Clinical trial NCT00513786
Conditions: Endometrial Cancer
Keywords: Endometrial Cancer Advanced Stage Endometrial Cancer Stage 3 or 4 endometrial cancer Cancer treatment gyn cancer
Sponsor: David O'Malley
Start date: 2007-08-01; Completion date: 2017-01-03
Enrollment: 38 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Purpose of this study is to determine the effectiveness of the drug combination carboplatin, paclitaxel, and bevacizumab(Avastin) in patients with advanced stage endometrial carcinoma.

Detailed description:
The purpose of this study is to test the effectiveness, safety, and tolerability of the drug combination carboplatin, paclitaxel, and bevacizumab(Avastin) in patients with advanced stage endometrial carcinoma. This is a phase II,open label,single center study. Patients will receive carboplatin, paclitaxel, and bevacizumab in an outpatient center by intravenous administration. The primary objectives is to study the progression free survival at 24 months after initiation of treatment and to determine the toxicity profile of the drug combinations. The secondary objectives are to estimate the overall survival and tumor response for this group of patients.

Arms: arm 1: A regimen of Carboplatin and paclitaxel combined with bevacizumab given every 21 days in patients with advanced stage endometrial cancer for a maximum of 6 cycles.
Interventions: intervention 1: Carboplatin intervention 2: Paclitaxel intervention 3: bevacizumab
Intervention descriptions: intervention 1: AUC (area under curve) 5 Intervenous (IV) over 30 minutes given every 21 days for a maximum of 6 cycles. intervention 2: 175 mg/m2 over 3 hours given every 21 days for a maximum of 6 cycles. intervention 3: 15 mg/kg intervenous (IV) given every 21 days for a maximum of 6 cycles.

Protocol documents:
Prot_SAP_000.pdf: 
A PHASE II EVALUATION OF 
CARBOPLATIN/PACLITAXEL/BEVACIZUMAB IN THE 
TREATMENT OF ADVANCED STAGE ENDOMETRIAL 
CARCINOMA 
 
Institution 
The OSU College of Medicine and Public Health 
Arthur G. James Cancer Hospital and Richard Solove Research Institute 
 
Study Drugs 
Bevacizumab (Genentech, Inc.) 
 
Support Provided By 
Genentech, Inc., San Francisco, California, USA 
 
Sponsor Investigator 
David O'Malley, MD 
Assistant Professor 
M210 Starling Loving Hall, 320 West 10th Avenue 
Columbus, Ohio 43210 
614-293-8737 
 
Co-Investigators 
Jeffrey M Fowler, MD;  David E Cohn, MD,  Larry Copeland, MD, Ritu Salani, MD, and 
Eric Eisenhauer, MD 
 
Research Coordinator 
Michele Vaughan 
Roberta Cobb, RN 
Lois Dial, RN 
 
 
 
 
 
Study Number: 
 
OSU06133 
 
Protocol Version Date: 
1.7.2010 
v 9.20.09 ( IRB approved 12.2.09) 
version 2.6.08amend1(IRB approved 4.18.08) 
Version 1.1.12.6.06 . 
 
1


TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.0 BACKGROUND 
1.1 
Disease Background ...................................................................... 5 
1.2 
Bevacizumab Clinical Experience .................................................. 8 
1.3 
Other Study Drug(s) Background…………………………….……..   9 
1.4 
Study Rationale .............................................................................. 12 
2.0 OBJECTIVES .............................................................................................. 13 
2.1  
Primary........................................................................................... 13 
2.2  
Secondary ...................................................................................... 13 
2.3  
Translational Research Objectives……………………………………13 
3.0 STUDY DESIGN.......................................................................................... 14 
3.1 
Description of the Study .................................................................... 14 
3.2 
Outcome Measures……………………………………………………….14 
3.2.1 
Primary Outcome Measures ................................................ 14 
3.2.2 
Secondary Outcome Measures ........................................... 14 
4.0 SAFETY PLAN ............................................................................................ 16 
4.1 
General Plan to Manage Safety…………………………………………16  
5.0 STUDY SUBJECTS..................................................................................... 17 
5.1 
Subject Selection............................................................................ 17 
5.2 
Inclusion Criteria............................................................................. 18 
5.3 
Exclusion Criteria............................................................................ 19 
6.0 Treatment 
6.1 
Treatment Plan............................................................................... 22 
6.2  
Accrual ........................................................................................... 22 
7.0 STUDY MEDICATION 
7.1 
Bevacizumab Dosage and Formulation ........................................... 23 
7.1.1 
Bevacizumab Administration................................................ 32 
7.1.2 
Bevacizumab Storage.......................................................... 33 
7.2 
Paclitaxel Chemotherapy .................................................................. 33 
7.3 
Carboplatin Chemotherapy ............................................................... 35 
7.4  
Methods of Chemotherapy Administration ........................................ 37 
 
7.4.1 Sequence and Timing of Drug Admin ...................................... 37 
 
7.4.2 Pre-Medication ......................................................................... 37 
 
 
2


TABLE OF CONTENTS (continued) 
8.0    Treatment Modifications.............................................................................. 40 
8.1 
Individual Dose Modifications............................................................ 40  
 
8.2 
Guidelines for Hematologic Toxicity.................................................. 40 
8.3 
Modifications for Hematologic Toxicity.............................................. 41 
8.4 
Modifications for Delayed Hematologic Recovery............................. 43 
8.5 
Adjustments for Non-Hematologic Toxicity ....................................... 44 
8.6 
Bevacizumab Dose Modifications and 
           Toxicity Management........................................................................ 49 
8.7     Concomitant Medications................................................................... 54 
9.0  CLINICAL AND LABORATORY EVALUATIONS......................................... 54 
10.0 STUDY DISCONTINUATION ...................................................................... 56 
11.0 STATISTICAL METHODS ........................................................................... 57 
11.1 Interim Analysis................................................................................. 57 
11.2 Planned Efficacy Evaluations............................................................ 57 
11.2.1 Primary Efficacy Variables................................................... 58 
11.2.2 Secondary Efficacy Variables .............................................. 58 
           11.4Methods of Analysis........................................................................... 59 
12.0 SAFETY REPORTING OF ADVERSE EVENTS 
12.1 Adverse Events Reporting and Definitions........................................ 59 
12.2 Reporting of Serious Treatment Emergent Adverse Events ............. 61 
12.3 Safety Reporting Requirements for Exempt IND Holders ................. 62  
 
13.0 Data Safety Monitoring Plan ...……………………………………………….63  
14.0 Retention of Records…………………………………………………………..63 
  
 
 
 
 
 
 
 
REFERENCES 
 
APPENDICES 
 
 
 
 
Appendix A: Study Flowchart/Schema 
Appendix B: NCI Common Toxicity Criteria 
Appendix C: FDA MedWatch 3500a Form 
Appendix D: NYHA Guidelines 
Appendix E: Procedure for Obtaining a Urine Protein / Creatinine Ratio 
Appendix F:  Collection and processes of samples 
Appendix G: GENETIC ALTERATIONS IN ENDOMETRIAL CANCERS 
Appendix H: Genotyping and analysis for allelic expression imbalance of 18 
tumor suppressor genes. 
 
3


 
STUDY FLOW CHART/SCHEMA 
 
Compressively surgically staged patients with advanced endometrial cancer 
(stage III-IV) with non-measurable or measurable disease 
 
Regimen 
Carboplatin (AUC 5) plus Paclitaxel (175 mg/m2 over 3 hours) plus 
Bevacizumab (15 mg/kg) every three weeks for 6 cycles 
 
Follow-up 
Measurable disease: CT-scan/MRI/US Following cycle 3 and 6 
Non-measurable disease: CT after 6 cycles 
All patients: Clinical exam every 3 months for 2 years then 6 months for an 
additional three years and annually thereafter 
 
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1.0 
BACKGROUND 
1.1 
DISEASE BACKGROUND  
In the United States, adenocarcinoma of the endometrium remains the 
most common malignancy of the female genital tract. Approximately 
40,880 new cases were diagnosed in 2005, resulting in about 7,310 
deaths. Five-year survival rates exceed 77% largely due to the ability to 
diagnose women with endometrial cancer at an early stage. Seventy-five 
percent of women are Stage I (tumor confined to the uterus) at the time of 
diagnosis. However, when the disease is diagnosed at more advanced 
stages or if the disease recurs, prognosis is very poor with cure rates less 
than 15%.1 Clearly improvements are needed in primary therapeutic 
strategies. 
After 
initial 
surgical 
diagnosis, 
the 
standard 
primary 
systemic 
chemotherapy for women with advanced endometrial cancer consists of 
chemotherapy. The optimal regimen remains under investigation. When 
endometrial cancer has spread beyond the uterine cavity and the draining 
lymph node basins, the regions at risk for recurrence expand greatly. 
Patients with positive pelvic washings or adnexal involvement and women 
with Stage IV or recurrent disease are at risk for tumor spread in a pattern 
similar to that of women with ovarian cancer. These patients are at 
significant risk for the development of intraabdominal carcinomatosis as 
well as hepatic, pulmonary, bone, and central nervous system 
metastases. Treatment strategies have therefore, focused on this 
extended area of risk and include: whole abdominal radiotherapy with or 
without sensitizing chemotherapy, hormonal therapy, and systemic 
cytotoxic chemotherapy. Prior to 2002, the control arm of collaborative 
group trials studying the treatment of women with measurable Stage III, 
Stage IV, and recurrent disease was cytotoxic chemotherapy utilizing 
cisplatin (50 mg/m2) and doxorubicin (60 mg/m2) for seven cycles. This 
was based on several clinical observations. First, the results of whole 
abdominal radiotherapy in women with measurable disease has been 
disappointing, particularly in women with residual tumor size greater than 
two centimeters.2,3 Secondly, the results of Gynecologic Oncology Group 
 
5


(GOG) Protocols #107 & #163 have suggested that the combination of 
cisplatin/doxorubicin is superior to single-agent therapy and not worse 
than all other combinations tested. In these two GOG chemotherapy trials, 
the combination of cisplatin/doxorubicin had a favorable toxicity profile 
with a response rate and PFS superior to doxorubicin as a single agent 
and similar to the combination of doxorubicin/paclitaxel. In GOG Protocol 
#107, the overall response rate to cisplatin/doxorubicin was 42% with a 
complete response rate of 19%. Median progression-free survival (PFS) 
was 4.8 months.4 In GOG Protocol #163, complete response to 
cisplatin/doxorubicin was 15% with an overall response rate of 40%.This 
was 
not 
significantly 
different 
from 
the 
experimental 
arm 
of 
doxorubicin/paclitaxel. Median PFS was 7.2 months. 5,6    
The most recently completed GOG trial in advanced and recurrent 
endometrial cancer (GOG 177) was published in 2004.7 In this trial, 
cisplatin/doxorubicin 
was 
compared 
to 
the 
triplet 
of 
cisplatin/doxorubicin/paclitaxel (TAP). The conclusions of this trial were 
that TAP improved progression-free survival as well as overall survival in 
patients with advanced and recurrent endometrial cancer compared to 
treatment with cisplatin/doxorubicin. The improvement in median 
progression free survival and median survival was 3 months in the TAP 
arm. In summary, the best expected result with current cytotoxic 
chemotherapies is a complete response rate of 22% and a median PFS of 
approximately nine months in patients with measurable and recurrent 
disease. In patients with advanced endometrial cancer without 
measurable disease (GOG 122) the regimen of cisplatin/ doxorubicin was 
compared to whole abdomen radiation. Chemotherapy was superior  to 
radiation with PFS and overall survival improved approximately 30%. The 
PFS at 24 months for the entire population studied was approximately 
50%. 7.5 
Simultaneous to the evolution of treatment for patients with advanced and 
recurrent endometrial cancer in the GOG trials described above, a 
significant segment of the community has adopted a treatment regimen 
consisting of carboplatin and paclitaxel.  Use of this combination is largely 
based on physicians’ vast experience with this regimen’s tolerability and 
efficacy in ovarian cancer. Although the regimen has not been tested in a 
 
6


phase III setting in endometrial cancer patients, its efficacy has been 
addressed in phase II studies.8-10 Hoskins and co-workers reported a 50% 
to 78% response rate in patients stratified by risk.8 In this study, toxicity 
was minimal and median failure-free interval varied by risk group (6 – 23 
months). Nakamura and coworkers reported similar results in their phase 
II trial.9 Their series of 18 patients with advanced endometrial carcinoma 
treated with carboplatin (AUC = 5 – 6) and paclitaxel (180 mg/m2), a 
complete response rate of 45% was achieved. Finally, Price and 
coworkers reported a 63% response rate in their series of 20 patients 
treated with carboplatin (AUC = 5) and paclitaxel (135 – 175 mg/m2).10 
Currently the GOG is conducting a randomized phase III trial evaluating 
doxorubicin/ cisplatin/paclitaxel versus carboplatin/paclitaxel in patients 
with stage III and IV or recurrent endometrial cancer. In addition, the GOG 
has supported a randomized prospective trial using carboplatin/paclitaxel 
as the standard therapy in high risk endometrial histological subtypes 
(three versus six cycles).  
Rationale for Angiogenesis -Targeted Therapeutics: 
Angiogenesis is one of the cardinal processes leading to invasion and 
metastasis of solid tumors. The angiogenic-signaling pathway may be 
triggered by the release of angiogenic promoters such as vascular 
endothelial growth factor (VEGF) from tumor cells into the local 
microenvironment. There is evidence that angiogenesis plays a role in 
endometrial cancer disease progression and prognosis.11-16 Given that a 
relationship has been demonstrated between the expression of 
biomarkers of angiogenesis and the behavior of endometrial cancer, it is 
logical to test pharmacological inhibitors of angiogenesis. Neutralizing 
anti-VEGF monoclonal antibodies have demonstrated therapeutic activity 
in a variety of pre-clinical solid tumor models.17, 18 The GOG has 
supported this concept with the design of phase II trial evaluating 
Bevacizumab in the treatment of recurrent or persistent endometrial 
carcinoma. 
Given the poor outcomes of advanced endometrial cancer patients with 
standard chemotherapy regimens and the pre-clinical evidence that 
angiogenesis plays a role in prognosis we have designed a Phase II 
 
7


protocol evaluating the regimen of Carboplatin (AUC 5) and paclitaxel 
(175 mg/m2 over 3 hrs) combined with bevacizumab (15 mg/kg) given 
every three weeks in patients with advanced stage endometrial cancer.  
 
1.2 
BEVACIZUMAB CLINICAL EXPERIENCE     
Bevacizumab has been studied in a multitude of Phase I, II, and III clinical 
trials in more than 5000 patients and in multiple tumor types. The following 
discussion summarizes bevacizumab’s safety profile and presents some 
of the efficacy results pertinent to this particular trial. ).  Approximately 
130,000 patients have been exposed to bevacizumab as a marketed 
product or in clinical trials.  The following discussion summarizes 
bevacizumab’s safety profile and presents some of the efficacy results 
pertinent to this particular trial. Please refer to the bevacizumab 
Investigator Brochure for descriptions of all completed Phase I, II, and III 
trials reported to date. (Bevacizumab Investigator Brochure, October 
2005) 
In a large phase III study (AVF2107g) in patients with metastatic colorectal 
cancer, the addition of bevacizumab, a monoclonal antibody directed 
against vascular endothelial growth factor (VEGF), to irinotecan/5-
fluorouracil/leucovorin (IFL) chemotherapy resulted in a clinically and 
statistically significant increase in duration of survival, with a hazard ratio 
of death of 0.67 (median survival 15.6 vs. 20.3 months; p < 0.001).  
Similar increases were seen in progression-free survival (6.2 vs. 10.6 
months; p < 0.001), overall response rate (35% vs. 45%; p < 0.01) and 
duration of response (7.1 vs. 10.4 months; p < 0.01) for the combination 
arm versus the chemotherapy only arm (bevacizumab Investigator 
Brochure, October 2005). 
Based on the survival advantage demonstrated in Study AVF2107g, 
bevacizumab was designated for priority review and was approved on 26 
February 2004 in the United States for first-line treatment in combination 
with IV 5-FU−based chemotherapy for subjects with metastatic colorectal 
cancer. (http://www.fda.gov/cder/foi/nda/2004/STN-125085_Avastin.htm) 
 
8


 
Additional data from Phase III trials in metastatic CRC (E3200), 
non−small cell lung cancer (NSCLC; E4599), and metastatic breast cancer 
(E2100) have also demonstrated clinical benefit from bevacizumab when 
added to chemotherapy.  In Study E3200, the addition of bevacizumab to 
FOLFOX chemotherapy resulted in improved overall survival compared 
with FOLFOX alone (13.0 vs. 10.8 months, respectively, HR = 0.75; 
p < 0.01) in a population of previously treated CRC patients.   
There was also improved overall survival in first-line NSCLC patients 
(E4599) treated with carboplatin/paclitaxel + bevacizumab compared with 
chemotherapy alone (12.3 vs. 10.3 months, respectively; HR = 0.80; 
p = 0.003).  The results from this trial were the basis for FDA approval of 
bevacizumab for use in combination with carboplatin + paclitaxel as first-
line treatment of patients with unresectable, locally advanced, recurrent or 
metastatic, non-squamous NSCLC in October 2006.  Finally, patients with 
untreated metastatic breast cancer (E2100) who received bevacizumab in 
combination with weekly paclitaxel had a marked improvement in PFS 
compared with chemotherapy alone (13.3 vs. 6.7 months, respectively; 
HR = 0.48; p < 0.0001) (see the Bevacizumab Investigator Brochure for 
additional details). 
 
