Tissue Pharmacokinetics of Intraoperative Gemcitabine in Adenocarcinoma of the Pancreas After Preoperative Chemoradiation Therapy
Phase: NA
Conditions: Pancreatic Cancer
Sponsor: M.D. Anderson Cancer Center

2011-0703 
August 30, 2011 
 
 
 
Tissue Pharmacokinetics of Intraoperative Gemcitabine in Adenocarcinoma  
of the Pancreas after Preoperative Chemoradiation Therapy 
 
 
 
Table of Contents 
 
List of Abbreviations 
1.0 
Objectives 
1 
 
2.0 
Background and Rationale  
 
 
 
 
 
1 
 
3.0 
Patient Eligibility 
3 
 
4.0 
Gemcitabine  
 
 
 
 
 
 
 
4 
 
5.0 
Gemcitabine Administration Plan  
 
 
 
 
6 
 
6.0 
Safety Monitoring 
 
 
 
 
 
 
 
8 
 
7.0 
Processing of Samples and Outcome Measurements 
 
 
9 
 
8.0 
Statistical Considerations  
 
 
 
 
 
11 
 
9.0 
Data and Protocol Management  
 
 
 
 
 
11 
 
10.0 
Guidelines for Monitoring Clinical Trials  
 
 
 
11 
 
11.0 
Confidentiality 
 
 
 
 
 
 
 
12 
 
References 
 
 
 
 
 
 
 
 
 
13 
 
Appendix 1    Karnofsky Performance Status Scale 
 
 
 
15 
 
Appendix 2    Reported Gemcitabine Toxicity 
 
 
 
 
16 
 
Appendix 3  Intraoperative Schema 
 
 
 
 
 
18 
 
Appendix 4  Anticipated Perioperative Complications  
 
 
19  
Appendix 5 
Photographs Depicting a Gross Pathologic Examination of  
 
the Pancreaticoduodenectomy Specimen 
 
 
20 
 
Appendix 6 
Assignment of Protocol Responsibilities  
 
 
21 
 
Appendix 7 
New York Heart Association Functional Classification  
22 

2011-0703 
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Study Chair:   
Gauri Varadhachary, MD 
 
 
 
Department of Gastrointestinal Medical Oncology 
 
Study Co-Chairs:  
Jason B. Fleming, MD 
 
 
Department of Surgical Oncology 
 
 
 
 
Vijaya Gottumukkala, MD 
 
 
 
 
 
 
Department of Anesthesiology and Perioperative Medicine 
 
 

2011-0703 
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List of Abbreviations 
 
AST/SGOT 
Aspartate aminotransferase 
ALT/SGPT 
Alanine aminotransferase 
CBC 
Complete blood count 
CDA 
Cytidine deaminase  
Cl 
Clearance 
dCDA 
Deoxycytidine deaminase (gemcitabine inactivating enzyme) 
dCK 
Deoxycytidine kinase (gemcitabine activating enzyme) 
dCTP 
Deoxycytidine triphosphate 
dFdC 
Gemcitabine (2’,2’-difluorodeoxycytidine) 
dFdCDP 
Difluorodeoxycytidine diphosphate 
dFdCTP 
Difluorodeoxycytidine triphosphate  
dFdU 
Difluorodeoxyuridine (a gemcitabine metabolite) 
DNA 
Deoxyribonucleic acid 
DVT 
Deep vein thrombosis 
ELISA 
Enzyme-linked immunosorbent assay 
EMR 
Electronic medical record 
FDR 
Fixed dose rate 
GI 
Gastrointestinal 
GV 
Gauri Varadhachary, MD 
hENT-1 
Human equilibrative nucleoside transporter 1 
Hh 
Hedgehog 
HPLC/MS 
High performance liquid chromotography-mass spectrometry 
HUS 
Hemolytic-uremic syndrome 
HW 
Huamin Wang 
IHC 
Immunohistochemistry 
JF 
Jason Fleming, MD 
LC/MS/MS 
Liquid chromatography-mass spectometry 
μmol  
Micromole  
m2  
Meters squared  
Mets 
metastases 
mg  
Milligram  
min 
Minute 
mL  
Milliliter  
mm  
Millimeter  
mol 
Mole 
mmol  
Millimole  

