
 
 
6.0 
TREATMENT MODIFICATIONS  FOR TOXICITY, AND CRITERIA FOR 
DISCONTINUATION FROM STUDY 
* Dose reductions and treatment delays only apply to gemcitabine
 
6.1  
Dose delay and modification on day 1 of each cycle based on toxicities on day of treatment
 
 
TABLE 3.  Day 1 dose delay or modification due to hematologic toxicity on day of     
       
treatment 
 
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ANC 
 
 
Platelet 
Delay, until 
ANC> 1500 
and Platelet > 
100,000 
Percent dose 
reduction of 
gemcitabine 
Percent dose 
reduction of 
genistein 
> 1,500 
>100,000 
None 
0 
0 
< 1 week 
           0 
0 
> 1 to 2 weeks 
25 
0 
<1,500 
<100,000 
> 2 weeks 
Off treatment 
  
 
 
TABLE 4.  Day 1 dose delay or modification due to non-hematologic toxicity on day of  
  
      
treatment (excludes alopecia, nausea, or vomiting, based on NCI Common Tox. Criteria 3.0) 
 
Grade 
 
Delay, until non-
hematologic 
toxicity = 0-1 
Percent dose 
reduction of 
gemcitabine 
Percent dose 
reduction of 
genistein 
0-1 
None 
0 
0 
0 to 1 week 
          0 
0 
> 1 to 3 weeks 
25 
0 
2 
> 3 weeks 
Off treatment 
0 to 1 week 
0 
0 
> 1 to 3 weeks 
25 
0 
3-4 
> 3 weeks 
Off treatment 
 
 
 
6.2   
Dose modification on day 1 of each cycle based on toxicity of the previous  
 
 
cycle
 
 
TABLE 5.   Dose modification on day 1 of each cycle based on previous cycle’s hematologic   
      
toxicity 
 
Worst toxicity during 
previous cycle 
Percent reduction in 
gemcitabine dose from the 
previous cycle 
Percent reduction of the 
genistein dose from 
previous cycle 
  ANC       or       Platelets 
 
 
  >0.250                     >40 
0 
0 
  <0.250                     <40 
25 
0 
 
Note:  Dose modifications should be repeated in subsequent cycles if hematologic toxicity recurs. 
 
 
 
TABLE 6.  Dose modification on day 1 of each cycle based on previous cycle’s non-  
 
       
hematological toxicity during previous cycle (excluding pulmonary toxicity, alopecia, nausea and 
vomiting) 
   
Worst toxicity during 
previous cycle 
Percent reduction in 
gemcitabine dose from the 
previous cycle 
Percent reduction of the 
genistein dose from 
previous cycle 
0 
0 
0 
1-2 
0 
0 
3-4 
25 
0 
 
 
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TABLE 7.  Dose modification on day 1 of each cycle based on previous cycle’s non-  
 
       
hematological pulmonary toxicity during previous  
   
Pulmonary toxicity 
Percent reduction in 
gemcitabine dose from the 
previous cycle 
Percent reduction of the 
genistein dose from 
previous cycle 
0 
0 
0 
1 
25 
0 
2-4 
Off treatment 
Off treatment 
 
 
If pneumonitis grade 2 or higher develops in a given cycle and is related to gemcitabine, gemcitabine 
should be promptly discontinued and the patient should be removed from protocol treatment.  Treatment with 
corticosteroids should be given according to established guidelines. 
 
6.3 
Dose modifications on day 8 of each cycle based on hematologic toxicities 
 
TABLE 8.  Dose modifications on day 8 of each cycle  
 
 
 
ANC 
Platelet (X 109) 
Percent dose 
reduction of 
gemcitabine 
Percent dose 
reduction of 
genistein 
> 1.0 
>75 
0 
0 
0.75-0.999 
50-74 
              25 
0 
0.50-0.749 
40-49 
50 
0 
<0.50 
<40 
100 
100 
 
6.4 
Discontinuation from the Study Treatment
 
A patient will be discontinued from the study under the following circumstances: 
 
 
6.4.1 
There is evidence of progressive disease. 
 
6.4.2 
The attending physician thinks the best interests of the patient require a change of therapy. 
 
 
6.4.3 
The patient requests discontinuation. 
 
 
6.4.4 
The drug exhibits unacceptable toxicity. 
 
6.5.5 
Patient becomes pregnant or fails to use adequate birth control (for those patients who   are 
potentially able to conceive). 
 