 
 
1.3 Translational Research Related to Anti-VEGF Therapy 
Markers of Angiogenesis 
Vascular endothelial growth factor (VEGF) and basic fibroblast growth 
factor (bFGF) are among the most well studied angiogenic growth factors. 
In tumors, angiogenesis has been studied by quantifying the tumor blood 
micro-vessel density (MVD) determined immunohistochemically using 
antibodies to CD31, a protein expressed on the surface of vascular 
endothelial cells. MVD has been shown to predict the response of gastric 
adenocarcinomas to taxane-based therapy.20 In addition to MVD, most 
angiogenesis studies also evaluate VEGF, which has been shown to 
promote neovascularization and stimulate endothelial cell survival. 21 
VEGF levels were also found to correlate with MVD in endometrial and 
cervical, but not ovarian cancers. 22,24 In ovarian cancer, higher  VEGF 
 
9


levels, but not MVD, were found to significantly correlate with decreased 
patient survival. 23 Multivariate analysis demonstrated that VEGF was an 
independent prognostic indicator of overall survival. 23 Additionally, resting 
and activated circulating endothelial cells (CECs) were found to be 
elevated in newly diagnosed cancer patients (lymphoma) and declined 
with curative therapy.25  Though this has not been studied in endometrial 
cancer patients, this is a potential novel surrogate marker for 
angiogenesis. 25 
Immunohistochemistry will be utilized to evaluate the expression of CD-31 
and VEGF in previously untreated primary or metastatic tumor tissue. 
Expression of these angiogenic markers will be examined in conventional 
unstained tumor sections compared with tissue microarrays (TMAs). An 
analysis will be undertaken to assess the relationship between tumor 
tissue expression of angiogenic markers and clinical outcome including 
tumor response, progression free survival and overall survival in this 
patient population in the TMAs created for this trial if appropriate. If not, 
conventional unstained tissue sections will be used to examine the 
relationship between the angiogenic markers and clinical outcome in 
patients participating in this treatment protocol. The exact choice of 
biomarkers to be evaluated and assays to be performed in tumor and/or 
serum specimens will be reevaluated based on evolving data in the field. 
Serum and plasma will be obtained prior to therapy and prior to each cycle 
of chemotherapy to evaluate if levels of angiogenin, bFGF, VEGF, and 
VCAM-1 can predict over time if these levels are related to therapy or 
response to therapy. This is an exploratory analysis to attempt to generate 
further hypothesis. 
 
Genomic Analysis: Possible role of tumor suppressor genes (TSGs) in 
endometrial cancer.   
Several TSGs have already been implicated in endometrial cancer, either 
in germ line or sporadic, such as pTEN, p53, MSH2, MLH1, and p16 
(Appendix H).  Polymorphisms that reduce expression of these genes 
could lead to increased risk.    Commonly, one assumes that both alleles 
must be lost in somatic cells before a TSG contributes to tumor 
 
10


transformation or progression.  However, with one allele yielding reduced 
levels, loss of the high-expressing allele may suffice for progression.  
Such amber mutations are difficult to detect, but in the case of 
adenomatous polyposis coli tumor suppressor gene (APC) 26, this concept 
has been validated in colon cancer.  Therefore Yan et al. 26 had called for 
the development of a rapid assay to measure such allelic expression 
imbalance (AEI), as a potential contributor to disease risk.  We have 
successfully developed such an assay, measuring the allelic ratios for 
genomic DNA and mRNA in tissue samples.  This has already led to the 
characterization of the functional polymorphisms in two genes, namely 
MDR1 and OPRM1. 27,28 In this project we will use AEI assays for 18 
TSGs – already fully developed and validated.  This has already revealed 
the presence of relatively frequent AEI for three genes in normal subjects 
(transformed B lymphocytes), one of which being p16 (5% frequency of 
AEI in normals). Even where no AEI is detectable in normal subjects (only 
15-30 subjects have been tested thus far for each gene),  we expect germ 
line AEI to more frequent in endometrial cancer patients if the TSG plays a 
role.    Moreover, we expect to find the high-expressing gene to be deleted 
in the tumor tissues as reported for APC.26 This specific set of predictions 
provides a powerful means of sorting out those functional polymorphisms 
and genes with an immediate role in endometrial cancer. 
 
Endometrial cancer is a monoclonal disease and is characterized by a 
high degree of genetic damage that is manifest both at the karyotypic and 
the molecular levels.  It is unclear whether the severity of these alterations 
reflects the need to inactivate multiple genes or is the result of wide 
spread loss of genomic stability.  Specific genes that are altered during the 
development of endometrial cancers are illustrated in Appendix H   
Since only 5% of endometrial cancers are hereditary, most of the 
molecular alterations involved in endometrial carcinogenesis are thought 
to be associated with sporadic tumors.  Alteration of the PTEN and p53 
tumor suppressor gene is the most frequent genetic event described thus 
far in endometrial cancers and has been associated a worse prognosis 
 
11


30,31 Extensive genotyping in both normal and tumor tissues can yield 
additional information on a patient’s tumor characteristics and prognosis. 
The availability of extensive genotyping panels at the Pharmacogenomics 
Laboratory affords the opportunity to search broadly for genetic variants 
affecting disease risk and treatment response. More than 180 
polymorphisms can be measured for pharmacogenetic studies, involving 
drug metabolizing enzymes, transporters, and drug targets.  Moreover the 
panels currently include 70 genes involved in tumor biology.  While we will 
broadly run these panels for most samples, the present project focuses on 
18 TSGs, a rich target implicated in all cancers but in need of novel 
approaches capable of revealing their true role in cancer and treatment.  
This TSG list does not include p53, because this gene harbors no germ 
line SNP in the transcribed region of sufficient frequency that can serve as 
a marker for AEI.  Also, MLH1 is as yet missing (Appendix H), but will be 
added for this project.  Focus on TSGs in this context promises maximal 
return, and provides the basis for more extensive studies in the future. 
 
1.4 
STUDY RATIONALE 
 
Limited numbers of patients have been treated previously on phase II 
trials with carboplatin/paclitaxel therefore  consideration was made to 
performing a randomized phase II to further characterize the efficacy of 
this treatment regimen. However, there is an ongoing GOG Phase III trial 
evaluating 
paclitaxel/cisplatin/doxorubicin 
compared 
to 
carboplatin/paclitaxel which will provide a contemporary treatment group 
that will permit a reasonable estimate of the contribution of bevacizumab 
to the treatment outcomes (eligibility criteria will be matched in the current 
trial to that ongoing phase III trial). Given the poor outcomes of advanced 
endometrial cancer patients with standard chemotherapy regimens and 
the pre-clinical evidence that angiogenesis plays a role in prognosis we 
have designed a Phase II protocol evaluating the regimen of Carboplatin 
(AUC 5) and paclitaxel (175 mg/m2 over 3 hrs) combined with 
bevacizumab (15 mg/kg) given every three weeks in patients with 
advanced stage endometrial cancer.  
 
 
12


 
2.0 
OBJECTIVES 
 
2.1 
Primary 
 
2.11 To assess the progression free survival at 24 months after initiating 
treatment 
for 
advanced 
endometrial 
cancer 
treated 
with 
carboplatin/paclitaxel/bevacizumab. 
2.12 
To 
determine 
the 
toxicity 
profile 
of 
carboplatin/ 
paclitaxel/bevacizumab as assessed by NCI Common Toxicity Criteria for 
Adverse Events Version 3.0 (CTCAE) in this cohort of patients. 
 
2.2 
Secondary  
 
2.21 To estimate overall survival for patients treated with this regimen. 
2.22 To assess objective tumor response using modified RECIST criteria 
(see section 3.2.2.1). 
 
2.3 
Translational Research Objectives (see Appendix G,H,I for description) 
 
2.31 Determine the levels of specific biomarkers 
2.311 Use immunohistochemistry assays to evaluate the levels of 
vascular endothelial growth factor (VEGF), CD-31, thrombospodin-
1 (TSP-1), and CD-105 (endoglin) in pre-treatment tumor tissue 
and correlate with clinical response in pre-treatment tissue (from 
recent surgery). 
2.312 Use enzyme-linked immunosorbent assays (ELISA) to 
quantify the level of angiogenin, basic fibroblast growth factor 
(bFGF), VEGF, and soluble vascular cell adhesion molecule-I 
(VCAM-I) in serum obtained prior to initiation of therapy, prior to 
each additional cycle of chemotherapy and through follow up (every 
3 months until recurrence or 3 years). This will be used to evaluate 
the changes in these levels over time.  
 
13


2.313 Use ELISA to measure the level of VEGF in plasma obtained 
prior to initiation of therapy prior to each additional cycle of 
chemotherapy and through follow up. This will be used to evaluate 
the changes in these levels over time. 
 
2.32 Perform exploratory data analysis 
2.321 Exploratory analysis to develop and test a high through-put 
system for the identification of genetic polymorphisms in endometrial 
cancer tissue and blood. (see Appendix I) 
 
3.0 
STUDY DESIGN  
3.1 
Description of the Study 
 
This is a single center open label single arm Phase II clinical trial with no 
control group.  A regimen of Carboplatin (AUC 5 IV over 30 minutes) and 
paclitaxel (175 mg/m2 over 3 hrs) combined with bevacizumab (15 mg/kg IV) 
given every 21 days in patients with advanced stage endometrial cancer for a 
maximum of 6 cycles. 
3.2  
Outcome Measures  
 
3.2.1 Primary Outcome Measures: 
 
3.2.1.1 
Progression-Free Survival is the period from study entry until 
disease progression or date of last contact. 
3.2.1.2 
Toxicity profile of the triple drug combination therapy as 
assessed by CTCAE v3.(Appendix C) 
3.2.2 Secondary Outcome Measures 
3.2.2.1 Measurable disease is defined as at least one lesion that can be 
accurately measured in at least one dimension (longest dimension to be 
recorded). Patients with measurable disease must have at least one 
“target lesion” to be used to assess response on this protocol as defined 
by RECIST (target lesion must be greater than 1 cm by spiral CT).  
Measurable disease, baseline documentation of “target” and “non-target” 
 
14


lesions, follow-up documentation of “target” and “non-target” lesions and 
documentation of the best response achieved (complete response, partial 
response, stable disease) or of progression of disease will be required. 
3.2.2.1.1 Complete Response (CR) is disappearance of 
gross evidence of disease with confirmation at least 4 weeks 
later. 
 
3.2.2.1.2 Partial Response (PR) is a 30% or greater 
reduction in measurement of longest dimension of each 
lesion with confirmation at least 4 weeks later..   
 
3.2.2.1.3 Progressive Disease (PD) at least a 20% increase 
in the sum of the longest dimension of target lesions, taking 
as reference the smallest sum of the longest dimension 
recorded since the treatment start or the appearance of one 
or more new lesions. 
3.2.2.1.4 Stable Disease is any condition not meeting the 
above criteria. 
 
3.2.2.3 Overall Survival is the duration of time from initiation of treatment to 
death or the date of last contact.  
Evaluation of best overall response will be evaluated as follows: 
The best overall response is the best response recorded from the start of the treatment until disease 
progression/recurrence (taking as reference for PD the smallest measurements recorded since the treatment 
started). In general, the patient's best response assignment will depend on the achievement of both 
measurement and confirmation criteria  
 
Target lesions 
Non-Target lesions 
New Lesions 
Overall response 
CR 
CR 
No 
CR 
CR 
Incomplete response/SD 
No 
PR 
PR 
Non-PD 
No 
PR 
SD 
Non-PD 
No 
SD 
PD 
Any 
Yes or No 
PD 
 
15


Any 
PD* 
No 
PD 
Any 
Any 
Yes 
PD 
 
 
 
 
 
*Unequivocal progression of existing non-target lesions, other than pleural effusions without 
cytological proof of neoplastic origin, in the opinion of the treating physician (in this circumstance 
an explanation must be provided) 
• 
Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be classified as having 
“symptomatic deterioration”. Every effort should be made to document the objective 
progression even after discontinuation of treatment.  
• 
In some circumstances it may be difficult to distinguish residual disease from normal tissue. 
When the evaluation of complete response depends on this determination, it is recommended 
that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the complete 
response status. 
 
 
 
4.0 
SAFETY PLAN 
See Section 4.1 for complete details of the safety evaluation for this study.  
Frequency and severity of adverse effects as assessed by CTCAE v3 for 
the triple drug combination.  The frequency of all toxicities will be tabulated 
from submitted case report forms and summarized for review. All serious 
and/or unexpected events will be communicated to FDA, Genentech, Inc 
and to the OSU Cancer IRB at the James Cancer Hospital and Solove 
Research Institute.  See section 12.2 for details. 
4.1 
GENERAL PLAN TO MANAGE SAFETY  
a. Bevacizumab-Specific 
A number of measures will be taken to ensure the safety of patients 
participating in this trial.  These measures will be addressed through 
exclusion criteria (see Section 5.3) and routine monitoring as follows. 
Patients enrolled in this study will be evaluated clinically and with standard 
laboratory tests before and at regular intervals during their participation in 
this study.  Safety evaluations will consist of medical interviews, recording 
 
16


of adverse events, physical examinations, blood pressure, and laboratory.  
Patients will be evaluated for adverse events (all grades), serious adverse 
events, and adverse events requiring study drug interruption or 
discontinuation at each study visit for the duration of their participation in 
the study.  Patients discontinued from the treatment phase of the study for 
any reason will be evaluated ~30 days (28–42 days) after the decision to 
discontinue treatment (see Section 7.1.3).   
Specific monitoring procedures are as follows: 
• Hypertension will be monitored through routine evaluation of blood 
pressure prior to each bevacizumab treatment.  Optimal control of 
blood pressure according to standard public health guidelines is 
recommended for patients on treatment with or without bevacizumab. 
• Proteinuria will be monitored by urine protein:creatinine (UPC) 
ratio.See  section 9.0 Clinical and Laboratory Evaluations and 
Appendix E: Procedure for Obtaining a urine protein/creatinine ratio. 
 
• If patients on treatment with bevacizumab require elective major 
surgery, it is recommended that bevacizumab be held for 4-8 weeks 
prior to the surgical procedure. Patients undergoing a major surgical 
procedure should not begin/restart bevacizumab until 4 weeks after 
that procedure (in the case of high risk procedures such as liver 
resection, thoracotomy, or neurosurgery, it is recommended that 
chemotherapy be restarted no earlier than 6 wk and bevacizumab no 
earlier than 8 wk after surgery). 
Please see Section 8.0 for detailed instructions for the management of 
study drug–related toxicities. 
5.0 
STUDY SUBJECTS 
5.1 
Subject Selection 
5.1.1 All patients with advanced endometrial carcinoma, of any histology 
including clear cell and serous papillary carcinomas. Surgical stage III and 
IV disease, including those patients with positive adnexa, tumor invading 
the serosa, positive pelvic and/or para-aortic nodes, involvement of bowel 
mucosa, intra-abdominal metastases, positive pelvic washings or vaginal 
involvement. 
 