2011-0703 
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MT 
Mark Truty, MD 
OR  
Operating room 
PBMC 
Peripheral blood mononuclear cells  
PC 
Pancreatic cancer 
PCR 
Polymerase chain reaction 
pK 
Pharmacokinetics 
RT-PCR 
Reverse transcription polymerase chain reaction 
SMA 
Superior mesenteric artery 
SMV 
Superior mesenteric vein 
SMPV 
Superior mesenteric portal vein 
SNPs 
Single-nucleotide polymorphisms  
TUNEL 
Terminal deoxynucleotidyl transferase dUTP nick end labeling 
UTI 
Urinary tract infection 
VTE 
Venous thromboembolism 
WHO 
World Health Organization 
WP 
William Plunkett, PhD 

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1.0 
Objectives 
 
1.1 
Primary Objective: 
 
• 
To quantifiably assess intratumoral gemcitabine levels in human pancreatic 
cancer tissue after a single intraoperative infusion in patients who have 
completed a course of preoperative chemotherapy and radiation therapy. 
 
1.2 
Secondary Objectives: 
 
• 
To measure intra-DNA gemcitabine (dFdC) levels using a novel assay 
LC/MS/MS, developed by Eli Lily, available through Advion BioServices. 
 
• 
To assess and validate previously described markers that may be predictive of 
gemcitabine sensitivity or resistance - examine the expression of molecules 
involved in gemcitabine metabolism (CDA, dCK, DCTD), and transport 
(hENT1, hCNT1, hCNT2, hCNT3) in the resected primary tumors.  
 
• 
To measure the impact of microvessel density and the molecular expression 
level of the Hh signaling pathway on gemcitabine delivery and DNA 
incorporation.  Blood vessels in resected tumors will be detected by IHC using 
antibodies to CD31 or vWF. The mRNA and protein expression of SHH, Gli 
and SMO will be measured by RT-PCR and IHC method, respectively. These 
markers will be correlated to the gemcitabine level. 
 
• 
To correlate intratumoral gemcitabine levels and its tumoricidal activity with 
Ki67 index and intratumoral apoptosis.  
 
 
2.0 
Background and Rationale 
 
2.1  
Gemcitabine is a nucleoside analogue that exhibits modest antitumor activity in 
numerous solid tumors, including pancreatic adenocarcinoma, and is currently the 
first-line agent for this malignancy.[1] There is no current data that demonstrates that 
this drug in fact penetrates tumor tissue despite the increasing use of this agent in the 
operable and locally advanced pancreatic cancer setting. This is even more critical 
a question for previously radiated tumors. Emerging evidence suggests that 
resistance to gemcitabine in pancreatic cancer may be related to several critical 
factors: a desmoplastic stroma with poor vascular permeability that inhibits drug 
delivery into cancerous tissue, and variable expression of key proteins involved in 
transport, activation, metabolism, and elimination of this agent.[2-6] 
 
2.2  
Previous clinical Phase I/II pharmacokinetic studies in pancreatic cancer have only 
measured gemcitabine levels within peripheral blood.[7-10] In these studies the 
efficacy of gemcitabine has been correlated with concentrations accumulated in 
peripheral blood mononuclear cells (PBMC), which are related to plasma 
concentrations of the drug. Classically these PBMC’s are used to quantify the levels 
of active gemcitabine and its metabolites in tumor tissue, however they are only a 
surrogate matrix. Unfortunately there is no data to suggest that gemcitabine or its 
active intracellular metabolite, gemcitabine triphosphate (dFdCTP), is present to any 

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Page 2 
 
significant level in human pancreatic cancer tissue despite this agent now being the 
standard-of-care. In patients with locally advanced pancreatic cancer, it is not 
unusual in clinical practice to give additional gemcitabine after completion of 
radiation without any knowledge that the drug actually enters radiated tissue. 
 
2.3 
Recent data suggests that the de novo resistance may be related to poor penetration 
of the therapeutic agent into the pancreatic tissue.  By way of example, many patients 
display resistance to even the standard first-line therapy – Gem. (2)  Some of the 
factors that have been described as contributing to resistance to Gem (dFdC) include: 
(i) Hypoperfusion (ii) Desmoplastic stroma (iii) Factors involved in its uptake, 
metabolism, and elimination - deoxycytidine kinase deficiency (dCK – Gem activating 
enzyme), higher deoxycytidine deaminase expression (dCDA - Gem inactivating 
enzyme) and hENT1 (transporter) deficiency in tumor cells. (13-16). Further detail into 
the action of these key regulatory genes and their role in gemcitabine activity within in 
vivo cancerous tissue warrants further investigation in a clinical trial setting. 
 