 
 
 
7.0 
CRITERIA FOR RESPONSE EVALUATION AND TOXICITY REPORTING 
 
7.1 
Definition of clinical response: 
 
7.1.1  Measurable lesions:  Lesions that can be measured in at least one dimension as > 20 mm with 
conventional techniques or as > 10 mm with spiral CT scan. 
7.1.2  Evaluable disease:  Includes unidimensionally measurable lesions, masses with poorly defined 
margins, palpable nodal disease (measured by two observers), lesions with diameter less than 0.5 cm. 
 
7.1.3 Non-measurable lesions:  All other lesions including small lesions (longest diameter < 20 mm 
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with conventional techniques) and other non-measurable lesions including:  pleural effusions, ascites, 
lesions on bone scans and disease documented by indirect evidence (e.g. biochemical abnormalities). 
Non-evaluable disease does not influence objective response assessment except for the determination 
of CR when all disease must be absent and in the determination of progression (if new non-evaluable 
disease develops). 
7.1.4  Target lesions:  All measurable lesions up to a maximum of 10 lesions. Target lesions are 
selected for their size and suitability for accurate repetitive measurements. The sum of the longest 
diameter of all target lesions will be calculated and reported as the baseline sum longitudinal diameter 
(LD). This will be used as a reference to further quantify objective response. 
 
7.1.5  Non-target lesions:  All other lesions are identified as non-target lesions and should be followed 
as present or absent. 
 
7.1.6  Objective response criteria:                 
Complete response (CR): Complete disappearance of all measurable and evaluable disease for at 
least 3 weeks without the appearance of any new lesions. Persistent effusions after therapy 
should be cytologically negative of malignant cells. 
Partial Response (PR): Greater than or equal to 30% reduction in the sum longest diameters of 
target lesions taking as reference the baseline sum of the longest diameters without the 
appearance of any new lesions. 
Progressive disease (PD): Greater than 20% increase in the sum of longest diameter of target 
lesions taking as reference the smallest sum of the longest diameter recorded since the treatment 
started OR appearance of new lesions. 
Stable disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD taking as reference the smallest sum of the longest diameters of target lesions 
since treatment started for a minimum interval of 6 weeks. 
Best objective response:  This will be determined from serial objective response assessments in 
a given patient using the response criteria mentioned above. For patients whose disease sites are 
assessed every three to six weeks, two consecutive objective CRs are required to assign the best 
response of CR.  Similarly, two successive evaluations documenting PR or SD are required 
before assigning the patient to either. 
Best overall response:  This is the best response from the start of the treatment until disease 
progression/recurrence. In general, the patient’s best response assignment will depend on the 
achievement of both measurement and confirmation criteria. 
7.1.7 
Evaluable Patients:  A participant who completes 2 cycles of therapy is 
evaluable for response. A patient who receives any amount of drug 
is evaluable for toxicity. 
 
 
 
 
 
 
TABLE 9.  Overall response definitions 
 
  Target lesions 
  Non-Target lesions 
    New lesions 
   Overall response 
        CR 
            CR 
          No 
            CR 
        CR 
   Non-CR/Non-PD 
          No 
            PR 
        PR 
         Non-PD 
          No 
            PR 
        SD 
         Non-PD 
          No 
            SD 
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        PD 
           Any 
    Yes or No 
            PD 
       Any 
            PD 
    Yes or No 
            PD 
       Any 
           Any 
    Yes or No 
            PD 
 
7.1.7 Duration of response:  Time from point at which response (PR or CR) is first noted, until first 
clinical or radiologic evaluation that shows progressive disease. 
 
7.1.8 Time to progression:  Time from date of registration to the date of documented progressive 
disease or death. 
 
7.1.9 Overall survival: Time from date of registration to date of death or last follow up. 
 
 
7.2 Toxicity definitions for evaluation of this protocol 
 
Toxicity reported for patients in this trial will be defined by NCI CTC 3.0 criteria.  
 
8.0 
SCHEDULED EVALUATIONS ON STUDY  
 
8.1  Within 4 weeks before study initiation all patients must have: 
• Bone scan. 
• Chest x-ray. 
• Tumor imaging to document measurable disease (Chest and abdominal CT scans, or MRI scans). 
 