17


5.1.2 Surgery must have included a hysterectomy, and bilateral salpingo-
oophorectomy. Selective pelvic and para-aortic lymph node sampling is 
optional however strongly encouraged. Chemotherapy will be initiated 
within 12 weeks after surgery and the patient must have fully recovered 
from surgery. 
5.1.3 All patients and/or guardians must have signed an informed consent 
prior to entry into the study. 
5.2 
Inclusion Criteria 
 
5.2.1 All patients with advanced endometrial carcinoma, of any histology 
including clear cell and serous papillary carcinomas. Surgical stage III and 
IV disease, including those patients with positive adnexa, tumor invading 
the serosa, positive pelvic and/or para-aortic nodes, involvement of bowel 
mucosa, intra-abdominal metastases, positive pelvic washings or vaginal 
involvement. 
5.2.2 Patients must have adequate end-organ function as follows: 
5.2.2.1 Bone marrow function: Absolute neutrophil count (ANC) 
greater than or equal to 1,500/mcl and WBC ≥ 3.0, equivalent to 
Common Terminology Criteria for Adverse Events (CTCAE v3.0) 
grade 1. Platelets greater than or equal to 100,000/mcl. 
5.2.2.2 Renal function: creatinine less than or equal to 1.5 x 
institutional upper limit normal (ULN), CTCAE v3.0 grade 1. 
5.2.2.3 Hepatic function: Bilirubin less than or equal to 1.5 x ULN 
(CTCAE v3.0 grade 1). SGOT and alkaline phosphatase less than 
or equal to 2.5 x ULN (CTCAE v3.0 grade 1). 
5.2.2.4 Urine Protein Creatinine: Urine protein creatinine (UPC) 
ratio must be < 1.0 gm. If UPC ratio > 1, collection of 24-hour urine 
measurement of urine protein is recommended.  
UPC ratio of spot urine is an estimation of the 24 urine 
protein excretion – a UPC ratio of 1 is roughly equivalent to a 
24-hour urine protein of 1 gm. UPC ratio is calculated using 
one of the following formulas: 
 
18


1. [urine protein]/[urine creatinine] – if both protein and 
creatinine are reported in mg/dL 
2. [(urine protein) x0.088]/[urine creatinine] – if urine 
creatinine is reported in mmol/L 
5.2.2.5 Blood coagulation parameters: Blood coagulation 
parameters: PT such that international normalized ratio (INR) is < 
1.5 (or an in-range INR, usually between 2 and 3, if a patient is on a 
stable dose of therapeutic warfarin) and a PTT < 1.5 times the 
institutional upper limit of normal. 
 
 
5.2.3 Patients must have a GOG Performance Status of 0, 1, or 2. 
5.2.4 Patients must have signed an approved informed consent and 
authorization 
5.2.5 Patients must be 18 years of age or older. 
5.2.6 Patients may have received radiation for the treatment of 
endometrial cancer.  At least four weeks should have elapsed since 
completion of RT involving the whole pelvis or over 50% of the spine. 
5.2.7 Patients must be enrolled between 1 and 12 weeks after initial 
surgery performed for the combined purpose of diagnosis, staging and 
cytoreduction (not to receive chemotherapy for at least 4 weeks following 
surgery). 
5.2.8 Patients with measurable and non-measurable disease are eligible. 
Patients may or may not have cancer-related symptoms. 
 
 
 
5.3  
Exclusion Criteria 
a. Disease-Specific Exclusions 
 
19


• Patients with a concomitant malignancy other than non-melanoma 
skin cancer. Patients with a prior malignancy who have been 
disease-free for less than 5 years will be excluded. 
• Patients with concomitant medical illness such as serious 
uncontrolled infection, uncontrolled angina, or serious peripheral 
neuropathy, which, in the opinion of the treating physician, make 
the treatments prescribed on this study unreasonably hazardous for 
the patient. 
• Patients whose circumstances will not permit study completion or 
adequate follow-up. 
• Patients who have received prior cytotoxic chemotherapy for the 
treatment of endometrial cancer, including chemotherapy used for 
radiation sensitization. Patients may have received prior hormonal 
therapy or therapy with biologic agents, but such therapies must be 
discontinued prior to entry on this study and prior to registration. 
 
b. General Medical Exclusions 
Subjects meeting any of the following criteria are ineligible for study entry:   
• Inability to comply with study and/or follow-up procedures 
• Current, recent (within 4 weeks of the first infusion of this study), or 
planned participation in an experimental drug study other than a 
Genentech Sponsored bevacizumab cancer study. 
• Life expectancy of less than 12 weeks. 
• Active malignancy, other than superficial basal cell and superficial 
squamous (skin) cell. 
 
 
20


c. Bevacizumab-Specific Exclusions 
• Inadequately controlled hypertension (defined as systolic blood 
pressure >150 and/or diastolic blood pressure > 100 mmHg) 
• Any prior history of hypertensive crisis or hypertensive 
encephalopathy 
• New York Heart Association (NYHA) Grade II or greater congestive 
heart failure (see Appendix E) 
• Known CNS disease, except for treated brain metastasis 
Treated brain metastases are defined as having no evidence of 
progression or hemorrhage after treatment and no ongoing 
requirement for dexamethasone, as ascertained by clinical 
examination and brain imaging (MRI or CT) during the screening 
period.  Anticonvulsants (stable dose) are allowed.  Treatment for 
brain metastases may include whole brain radiotherapy (WBRT), 
radiosurgery (RS; Gamma Knife, LINAC, or equivalent) or a 
combination as deemed appropriate by the treating physician.  
Patients with CNS metastases treated by neurosurgical resection or 
brain biopsy performed within 3 months prior to Day 1 will be 
excluded 
• Significant vascular disease (e.g., aortic aneurysm, requiring 
surgical repair or recent peripheral arterial thrombosis) within 6 
months prior to Day 1. 
• History of hemoptysis (>1/2 teaspoon of bright red blood per 
episode) within 1 month prior to Day 1. 
• Symptomatic peripheral vascular disease  
• Evidence of bleeding diathesis or significant coagulopathy ( in the 
absence of therapeutic anticoagulation) 
• Major surgical procedure, open biopsy, or significant traumatic 
injury within 28 days prior to Day 1  or anticipation of need for major 
surgical procedure during the course of the study (Enrollment is 
 
21


acceptable however chemotherapy should not be initiated earlier 
than 28 days following surgery) 
• Core biopsy or other minor surgical procedure within 7 days prior to 
study enrollment (patients may have had minor surgical procedures 
but will need to allow for 7 days prior to enrollment). excluding 
placement of a vascular access device (no delay needed) 
• History of abdominal fistula, gastrointestinal perforation, or intra-
abdominal abscess within 6 months prior to Day 1. 
• Serious, non-healing wound, ulcer, or bone fracture 
• Proteinuria at screening as demonstrated by either 
• Urine protein:creatinine (UPC) ratio  ≥ 1.0 at screening OR  
• Urine dipstick for proteinuria ≥ 2+ (patients discovered to 
have ≥2+ proteinuria on dipstick urinalysis at baseline should 
undergo a 24 hour urine collection and must demonstrate ≤ 
1g of protein in 24 hours to be eligible). 
• Known hypersensitivity to any component of bevacizumab  
• any history of stroke or transient ischemic attack at any time 
• Pregnant (positive pregnancy test) or lactating. Use of effective 
means of contraception (men and women) in subjects of child-
bearing potential is required. 
• history of myocardial infarction or unstable angina within 12 months 
of study enrollment  
• Patients who have received prior therapy with any anti-VEGF drug, 
including Bevacizumab. 
 
6.0 
TREATMENT PLAN 
 
6.1 
Treatment Plan 
 
 
This is a single center open label single arm Phase II clinical trial with no control 
group.  A regimen of Carboplatin (AUC 5 IV over 30 minutes) and paclitaxel (175 
mg/m2 over 3 hrs) combined with bevacizumab (15 mg/kg IV) given every 21 
 
22


days in patients with advanced stage endometrial cancer for a maximum of 6 
cycles 
 
6.2  
Accrual 
Thirty-eight patients will be treated on protocol (inevaluable patients to be 
replaced at one to one). There are currently five full-time gynecologic oncologists 
at our institution performing over five hundred major oncology surgeries annually. 
Approximately 30-40% are endometrial malignancy cases, yielding about 150-
200 primary endometrial cancer patients annually.  Approximately 20-30 (15%) of 
these patients will be diagnosed with stage III-IV endometrial cancer. In addition, 
two other academic gynecologic practices with high clinical volumes have 
committed to the current protocol if single institution recruitment does not meet 
goals (minimum of 12 patients annually). We estimate recruitment to be 
completed in approximately 2 years. Patients will be followed for a maximum of 5 
years or until death. Approximate date of completion is 2013, but mature data 
should be available by 2010. 
 
 
 
 
7.0 
STUDY MEDICATION  
 
7.1  
BEVACIZUMAB DOSAGE AND FORMULATION 
Bevacizumab is to be given at 15 mg/kg every 3 weeks for 6 cycles. 
Bevacizumab is a clear to slightly opalescent, colorless to pale brown, sterile 
liquid concentrate for solution for intravenous (IV) infusion.  Bevacizumab may be 
supplied in 5-cc (100-mg), 20-cc (400-mg), and 50-cc (1000-mg) glass vials 
containing 4 mL, 16 mL, or 40 mL of bevacizumab, respectively (all at 25 
mg/mL).  Vials contain bevacizumab with phosphate, trehalose, polysorbate 20, 
and Sterile Water for Injection (SWFI), USP.  Vials contain no preservative and 
are suitable for single use only. 
 
23


a. Safety Profile 
In the initial Phase I and II clinical trials, four potential bevacizumab-
associated safety signals were identified: hypertension, proteinuria, 
thromboembolic events, and hemorrhage.  Additional completed Phase II 
and Phase III studies of bevacizumab as well as spontaneous reports 
have further defined the safety profile of this agent. Bevacizumab-
associated adverse events identified in phase III trials include congestive 
heart 
failure 
(CHF), 
gastrointestinal 
perforations, 
wound 
healing 
complications, and arterial thromboembolic events (ATE). These and other 
safety signals are described in further detail as follows and in the 
bevacizumab Investigator Brochure. 
Hypertension: An increased incidence of hypertension has been 
observed in patients treated with bevacizumab.  Grade 4 and 5 
hypertensive events are rare. Clinical sequelae of hypertension are rare 
but have included hypertensive crisis, hypertensive encephalopathy, and 
reversible posterior leukoencephalopathy syndrome (RPLS) (Ozcan et al., 
2006; Glusker et al., 2006). RPLS may include signs and symptoms of 
headache, altered mental function, seizures, and visual disturbances / 
cortical blindness and requires treatment, which should include control of 
hypertension, management of specific symptoms, and discontinuation of 
bevacizumab. 
There is no information on the effect of bevacizumab in patients with 
uncontrolled hypertension at the time of initiating bevacizumab therapy.  
Therefore, caution should be exercised before initiating bevacizumab 
therapy in these patients.  Monitoring of blood pressure is recommended 
during bevacizumab therapy.  Optimal control of blood pressure according 
to standard public health guidelines is recommended for patients on 
treatment with or without bevacizumab.   
Temporary interruption of bevacizumab therapy is recommended in 
patients with hypertension requiring medical therapy until adequate control 
is achieved.  If hypertension cannot be controlled with medical therapy, 
bevacizumab therapy should be permanently discontinued.  Bevacizumab 
should be permanently discontinued in patients who develop hypertensive 
crisis or hypertensive encephalopathy. 
 
24


 
Proteinuria: An increased incidence of proteinuria has been observed in 
patients treated with bevacizumab compared with control arm patients.  In 
the bevacizumab-containing treatment arms of clinical trials (across all 
indications), the incidence of proteinuria (reported as an adverse event) 
was up to 38% (metastatic CRC Study AVF2192g). The severity of 
proteinuria has ranged from asymptomatic and transient events detected 
on routine dipstick urinalysis to nephrotic syndrome; the majority of 
proteinuria events have been grade 1. NCI-CTC Grade 3 proteinuria was 
reported in up to 3% of bevacizumab-treated patients, and Grade 4 in up 
to 1.4% of bevacizumab-treated patients.  The proteinuria seen in 
bevacizumab clinical trials was not associated with renal impairment and 
rarely required permanent discontinuation of bevacizumab therapy.  
Bevacizumab should be discontinued in patients who develop Grade 4 
proteinuria (nephrotic syndrome) 
Patients with a history of hypertension may be at increased risk for the 
development of proteinuria when treated with bevacizumab.  There is 
evidence from the dose-finding, Phase II trials (AVF0780g, AVF0809s, 
and AVF0757g) suggesting that Grade 1 proteinuria may be related to 
bevacizumab dose. 
Proteinuria will be monitored by urine protein:creatinine (UPC) ratio at 
least every 6 weeks.  If the UPC ratio is not available, a dipstick urinalysis 
may be used to allow treatment to proceed. 
  
 
Thromboembolic Events: Both venous and arterial thromboembolic (TE) 
events, ranging in severity from catheter-associated phlebitis to fatal, have 
been reported in patients treated with bevacizumab in the colorectal 
cancer trials and, to a lesser extent, in patients treated with bevacizumab 
in NSCLC and breast cancer trials.  
 
 
25


Venous thromboembolism (including deep venous thrombosis, pulmonary 
embolism, and thrombophlebitis): In the phase III pivotal trial in metastatic 
CRC, there was a slightly higher rate of venous TE events that was not 
statistically significant in patients treated with bevacizumab plus 
chemotherapy compared with chemotherapy alone (19% vs. 16%). 
In Study AVF2107g, a Phase III, pivotal trial in metastatic CRC, VTE 
events, including deep venous thrombosis, pulmonary embolism, and 
thrombophlebitis, occurred in 15.2% of patients receiving chemotherapy 
alone and 16.6% of patients receiving chemotherapy + bevacizumab.  
The incidence of NCI-CTC Grade ≥ 3 venous VTE events in one NSCLC 
trial (E4599) was higher in the bevacizumab-containing arm compared to 
the chemotherapy control arm (5.6% vs. 3.2%).  One event (0.2%) was 
fatal in the bevacizumab-containing arm; not fatal events were reported in 
the carboplatin/paclitaxel arm (see Bevacizumab Investigator Brochure).  
In metastatic CRC clinical trials, the incidence of VTE events was similar 
in patients receiving chemotherapy + bevacizumab and those receiving 
the control chemotherapy alone.  
In clinical trials across all indications the overall incidence of VTE events 
was 2.8%−17.3% in the bevacizumab-containing arms compared with 
3.2%−15.6% in the chemotherapy control arms.  The use of bevacizumab 
with chemotherapy does not substantially increase the risk of VTE event 
compared with chemotherapy alone.  However, patients with metastatic 
CRC who receive bevacizumab and experienced a VTE event may be at 
higher risk for recurrence of VTE event. 
 
Arterial Thromboembotic Events: :  An increased incidence of ATE events 
was observed in patients treated with bevacizumab compared with those 
receiving control treatment.  ATE events include cerebrovascular 
accidents, myocardial infarction, transient ischemic attacks (TIAs), and 
other ATE events.  In a pooled analysis of data from five randomized 
Phase II and III trials (mCRC [AVF2107g, AVF2192g, AVF0780g]; locally 
advanced or metastatic NSCLC [AVF0757g]; metastatic breast cancer 
[AVF2119g]), the incidence rate of ATE events was 3.8% (37 of 963) in 
 
26


patients who received chemotherapy + bevacizumab compared with 1.7% 
(13 of 782) in patients treated with chemotherapy alone.  ATE events led 
to a fatal outcome in 0.8% (8 of 963) of patients treated with 
chemotherapy + bevacizumab and 0.5% (4 of 782) of patients treated with 
chemotherapy alone.  Cerebrovascular accidents (including TIAs) 
occurred in 2.3% of patients treated with chemotherapy + bevacizumab 
and 0.5% of patients treated with chemotherapy alone.  Myocardial 
infarction 
occurred 
in 
1.4% 
of 
patients 
treated 
with 
chemotherapy + bevacizumab compared with 0.7% of patients treated with 
chemotherapy alone (see the Bevacizumab Investigator Brochure for 
additional details). 
 