2.4  
We currently have a novel pharmacokinetic human clinical trial (2010-0371) in which 
patients undergoing curative resection of pancreatic adenocarcinoma receive an 
intraoperative single infusion of gemcitabine. The purpose of this clinical and 
translational science is to specifically assess and quantify drug incorporation within in 
vivo cancerous tissues in order to develop a novel and effective model for Phase 0/1 
trials in the neoadjuvant setting to enable us to ask mechanistic questions pertaining 
to drug delivery and discovery in pancreatic cancer. Gemcitabine is a safe, well-
studied, and commonly used drug in this tumor model – and currently 7 patients have 
completed the study (2010-0371) without any drug related toxicity or concerns.  We 
plan to extend this approach to additional patients who have received preoperative 
chemoradiation and undergoing tumor resection.  
 
2.5 
Previous preclinical, biochemical, and clinical investigations have strongly suggested 
that the rate of gemcitabine triphosphate accumulation is saturated at 10 to 20 mol/L 
of gemcitabine in plasma or medium. This concentration of gemcitabine is achieved 
in plasma when gemcitabine is infused at a dose rate of 10 mg/m2/min (Fixed dose 
rate, FDR). A phase II trial by Tempero et al compared FDR to standard infusion and  
pharmacokinetic analyses demonstrated a two-fold increase in intracellular 
gemcitabine triphosphate concentration in the FDR arm (P =.046).  The selected 
dose for this trial will be lower than the full dose of 1000mg/m2/min since in practice 
after completion of RT, the dose is often decreased and most patients are given up 
to 750mg/m2.  After safety is established with a lower dose of 500mg/m2, we will 
increase the dose to 750mg/m2.  
 
2.6 
Quantification of gemcitabine incorporation into human DNA by liquid 
chromatography-mass spectometry (LC/MS/MS) is a novel assay which is available 
through Advion BioServices.. These methods will normalize the amount of dFdC 
released relative to the native (DNA) nucleoside dG and simultaneously quantify the 
signal of both analytes by LC/MS/MS by extensive detuning of the signal for dG. This 
method represents an improved surrogate for quantitating gemcitabine target 
engagement and results will be compared to intracellular gemcitabine (metabolite) 
levels in circulating PBMCs and PC tumor tissue and will be correlated to the 
markers in the secondary objectives. 
 

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2.7  
With preliminary data demonstrating the feasibility and promise of this approach, we 
will address important issues including impact of therapeutic intervention, including 
pre-operative radiation on drug penetration (potentially change the treatment 
paradigm for borderline and locally advanced pancreatic cancer). 
 
 
3.0 
Patient Eligibility 
 
3.1 
Inclusion Criteria: 
 
3.1.1 Cytologic or histologic proof of adenocarcinoma of the pancreas is required. 
Patients with Islet cell tumors are not eligible. 
 
3.1.2 Patients do not have known metastases.  
 
3.1.3 Patients must have potentially resectable or borderline resectable pancreatic 
cancer and have agreed to undergo surgical resection at MD Anderson Cancer 
Center if operable. They will have undergone staging (physical examination, 
chest x-ray, contrast enhanced CT or MRI (if CT contraindicated) and/or 
angiogram) to determine resectability.   
 
3.1.4 Patients have completed radiation and chemotherapy with either capecitabine 
or gemcitabine as a radiosensitizing agent as part of their preoperative 
therapy.  Previous systemic chemotherapy alone is not allowed. Preoperative 
therapy will be completed at least 4 weeks prior to surgery.     
 
3.1.5 Patients with Karnofsky performance status > 70 are eligible. (Appendix 1)  
 
3.1.6 There will be no upper age restriction. Patients less than 18 years of age are 
excluded from the protocol because adenocarcinoma of the pancreas is rarely 
seen in the pediatric population.  
 
3.1.7 Adequate renal and bone marrow function: 
 
 
 
 
• 
Leukocytes >= 3,000/uL 
• 
Absolute neutrophil count >= 1,500/uL 
• 
Platelets >= 100,000/Ul 
• 
Serum creatinine <= 2.0 mg/dL  
• 
Creatinine clearance >= 60 ml/min (calculated by the Cockroft-Gault 
equation) 
 
3.1.8 Adequate Hepatic function (endoscopic or percutaneous drainage as needed) 
• 
Total bilirubin < = 3 X institutional upper limits of normal (ULN) 
• 
AST (SGOT)/ALT (SGPT) <= 5 X institutional ULN 
 
3.1.9 Patients must have no fever or evidence of infection or other coexisting medical 
condition that would preclude administration of gemcitabine. Patients with 
uncontrolled congestive heart failure, unstable angina and myocardial 
infarction within 3 months will be excluded 
 

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Page 4 
 
3.1.10 Patient is not pregnant.  Women of childbearing potential (i.e., women who are 
pre-menopausal or not surgically sterile) must use acceptable contraceptive 
methods (abstinence, intrauterine device [IUD], oral contraceptive or double 
barrier device) and refrain from breast-feeding, as specified in the informed 
consent.   
 