 
8.2  Within two weeks before study initiation all patients must have: 
• History and physical exam, including height and weight. 
• Performance status. 
• CBC with differential white cell and platelet counts 
• Serum chemistries:  electrolytes, BUN, creatinine, LDH, alk. phosphatase, total bilirubin, 
AST(GOT), ALT(GPT), magnesium, calcium, phosphate and albumin. 
• Amylase and lipase. 
• Tumor biopsy from easily accessible tumor, before first genistein dose (Optional – if patient has 
easily accessible tumor and patient agrees to procedure). 
 
 
 
 
 
8.3  On treatment evaluations: 
 
8.3.1 
On cycle 1, day –7 
 
5 ml plasma for genistein level, before A.M. genistein dose 
 
Tumor biopsy before A.M. dose, or earlier (Optional) 
 
8.3.2 
On cycle 1, day –1, on day 7 of genistein alone (before cycle 1, day 1) 
 
5 ml plasma for genistein level, before A.M. genistein dose, and 4 h after A.M. dose 
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 
Tumor biopsy from same site as before (Optional) 
 
8.3.3 
On cycle 2 only, day 1 
    5 ml plasma for genistein level, before A.M. genistein dose, and 4 h after A.M. dose. 
 
8.3.3  On cycles 2 through cycle N, day 1 (before genistein and gemcitabine administration): 
 
History and physical examination. 
 
Performance status. 
 
Toxicity evaluation using NCI-CTC criteria. 
 
CBC with differential white cell and platelet counts. 
 
Electrolytes, SGOT (AST), SGPT (ALT), alkaline phosphatase, bilirubin, creatinine, calcium, 
phosphate. 
 
Amylase and lipase. 
 
8.3.4  On day 8 and 15 of every cycle: 
   CBC with differential white cell and platelet counts.  Therefore all patients will have weekly 
CBC and differential throughout their treatment period. 
 
8.3.5  After every 2 cycles:   
 Repeat all imaging tests to evaluate objective tumor response 
 
9.0      CORRELATIVE STUDIES:   INSTRUCTIONS  
 
For schedule please see Appendix, Study Calendar. 
 
9.1  Blood samples for plasma genistein and other isoflavone levels: 
 
• Plasma will be collected: 
• 
before first dose of genistein on cycle 1, day –7 (or earlier) 
• 
cycle 1, day –1 (on day 7 of genistein alone) before A.M. dose, then 4 h after A.M. dose 
• 
cycle 2, day 1 before A.M. dose, then 4 h after A.M. dose 
• Blood will be collected into (1) 10 ml or (2) 5 ml citrated tubes. Within 2 hours of collection tubes should be 
spun at 1000g for 10 m and plasma stored at -70° C in (2) cryovials, each with ~2 ml aliquots. 
 
• Cryovials should be labeled with: 
 
• 
Institution name 
• 
patient number (assigned at registration) 
• 
plasma 
• 
date and time collected 
• 
cycle number 
• 
either as “cycle 1, day-7” or “cycle  1, day-1” or “cycle 2, day 1” 
• 
either as “pre-AM dose” or “4-hour post AM dose”   
 
• Samples should be sent on dry ice by next AM express mail to: 
 
Translational Research Lab (Dr. Sarkar’s lab) 
HWCRC, room 703 
Karmanos Cancer Institute 
   
   
Lab Phone:  (313) 576-8314 or (313) 576-8315 
 
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• Contact Dr. F. Sarkar’s lab at (313) 576-8314 or( 313) 576-8315, for questions or sample pick up.   
 
 
9.2  Tumor tissue samples to assess effects of genistein alone on Akt and NF-κB activation and on potential  cDNA 
biomarkers: 
 
• Patients with tissue easily accessible for biopsy (i.e. skin metastasis) should be requested to volunteer to 
biopsy for tumor tissue.  This is a recommended but optional study component. 
 
• Tumor tissue collection should be arranged at least 2 days in advance of treatment initiation by calling Dr. 
Sarkar’s lab at (313) 576-8315, or (313) 576-8314.   
 
• Tumor tissue will be obtained, before first genistein on cycle 1, day –7 (or earlier), and on cycle 1 day–1 (day 
7 of genistein alone).   
• At least 0.1 g of tumor should be collected (equivalent to a 5 mm cube of tumor, or 3 core (not FNA) cutting 
needle biopsies should be taken at each time point. 
 