Aspirin is a standard therapy for primary and secondary prophylaxis of 
arterial thromboembolic events in patients at high risk of such events, and 
the use of aspirin ≤ 325 mg daily was allowed in the five randomized 
studies discussed above. Use of aspirin was assessed routinely as a 
baseline or concomitant medication in these trials, though safety analyses 
specifically regarding aspirin use were not preplanned. Due to the 
relatively small numbers of aspirin users and arterial thromboembolic 
events, retrospective analyses of the ability of aspirin to affect the risk of 
such events were inconclusive. However, similarly retrospective analyses 
suggested that the use of up to 325 mg of aspirin daily does not increase 
the risk of grade 1-2 or grade 3-4 bleeding events, and similar data with 
respect to metastatic colorectal cancer patients were presented at ASCO 
2005 (Hambleton et al., 2005). Further analyses of the effects of 
concomitant use of bevacizumab and aspirin in colorectal and other tumor 
types are ongoing. 
Gastrointestinal perforation Patients with metastatic carcinoma may be 
at increased risk for the development of gastrointestinal perforation when 
treated with bevacizumab and chemotherapy. Bevacizumab should be 
permanently discontinued in patients who develop gastrointestinal 
perforation. A causal association of intra-abdominal inflammatory process 
and gastrointestinal perforation to bevacizumab has not been established. 
Nevertheless, caution should be exercised when treating patients with 
 
27


intra-abdominal 
inflammatory 
processes 
with 
bevacizumab. 
Gastrointestinal perforation has been reported in other trials in non-
colorectal cancer populations (e.g., ovarian, renal cell, pancreas, and 
breast, and NSCLC) and may be higher in incidence in some tumor types. 
Wound healing complications: Wound healing complications such as 
wound dehiscence have been reported in patients receiving bevacizumab. 
In an analysis of pooled data from two trials in metastatic colorectal 
cancer, patients undergoing surgery 28-60 days before study treatment 
with 5-FU/LV plus bevacizumab did not appear to have an increased risk 
of wound healing complications compared to those treated with 
chemotherapy alone (Scappaticci et al., 2005). Surgery in patients 
currently receiving bevacizumab is not recommended. No definitive data 
are available to define a safe interval after bevacizumab exposure with 
respect to wound healing risk in patients receiving elective surgery; 
however, the estimated half life of bevacizumab is 20 days. Bevacizumab 
should be discontinued in patients with severe wound healing 
complications. 
If patients receiving treatment with bevacizumab require elective major 
surgery, it is recommended that bevacizumab be held for 4–8 weeks prior 
to the surgical procedure.  Patients undergoing a major surgical procedure 
should not begin or restart bevacizumab until 4 weeks after that procedure 
(in the case of high-risk procedures such as liver resection, thoracotomy, 
or neurosurgery, it is recommended that chemotherapy be restarted no 
earlier than 6 weeks and bevacizumab no earlier than 8 weeks after 
surgery). 
 
Hemorrhage: Overall, grade 3 and 4 bleeding events were observed in 
4.0% of 1132 patients treated with bevacizumab in a pooled database 
from eight phase I, II, and III clinical trials in multiple tumor types 
(bevacizumab Investigator Brochure, October 2005). The hemorrhagic 
events that have been observed in bevacizumab clinical studies were 
predominantly tumor-associated hemorrhage (see below) and minor 
mucocutaneous hemorrhage.  
 
28


Tumor-associated hemorrhage – Major or massive pulmonary 
hemorrhage or hemoptysis has been observed primarily in patients with 
NSCLC.  Life-threatening and fatal hemoptysis was identified as a 
bevacizumab-related adverse event in NSCLC trials.  These events 
occurred suddenly and presented as major or massive hemoptysis.  
Among the possible risk factors evaluated (including squamous cell 
histology, treatment with anti-rheumatic/anti-inflammatory drugs, treatment 
with anticoagulants, prior radiotherapy, bevacizumab therapy, previous 
medical history of atherosclerosis, central tumor location, and cavitation of 
tumors during therapy), the only variables that showed statistically 
significant correlations with bleeding were bevacizumab therapy and 
squamous cell histology. 
Of patients experiencing pulmonary hemorrhages requiring medical 
intervention, many had cavitation and/or necrosis of the tumor, either 
preexisting or developing during bevacizumab therapy.  Patients 
developing lung cavitation on treatment should be assessed by the 
treating physician for risk-benefit. 
In Study E4599, in which squamous cell carcinoma was excluded, the rate 
of any type of Grade ≥ 3 hemorrhage was 1.0% in the control arm 
(carboplatin and paclitaxel) versus 4.1% in the carboplatin and 
paclitaxel + bevacizumab arm (Sandler et al. 2006).   
GI hemorrhages, including rectal bleeding and melena have been reported 
in patients with CRC, and have been assessed as tumor-associated 
hemorrhages. 
Tumor-associated hemorrhages were also seen rarely in other tumor 
types and locations, including a case of CNS bleeding in a patient with 
hepatoma with occult CNS metastases and a patient who developed 
continuous oozing of blood from a thigh sarcoma with necrosis. 
Mucocutaneus Hemorrage:  Across all bevacizumab clinical trials, 
mucocutaneous hemorrhage has been seen in 20%-40% of patients 
treated with bevacizumab. These were most commonly NCI-CTC Grade 1 
epistaxis that lasted less than 5 minutes, resolved without medical 
 
29


intervention and did not require any changes in bevacizumab treatment 
regimen.  
There have also been less common events of minor mucocutaneous 
hemorrhage in other locations, such as gingival bleeding and vaginal 
bleeding.  
Reversible Posterior Leukoencephalopathy Syndrome:  There have 
been rare reports of bevacizumab-treated patients developing signs and 
symptoms that are consistent with RPLS, a rare neurologic disorder that 
can present with the following signs and symptoms (among others):  
seizures, headache, altered mental status, visual disturbance, or cortical 
blindness, with or without associated hypertension.  Brain imaging is 
mandatory to confirm the diagnosis of RPLS.  In patients who develop 
RPLS, treatment of specific symptoms, including control of hypertension, 
is recommended along with discontinuation of bevacizumab.  The safety 
of reinitiating bevacizumab therapy in patients previously experiencing 
RPLS is not known (Glusker et al. 2006; Ozcan et al. 2006).   
 
Congestive heart failure: In clinical trials CHF was observed in all cancer 
indications studied to date, but predominantly in patients with metastatic 
breast cancer.  In the Phase III clinical trial of metastatic breast cancer 
(AVF2119g), 7 (3%) bevacizumab-treated patients experienced CHF, 
compared with two (1%) control arm patients.  These events varied in 
severity from asymptomatic declines in left ventricular ejection fraction 
(LVEF) to symptomatic CHF requiring hospitalization and treatment.  All 
the patients treated with bevacizumab were previously treated with 
anthracyclines (doxorubicin cumulative dose of 240−360 mg/m2).  Many of 
these patients also had prior radiotherapy to the left chest wall.  Most of 
these patients showed improved symptoms and/or left ventricular function 
following appropriate medical therapy (Miller et al. 2005). 
In a randomized, Phase III trial of patients with previously untreated 
metastatic breast cancer (E2100), the incidence of LVEF decrease 
(defined as NCI-CTC Grade 3 or 4) in the paclitaxel + bevacizumab arm 
was 0.3% versus 0% for the paclitaxel alone arm  
 
30


No information is available on patients with preexisting CHF of New York 
Heart Association (NYHA) Class II−IV at the time of initiating bevacizumab 
therapy, as these patients were excluded from clinical trials. 
Prior anthracyclines exposure and/or prior radiotherapy to the chest wall 
may be possible risk factors for the development of CHF.  Caution should 
be exercised before initiating bevacizumab therapy in patients with these 
risk factors. 
A Phase II trial in patients with refractory acute myelogenous leukemia 
reported 5 cases of cardiac dysfunction (CHF or LVEF decrease to < 40%) 
among 48 patients treated with sequential cytarabine, mitoxantrone, and 
bevacizumab.  All but 1 of these patients had significant prior exposure to 
anthracyclines as well (Karp et al. 2004).  
 
Two additional studies investigated concurrent administration of 
anthracyclines and bevacizumab.  In 21 patients with inflammatory breast 
cancer treated with neoadjuvant docetaxel, doxorubicin, and 
bevacizumab, no patients developed clinically apparent CHF; however, 
patients had asymptomatic decreases in LVEF to < 40% (Wedam et al. 
2004).  In a small Phase II study in patients with soft tissue sarcoma, 2 of 
the 17 patients treated with bevacizumab and high-dose doxorubicin (75 
mg/m2) developed CHF (one Grade 3 event after a cumulative doxorubicin 
dose of 591 mg/m2, one Grade 4 event after a cumulative doxorubicin 
dose of 420 mg/m2); an additional 4 patients had asymptomatic decreases 
in LVEF (D’Adamo et al. 2004). 
Other studies in patients with various tumor types and either a history of 
anthracycline exposure or concomitant use with bevacizumab are 
ongoing.  
Patients receiving concomitant anthracyclines or with prior exposure to 
anthracyclines should have a baseline MUGA scans or echocardiograms 
(ECHOs) with a normal LVEF. 
Neutropenia:  Increased rates of severe neutropenia, febrile neutropenia, 
or infection with severe neutropenia (including some fatalities) have been 
 
31


observed in patients treated with some myelotoxic chemotherapy 
regimens plus bevacizumab in comparison to chemotherapy alone 
(Sandler et al. 2006). 
 
Additional Adverse Events: See the bevacizumab Investigator Brochure 
for additional details regarding the safety experience with bevacizumab. 
 
7.1.1 Bevacizumab Administration 
Bevacizumab will be diluted in 100ml of 0.9% Sodium Chloride Injection..  
Administration will be as a continuous IV infusion.  Anaphylaxis precautions 
should be observed during study drug administration.  It is not necessary to 
correct dosing based on ideal weight. 
The initial dose will be delivered over 90±15 minutes.  If the first infusion is 
tolerated without infusion-associated adverse events (fever and/or chills), the 
second infusion may be delivered over 60±10 minutes.  If the 60-minute infusion 
is well tolerated, all subsequent infusions may be delivered over 30±10 minutes.   
If a subject experiences an infusion–associated adverse event, she may be 
premedicated for the next study drug infusion; however, the infusion time may 
not be decreased for the subsequent infusion.  If the next infusion is well 
tolerated with premedication, the subsequent infusion time may then be 
decreased by 30±10 minutes as long as the subject continues to be 
premedicated.  If a subject experiences an infusion-associated adverse event 
with the 60-minute infusion, all subsequent doses should be given over 
90±15 minutes.  Similarly, if a subject experiences an infusion-associated 
adverse event with the 30-minute infusion, all subsequent doses should be given 
over 60±10 minutes. 
 
32


7.1.2 Bevacizumab Storage 
Upon receipt of the study drug, vials are to be refrigerated at 2°C–8°C  
(36°F–46°F) and should remain refrigerated until just prior to use.  DO NOT 
FREEZE.  DO NOT SHAKE.   Vials should be protected from light. 
Opened vials must be used within 8 hours.  VIALS ARE FOR SINGLE USE 
ONLY.  Vials used for 1 subject may not be used for any other subject.  Once 
study drug has been added to a bag of sterile saline, the solution must be 
administered within 8 hours. 
7.2 
PACLITAXEL (NSC #673089) 
Formulation: Paclitaxel is a poorly soluble plant product from western yew, 
Taxus brevifolia. Improved solubility requires a mixed solvent system with 
further dilutions of either 0.9% sodium chloride or 5% dextrose in water. 
Supplier/How Supplied: Commercially available. A sterile solution 
concentrate, 6 mg/ml in 5 ml vials (30 mg/vial) in polyoxyethylated castor 
oil (Cremophor EL) 50% and dehydrated alcohol, USP, 50%. The contents 
of the vial must be diluted just prior to clinical use. It is also available in 
100 and 300 mg vials. 
Solution Preparation: Paclitaxel, at the appropriate dose, will be diluted in 
500-1000 cc of 0.9% Sodium Chloride injection, USP or 5% Dextrose 
injection, USP (D5W) (500 cc’s is adequate if paclitaxel is a single agent). 
Paclitaxel must be prepared in glass or polyolefin containers due to 
leaching of diethylhexlphthalate (DEHP) plasticizer from polyvinyl chloride 
(PVC) bags and intravenous tubing by the Cremophor vehicle in which 
paclitaxel is solubilized. 
NOTE: Formation of a small number of fibers in solution (within acceptable 
limits established by the USP Particulate Matter Test for LVPs) have been 
observed after preparation of taxol. Therefore, in-line filtration is necessary 
for administration of paclitaxel solutions. In-line filtration should be 
accomplished by incorporating a hydrophilic, microporous filter of pore 
size not greater than 0.22 microns (e.g.: IVEX-II, IVEX-HP or equivalent) 
into the IV fluid pathway distal to the infusion pump   
 
33


Although particulate formation does not indicate loss of drug potency, 
solutions exhibiting excessive particulate matter formation should not be 
used. 
Storage: The intact vials can be stored in a temperature range between 2-
25oC (36-77°F). 
Stability: Commercially available paclitaxel will be labeled with an 
expiration date. All solutions of paclitaxel exhibit a slight haziness directly 
proportional to the concentration of drug and the time elapsed after 
preparation, although when prepared as described above, solutions of 
paclitaxel (0.3-1.2 mg/ml) are physically and chemically stable for 48 
hours. 24 
Administration of Paclitaxel: Paclitaxel, at the appropriate dose and 
dilution, will be given as a 3-hour continuous IV infusion. Paclitaxel will be 
administered via an infusion control device (pump) using non-PVC tubing 
and connectors, such as the IV administration sets (polyethylene or 
polyolefin) which are used to infuse parenteral Nitroglycerin. Nothing else 
is to be infused through the line where paclitaxel is being administered. 
 
Adverse Effects: 
Hematologic: Myelosuppression 
Gastrointestinal: Nausea and vomiting, diarrhea, stomatitis, mucositis, 
pharyngitis, typhlitis, ischemic colitis, neutropenic enterocolitis 
Heart: arrhythmia, heart block, ventricular tachycardia, myocardial 
infarction (MI), bradycardia, atrial arrhythmia 
Pulmonary: Pneumonitis 
Blood Pressure: Hypotension, hypertension (possibly related to 
concomitant medication--Dexamethasone) 
Neurologic: Sensory (taste), peripheral neuropathy, seizures, mood 
swings, hepatic encephalopathy, encephalopathy 
 
34


Skin: Infiltration: erythema, induration, tenderness, rarely ulceration, 
radiation recall reactions 
Allergy: Anaphylactoid and urticarial reactions (acute), flushing, rash, 
pruritus 
Liver: Increased SGOT, SGPT, bilirubin and alkaline phosphatase, hepatic 
failure, hepatic necrosis 
Other: Alopecia, fatigue, arthralgia, myalgia, light-headedness, myopathy 
Other, Vision: Sensation of flashing lights, blurred vision, scintillating 
scotomata 
*Refer to Package Insert for additional information Paclitaxel is a poorly 
soluble plant product from western yew, Taxus brevifolia. Improved 
solubility requires a mixed solvent system with further dilutions of either 
0.9% sodium chloride or 5% dextrose in water. 
7.3  
CARBOPLATIN (Paraplatin®, NSC # 241240) 
Formulation: Carboplatin is supplied as a sterile lyophilized powder 
available in single-dose vials containing 50 mg, 150 mg and 450 mg of 
carboplatin for administration by intravenous infusion. Each vial contains 
equal parts by weight of carboplatin and mannitol. 
Solution Preparation: Immediately before use, the content of each vial 
must be reconstituted with either sterile water for injection, USP, 5% 
dextrose in water, or 0.9% sodium chloride injection, USP, according to 
the following schedule: 
Vial Strength
Diluent Volume
 
 
50 mg 
5 ml 
150 mg 
15 ml 
450 mg 
45 ml 
These dilutions all produce a carboplatin concentration of 10 mg/ml. 
 
35


NOTE: Aluminum reacts with carboplatin causing precipitate formation 
and loss of potency. Therefore, needles or intravenous sets containing 
aluminum parts that may come in contact with the drug must not be used 
for the preparation or administration of carboplatin. 
Storage: Unopened vials of carboplatin are stable for the life indicated on 
the package when stored at controlled room temperature and protected 
from light. 
Stability: When prepared as directed, carboplatin solutions are stable for 
eight hours at room temperature. Since no antibacterial preservative is 
contained in the formulation, it is recommended that carboplatin solutions 
be discarded eight hours after dilution. 
Supplier: Commercially available from Bristol-Myers Squibb Company. 
Administration: Carboplatin will be administered as a 30 minute infusion.  
Adverse effects: 
Hematologic: Myelosuppression 
Gastrointestinal: Nausea, vomiting, diarrhea, abdominal pain, constipation 
Neurologic: Peripheral neuropathy, ototoxicity, visual disturbances, 
change in taste, central nervous system symptoms 
Renal: Abnormal renal function test results including serum creatinine, 
blood urea nitrogen, and creatinine clearance 
Hepatic: Abnormal liver function tests including bilirubin, SGOT, and 
alkaline phosphatase 
Electrolyte Changes: Abnormally decreased serum electrolyte values 
reported for sodium, potassium, calcium, and magnesium 
Allergic Reactions: Rash, urticaria, erythema, pruritus, and rarely 
bronchospasm and hypotension  
Injection Site Reactions: Redness, swelling, pain; necrosis associated with 
extravasation has been reported. 
 
36


Other: Pain, asthenia, alopecia. Cardiovascular, respiratory, genitourinary, 
and mucosal side effects have occurred in 6% or less of the patients. 
Cardiovascular events (cardiac failure, embolism, cerebrovascular 
accidents) were fatal in less than 1% of patients and did not appear to be 
related to chemotherapy. Cancer associated hemolytic-uremic syndrome 
has been reported rarely. Malaise, anorexia, and hypertension have been 
reported as part of post-marketing surveillance. 
*See FDA-approved package insert for a comprehensive list of adverse 
events associated with carboplatin. 
 