3.1.11 Patients must sign a study-specific consent form. 
 
 
3.2 
Exclusion criteria: 
 
 
3.2.1 Major cardiovascular or pulmonary comorbidity that precludes use of general 
anesthesia (NYHA [New York Heart Association] Class III and IV) (Appendix 
7) 
 
3.2.2 Identification of metastatic disease. 
 
3.2.3 Patients with a known hypersensitivity to Gemcitabine. 
 
3.2.4 Pregnant women 
 
3.2.5 Inability to comply with study and/or follow-up procedures. 
 
3.2.6 Patients < 18 years of age. 
 
 
4.0 
Gemcitabine 
 
4.1 
Pharmacology 
 
4.1.1 Gemcitabine is commercially available from Eli Lilly and Company, 
Indianapolis, Indiana. Drug supply for this study will be purchased with 
Institutional Research Grant funds through Investigational Pharmacy 
Services. Investigational Pharmacy Services will dispense the drug.  Patients 
will not be charged for the cost of the drug. 
 
4.1.2 Gemcitabine HCl is 2-deoxy-2, 2-difluorocytidine monohydrochloride (beta 
isomer) nucleoside analogue.  
 
4.1.3 The main cytotoxic effect of gemcitabine involves the competitive incorporation 
of gemcitabine triphosphate into replicating DNA leading to masked chain 
termination, which inhibits DNA synthesis and leads to cell death.[17-19] 
Gemcitabine exhibits cell phase specificity, primarily killing cells undergoing 
DNA synthesis (S-phase) and also blocking the progression of cells through 
the G1/S-phase boundary. Gemcitabine is a pro-drug that must be 
phosphorylated to its active metabolites, difluorodeoxycytidine diphosphate 
(dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP) nucleosides, 
intracellularly by nucleoside kinases to have biological activity. The cytotoxic 
effect of gemcitabine is attributed to a combination of two actions of the 

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diphosphate and the triphosphate nucleosides, which leads to inhibition of DNA 
synthesis. First, gemcitabine diphosphate inhibits ribonucleotide reductase, 
which is responsible for catalyzing the reactions that generate the 
deoxynucleoside triphosphates for DNA synthesis. Inhibition of this enzyme by 
the diphosphate nucleoside causes a reduction in the concentrations of 
deoxynucleotides, including dCTP. Second, gemcitabine triphosphate 
competes with dCTP for incorporation into DNA. Gemcitabine diphosphate 
(dFdCDP) induces a self-potentiating mechanism by inhibiting ribonucleotide 
reductase, thereby reducing the amount of intracellular dCTP. After the 
gemcitabine nucleotide is incorporated into DNA, only one additional 
nucleotide is added to the growing DNA strands. After this addition, there is 
inhibition of further DNA synthesis. DNA polymerase epsilon is unable to 
remove the gemcitabine nucleotide and repair the growing DNA strands 
(masked chain termination) after which the DNA synthesis is inhibited and cell 
death occurs.  
 
 
4.2 
Pharmacokinetics 
 
• 
Gemcitabine pharmacokinetics are linear and are described by a 2-
compartment model.  Population pharmacokinetic analyses of combined 
single-and multiple-dose studies showed that the volume of distribution of 
gemcitabine was significantly influenced by duration of infusion and gender. 
Volume of distribution is 50 L/m2 – 370 L/m2 depending on length of infusion 
and protein binding is negligible. 
 
• 
Clearance is dependent on age and gender and is primarily renal. Cl is 75.7 – 
92.2 L/h/m2 (men) and 57 – 69.4 L/h/m2 (women). In elderly populations, Cl is 
55.1 L/h/m2 (men) and 41.5 L/h/m2 (women). Half-life of drug is 42 (short 
infusion) – 638 (long infusion) minutes depending on length of infusion time. 
 