• Tumor will be immersed immediately into sterile plastic containers with at least 4 volumes of RNAlater 
solution (Ambion, 800-888-8804).     
 
• The container should be labeled with: 
 
• 
Institution name 
• 
patient number (assigned at registration) 
• 
metastatic site biopsied 
• 
date and time 
• 
“pre-genistein” or “post 7 days of genistein.” 
 
• After immersion in RNAlater, the tumor samples must be refrigerated or iced until brought to the 
Translational Lab (Room 703 HWCRC, (313) 576-8314, or (313) 576-8315) before 5 pm the day of 
collection.  From outside institutions, they should be stored in ice or 4C and delivered the same day, or 
express shipped for delivery the morning after the biopsy in a container containing  4C cold packs).   
 
• Upon arrival to the lab: 
 
• 
For core biopsies, one will be sent to pathology for routine H and E staining.  The other two cores 
will be transferred to one labeled 2 ml cryovial with 4 volumes RNAzol and stored at –70C.   
• 
For incision or excision biopsies, they will be divided in 3 parallel sections, and the middle 1/3 of the 
sample will be sent to pathology for routine H and E staining, while the two flanking 1/3 pieces will 
be transferred to one labeled 2 ml cryovial with 4 volumes RNAzol and stored at –70C 
 
 
• Shipping address: 
 
  
 
 
 
    Karmanos Cancer Institute  
Translational Research Lab 
 
4100 John R 
HWCRC, room 703 
Detroit, MI  48201 
 
 
Lab Phone:  (313) 576-8314 or (313) 576-8315 
 
 
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• 
Histologic analysis of tumor biopsies taken before and on day 7 of genistein alone:   
• 
Routine H and E staining 
• 
TUNEL staining for determination of percent apoptotic tumor cells (Calbiochem) 
• 
IHC will be performed using established staining methods for: 
• 
Ki67 to estimate percent cells in G1-S phase using specific antibody (Dako, #Ki-S5). 
• 
Expression of activated Akt using anti-phosphoserine473-Akt antibody (Cell Signaling, 
#9277).  
• 
Expression of activated NF-κB using an antibody specific for the activated form of NF-κB 
p65 that is free of IκB (Chemicon, #12H11). 
• 
Interpretation of IHC for activated Akt or NF-κB: 
 
  
 
For p-Ser473-Akt and activated NF-κB, IHC each stain will be interpreted based a 
composite score of two parameters:  percent tumor cell staining (0 = 0 - 9%, 1 = 10 - 49%, 
2 =  > 50%), and relative staining intensity (0 = absent - minimal, 1 = moderate, 2 = strong 
staining).  Appropriate controls will include tumor xenografts that are known to overexpress 
either marker.  A tumor will be positive for expression only it scores 1 or 2 for both percent 
and intensity parameters; otherwise expression will be considered negative.  In addition, the 
sum score of the parameters for each biomarker will be recorded (i.e., 0 – 4).    
 
• 
CDNA microarray analysis of tumor biopsies taken before and on day 7 of genistein alone 
• 
Paired analysis of each patient’s pre- and post- gensistien effects will be done if the H and E 
staining demonstrates  >50 % tumor cells.   
• 
The –70C frozen tumor specimens will be crushed on dry ice, then processed in RNAzol to 
collect total RNA.  If the quantity and quality of the pair of tumor RNAs is confirmed (each 
specimen yielding > 10 ug of non-degraded RNA gel analysis), pairs of matched RNAs will be 
subjected to microarray analysis. 
• 
Microarray analysis will be performed using established methods ((60) and manuscript in 
preparation). 
  
 
10.0  
REGISTRATION PROCEDURE
 
All patients entering into this study will be registered with the Clinical Trials Office (CTO) at the 
Karmanos Cancer Institute, Detroit, Michigan, within 7 days prior to starting treatment (313) 576-8994.  
Each patient will be assigned an alphanumeric code, indicating the overall number of the patient on 
study, and the institution acronym (e.g., “05-KCI” would indicate the fifth patient registered, and he/she 
was from KCI. 
 
 
 
11.0 
REPORTING OF ADVERSE EVENTS  
 
11. 1  General principles: 
 
• Reporting of adverse events will include those that occur within 60 days of last treatment (FDA 
requirement is 30 days). 
 