7.4 Methods of Chemotherapy Administration 
Biometric considerations in dose calculation 
Maximum body surface area used for Carboplatin, Paclitaxel and dose 
calculations will be 2.0 m2. 
Bevacizumab will be dosed at 15 mg/kg, with no maximum to total mg. 
7.4.1 Sequence and timing of drug administration: 
• Paclitaxel will be infused over 3 hours. Due to the risk of 
immediate hypersensitivity reaction, paclitaxel should always 
be the first drug to be infused during any combination.  
• Carboplatin will be administered as a 30 minute infusion, 
following paclitaxel administration. 
• Bevacizumab administration will be as a continuous 
intravenous infusion following carboplatin infusion. 
Anaphylaxis precautions should be observed during 
Bevacizumab administration DO NOT ADMINISTER AS AN 
IV PUSH OR BOLUS. The initial dose should be delivered 
over 90 minutes as an IV infusion. If the first infusion is well 
tolerated, the second infusion may be administered over 60 
minutes. If the 60- minute infusion is well tolerated, all 
subsequent infusions may be administered over 30 minutes. 
7.4.2 Pre-Medication: 
 
37


7.4.2.1 Paclitaxel 
For all courses where paclitaxel is to be administered, it is 
recommended that a preparative regimen be employed one hour 
prior to the treatment regimen on that day, to reduce the risk 
associated with hypersensitivity reactions to these drugs. This 
regimen should include a standard dose of Dexamethasone (either 
IV or PO), an anti-histamine H1 (diphenhydramine 25-50 mg IVP 
or orally, or an equivalent dose of an alternate H1 blocker such as 
loratadine or fexofenadine), and a standard dose of antihistamine 
H2 IVP (such as cimetidine, ranitidine, or famotidine). 
7.4.2.2 In the event of a prior Bevacizumab hypersensitivity reaction, 
the prophylactic regimen should be modified as suggested in 7.4.2.2.1 
below. 
7.4.2.2.1 Suggested Prophylaxis in Event of Prior Bevacizumab 
Infusion Reaction 
In the event of a prior Bevacizumab hypersensitivity reaction, the 
following recommended prophylactic regimen is recommended 
upon re-exposure: 
• H1 blocker (diphenhydramine 25-50 mg IVP or orally one 
hour prior to injection; or an equivalent dose of an alternate 
H1 blocker such as loratadine 10 mg or fexofenadine 60 
mg). 
• H2 blocker (famotidine 20 mg IVP or orally one hour prior 
to injection; or an equivalent dose of an alternate H2 
blocker). 
• Dexamethasone (10 mg administered PO 12 and 6 hours 
prior to Bevacizumab injection). 
7.4.2.3 Antiemetic Regimens 
It is anticipated that nausea and vomiting may be a significant side 
effect. The following representative antiemetic regimens are 
suggested: 
 
38


• Ondansetron 8-32 mg IV 30 minutes prior to 
administration of chemotherapy and dexamethasone 10-20 
mg IV 30 minutes prior to drug administration or, 
• Granisetron 10 mcg/kg IV (or 2 mg PO) 30 minutes prior to 
chemotherapy, with or without lorazepam 0.5-2.0 mg IV 30 
minutes prior to chemotherapy. 
7.4.2.4 Dosing of Carboplatin 
The carboplatin dose will be calculated to reach a target area under 
the curve (AUC) of concentration x time according to the Calvert 
formula using an estimated glomerular filtration rate (GFR) from the 
Jelliffe formula. 
The initial dose of carboplatin must be calculated using GFR. In the 
absence of new renal obstruction or other renal toxicity greater than 
or equal to CTC Grade 2 (serum creatinine >1.5 x ULN), the dose 
of carboplatin will not be recalculated for subsequent cycles, but will 
be subject to dose modification as noted. 
In patients with an abnormally low serum creatinine (less than or 
equal to 0.6 mg/dl), due to reduced protein intake and/or low 
muscle mass, the creatinine clearance should be estimated using a 
minimum value of 0.6 mg/dl. If a more appropriate baseline 
creatinine value is available within 4 weeks of treatment,which may 
also be used for the initial estimation of GFR. 
CALVERT FORMULA: 
Carboplatin dose (mg) = target AUC x (GFR + 25) 
For the purposes of this protocol, the GFR is considered to be 
equivalent to the creatinine clearance. The creatinine clearance is 
calculated by the method of Jelliffe using the following formula: 
Ccr = 0.9 x ____{98 - [0.8 (age - 20)]} 
Scr 
Where: Ccr = creatinine clearance in ml/min 
 
39


Age = patient's age in years (from 20-80) 
Scr = serum creatinine in mg/dl 
7.4.2.5 Dosing of Bevacizumab 
Bevacizumab will be administered at 15 mg/kg IV. Patient weight 
prior to each cycle will be used to determine the Bevacizumab dose. 
 
8.0 TREATMENT MODIFICATIONS 
In order to maintain dose-intensity and cumulative dose-delivery on this 
study, reasonable efforts will be made to minimize dose reduction and 
treatment delays as specified. No dose escalation is planned for this 
study. 
8.1 Individual Dose Modification Levels 
All modifications are relative to the actual starting doses for the specific 
Regimen. For application of individual dose modifications, see specific 
guidelines below. Allowable drug dose levels and instructions are 
summarized in Tables A, B, and C. 
• General Guidelines for Hematologic Toxicity (Section 8.2) 
• Hematologic Nadirs, Table A (Section 8.3) 
• Delayed Hematologic Recovery, Table B (Section 8.4) 
• Non-Hematologic Toxicity Table C (Section 8.5) 
8.2 General Guidelines for Hematologic Toxicity 
8.21 Initial treatment modifications will consist of cycle delay and/or 
dose reduction as directed. 
8.22 Treatment decisions will be based on the absolute neutrophil 
count (ANC) rather than the total white cell count (WBC). 
8.23 Subsequent cycles of therapy will not begin until the ANC is ≥ 
1500 cells/mm3 (CTCAE Grade 1) and the platelet count is ≥ 
100,000/mm3. 
 
40


Therapy may be delayed for a maximum of three weeks until these 
values are achieved. Patients who fail to recover adequate counts 
within a three-week delay will no longer receive protocol directed 
cytotoxic chemotherapy 
8.24 Use of Hematopoietic Cytokines and Protective Agents 
The use of hematopoietic cytokines and protective reagents are restricted 
as noted: 
8.241 In general, patients will NOT receive prophylactic filgrastim 
(G-CSF), PEG-filgrastim (Neulasta), or sargramostim (GM-CSF) 
unless they experience treatment delays or recurrent neutropenic 
complications after treatment modifications as specified. However, 
this is not disallowed in the current protocol. Patients may also 
receive growth factors for management of neutropenic 
complications in accordance with clinical treatment guidelines. If 
required, it is recommended that growth factors be initiated the day 
after the last dose of chemotherapy and typically continuing for a 
minimum of 10 days or until the ANC is sustained above 
>1000/mm3. Growth factors should be discontinued if the ANC 
exceeds 10,000/ mm3 and should not be used within 72 hours of a 
subsequent dose of chemotherapy. 
8.242 Patients will NOT receive prophylactic thrombopoietic agents 
unless they experience recurrent Grade 4 thrombocytopenia after 
treatment modifications as specified below. 
8.243 Patients may receive erythropoietin (EPO), iron supplements, 
and/or transfusions as clinically indicated for management of 
anemia. 
8.244 Patients may receive amifostine or other protective reagents. 
8.25 Dose Modifications for Bevacizumab 
There will be no dose reduction for Bevacizumab. Treatment should be 
interrupted or discontinued for certain adverse events, as described 
above. 
8.3 Modifications for Hematologic Toxicity (Nadirs) 
 
41


8.31 Initial occurrence of dose-limiting neutropenia (defined in 8.32) 
and dose limiting thrombocytopenia (defined in 8.33) will be 
performed according to Table A. 
8.32 Dose-Limiting Neutropenia (DLT-ANC) is defined by the 
occurrence of febrile neutropenia or prolonged Grade 4 neutropenia 
persisting ≥ 7 days. There will be no modifications for 
uncomplicated Grade 4 neutropenia lasting less than 7 days. 
Febrile neutropenia is defined within the CTCAE as fever with or 
without clinically or microbiologically documented infection with 
ANC less than 1,000 /mm3 and fever greater than or equal to 
38.5°C. 
8.33 Dose-limiting thrombocytopenia (DLT-PLT) is defined by any 
occurrence of Grade 4 thrombocytopenia (<25,000/mm3) or 
bleeding associated with Grade 3 thrombocytopenia (25,000 to 
<50,000/mm3). There will be no modifications for uncomplicated 
Grade 3 thrombocytopenia. 
 
42


 
Table A: Modification Instructions for Dose-Limiting Hematologic Toxicity  
DLT 
ANC 
DLT 
PLT 
First Occurrence 
Second 
Occurrence 
Third 
Occurrence 
Yes 
No 
Reduce carboplatin one 
AUC unit  
 
Add G-CSF and 
maintain all current 
drug doses 
Off Study 
Yes 
Yes 
Reduce carboplatin one 
AUC unit  
Add G-CSF and 
decrease carboplatin 
one AUC  
Off Study 
No 
Yes 
Reduce carboplatin one 
AUC unit  
decrease carboplatin 
one AUC  
Off Study 
 
 
8.4 Modifications for Delayed Hematologic Recovery: 
8.41 Delay on the basis of neutropenia (Delay-ANC) is defined if 
the ANC is less than 1,500 cells/mm3 (CTCAE Grade 2 or worse) 
within 24 hours prior to scheduled therapy, or less than 1,000 cells/ 
mm3, if the patient received G-CSF during the previous cycle. 
8.42 Delay on the basis of thrombocytopenia (Delay-PLT) is 
defined if the platelet count is less than 100,000/ mm3 within 24 
hours prior to scheduled therapy. 
8.43 Modifications noted below are only required for management 
of delays in the absence of dose reductions stipulated by nadir 
DLT-ANC and/or DLT-PLT (as noted above). In other words, if the 
patient experiences DLT-ANC and Delay- ANC, make the 
modifications as indicated for the nadir counts without additional 
modifications based on delayed recovery. 
 
43


 
Table B. Modifications for Delayed Hematologic Recovery 
Category  
Delay (days) 
Modification 
1-7 
No Change 
8-21 
Add G-CSF with next cycle and 
decrease carboplatin one AUC 
Delay-ANC 
>21 
Off Study (see Section 8.7) 
1-7 
No Change 
8-21 
Decrease carboplatin one AUC  
Delay-PLT 
>21 
Off study 
 
8.5 Adjustments for Non-Hematologic Toxicity 
Table C: Modifications for Non-Hematologic Toxicity 
Drug 
Regimen 
-2 Level 
Regimen 
-1 Level 
Regimen Starting 
Dose 
Paclitaxel 
110 mg/m2 
135 mg/m2 
175 mg/m2 
Carboplatin (AUC) 4.0 
5.0 
6.0 
Bevacizumab 
Not applicable 
Not applicable 
Not applicable 
 
8.5.1 Peripheral neuropathy Grade 2 (or greater) requires reduction of 
one dose level in paclitaxel and delay in subsequent therapy for a 
maximum of three weeks until recovered to Grade 1. If peripheral 
neuropathy fails to recover to Grade ≤ 2 by a maximum delay of three 
weeks from time therapy is due, then paclitaxel only should be reduced 1 
treatment level. 
 
44


8.5.2 Hypertension. Patients receiving Bevacizumab should be monitored 
prior to each dose with measurement of blood pressure. Medication 
classes used for management of patients with Grade 3 hypertension 
receiving Bevacizumab included angiotensin-converting enzyme inhibitors, 
beta blockers, diuretics, and calcium channel blockers. The goal for blood 
pressure control should be consistent with general medical practice 
guidelines (i.e. < 140/90 mmHg in general and < 130/80 mmHg for 
patients with diabetes). 
• For controlled hypertension, defined as systolic ≤ 150 mm 
Hg and diastolic ≤ 90 mm Hg, continue Bevacizumab 
therapy. 
• For uncontrolled hypertension (systolic > 150 mm Hg or 
diastolic > 90) or symptomatic hypertension less than 
CTCAE Grade 4, hold Bevacizumab treatment, with anti-
hypertensive therapy initiated or continued, as in 8.52. 
• During the period of combination chemotherapy with 
Bevacizumab, if hypertension is controlled and symptomatic 
hypertension has resolved by one week after holding 
treatment, continue all therapy. 
• During the period of combination chemotherapy with 
Bevacizumab, if hypertension remains uncontrolled or 
symptomatic hypertension, less than CTCAE Grade 4, 
persists one week after holding treatment, the next treatment 
cycle should contain paclitaxel and Carboplatin only, if 
applicable, as otherwise indicated in the protocol, with 
Bevacizumab omitted. 
• Bevacizumab should be discontinued for the remainder of 
the study in any patient developing CTCAE Grade 4 
hypertension. 
8.5.3 Proteinuria. Patients receiving Bevacizumab should be monitored 
by urine analysis for urine protein:creatinine (UPC) ratio prior to every 
other dose of Bevacizumab: 
 
45


UPC ratio < 3.5 
Continue Bevacizumab 
UPC ratio ≥ 3.5 
Hold Bevacizumab until UPC 
ratio recovers to < 3.5. If 
therapy is held for > 2 months 
due to proteinuria, discontinue 
Bevacizumab. 
Grade 4 or 
nephrotic syndrome 
Discontinue Bevacizumab 
 
 
8.5.4 Hemorrhage. Bevacizumab will be discontinued in patients with 
CTCAE Grade 3 hemorrhage and receiving full-dose anticoagulation. For 
all other patients with CTCAE Grade 3 hemorrhage, Bevacizumab should 
be held until ALL of the following criteria are met: 
8.541 bleeding has resolved 
8.542 blood hemoglobin level is stable 
8.543 there is no bleeding diathesis that would increase the risk of 
therapy 
8.544 there is no anatomical or pathologic condition that can 
increase the risk of hemorrhage recurrence. 
Patients who experience delay of resolution according to the above criteria 
for >3 weeks, recurrence of Grade 3 hemorrhage, or any CTCAE Grade 4 
hemorrhage will be taken off Bevacizumab therapy. 
8.5.5 Thrombosis. 
8.5.51 Arterial Thrombosis 
Bevacizumab will be discontinued for ≥ CTCAE Grade 3 arterial 
thrombotic events (including cerebrovascular ischemia, transient 
ischemic attack, cardiac ischemia/infarction, peripheral or visceral 
 
46


arterial ischemia) or CTCAE Grade 2 arterial thrombotic events new 
or worsened since beginning Bevacizumab therapy. 
8.5.52 Venous Thrombosis 
Treatment with Bevacizumab will be held for CTCAE Grade 3 or 
asymptomatic CTCAE Grade 4 venous thrombosis. For patients on 
therapeutic anticoagulation, PT INR or PTT (whichever appropriate) 
should be monitored closely during Bevacizumab therapy. If the 
planned duration of full-dose anticoagulation is ≤ 3 weeks, 
Bevacizumab should be held until the full-dose anticoagulation 
period is over. If the planned duration of full-dose anticoagulation is 
> 3 weeks, Bevacizumab may be resumed during the period of full 
dose anticoagulation if ALL of the following criteria are met 
(otherwise such patients will be taken off Bevacizumab therapy): 
8.5.52.1 The patient must have an in-range INR (usually 
between 2 and 3) on a stable dose of warfarin (or other 
anticoagulant) or on stable dose of heparin prior to restarting 
treatment. 
8.5.52.2 The subject must not have pathological conditions 
that carry high risk of bleeding (e.g. tumor involving major 
vessels). 
8.5.52.3 The subject must not have had hemorrhagic events 
while on study. 
8.5.52.4 The patient is benefiting from treatment (no 
evidence of disease progression). 
Patients with symptomatic CTCAE Grade 4, or recurrent/worsening 
venous thromboembolic events after resumption of Bevacizumab 
treatment, will be taken off Bevacizumab therapy. 
8.5.6 Coagulopathy. For CTCAE Grade 3 or 4 coagulopathy: hold 
treatment, until PT/PTT resolve to Grade 1. For patients with PT/INR 
greater than the intended therapeutic range while on therapeutic warfarin, 
treatment with Bevacizumab will be held at the discretion of the treating 
physician in discussion with the principal investigator. Patients 
 