4.3 
Gemcitabine Preparation, Administration, Storage, and Disposal: 
 
4.3.1 Gemcitabine is a white to off-white lyophilized powder available in sterile 
single-use vials containing 200mg or 1000mg gemcitabine. 
 
4.3.2 Gemcitabine will be prepared according to the institutional standard. Caution 
should be exercised in handling and preparing gemcitabine solutions and the 
use of gloves is recommended. Drug vials will be reconstituted by the 
institutional inpatient operating room pharmacy with 0.9% sodium chloride 
without preservatives to a maximum concentration of 38mg/ml due to solubility 
considerations. A predetermined amount of drug (see gemcitabine 
administration plan for dosing) will be prepared for administration as a 
continuous infusion. Once the drug has been reconstituted, it should be stored 
at room temperature and used within 24 hours. 
 
4.3.3 Gemcitabine will be administered by the anesthesiologist.  Gemcitabine 
will be infused at a fixed dose rate of 10mg/m2/min. Previous preclinical, 
biochemical, and clinical investigations have strongly suggested that the rate 
of gemcitabine triphosphate accumulation is saturated at 10 to 20 mol/L of 

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gemcitabine in plasma or medium.[9, 12, 20] This concentration of gemcitabine 
is achieved in plasma when gemcitabine is infused at a dose rate of 
10mg/m2/min. Although at present the data in the target tumor tissue are 
lacking for such comparisons, the data in surrogate PBMC’s strongly suggest 
the benefit of this pharmacologically based gemcitabine infusion rate and 
serves as a dose rate to provide plasma concentrations (approximately 20 
mol/L) that are sufficient to maximize the rate of gemcitabine triphosphate 
accumulation within cancerous tissue. 
 
4.3.4 Stored at controlled room temperature (20-25°C), gemcitabine is stable for 24 
hours.  Solutions of reconstituted gemcitabine will not be refrigerated. No 
incompatibilities have been observed with infusion bottles or polyvinyl chloride 
bags and administration sets. All unused portions will be handled and disposed 
of in a manner consistent with other anti-cancer drugs according to institutional 
policies.  
 
4.4 
Gemcitabine Precautions and Toxicities:  
 
4.4.1 Gemcitabine is contraindicated in those with a known hypersensitivity to the 
drug. 
 
4.4.3 Previous studies using a similar intraoperative approach in central nervous 
system cancers, found no complications related to single-dose infusion of 
Gemcitabine.[21] Earlier Phase I/II trials at this institution found little toxicity 
with initial single-dose therapy. Our current clinical trial using intraopeartive 
Gemcitabine is accruing steadily and no major complications related to 
gemcitabine use have been reported (IRB 2010-0371).  
 
See Appendix 2 for a full list of Gemcitabine related side effects as mentioned 
in the package insert. 
 
 
5.0 
Gemcitabine Administration Plan 
 
This study is limited to patients who have been determined previously to have 
potentially resectable or more likely, borderline resectable pancreatic cancer, 
according to current surgical practice at MD Anderson.  The preoperative and 
postoperative tests/evaluations referenced in this study are standard of care for this 
patient population and would be performed regardless of whether a patient enrolled in 
this study or not.  The tests/evaluations are described in this study to demonstrate 
good clinical practices and because the results will be used to document eligibility for 
this study or as part of postoperative safety monitoring.   
 
 
5.1 
Pre surgical Evaluation: 
 
5.1.1 Patients will have the routine laboratory and radiologic studies required for their 
surgery. 
 
5.2 
Intraoperative Gemcitabine Administration (Appendix 3): 

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5.2.1 Surgery will be planned within 4 weeks of enrollment. 
 
5.2.2 There will be no increase in the anesthesia or operative time, nor will the 
operative procedure be increased in complexity as a result of this protocol. 
Standard operating room procedures and intravenous fluid and medication 
administration will be performed for pancreatic resection per anesthesiology 
and surgical teams. 
 
5.2.3 Diagnostic laparoscopy will be performed in all patients. Patients will be 
excluded from the protocol if metastases are found during laparoscopy. 
 
5.2.4 Following the initial laparoscopic exploration patients will proceed to formal 
laparotomy to allow for inspection of all peritoneal surfaces and viscera 
including liver. If on laparotomy, the tumor is found to be locally advanced and 
unresectable; the patient will be excluded from the protocol. 
 
5.2.5 In the absence of identified metastases, full tumor mobilization and surgical 
resection will be performed according to surgical oncology principles.  
 