• All adverse events that warrant reporting should use the Wayne State University (WSU) adverse 
event (AE) form, available on the Human Investigations Committee (HIC) web site:  
www.hic.wayne.edu).   
 
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• Note that certain events require reporting within 3 calendar days of awareness of adverse event, 
while less urgent events are to be reported within 10 days of awareness.  These deadlines may be met 
by verified fax, with the original sent by mail.  In addition, for deaths and immediately life 
threatening events, the Principle Investigator (PI) and WSU Clinical Trials Office (CTO) should be 
contacted by phone immediately so that compliance with contacting other agencies within 24 h can 
be facilitated. 
 
• The WSU CTO staff will coordinate the reporting process between the PI and the WSU human 
Investigations Committee (HIC, a.k.a. IRB) as well as other applicable reporting agencies (FDA, 
CTEP, NIH, OBA, and industry).  Copies of all related correspondence and reporting documents will 
be maintained in the regulatory file.  All deaths, immediately life threatening, and reportable serious 
adverse events will be reported and documented on Form FDA 3500 A (MedWatch Form) and 
forwarded directly to Eli Lilly.  This includes serious, related, expected and serious, related, 
unexpected events.  Reports should be sent to Eli Lilly, US Pharmacovigilance Department by fax at 
908-243-6800, within 24 hours of receipt by the investigator. Fax transmission should include the 
Grant-In-Aid Study Number, Study Title, and name of Principal Investigator. 
 
• Reporting of AEs:  Contact information: 
 
• Study PI of the lead institution, WSU/KCI: 
 
Amy M. Weise, D.O. 
 
 
  Phone:  (313) 576-8952 
 
E-mail:  weise@karmanos.org 
 
Pager:  (313) 745-5111 #0585 
 
Fax:  (313) 576-8767 
 
 
 
 
Address: 
Karmanos Cancer Institute 
4100 John R 
4-HWCRC 
Detroit, MI  48201 
 
• Data Manager, Clinical Trials Office (CTO) at WSU/KCI: 
Matthew Gretkierewicz 
 
 
Phone:  (313) 576-8994 
 
E-mail:  gretkiem@karmanos.org 
Pager:  (313) 745-5111 #98026 
 
Fax:  (313) 576-8368 
 
 
Address: 
 
Clinical Trials Office 
 
Harper Professional Bldg., Room 711 
Karmanos Cancer Institute 
4160 John R 
Detroit, MI  48201 
 
 
11.2 Events to be reported by phone within 24 h, and within 3 calendar days, by WSU AE form.   
 
• Deaths 
 
Death from any cause must be reported by phone to the Karmanos Cancer Institute Clinical 
Trials Office within 24 hours of the death (while receiving this treatment or up to 60 days from 
the last dose of treatment).  Any death that occurs after 60 days of the last treatment that is felt 
to be a drug-related toxicity should also be reported.  An WSU AE form must be faxed/sent to 
the WSU Clinical Trials Office within 3 calendar days.  In addition, the PI and CTO will be 
responsible for reporting deaths to the HIC of WSU/KCI (phone 313-577-1628, fax 313-993-
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7122), with formal written notification faxed within 3 days of the event.  Formal notification 
includes the FDA and/or sponsor AE reporting forms attached to the completed WSU AE 
reporting form.  Deaths must be reported to the FDA within 3 days. 
 
• Immediately life threatening events  
 
These events must be reported by phone to the Karmanos Cancer Institute Clinical Trials 
Office within 24 hours of the event (while receiving this treatment or up to 60 days from the 
last dose of treatment).  An WSU AE form must be sent to the WSU Clinical Trials Office 
within 3 days.  In addition, the PI and CTO will be responsible for reporting of immediately 
life threatening events to the HIC of WSU/KCI (phone 313-577-1628, fax 313-993-7122), with 
formal written notification within 3 days of the event.  Formal notification includes the FDA 
and/or sponsor AE reporting forms attached to the completed WSU AE reporting form.  Deaths 
must also be reported to the FDA within 3 days.   
 
 
11.3 Events to be reported within 3 calendar days, by WSU AE form.   
 
• Serious Adverse Events (SAEs) that are grade 3 and 4, and:  
 
• Unexpected, and/or  
• Expected, but definitely or probably related to protocol therapy (i.e., relationship to 
treatment cannot be ruled out) 
 
These SAEs should reported to the PI and CTO of WSU within 3 days of awareness. 
 