47


experiencing treatment delay greater than three weeks because of failure 
to meet the above criteria will be taken off Bevacizumab therapy. 
8.5.7 Wound Disruption/Bowel Perforation, Fistula, or GI Leak: 
Bevacizumab will be discontinued in the event of wound disruption 
requiring surgical intervention. Bevacizumab will be discontinued in the 
event of new development of bowel perforation, fistula, or GI leak  
8.5.8 Renal toxicity (associated with reduction in GFR) is not expected as 
a direct complication of chemotherapy in this previously untreated patient 
population using the prescribed dose and schedule of each regimen. As 
such, there are no specific dose modifications for renal toxicity. However, 
the target AUC dose of carboplatin must be recalculated each cycle in any 
patient who develops renal toxicity, defined by serum creatinine greater 
than 1.5 x institutional upper limit normal (ULN), CTCAE Grade ≥ 2. 
8.5.9 Intestinal obstruction. Bevacizumab will be held for occurrence of 
CTCAE Grade 3 toxicity, until resolution to ≤CTCAE Grade 1 and will be 
permanently discontinued for occurrence of CTCAE Grade 4 toxicity. 
8.5.10 Hepatic toxicity is not expected as a direct complication of 
chemotherapy in this untreated patient population using the prescribed 
dose and schedule for each regimen. However, the development of Grade 
3 (or greater) elevations in SGOT (AST), SGPT (ALT), alkaline 
phosphatase or bilirubin requires reduction of one dose level in paclitaxel 
and withholding of Bevacizumab, and delay in subsequent therapy for a 
maximum of three weeks until recovered to Grade 1. 
8.5.11 There will be no dose modifications for alopecia, nausea, 
constipation, or diarrhea. It is recommended that routine medical 
measures be employed to manage nausea, constipation, and diarrhea. 
8.5.12 In general, with the occurrence of a hypersensitivity reaction to 
paclitaxel, carboplatin, Bevacizumab is not considered a dose-limiting 
toxicity. Patients may be retreated at full doses after administration of 
medication to prevent hypersensitivity reactions, and adjustments in 
infusion rates should be made. However, if despite these safety measures 
repeat attempt at infusion of the inciting drug results in a recurrent 
 
48


hypersensitivity reaction, the inciting drug should be discontinued for the 
remainder of the study. In the event of any CTCAE Grade 3 or 4 allergic or 
infusional reaction to Bevacizumab, Bevacizumab will be permanently 
discontinued.  
Also, please see Appendix J for management of suspected 
hypersensitivity reactions to Bevacizumab. 
8.5.13 Potential modifications for other non-hematologic toxicities with an 
impact on organ function of Grade 2 (or greater) require discussion with 
the principal investigator except where noted below in Section 8.631. 
8.5.14 Special Modifications in Study Treatment 
8.6311 For any CTCAE Grade 3 non-hematologic adverse event 
(except controllable nausea/emesis), Bevacizumab should be held 
until symptoms resolve to ≤ CTCAE Grade 1. If a CTCAE Grade 3 
adverse event persists for > three weeks or recurs after resumption 
of Bevacizumab, the patient will be taken off all protocol therapy. 
8.6312 For any CTCAE Grade 4 non-hematologic adverse event 
(except controllable nausea/emesis), the patient will be taken off all 
protocol therapy. 
8.6 
Bevacizumab Dose Modification and Toxicity Management 
There are no reductions in the bevacizumab dose. If adverse events occur that 
require holding bevacizumab, the dose will remain the same once treatment 
resumes. 
Any toxicities associated or possibly associated with bevacizumab treatment 
should be managed according to standard medical practice. Bevacizumab has a 
terminal half-life of 2 to 3 weeks; therefore, its discontinuation results in slow 
elimination over several months. There is no available antidote for bevacizumab.  
Subjects should be assessed clinically for toxicity prior to, during, and after each 
infusion. If unmanageable toxicity occurs because of bevacizumab at any time 
during the study, treatment with bevacizumab should be discontinued.  
Infusion Reaction: Infusion of bevacizumab should be interrupted for subjects 
who develop dyspnea or clinically significant hypotension. Subjects who 
 
49


experience a NCI CTCAE v. 3.0 Grade 3 or 4 allergic reaction / hypersensitivity, 
adult respiratory distress syndrome, or bronchospasm (regardless of grade) will 
be discontinued from bevacizumab treatment. 
The infusion should be slowed to 50% or less or interrupted for subjects who 
experience any infusion-associated symptoms not specified above. When the 
subject’s symptoms have completely resolved, the infusion may be continued at 
no more than 50% of the rate prior to the reaction and increased in 50% 
increments every 30 minutes if well tolerated. Infusions may be restarted at the 
full rate during the next cycle.  
Adverse events requiring delays or permanent discontinuation of bevacizumab 
are listed in Table 1  
Regardless of the reason for holding study drug treatment, the maximum 
allowable length of treatment interruption is 2 months (for events not treatment 
related).  The maximum allowable length for treatment interruptions secondary to 
treatment related toxicities  3 weeks as described in Section 8.0 
 
50


Table1: Bevacizumab Dose Management Due to Adverse Events 
 
Event 
Action to be Taken 
Hypertension 
No dose modifications for grade 1/2 events 
Grade 3 
If not controlled to 150/100 mmHg with medication, hold 
bevacizumab. 
Grade 4 (including 
RPLS (confirmed by 
MRI) or hypertensive 
encephalopathy) 
Discontinue bevacizumab. 
Hemorrhage 
No dose modifications for grade 1/2 nonpulmonary and non-CNS events 
Grade ≥ 2 pulmonary 
or CNS hemorrhage 
Discontinue bevacizumab. 
Grade 3  
nonpulmonary and 
non-CNS hemorrhage 
Subjects who are also receiving full-dose anticoagulation will 
be discontinued from receiving bevacizumab.   
All other subjects will have study treatment held until all of the 
following criteria  are met: 
• The bleeding has resolved and hemoglobin is stable. 
• There is no bleeding diathesis that would increase 
the risk of therapy. 
• There is no anatomic or pathologic condition that 
significantly increases the risk of hemorrhage 
recurrence. 
Subjects who experience a repeat Grade 3 hemorrhagic event 
will be discontinued from receiving bevacizumab. 
Grade 4 
Non pulmonary or 
non-CNS hemorrhage 
Discontinue bevacizumab. 
Venous Thrombosis 
[Note: Subjects with lung cancer placed on anticoagulant therapy for a thrombotic event 
should be discontinued from receiving bevacizumab] 
No dose modifications for grade 1/2 events 
 
51


Grade 3/ 
Asymptomatic Grade 
4 
Hold study drug treatment.  If the planned duration of full-dose 
anticoagulation is <2 weeks, study drug should be held until the 
full-dose anticoagulation period is over.  If the planned duration 
of full-dose anticoagulation is >2 weeks, study drug may be 
resumed during the period of full-dose anticoagulation if all of 
the following criteria  are met: 
• The subject must have an in-range INR (usually 
between 2 and 3) on a stable dose of warfarin (or 
other anticoagulant) prior to restarting study drug 
treatment. 
• The subject must not have had a Grade 3 or 4 
hemorrhagic event while on anticoagulation. 
• The subject must not have had evidence of tumor 
involving major blood vessels on any prior CT scan. 
Symptomatic Grade 4 
Discontinue bevacizumab. 
 
52


 
Table 1 
Bevacizumab Dose Management due to Adverse Events (continued) 
 
Arterial Thromboembolic event 
(Angina, myocardial infarction, transient ischemic attack, cerebrovascular accident, 
pulmonary embolus and any other arterial thromboembolic event) 
 Any grade 
Discontinue bevacizumab. 
Congestive Heart Failure (Left ventricular systolic dysfunction) 
No dose modifications for grade 1/2 events 
Grade 3 
Hold bevacizumab until resolution to Grade ≤ 1. 
Grade 4 
Discontinue bevacizumab. 
Proteinuria 
No dose modifications for grade 1/2 events 
Grade 3 
(UPC> 3.5, urine 
collection > 3.5 g/24 
hr, or dipstick 4+)   
 
Hold bevacizumab treatment until ≤ Grade 2, as determined by 
either UPC ratio ≤ 3.5 or 24 hr collection ≤ 3.5 g 
 
Grade 4 (nephrotic 
syndrome) 
Discontinue bevacizumab 
GI Perforation 
 
Discontinue bevacizumab. 
Bowel Obstruction 
Grade 1 
 
Continue patient on study for partial obstruction NOT requiring 
medical intervention. 
Grade 2 
Hold bevacizumab for partial obstruction requiring medical 
intervention.  Patient may restart upon complete resolution. 
Grade 3/4 
Hold bevacizumab for complete obstruction.  If surgery is 
necessary, patient may restart bevacizumab after full recovery 
from surgery, and at investigator’s discretion. 
Wound dehiscence 
requiring medical or 
surgical therapy 
Hold bevacizumab at the discretion of the treating physician and 
principle investigator. 
Other Unspecified Bevacizumab-Related Adverse Events 
Grade 3 
 
Grade 4 
Hold bevacizumab until recovery to ≤ Grade 1 
 
Discontinue bevacizumab. 
Any Grade 
 
Reversible Posterior Leukoencephalopathy 
Discontinue Bevacizumab 
 
 
53


8.7 
CONCOMITANT MEDICATIONS 
Low-dose aspirin (≤ 325 mg/d) may be continued in subjects at higher risk for 
arterial thromboembolic disease. Subjects developing signs of arterial ischemia 
or bleeding on study should be evaluated for possible bevacizumab 
discontinuation per Table 1, Bevacizumab Dose Management Due To Adverse 
Events. 
 
9.0 
CLINICAL AND LABORATORY EVALUATIONS 
9.1 Pretreatment/During Treatment/Post-Treatment Evaluations: 
The following observations and tests are to be performed and recorded on the 
appropriate form(s).  
The following observations and tests are to be performed and recorded: 
 
54


 
Parameter 
pre-
treat 
Nadir 
Labs 
Prior 
to 
every 
cycle 
prior 
to 
every 
other  
cycle 
 Prior 
to cycle 
4 after 
cycle 6 
After 
Treatment 
and every 3 
months for 2 
years than 
every 6 
months x 3 
yrs 
History  and 
Physical 
X4 
 
X 
 
 
X 
Blood pressure 
X4 
 
 X 
 
 
X 
Toxicity 
Assessment 
X4 
 
X 
 
 
X  
Clin. Tumor 
Measurement 
X4 
 
 
 
      X 
X 
CT-scan/MRI/US 
X 2 
 
 
 
X 
X  
CBC/Diff./ 
Platelets 
X 4 
X6, 7 
X 5,6 
 
 
 
X 8 
Electrolytes, BUN 
Creat, Ca, Mg, 
PO4,  
 
X 4 
 
 
X 5 
 
 
 
 
X 8 
Bilirubin, SGOT,  
Alkaline 
Phosphatase 
 
X 4 
 
 
 
X  
 
 
PT/INR,PTT 
 
X 4 
 
X 
 
 
 
 
 
Chest x-ray 
X 1 
 
 
 
 
 
CA-125  
 
X 4 
 
X 3 
 
 
X3 
Serum for 
Translational  
studies 
X 4 
 
 
 
X 
X  
UPC ratio 
X 
 
 
X 
 
 
Notes: 
1. Repeat chest x-ray if initially abnormal or if required to monitor tumor 
response. 
2. CT scan, ultrasound, or MRI as appropriate for consistent measurement of 
tumor response at the physician discretion within 28 days of initial therapy. 
3. To be followed as an adjuvant marker. 
4. Must be obtained within 14 days prior to initiating protocol therapy. 
5. CBC/Diff./Platelets and UPC ratio must be obtained within 4 days of re-
treatment with protocol therapy 
 
55


6. If grade 4 neutropenia is documented (ANC<500/mm3), obtain twice weekly 
to weekly until resolved to grade 3. 
7. Nadir Labs to be obtained approximately 10 days after chemotherapy (+/- 2 
days) 
8. Post-Treatment visit only (at the completion of chemotherapy) should be 
completed 30days after the last treatment date. 
 
 
10.0 SUBJECT DISCONTINUATION  
Subjects who meet the following criteria should be discontinued from study 
treatment:  
• Grade 4 hypertension or reversible posterior leukoencephalopathy 
syndrome (RPLS) 
• Nephrotic syndrome 
• Grade ≥ 2 pulmonary or CNS hemorrhage; any Grade 4 hemorrhage 
• Symptomatic Grade 4 venous thromboembolic event (for lung 
protocols: any venous thromboembolic event requiring full dose 
warfarin or equivalent (i.e., unfractionated or low molecular weight 
heparin)  
• Any grade arterial thromboembolic event 
• Grade 4 congestive heart failure 
• Gastrointestinal perforation  
• Tracheoesophageal fistula (any grade) or Grade 4 fistula 
• Wound dehiscence requiring medical or surgical intervention 
• Bowel obstruction that has not fully recovered despite medical or 
surgical intervention. 
• Wound dehiscence requiring medical or surgical intervention 
• Inability of subject to comply with study requirements 
• Determination by the investigator that it is no longer safe for the 
subject to continue therapy 
• All Grade 4 events thought to be related to bevacizumab by the 
investigator 
• Inability to tolerate the lowest doses (Carboplatin AUC4 or paclitaxel 
110 mg/m2) because of toxicity. 
 
56


• The patient may withdraw from the study at any time for any reason. 
• Patients with evidence of progressive disease or patients with 
significant side effects or deterioration of performance status may be 
removed from study at the investigator’s discretion. 
• All the patients will be followed until death or maximum of 5 years after 
receiving first treatment. 
 
Patients who have an ongoing bevacizumab-related Grade 4 or serious adverse 
event at the time of discontinuation from study treatment will continue to be 
followed until resolution of the event or until the event is considered irreversible  
 
11.0 STATISTICAL METHODS 
A Fleming single stage Phase II design will be used to assess whether the 24 
month failure rate is at most 50% against the alternative that the 24 month failure 
rate is at least 70% (alpha of 0.1, Beta of 0.1, power of 90%). Data will be 
reported for all patients treated under the protocol. Toxicity, overall survival, 
objective tumor response, correlative endpoints will be examined. All patients will 
who start treatment will be included in the analysis. If a patient enrolls in the 
study and does not receive any of the protocol chemotherapy will be declared 
“cancelled” and not included in the analysis. These patients will be replaced at a 
one to one basis. 
11.1 Interim Analysis: 
No planned interim analysis planned of efficacy data. However, annual 
review will be performed to assure annual recruitment goals are met 
(minimum of 12 patients annually). Safety will be continually evaluated 
(see Safety section) 
11.2 Planned Efficacy Evaluations 
Efficacy Endpoints:  Parameters employed to evaluate treatment efficacy 
and toxicity are:  
11.2.1 Primary Efficacy Variables 
Primary Endpoints:  
 
57


• 
24 month failure-free rate (events are defined as disease 
progression, death without progression, or discontinue due to 
progression, death, or unacceptable toxicity) is calculated by the 
Kaplan Meier method.  Patients without a defined event will be 
censored at the last tumor assessment date. 
• 
Frequency and severity of adverse effects as assessed by 
CTCAE v3. 
 
11.2.2 Secondary Efficacy Variables 
11.2.2.1 Progression-free survival and overall survival  
11.2.2.2 Time to progression will be reported using Kaplan 
Meier method. 
11.2.3 Translational Research Endpoints (Analysis to be 
exploratory as hypothesis generating): 
11.2.3.1 Levels of VEGF, CD-31, TSP-1, and CD-105 in 
tumor tissue as determined by IHC at baseline will be 
reported in frequency tables with 24 month failure free rate 
as an end point to attempt to determine if the levels predict 
response to the study drug.  
11.2.3.2 Levels of angiogenin, bFGF, VEGF, and VCAM-I in 
serum as determined with ELISA. These values will be 
evaluated over time in a time series plot to evaluate if these 
change over time with treatment or with recurrence. If 
deemed appropriate further statistical analysis will be 
performed. 
11.2.3.3 ELISA measures of VEGF in plasma will be 
performed. These values will be evaluated over time in a 
time series plot to evaluate if these change over time with 
treatment or with recurrence. If deemed appropriate further 
statistical analysis will be performed. 
 