5.2.6 Gemcitabine will be administered intravenously as a dose of 500mg/m2 at a 
fixed dose rate of 10mg/m2/min for the first 5 patients (to validate hematologic 
safety).  Next 15 subsequent patients will receive 750mg/m2 at a fixed dose 
rate of 10mg/m2/min. The drug infusion will be started 50-75 minutes prior to 
complete gross tumor removal (timing dependent on dose) in order to have 
drug administration complete at tumor removal ( +/- 10 minutes). Appendix 3 
describes the intraoperative time points. Any deviations in the time window 
allowed for chemotherapy administration will not be considered a protocol 
deviation. 
 
5.2.7 Blood will be drawn for pK and correlative studies at various time points during 
the procedure as described in Appendix 3. 
 
5.2.8 Following tumor removal, specimen will be taken to Pathology lab for 
subsequent analysis and preparation for correlative studies. 
 
5.3 
Standard Anesthesia Management Plan  
 
Drugs commonly used during pancreatic cancer surgery are listed below. These drugs 
have no reported interactions with gemcitabine. In the event any drugs not on this list 
are to be given to the patient, the anesthesiologist will confirm that there are no 
reported interactions with gemcitabine and the drug prior to administration.    
• 
Amiodarone 
• 
Antiemetics (both seratonin inhibitors and dopamine antagonists) 
• 
Bupivicaine 
• 
Calcium chloride  
• 
Cisatracurium  
• 
Dopamine 

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Page 8 
 
• 
Ephedrine 
• 
Etomidate 
• 
Fentanyl  
• 
H2 blockers (cimetidine, ranitidine, famotidine, nizatidine) 
• 
Hydromorphone  
• 
Lidocaine  
• 
Metoprolol 
• 
Phenylephrine 
• 
Propofol   
• 
Rocuronium 
• 
Ropivicaine 
• 
Sufentanil  
  
 
6.0 
Safety Monitoring 
 
 
6.1 
Postoperative Patient Evaluation: 
 
6.1.1 Examinations, vital signs, laboratory tests and any additional imaging will follow 
routine practice guidelines post pancreaticoduodenectomy.  
 
 
Patients will be admitted post-operatively and followed closely as per standard 
of care post-pancreatic resection.  This includes blood work, with CBC at least 
every other day.  We will terminate the study if we observe >33% of the first 10 
patients with grade 3 or 4 adverse events that are thought to be related to 
gemcitabine except for transient (lasting <= 48 hours) grade 3-4 leukopenia 
and/or neutropenia.  
 
 
Patients will be closely monitored but we do not anticipate any significant 
toxicity after one dose/infusion of gemcitabine. The drug labeling and literature 
reports of gemcitabine toxicity are on patients who have received multiple 
infusions of gemcitabine (at least one–two full cycles which is 3-6 infusions of 
gemcitabine and at a higher weekly dose). 
Patients will be followed for adverse events for 30 days post surgery. Patients 
will be followed daily while in the hospital.  Additional follow-up will occur at the 
time of routine clinic visits, or by a phone call to the patient at least once weekly.   
6.1.2 Our institution and study investigators have extensive experience in 
preoperative therapy and pancreatic resection for adenocarcinoma of the 
pancreas.[22, 23] Appendix 4 lists incidence of all major and minor 
complications. Any significant deviations from expected morbidity or mortality 
will be reviewed by the Study Chair, Co-Chairs, and Dr. James Yao prior to 
proceeding with enrollment of additional patients to the protocol. Dr. Yao will 
not enroll patients on this study.  His role is to provide additional safety 
monitoring.   
 
6.1.3 Meetings will be held after a cohort of 3 patients who have undergone resection 
and received intraoperative gemcitabine to monitor safety and review 

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correlative data.   These will be attended by the Chair, Co-Chairs, and the 
research team (research nurses, data manager, and laboratory research 
personnel).  Data will be compiled in a comprehensive research overview table 
and shared at each meeting.   In addition, laboratory values and adverse 
events (AE) will be documented for all patients who receive gemcitabine. 
 
 
Weekly registration reports will be generated to monitor patient accruals.   
Routine data quality reports will be generated to assess missing data and 
inconsistencies.   Accrual rates will be monitored periodically throughout the 
study period and potential problems will be brought to the attention of the study 
team for discussion and action. 
 
6.1.4 Only 
the 
designated 
clinical 
investigators 
will 
obtain 
informed 
consent/authorization. The research nurse will confirm eligibility and 
compliance of the consent process before entering patients into the CORe 
system.  
 