 
Serious adverse events that are expected, but a relationship to the study treatment is ruled out 
do not have to be reported. 
 
 
 
11.4  Non-serious adverse events that are to be reported within 10 days by WSU AE form. 
 
 
• Non-serious adverse events (generally grade 1 – 2) that are: 
• Unexpected, and/or 
• Expected, but more intense, longer in duration, or permanent, and definitely, or 
probably related to protocol therapy (i.e., relationship to treatment cannot be ruled 
out) 
 
Non-serious adverse events that are expected, but a relationship to the study treatment is ruled 
out, do not have to be reported. 
 
 
11.5  If the adverse event requires modification of the study protocol and the informed consent 
document, then they should be provided to the local IRB with the report of the adverse event. 
Consent revisions for sponsored research studies must receive sponsor approval prior to 
submission to the local IRB.   
 
 
11.6  Any serious clinical adverse event or laboratory value occurring during the course of the study 
even if unrelated to the therapy should be reported to the Karmanos Cancer Institute Clinical 
Trials Office.  All adverse events will be noted in the case report forms.  
 
 
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DATA AND SAFETY MONITORING 
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12.1  Scheduled meetings or conference calls will occur every month.  These meetings will include the 
protocol principal investigators and any data managers involved with the conduct of the protocol.  
 
 
12.2  During these meetings the investigators will discuss: 
 
1. Safety of protocol participants (AE reporting and regulatory compliance) 
2. Monitoring for excessive toxicity after the first 9, 11, 17 and 20 patients enrolled and treated, 
as described in Section 13.2; 
3. Validity and integrity of the data; 
4. Enrollment rate relative to expectations and the characteristics of participants; 
5. Retention of participants and adherence to the protocol (potential or real protocol violations); 
6. Completeness of collected data. 
 
 
12.3 Data and Safety Monitoring Reports of these regular meetings will be kept on file in the 
Karmanos Cancer Institute Clinical Trials Office.  The data manager assigned to the trial will be 
responsible for completing the report. These reports will be signed by the PI or one of the Co-PI’s. 
 
13.0 
STATISTICAL CONSIDERATIONS 
 
13.1 
Primary Objective:  To estimate the objective response rate of patients with metastatic breast 
cancer treated with the combination of gemcitabine and genistein. 
 
13.1.1 Assumptions/Hypothesis  
The primary statistical endpoint is the objective (complete plus partial) response rate.   We 
would consider this regimen not promising if the true response rate was 0.20 in these pretreated 
patients, since the average response rate of the 5 phase II studies in Table 1 that have an average 
of 1 prior chemotherapy treatment, is 0.23.  The regimen would be promising for further study 
if the true response rate were at least 0.40.   Only response evaluable (RE) patients will 
contribute to this assessment.  In designing this Phase II trial, we have set significance level = 
0.05, and power = 0.80. 
 
13.1.2 Estimation of response rate: 
Based upon the Simon minimax design (61), stage 1 will accrue 18 response RE patients.   If 4 
or fewer responses are observed, we will recommend the trial for early termination and 
conclude that the regimen has a true response rate of 0.20 or less. If there are > 5 responders, we 
will proceed to stage 2 and enroll and additional 15 RE patients.   If at least 11 responders are 
observed among the 33 RE patients, we will conclude that the regimen has a true response rate 
of 0.40 or more.   Otherwise, we will conclude that the true response rate is 0.20 or less. 
 