58


11.2.3.4. Gene Analysis Statistics and sample cohorts 
needed.(see appendix I)  In genes where we do not see any 
AEI in normal subjects (such as APC; our results are 
identical to those of Yan et al.), the presence of even a 
single AEI patient can be followed mechanistically by 
comparing allelic ratios in normal and tumor tissues (as 
done by Yan et al32. 
11.3 Sample Size Considerations 
A sample size of 38 patients, a one sample test of proportions will have a 
power of 90% and a one-sided alpha=0.10 to detect that the 24 month 
Failure free rate is greater than 50% when the true 24 month is at leas 
70%. A 90% confidence interval will be constructed using properties of 
binomial distribution. 
11.4 Methods of Analysis 
Progression-free survival and overall survival will be estimated using 
Kaplan-Meier method.  Estimates of rates of clinical response (defined as 
complete or partial response) will be calculated along with corresponding 
95% confidence intervals.  Safety analyses will include listing and 
tabulating frequencies of adverse events (AE) and serious adverse events 
(SAE) resulting from both clinical observation and laboratory tests.  Each 
AE will be graded from 1 to 4 based on criteria established by NCI CTCAE 
v3.  Abnormal laboratory parameters in data listings will be flagged.  
Reports will present tables of all AEs as well as only those related to the 
treatment regimen.  . 
 
12.0 SAFETY REPORTING OF ADVERSE EVENTS 
 
12.1 ADVERSE EVENT REPORTING AND DEFINITIONS 
 
In the event of an adverse event the first concern will be for the safety of 
the subject.   
Investigators are required to report to Genentech Drug Safety ANY 
serious treatment emergent adverse event (STEAE) as soon as possible.   
 
59


A STEAE is any sign, symptom or medical condition that emerges during 
Bevacizumab treatment or during a post-treatment follow-up period that 
(1) was not present at the start of Bevacizumab treatment and it is not a 
chronic condition that is part of the patient’s medical history, OR (2) was 
present at the start of Bevacizumab treatment or as part of the patient’s 
medical history but worsened in severity and/or frequency during therapy, 
AND that meets any of the following regulatory serious criteria: 
• Results in death  
• Is life-threatening  
• Requires or prolongs inpatient hospitalization  
• Is disabling  
• Is a congenital anomaly/birth defect  
• Is medically significant or requires medical or surgical intervention to 
prevent one of the outcomes listed above. 
 
Serious adverse events occurring after a patient is discontinued from the 
study will NOT be reported unless the investigator feels that the event may 
have been caused by the study drug or a protocol procedure.  Study-specific 
clinical outcomes of death because of disease progression are exempt from 
serious adverse event reporting, unless the investigator deems them related 
to use of the study drug.  Hospitalization for study drug administration is not a 
serious adverse event. 
In general, serious adverse events assessed as clearly being due to disease 
progression and not due to study drug(s) should be excluded from adverse 
event reporting. However, in cases where the specificity or severity of an 
event is not consistent with the risk information, the event should be reported.  
If clearly related to the commercial agent(s), adverse effects that are either 
severe or for which the cause is unknown must be reported using the rules 
listed below. 
The study will utilize the NCI Common Terminology for Adverse Reporting 
Version 3.0. 
 
Toxicity Grade 
 
Type (a) 
4,5 
Unknown 
5 
Known 
2,3 
Unknown 
4 (non-myelo) 
Known 
4 (myelo  b) 
Known 
 
60


(a)Type is based on toxicities included in the NCI list of known toxicities included 
in the known toxicities associated with the study drug(s). 
(b)Myelosoppression, which includes neutropenia, anemia, and 
thrombocytopenia. 
 
12.2 Reporting of Serious Treatment Emergent Adverse Events  
All STEAEs should be recorded on a MedWatch 3500 Form and faxed to: 
Genentech Drug Safety  
Fax: (650) 225-4682 or (650) 225-4683 
 
(Please use the safety reporting fax cover sheet attached to this document for 
your fax transmission) 
AND: 
David O’Malley, MD (Principal Investigator) 
Lois Dial, RN fax #614-293-0058(Research Center) 
Bobbi Cobb, RN, OCN, CCRP.  fax # 614-2933078 (Study Coordination Center) 
 
AND: 
 
Ohio State University IRB 
 Beth Wiley, Tel. 614-292-0243. fax # 614-688-0366 
 
MedWatch 3500 Reporting Guidelines: 
In addition to completing appropriate patient demographic and suspect 
medication information, the report should include the following information within 
the Event Description (section 5) of the MedWatch 3500 form: 
• Treatment regimen (dosing frequency, combination therapy) 
• Protocol description (and number, if assigned) 
• Description of event, severity, treatment, and outcome, if known 
• Supportive laboratory results and diagnostics 
• Investigator’s assessment of the relationship of the adverse event to each 
investigational product and suspect medication 
 
Follow-up information:   
Additional information may be added to a previously submitted report by any of the 
following methods: 
• Adding to the original MedWatch 3500 report and submitting it as follow-
up 
 
61


• Adding supplemental summary information and submitting it as follow-up 
with the original MedWatch 3500 form  
• Summarizing new information and faxing it with a cover letter including 
subject identifiers (i.e. D.O.B. initial, subject number), protocol description 
and number, if assigned, suspect drug, brief adverse event description, 
and notation that additional or follow-up information is being submitted  
(The subject identifiers are important so that the new information is added 
to the correct initial report) 
Occasionally Genentech may contact the reporter for additional information, 
clarification, or current status of the subject for whom and adverse event was 
reported.  
Assessing Causality: 
Investigators are required to assess whether there is a reasonable possibility that 
bevacizumab caused or contributed to an adverse event.  The following general 
guidance may be used. 
Yes: if the temporal relationship of the clinical event to bevacizumab 
administration makes a causal relationship possible, and other drugs, therapeutic 
interventions or underlying conditions do not provide a sufficient explanation for 
the observed event. 
No: 
if the temporal relationship of the clinical event to bevacizumab 
administration makes a causal relationship unlikely, or other drugs, therapeutic 
interventions or underlying conditions provide a sufficient explanation for the 
observed event. 
12.3 
Safety Reporting Requirements for IND Exempt Studies 
For Investigator Sponsored IND Exempt Studies, there are some reporting 
requirements for the FDA in accordance with the guidance set forth in 21 CFR 
314.80. 
 
Postmarketing 15-Day “Alert Report”: 
The Sponsor-Investigator is required to notify the FDA of any fatal or life-
threatening adverse event that is unexpected and assessed by the 
investigator to be possibly related to the use of Bevacizumab. An 
unexpected adverse event is one that is not already described in the Investigator 
Brochure. Such reports are to be submitted to the FDA (2 copies) at the following 
address: Central Document Room, 12229 Wilkins Avenue, Rockville, MD 20852. 
 
All Postmarketing 15-Day “Alert Reports” submitted to the FDA by the Sponsor-
Investigator must also be faxed to: Genentech Drug Safety  
 
Fax: (650) 225-4682 or (650) 225-5288 (Please use the safety reporting fax 
cover sheet attached to this document for your fax transmission) 
 
For questions related to safety reporting, contact: 
 
Genentech Drug Safety 
Tel: 
1-888-835-2555 
 
62


         or 
Fax: (650) 225-4682 or (650) 225-5288 
(Please use the safety reporting fax cover sheet attached to this document for 
your fax transmission) 
 
13.0 
 DATA AND SAFETY MONITORING 
Overview: 
Data Safety Monitoring Board is to determine the safe and effective conduct of this 
clinical trial. This committee will recommend conclusions of the clinical trial. Risk 
associated with participation in research must be minimized to the extent practical. 
Monitoring will be conducted in toxicity, dose-finding studies, efficacy, effectiveness and 
comparative trials involving greater than minimal risk to participants to assure the safety 
and welfare of the research subjects.  
 
Data Safety Monitoring Plan 
1. Summary of main findings,  
2. Discussion of issues or problems 
3. List of participants of the trial, data personal and data centers. 
4. Report and review accrual and multi site participation. 
5. Review unanticipated problems, adverse events, and serious adverse events as 
they are reported in the data. Ensure proper reporting of each event (ex. FDA and 
sponsor) 
6. Review data quality, timeliness, patient recruitment, accrual and retention 
7. Review protocol violations-deviations from protocol treatment, monitoring or 
study calendar 
8. Discuss protocol amendments as they would relate to the data collection results. 
9. Written report will document the results of the meeting.  
 
Data Safety Monitoring Committee for Gyn Oncology 
Gyn Oncology Data Safety Monitoring Committee will include the principal investigator, 
data manager, clinical trials manager, research nurse, and disease specific committee 
director. A written report of the Disease Specific Committee’s recommendations of the 
Data Safety Monitoring Monthly report for this clinical trial will be signed by the 
principle investigator and Disease Specific Committee Director. Multi site participation 
of principal investigators can be accomplished by teleconference. Sub investigators will 
be sent the written report on a monthly basis. All data safety monitoring committee 
reports will be sent to the IRB with the continuing review submission. 
 
14.0 RETENTION OF RECORDS 
 
The Federal Privacy Act protects the confidentiality of OSUMC medical records. 
Clinical records are kept in the office of the Division of Gynecologic Oncology (M 
210 Starling-loving Hall).Division of Gynecologic Oncology clinical trial personnel 
is the only people with the authority to have access to these records. The Act 
does allow release of some information from the medical records without 
 
63


permission, for example if the Food and Drug Administration (FDA) or the 
Sponsor of this study (Genentech) requires it. When the results of the research 
study are reported in medical journals or at scientific meetings the people who 
take part are not named or identified. 
 
All documentation of adverse events, records of study drug receipt and dispensation, 
and all IRB correspondence will be retained for at least 2 years after the investigation 
is completed.  
 
64


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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF 
ADVANCED STAGE ENDOMETRIAL CARCINOMA 
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12. Holland CM. Day K. Evans A. Smith SK. Expression of the VEGF and 
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13. Sivridis, E. Angiogenesis and endometrial cancer. Anticancer Research. 
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15. Hirai, M. Nakagawara, A. Oosaki, T. Hayashi, Y. Hirono, M. Yoshihara, 
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16. Yokoyama, Y. Sato, S. Futagami, M. Fukushi, Y. Sakamoto, T. Umemoto, M. 
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28. Wang D, Johnson AD, Papp A, Kroetz DL, Sadée W.  Multidrug Resistance 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF ADVANCED 
STAGE ENDOMETRIAL CARCINOMA 
 
APPENDIX A 
STUDY FLOW CHART/SCHEMA 
 
Compressively surgically staged patients with advanced endometrial cancer 
(stage III-IV) with non-measurable or measurable disease 
 
Regimen 
Carboplatin (AUC 5) plus Paclitaxel (175 mg/m2 over 3 hours) plus 
Bevacizumab (15 mg/kg) every three weeks for 6 cycles 
 
Follow-up 
Measurable disease: CT-scan/MRI/US Following cycle 3 and 6 
Non-measurable disease: CT after 6 cycles 
All patients: Clinical exam every 3 months for 2 years then 6 months for an 
additional three years and annually thereafter 
 
70


A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF ADVANCED 
STAGE ENDOMETRIAL CARCINOMA 
APPENDIX B 
 
 
NCI COMMON TOXICITY CRITERIA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF ADVANCED 
STAGE ENDOMETRIAL CARCINOMA 
 
APPENDIX C 
 
FDA MEDWATCH FORM 3500a 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF ADVANCED 
STAGE ENDOMETRIAL CARCINOMA 
 
APPENDIX D 
 
 
NYHA GUIDELINES 
 
NYHA Classification 
 
 
Excerpted from Oxford Textbook of Medicine. Vol 2, p.2228. Oxford Press.1997. 
Class 
Description 
I 
Subjects with cardiac disease but without resulting limitations of 
physical activity.  Ordinary physical activity does not cause 
undue fatigue, palpitation, dyspnea, or anginal pain. 
II 
Subjects with cardiac disease resulting in slight limitation of 
physical activity.  They are comfortable at rest.  Ordinary 
physical activity results in fatigue, palpitation, dyspnea, or 
anginal pain. 
III 
Subjects with cardiac disease resulting in marked limitation of 
physical activity.  They are comfortable at rest.  Less than 
ordinary physical activity causes fatigue, palpitation, dyspnea, 
or anginal pain. 
IV 
Subjects with cardiac disease resulting in inability to carry on 
physical activity without discomfort.  Symptoms of cardiac 
insufficiency or of the anginal syndrome may be present event 
at rest.  If any physical activity is undertaken, discomfort is 
increased. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF 
ADVANCED STAGE ENDOMETRIAL CARCINOMA 
 
 
APPENDIX E 
 
PROCEDURE FOR OBTAINING A URINE PROTEIN / 
CREATININE RATIO 
 
 
 
1)  Obtain at least 4 ml of a random urine sample (does not have to be a 24 
hour urine)  
2)  Determine protein concentration (mg/dL)  
3)  Determine creatinine concentration (mg/dL)  
4)  Divide #2 by #3 above:      urine protein / creatinine ratio = protein 
concentration (mg /dL) / creatinine concentration (mg /dL) 
 
The UPC directly correlates with the amount of protein excreted in the urine per 
24 hrs (i.e. a UPC of 1 should be equivalent to 1g protein in a 24hr urine 
collection) 
                                            
Protein and creatinine concentrations should be available on standard reports 
of urinalyses, not dipsticks. If protein and creatinine concentrations are not 
routinely reported at an Institution, their measurements and reports may need 
to be requested. 
 
 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF 
ADVANCED STAGE ENDOMETRIAL CARCINOMA 
 
APPENDIX F 
 
COLLECTION AND PROCESSES OF SAMPLES 
 
1. Blood Samples: Following patient counseling and consent, patients will have 
approximately 5 ml of blood collected into a red top and 5 ml of blood into a purple top 
Vacutainer tube for serum and DNA processing. The total amount of blood taken for research 
purposes will be approximately 15 ml.  The blood will be drawn during the patients pre-
operative office visit or in the pre-operative area prior to surgery at the James Cancer 
Hospital Surgical Area.   
2. Tissue Specimens: Tissue specimens will be harvested by Ohio State University 
pathologists following removal of the specimen from the patient in surgery.  Only tissues that 
are removed as part of the scheduled operative procedure will be considered for sampling.  
In the case of cancer, metastatic specimens will be obtained for comparison with primary 
tumors when there is gross evidence of extended spread.  Normal appearing portions of the 
surgical specimen may also be sampled for comparison with cancer-involved areas.  In the 
cases of benign gynecologic disease, the portion of the diseased tissue as well as normal 
tissue will be sampled from the excised surgical specimen.  All specimens will be frozen 
within 60 minutes of removal from the patient in order to maximize the quality of molecular 
composition.  The specimens will be temporarily stored at –80 until the final diagnosis has 
been confirmed by The Department of Pathology at The Ohio State University (OSU) for 
purposes of patient management.  If there is any question regarding diagnostic issues, the 
tissue collected for research purposes can be recalled to further delineate problems in 
making the clinical diagnosis.  Once the formal pathology report has been rendered as part of 
the patient’s care, each “research specimen” will be processed for permanent storage in The 
Ohio State University Gynecologic Oncology Tissue and Serum Bank, maintained by The 
Department of Pathology at The Ohio State University.  The patient’s diagnosis and 
determination of stage will not be affected by the acquisition of tissue for our research 
purposes. Only remnant tissue samples not needed for diagnosis will be selected for the 
tissue bank. The Ohio State University Gynecologic Oncology Tissue and Serum Bank will 
be maintained by the Human Tissue Resource Network a division in the Department of 
Pathology at OSU.  
 