 
7.0 
Processing of Samples and Outcome Measurements 
 
7.1 
Blood Samples: 
 
7.1.1 Blood samples will be collected, processed, and analyzed in a single 
institutional laboratory (Dr. William Plunkett, T6.3916). Patients will not be 
charged for any costs for processing of blood and tissue samples related to 
this specific protocol. 
 
7.1.2 From each patient studied, 5-10 mL of blood will be taken into sterile 
heparinized tubes containing tetrahydrouridine (in order to inhibit degradation 
of active gemcitabine) at predetermined timepoints depending on the total 
gemcitabine dose. Plasma will be obtained by centrifugation of a portion of 
blood samples at 4°C and stored at −20°C until required for analysis of 
circulating plasma levels of gemcitabine and its metabolite. Gemcitabine and 
dFdU in tetrahydrouridine-treated human plasma are stable for at least half a 
year at −20°C.[24] The Ficoll-gradient method will be used for isolation of 
mononuclear blood cells. These cells will be immediately frozen in liquid 
nitrogen and subsequently stored -80°C until analysis of intracellular 
gemcitabine.  
 
 
7.2 
Tissue Samples: 
 
7.2.1 Tissue obtained will be from freshly excised tumor (superficial and deep), 
adjacent normal pancreas, and normal duodenal mucosa as shown in 
Appendix 5. These tissue specimens will be immediately frozen in liquid 
nitrogen and subsequently stored at -80°C until analysis (Dr. William Plunkett’s 
laboratory, T6.3916). Previous studies have found that such samples remain 
stable for analysis for 2–3 years under these storage conditions.[25] The 
remaining resected cancer specimen will undergo full pathological evaluation. 
 
 
7.3 
Correlative Studies on Blood and Tissue Samples: 

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Page 10 
 
 
7.3.1 The quantification of serum, PBMC, and cancer tissue levels of gemcitabine 
from frozen samples will be assessed using standardized techniques in high 
performance liquid chromatography-mass spectrometry (HPLC/MS).[24, 25] 
 
7.3.2 Blood vessels in resected tumors will be detected by immunohistochemistry 
(IHC) using antibodies to CD31 or vWF.  The mRNA and protein expression of 
SHH, Gli and SMO will be measured by RT-PCR and IHC method, 
respectively.  The range of levels in patients with PC will be established.  We 
will also correlate these markers to the gemcitabine levels measured in the 
cancerous tissue.  
 
7.3.3. The expression level of genes involved in gemcitabine metabolism (CDA, 
dCK, DCTD) and transport (hENT1, hCNT1, hCNT2, hCNT3) in the resected 
primary tumors will be examined. The mRNA level will be measured using 
real time PCR and protein expression by IHC.  Gene expression level will be 
correlated to the gemcitabine measurements.  Additionally, we will measure 
markers of proliferation and apoptosis by p21 and Ki67 IHC and fluorescent 
TUNEL analysis.  
 
7.3.4 Gemcitabine incorporation in DNA will be quantified by a new proprietary 
LC/MS/MS method developed by Eli Lilly (available through Advion 
BioServices) and performed by Advion BioServices. DNA extracted from the 
tissue samples will be sent to Advion (in batches of 50 samples including 
normal tissue). 
 
 
 
Advion BioServices, Inc.: 
 
Scientific Contact:    
 
Gary A. Schultz, PhD 
 
 
 
 
 
Senior Director, Biomarkers 
 
 
 
 
 
Advion BioServices, Inc. 
 
 
 
 
 
 
19 Brown Road 
 
 
 
 
 
 
 
Ithaca, NY 14850 
 
 
 
 
 
 
Phone: 607-330-9866 
 
 
 
 
 
 
Email: SchultzG@advion.com 
 
 
Shipping Address for Samples: 
Shawn P. Bode 
 
 
 
 
 
 
Manager of Laboratory Operations 
 
 
 
 
 
 
Advion BioServices, Inc. 
 
 
 
 
 
 
19 Brown Road 
 
 
 
 
 
 
Ithaca, NY  14850 
 
 
 
 
 
 
Phone: 607-266-0665 ext. 175 
 
 
 
 
 
 
Email:  Bodes@advion.com 
 
 
 
8.0 
Statistical Considerations 
 
 
8.1 
Methodology and Sample Size: 
 

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Page 11 
 
This pilot protocol is a non-randomized, non-blinded regimen.  
 
We plan to have 20 patients in the study (i.e., those who will undergo potentially 
curative resection and receive gemcitabine infusion).   
 