13.1.3 Sample size, accrual rate, and follow-up: 
In this two-stage Simon minimax design 18 RE patients will be accrued in stage 1, and 15 RE 
patients will be accrued in stage 2 (if necessary), for a total of 33 RE patients.  Three extra 
patients should insure 33 RE patients.  Therefore, 36 total patients are anticipated for accrual at 
a combined rate equivalent to about 36 patients over 18 months (1.5 years) (i.e., about 
2/month).  We anticipate accrual from two other institutions so that each institution would 
contribute 12 patients over 18 months, or 8 patients per 12 months (or 0.5 – 1 per month).  
Thus, we expect to enroll the 18 patients needed for Stage 1 in about 9 months, and (if 
necessary) the additional 15 – 18 patients for Stage 2 in about 9 more months.   Additional time 
(a few months beyond 18 months) may be required to assess response in the last few patients, 
and because some patients will not become response evaluable until after registration and 
treatment is started.  Patients will be followed for 60 days after the development of disease 
progression. 
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13.2 
Monitoring for excessive toxicity 
Using exact upper 1-sided 80% confidence limits (computed in StatXact 4 via the Casella 
methodology (62)), we have determined a monitoring plan for excessive toxicity for the first 20 
patients entered.  From prior studies of gemcitabine alone, we expect about a 10% rate of grade 
4 toxicity (of any type).  We wish to have 80% confidence that the true grade 4 toxicity rate of 
this new regimen is less than 30%.   That will be assured if, among the first 9 patients there is at 
most 1 patient with a grade 4 toxicity, and if among the first 11 patients at most 2 Grade 4 
toxicities, and if among the first 17 patients at most 3 grade 4 toxicities, and if among the first 
20 patients at most 4 grade 4 toxicities. If at any of these accrual points, these respective 
numbers of grade 4 toxicity are exceeded, then the potential incidence of grade 4 toxicity would 
be > 30% and enrollment will be terminated. 
 
13.3 
Secondary endpoints: 
 
Since all the secondary endpoints are exploratory and intended for hypothesis generation, the 
results will primarily descriptive, and no formal hypothesis testing is presented. 
 
13.3.1 Overall survival:  Patients will be followed from the time the last patient comes off 
study treatment for one year to monitor survival 
 
13.3.2 Duration of response:  For patients with an objective response, this will be measured 
from the time when response is first documented, to the last restaging that continues to 
confirm the response. 
 
13.3.3 Time to disease progression:  This will be measured from the time that treatment is 
initiated until the time restaging indicates progressive disease. 
13.3.4 Explore if there is an association between plasma genistein levels and responses.    
• 
Exploratory associations:  The response evaluable patients will be grouped by 
whether the cycle 1 day –1, or cycle 2 day 1 trough (if elevated) or 4 h peak fall 
above (“high genistein”) or below the median values (“low genistein”).  The 
number of responses will be determined for either high or low level genistein 
groups.  The possible association of genistein levels with responses will be tested 
by the Fisher exact test. 
 
13.3.5 Explore the in vivo effects of genistein on human breast cancer tumor biomarkers 
(Ki67, TUNEL, p-Akt and activated NF-κB immunohistochemistry (IHC), and cDNA 
expression by microarray analysis. 
 
• 
Exploratory associations:  Genistein treatment for 7 days will be associated with:  1.  
Decreased tumor cell cycling by Ki67 staining; 2.  Increased apoptosis as detected 
by TUNEL staining; 3.  Decreased expression of activated Akt or NF-κB by IHC; 
and 4. Modulation in expression of other potential cDNA biomarkers. 
• 
Methods: 
• 
The mean and S.D. of the Ki67 or TUNEL percentages all pre-genistein versus 
post-genistein tumor biopsies will be calculated, along with 95% confidence 
intervals.   
• 
The mean and S.D. of the sum expression of p-Akt or NF-κB of all pre-
genistein versus post-genistein tumor biopsies will be calculated, along with 
95% confidence intervals.   
• 
For each tumor after normalization for control housekeeping gene expression, 
genes that have > 3 fold induction or repression by genistein will be identified. 
Those genes which are found to be modulated consistently in all of the paired 
specimens (e.g., occurring in 5 out of 5 patients in whom paired tumor biopsies 
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are collected and technically successful cDNA array analysis is done) will be 
preliminarily considered as a significant results.  These results are to be used in 
association with ongoing in vitro cell culture and animal models, as well as for 
hypothesis generation for future research. 
 