75


Approximately 25% of each tissue specimen will be embedded in OCT and stored at –80°C 
to avoid artifact that can result from storage of OCT embedded at lower temperatures.  The 
remainder of the tissue specimen will be stored in cryotubes at -180°C in a vapor phase 
nitrogen freezer (MVE chart).  Each OCT embedded specimen will have a hematoxylin and 
eosin stained slide prepared that will be reviewed by a board certified gynecologic 
pathologist.  This microscopic image will be photographed using the Aperio Scanscope and 
stored into database for future use.  In the case of a large volume tumor, tissue specimens 
from the primary tumor will be minced into fragments and placed into separate cryotubes.   
In cases in which there is less than 75% of the tissue specimen composed of tumor, 
specimens may also be processed using laser capture microscopy to isolate focal aspects of 
a particular tissue specimen.  Molecular extracts (i.e. DNA, RNA, and protein) will be 
prepared according to the techniques described below and stored in the molecular library 
maintained by the Human Tissue Resource Network a division in the Department of 
Pathology at OSU.  
Refrigeration and freezer units will be equipped with continuous chart recording of 
temperature.  The laboratory technicians for the Human Tissue Resource Network a division 
in the Department of Pathology at OSU will handle the quality assurance management of the 
equipment.  The refrigerator/freezer unit will be locked for security and keys will only be 
distributed to the laboratory technicians.  
3. Dissection and Acquisition of Cancer Cells:  Specimens of ascitic fluid will be centrifuged 
for collection of tumor cells prior to extraction of DNA/RNA or preparation of cell cultures.  
The white blood cell buffy coat will be similarly collected after centrifuging of blood 
specimens, with collection of genomic DNA.  Both gross and laser capture microdissection 
(LCM) will be performed on the frozen tissue specimens in order to maximize the yield of 
tumor cells prior to DNA and RNA extraction. Morphologically defined normal and tumor 
epithelial cells will be dissected out by laser capture micro-dissection.  In instances in which 
blood was not collected from a patient, genomic DNA will be extracted from paraffin 
embedded normal tissue specimens.  If paraffin tissue is used, sections (5 μm) will be treated 
with xylene initially to remove paraffin and continued as per frozen sections.  All tissue 
microdissection will be performed in a laser capture microscopy core facility located in the 
Department of Pathology at OSU.  Microdissected specimens will undergo DNA, RNA, and 
protein extraction according to procedures listed below.  All “extracts” will be stored in the 
 
76


Ohio State University Gynecologic Oncology Tissue and Serum Bank using bar codes that 
correspond with those that are used to label the tissue specimens that are permanently 
maintained by The Ohio State University.  
4. Sample preparation. Genomic DNA and RNA will be prepared from 100 mg of frozen tissues.  For 
tissue extractions, frozen tissue samples are pulverized under liquid nitrogen. The frozen powder is 
portioned into aliquots for DNA and RNA extractions.  DNA is prepared by digestion of the pellet or 
frozen powder with SDS and proteinase K followed by NaCl “salting out” precipitation of proteins. The 
DNA in the supernatant is further purified and recovered by ethanol precipitation. For RNA 
preparation, the starting material is homogenized in Trizol ™ reagent.  Chloroform is added to 
partition the solution into an organic phase containing the proteins, a DNA interface, and an aqueous 
layer containing the RNA. The RNA is recovered by precipitation with isopropanol followed by 
centrifugation. For additional purification, the RNA precipitate is dissolved in RNase free water or 
Qiagen buffer, then extracted using Qiagen RNeasy ™ columns according to the manufacturers 
instructions. Complementary DNA (cDNA) is generated from the mRNA by Superscript II reverse 
transcriptase (Invitrogen) employing oligo dT as a primer.  This ensures that all polyA gene transcripts 
are represented at a rate that reflects the original abundance of each gene product.  However, the 
oligo dT priming often fails in autopsy brain tissues, particularly where the marker SNP for AEI 
analysis resides far upstream of the polyadenylation site.  Therefore we use gene specific oligos 
targeting a region immediately 3’ of the marker SNP to prime cDNA synthesis.  Since tissue samples 
are often in limited supply, we have multiplexed up to 25 primers to permit 25 RT-PCR or AEI assays 
per cDNA preparation.  Comparisons between single and multiple primers showed no significant 
differences for the mRNA levels and AEI ratios.   We have been successful in extracting DNA and 
RNA from small frozen brain sections on slides, under protocols we have established for optimal 
results with SNaPshot, SNPlex, and other genotyping assays.  Since most of our assays are highly 
multiplexed, minimum requirements for DNA and RNA are in the order of 100 ng for DNA and 1 µg for 
RNA (depending upon the quality).  However, for multiple and diverse assays as proposed here we 
need several micrograms of DNA and RNA.  Where needed we apply whole-genome 
preamplification, with phi-29 DNA polymerase with random hexamer primers, using as little as 10 ng 
DNA.  This however does introduce additional error and is not useful for quantitation or methylation 
analysis. 
5. Serum extraction:  Blood samples will be allowed to clot for 30-45 minute prior to 
centrifuge at 1000 x 10 min under refrigeration at 4°C x 2.  The supernatent will be removed 
for storage at -180°C in a vapor phase nitrogen freezer   
 
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6. DNA extraction:  DNA will be extract from white blood cell buffy coat and/or tissue using 
the Qiagen nucleic acid isolation kits for DNA using the robotic M48 equipment. This 
technology uses a magnetic bead system. The integrity and concentration of DNA from 
tissue will be determined spectrophotometrically.  The DNA will be dissolved in pure DNase 
free and stored at -180°C in a vapor phase nitrogen freezer   
7. RNA extraction:  Tissues will be homogenized in RNAzol B followed by 
chloroform/isopropanol extraction and ethanol precipitation.  The RNA will be dissolved in 
filtered DEPC water.  Contamination of any genomic DNA will be removed by the treatment 
of RNA preparation with RNase free DNase.  Blood collected in PaxGene tubes will be 
processed for RNA using the RNA extraction kit that is commercially available.  Quality and 
quantity of RNA will be checked on formaldehyde-agarose gels and there will be a 
spectrophotometric determination of concentration prior to storage at -180°C in a vapor 
phase nitrogen freezer. 
8 ELISA:  Vascular endothelial growth factor (VEGF), CD-31, thrombospodin-1 (TSP-1), and 
CD-105 (endoglin) levels will be obtained by ELISA. ELISA will be performed using standard 
practices and commercially available kits. 
9. Recording. All tissue specimens as well as extracted components will be bar coded to 
maintain confidentiality.  The exact location of the sample in the freezer will be stored in 
database (i.e. rack 1, box 3, row 4, 6th sample left-right) using am MS SQL database program 
for specimen inventory.  The amount of tissue as well as extracted components will also be 
listed within the database.  For example, there may 1 vial of tissue, as well as 1 DNA sample, 
1 RNA sample and 1 serum sample that were broken into 25% aliquots.  This would result in 
13 tubes of material that would be recorded within the bank.  Samples (and quantity) added 
or removed from the bank will be recorded.  The audit trail will record aliquot changes and 
deletions as well.  Security levels and passwords that protect the data meet HIPPA and HHS 
requirements.    
10. Security. The tissues will remain in the bank maintained by The Ohio State University 
Gynecologic Oncology Tissue and Serum Bank until they have been depleted as a result of 
use in research studies. The director of tissue procurement will be in charge of maintaining 
the security of the tissue for The Ohio State University Gynecologic Oncology Tissue and 
Serum Bank.   Future studies that utilize the molecular extracts must undergo approval by 
 
78


The Ohio State University Gynecologic Oncology Tissue and Serum Bank Oversight 
Committee. Access into the laboratory in The Ohio State University Gynecologic Oncology 
Tissue and Serum Bank will be obtained through a locked door that is only accessible to the 
laboratory technician and authorized personnel that work in The Ohio State University 
Gynecologic Oncology Tissue and Serum Bank.  All molecular extracts will be maintained in 
a locked freezer with keys only available to the laboratory technician/honest broker for the 
GDP.  In addition, video surveillance of the building will guarantee the security of the tissue 
bank as well as the equipment located there.   
11 Patient Confidentiality: All data along with an inventory of tissue and serum specimens 
and the master link between patient identities band the patient’s code numbers will be 
maintained at The Ohio State Gynecologic Oncology Tissue and Serum Bank. The identity 
code will not include patient’s social security number.  All information will be gathered by the 
research staff (principal investigators, data managers, research assistants, research 
administrators, research nurses) of the Division of Gynecologic Oncology.  Only the honest 
broker, specified by the principal investigator, will have access to the link between patient 
identity and coded number used to label tissue specimens and data forms.  Documents will 
be destroyed 20 years after the close of the study.  Retrospective data will be destroyed 
immediately upon completion of the databases for that year. Agencies receiving de-identified 
data will be governed by memorandums of understanding, which outline confidentiality 
requirements are necessary.  Institutions are also bound by federal guidelines regarding this 
data. All patients will be asked to sign HIPAA consent form. Patients can withdraw from the 
study at anytime. Patients will be instructed during time of consent to contact the principal 
investigator (David O'Malley, MD at 614-293-8737) if they wish to withdraw from the study. At 
this time the patient may request that any samples that they have donated by destroyed or 
that their identification be removed without destroying the samples.  Data collected before the 
patients withdraw from the study will be kept private and will not affect the patient’s medical 
care. 
 
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 PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF 
ADVANCED STAGE ENDOMETRIAL CARCINOMA 
 
APPENDIX G 
 
GENETIC ALTERATIONS IN ENDOMETRIAL CANCERS 
GENETIC ALTERATIONS IN ENDOMETRIAL CANCERS 
 
 
 
 
 
CLASS 
 
 
ACTIVATION 
 
FREQUENCY 
Hereditary 
BRCA1 
 
tumor suppressor 
 
mutation/deletion 
 
 
1% 
BRCA2 
 
tumor suppressor 
 
mutation/deletion 
 
 
1% 
MSH2  
 
DNA repair 
 
 
mutation 
 
 
 
1-3% 
 
 
Sporadic 
PTEN  
 
tumor suppressor 
 
mutation/allelic loss  
50% 
 
 
K-ras  
 
G protein 
 
 
mutation 
 
 
 
10-30%  
  
BRAF  
 
G protein 
 
 
mutation 
 
 
 
20% 
 
Β-catenin 
 
signal transduction  
overexpression 
 
 
20% 
 
p53 
 
 
tumor suppressor 
 
mutation/deletion 
 
Type I: 10-20% 
 
 
 
transcription factor 
 
overexpression 
 
Type II: 80-90% 
 
HER-2/neu 
 
tyrosine kinase 
 
amplification/  
 
10-30%   
 
 
 
 
 
 
 
Overexpression 
 
RB1 
 
 
transcription factor 
 
allelic loss 
 
 
 
20% 
 
 
 
p16 
 
 
tumor suppressor 
 
homozygous  
 
 
10-15%  
cdk inhibitor  
 
deletion 
 
c-myc  
 
transcription  
 
overexpression 
 
 
10-15% 
 
 
 
factor 
 
 
 
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A PHASE II EVALUATION OF CARBOPLATIN/  PACLITAXEL/ BEVACIZUMAB IN THE TREATMENT OF 
ADVANCED STAGE ENDOMETRIAL CARCINOMA 
APPENDIX H 
 
GENOTYPING AND ANALYSIS FOR ALLELIC EXPRESSION 
IMBALANCE OF 18 TUMOR SUPPRESSOR GENES 
Genotyping and analysis for allelic expression imbalance of 18 tumor suppressor 
genes. 
 
Approach.  Using SNPlex, we measure 3-7 SNPs per gene to assess genetic variability 
(including haplotype information) and to determine which samples are heterozygous for the 
marker SNP(s) in the transcribed region of the 18 TSG genes.  We   then perform SNaPshot 
analysis for those samples heterozygous for the marker SNPs, measuring allelic ratio in 
genomic DNA and mRNA.  The presence of an allelic expression imbalance is detected by a 
difference in the allelic ratios for DNA and mRNA.  AEI analysis will be performed in PBLs 
(germ line) and cancer plus adjacent normal tissues.    The pattern of AEI in these samples 
alerts us to a possible role of the respective gene in ovarian cancer.  This may also predict 
treatment outcome.  Once we have established the pattern and frequency of AEI, we can use 
AEI as the phenotype to search for the function polymorphism (23,24).  However this 
exceeds the scope of the present grant application, but rather can form the basis for R01 
applications.    
 
Methods.  Selection of marker SNPs.  Marker SNPs must be in the transcribed region and 
sufficiently abundant (because we can perform allelic assays only in heterozygous 
individuals).  Table 2 contains the marker SNPs we have selected.  For two genes the 
marker SNPs were of unexpected low abundance and other SNPs will need to be used.     
 
 
Table 1.  Eighteen tumor suppressor genes included with this study.  Also provided are the marker 
SNPs we have selected for developing the AEI assays. For some genes more than one marker are used 
to increase the number of heterozygous samples and provide internal controls.  These SNPs are also in 
the rapid genotyping panels, so that we can rapidly identify heterozygous carriers for each gene. 
APC_rs42427       
APC_rs465899 
ATM_rs1801516 
BRCA1_rs1799966     
BRCA2_rs144848 
BLM_rs11852361      
 
HPC1_rs486907 
RB1_rs4151539        
p16_rs11515   
p16_3088440   
p27_rs2066827 
p27_rs34330  
p21_rs1801270 
 
TP73_rs1801174 
TP53_rs1042522 
VHL_rs1642742 
NF1_rs1801052 
NF2_rs1008515 
NF2_rs1034880 
 
MLH1_rs1799977 
RAD51_rs1801174
 XPC_rs2228000  
WRN_ rs1346044 
WRN_rs1800392 
 
 
Genotyping.  We use either SNPlex (ABI) or SNaPshot (ABI) for genotyping.  SNPlex permit 
the simultaneous analysis of 48 SNPs per run, with a throughput of 5-10,000 SNPs per hour.    
(We have already determined over 150,000 genotypes with these methods, and have 
controlled for quality with SNaPshot.  Panels for the tumor suppressor genes are available, 
 
81


with frequent htSNPs, any known functional SNPs, and the marker SBPs in Table 2. 
Genotypes not amenable to SNPlex analysis are done by SNaPshot or other methods.     
SNaPshot methodology for allele-specific mRNA assays.  The method has been described 
by us in detail (23, 24) It is understood that this type of analysis is challenging.  Therefore, 
each AEI assay requires rigorous validation and quality control, and every single step in the 
process must be optimized.  Main causes of analytical variability include cDNA formation and 
PCR amplification (see alternative methods below).  In contrast to absolute levels of mRNA 
which are also measured in all samples by real-time RT-PCR, allelic ratio analysis takes 
advantage of the fact that one allele serves as the control for the other.  We determine 
whether the DNA and mRNA ratios differ statistically for each single sample (requiring 
independent repeats, 3 repeats each measured twice).  In this fashion we can use AEI as the 
individual phenotype to scan the gene locus for the functional polymorphisms.  For most 
genes, we can reliably detect a 10-20% difference in mRNA levels generated from each of 
the two alleles present.  The level of AEI required for functional consequences is likely to 
vary from one gene to the next.  We have selected SNaPshot (ABI) as the assay system, 
involving PCR and fluorescence primer extension analysis.  
SNaPshot assay procedure. The details of this assay for detecting AEI (measuring bot DNA 
and mRNA) have been published (23,24).  Briefly, a stretch of genomic DNA (~70 bp's), or 
cDNA made from the same tissue, is amplified by polymerase chain reaction (PCR), and the 
polymorphism is measured by primer extension using fluorescently labeled terminator 
nucleotides. The products are analyzed using an ABI 3730 capillary electrophoresis DNA 
instrument, and calculated with Gene Mapper TM 3.0 (ABI) software. This permits injection of 
48 samples every 20 min. The data for each incorporated fluorescently labeled nucleotide is 
measured as a peak area.  Success rate for assay development is >90%, and alternative 
SNPs can be selected to overcome problems.  For maximum precision, we dilute samples as 
needed so that DNA and mRNA thresholds are similar.  Using cloned DNA, we establish the 
number of mRNA molecules present (1,000 or greater for optimal results) using standard 
curves.  S.D.s for heterozygous genomic DNA samples range from 3-7%.  mRNA/cDNA 
ratios (triplicate samples each assayed twice) show higher standard deviations (5-15%), so 
that detectability of AEI varies with the quality of the sample and the SNP analyzed.   We 
have developed numerous controls, such as standard curves using cloned gene fragments 
containing the marker SNP, the use of more than one marker SNP per gene, use of 
extension primers in both directions, 3 independent cDNA preparations, and use of gene 
specific primers for cDNA preparation.   
Potential problems.  AEI can also be caused by epigenetic factors.  If we suspect such factors 
to play a role, CpG island methylation is measured using methylation-sensitive restriction 
enzymes and PCR.  In tumor samples the allelic DNA ratios can vary (LOH, gene 
amplification).  This is addressed by normalizing the mRNA ratios to the DNA ratios, set as 1.  
However, significantly different DNA ratios in some samples indicate the presence of 
chromosomal instability, which is also to be expected if one allele (the high-expressing one) is 
selectively deleted thereby yielding growth advantage to the cancer cells.  We also expect the 
tumor samples not to be homogenous; this problem is partially overcome by the precision of 
the AEI analysis, whereby we can detect even small differences in allelic ratios (assuming a 
mixture of tumor and stroma). Additionally, the use of microdissection techniques (Appendix A-
section 3) will be used were applicable. 
 
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Statistics and sample cohorts needed.  In genes where we do not see any AEI in normal 
subjects (such as APC; our results are identical to those of Yan et al.), the presence of even 
a single AEI patient can be followed mechanistically by comparing allelic ratios in normal and 
tumor tissues (as done by Yan et al.).  In the case of p16, with 5% AEI frequency in normal 
subjects, regular power analyses can yield the number of cases needed to show a statistical 
association with ovarian cancer.  More importantly however, we can use each patient 
separately by comparing AEI in PBL/normal tissue versus tumor tissues.  The requirement 
that the high-expressing allele be deleted in tumors is highly specific, thus reducing the 
number of subject needed drastically.  
 
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