14.0 
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15.0 
APPENDICES 
 
Appendix I:  Study Schema 
 
Metastatic Breast Cancer  
any prior chemotherapies for metastatic disease 
Except Gemcitabine 
 
Pretreatment baseline studies 
 
Registration 
 
Baseline genistein plasma levels  
Baseline Tumor biopsy (optional) 
 
 
Genistein 100 mg PO BID x 7 days (Day –7 to –1*) 
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Day –1*  
Genistein plasma levels (trough and 4h peak) 
Tumor biopsy (optional) 
 
 
Cycle 1, Day 1 
Gemcitabine 1000 mg/m2 IV days 1 and 8, q 21 days 
Genistein 100 mg PO BID days 1-21 
Repeat cycle q 21 days 
 
 
Cycle 2, Day 1 
Genistein plasma trough level 
 
 
 
 
 
 
 
 
 
Restage for response evaluation every 2 cycles 
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Progressive disease  
      SD, PR, or CR 
     or unacceptable toxicity 
 
 
Off protocol, event monitoring for 60 days 
 
 
 
(*Day-1 = day before cycle 1 day 1 of Gemcitabine and Genistein) 
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Appendix II:  Study Calendar 
 
 
Pre-
study 
Cycle 1 
Day-7  
(Day 1 of 
genistein 
alone) 
Cycle 1 
Day-11  
(Day 7 of 
genistein 
alone) 
Cycle 1,  
Day 1 
(gemcitabine 
and genistein) 
Cycle N, 
Day 1 
Cycle N, 
Day 8 and 
15 
After 
every 2 
cycles 
History and Physical  
X2
 
 
X 
X 
 
 
Performance status 
X2
 
 
X 
X 
 
 
Height and Weight 
X2
 
 
X 
X 
 
 
CBC, differential and 
platelets 
X2
 
 
X 
X 
X 
(weekly) 
 
Serum Chemistry4
X2
 
 
X 
X 
 
 
Amylase and Lipase 
X2
 
 
X 
X 
 
 
CXR 
X3
 
 
 
 
 
 
Bone Scan 
X3
 
 
 
 
 
 
Tumor site(s) 
imaging5
X3
 
 
 
 
 
X 
Toxicity assessment 
 
 
 
X 
X 
 
 
Genistein plasma level 
before a.m. dose6
 
X 
X 
 
X7
 
 
Genistein peak plasma 
level 4 h after a.m. 
dose6
 
 
X 
 
X7
 
 
Tumor biopsy8
 
X9
X9
 
 
 
 
 
1. Day –1 = day before Cycle 1, Day1 of gemcitabine and genistein 
2. Within two weeks of registration. 
3. Within four weeks of registration. 
4. Serum chemistry: electrolytes, BUN, creatinine, LDH, alk. phosphatase, total bilirubin, AST(SGOT), 
ALT(SGPT), magnesium, calcium, phosphate and albumin. 
5. Radiologic studies to document measurable disease; i.e., CT and/or MRI scans. 
6. Collection of 5 ml of plasma and store at –70C, then shipped on dry ice to Translational Lab, 703 HWCRC, 
KCI, 4100 John R, Detroit, MI  48201; (313) 576-8314, or (313) 576-8315. 
7. Cycle 2 only. 
8. Optional.  Pre-arrange tissue collection > 1 day prior, by calling (313) 576-8314, or (313) 576-8315.  Tissue 
must be immediately placed in RNAlater solution, then kept at 4C or on ice until transported same day, or 
next-A.M. express shipped at 4°C to Translational Lab, 703 HWCRC, KCI, 4100 John R, Detroit, MI  
48201; (313) 576-8314, or (313) 576-8315. 
9. Biopsies are prior to first dose of genistein alone (on cycle 1, day-7, or earlier), and then on  day 
7 
of 
genistein alone (on day-1). 
 
 
 
 
 
 
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Appendix III:  Performance Status 
 
SWOG Scale 
 
 
 
 
 
 
 
Grade  
Scale 
0 
 
Fully active; able to carry on all pre-disease activities without restriction.  
 
(Karnofsky 90-100) 
 
1 
 
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.    
 
 
(Karnofsky 70-80) 
 
2 
 
Ambulatory and capable of all self-care but unable to carry out any work  
 
activities.  Up and about more than 50% of waking hours. (Karnofsky 50-60) 
 
3 
 
Capable of only limited self-care; confined to bed or chair.  Up and about less  
 
 
than 50% of waking hours (Karnofsky 30-40) 
 
4 
 
Completely disabled.  Cannot carry on any self-care.  Totally confined to bed or   
 
chair.  (Karnofsky 10-20) 
 
5 
 
Dead 
 
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APPENDIX IV:  NCI-CTEP COMMON TOXICITY CRITERIA (CTC version 3.0) 
   
 
(see Internet Website http://www.ctep.info.nih.gov) 
 
 
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