To reach the planned 20 patients, up to 40 patients may need to be enrolled 
(consented) at a rate of 2-3 patients/month.  Those patients who do not undergo 
curative PC surgery and receive gemcitabine infusion will be deemed ineligible and 
excluded from the analysis. The reason for enrolling up to double the number of 
patients is that since a significant portion of these patients may have borderline 
resectable disease, only 50-60% of the enrolled patients’ cancers may be finally 
resected (data based on our large retrospective study).   
 
If we observe >= 2 patients in the 500mg/m2 lower dose group (5 patients) with 
grade 3 or 4 adverse events that are thought to be related to gemcitabine except 
for transient (lasting <= 48 hours) grade 3-4 leukopenia and/or neutropenia, the 
study will be halted and will be amended to lower the dose/rate of infusion. 
We will terminate the study if we observe >33% of the first 10 patients with grade 
3 or 4 adverse events that are thought to be related to gemcitabine except for 
transient (lasting <= 48 hours) grade 3-4 leukopenia and/or neutropenia.   
 
An institutional departmental staff statistician will approve and oversee all statistical 
matters regarding current protocol and any subsequent analyses and publications. 
 
The primary objective is a feasibility objective and is not amenable to statistical 
analysis.   Analyses for the secondary objectives are exploratory in nature and will be 
conducted via the Spearman’s Rank Correlation Coefficient. 
 
 
9.0 
Data and Protocol Management 
 
After obtaining informed consent, patients will be registered on the MDACC computerized 
Clinical Oncology Research (CORe) System. Appendix 6 shows assignment of individual 
protocol responsibilities. 
 
 
10.0 
Guidelines for Monitoring Clinical Trials 
 
10.1 
Reporting of adverse events will be according to MD Anderson Guidelines for AE 
Reporting. 
 
10.2 
This study will utilize the Common Terminology Criteria for Adverse Events 
(CTCAE) Version 4.0 for toxicity grading and adverse event reporting. A copy of 
the CTCAE version 4.0 can be downloaded from the CTEP home page 
(http://ctep.cancer.gov).  
 
 
11.0 
Confidentiality 
 

2011-0703 
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This study will be conducted in accordance with all applicable privacy laws, rules and 
regulations.  The Principal Investigator will take steps to guard against any loss of 
confidentiality. Only the Principal Investigator, Study Co-Chairs, and the authorized research 
team will have access to the identifiable information from this study.   
 
Identifiers (such as name and medical record number) will be collected but will be replaced 
by study numbers in the analytic file. The key linking to these numbers will be retained in a 
secure computer file. Access to this file will be limited to the Principal Investigator, Study Co-
Chairs, and the authorized research team. All computer files are password-protected and 
stored on institution computers behind the institution firewall to further ensure database 
security and all records are kept confidential. Any reports or publications resulting from this 
study will not include any personal identifiers. 
 
Procedures for patients who consent to the optional banking of any left-over blood and/or  
tissue samples for future research:  IRB approval will be obtained prior to the use of the 
banked samples for any research not described in this protocol. The samples will be given a 
code number.  No identifying information will be directly linked to the samples.  Only the 
researcher team in charge of the bank will have access to the code numbers and be able to 
link the samples to the subject. If a patient withdraws his/her consent for banking of samples, 
the banked samples will be destroyed. However, if any samples were previously released for 
research prior to the withdrawal of consent, the samples will not be able to be destroyed.      

2011-0703 
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References: 
1. 
Burris, H.A., 3rd, et al., Improvements in survival and clinical benefit with 
gemcitabine as first-line therapy for patients with advanced pancreas cancer: a 
randomized trial. J Clin Oncol, 1997. 15(6): p. 2403-13. 
2. 
Achiwa, H., et al., Determinants of sensitivity and resistance to gemcitabine: the 
roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in 
non-small cell lung cancer. Cancer Sci, 2004. 95(9): p. 753-7. 
3. 
Bergman, A.M., H.M. Pinedo, and G.J. Peters, Determinants of resistance to 2',2'-
difluorodeoxycytidine (gemcitabine). Drug Resist Updat, 2002. 5(1): p. 19-33. 
4. 
Nakano, Y., et al., Gemcitabine chemoresistance and molecular markers associated 
with gemcitabine transport and metabolism in human pancreatic cancer cells. Br J 
Cancer, 2007. 96(3): p. 457-63. 
5. 
Olive, K.P., et al., Inhibition of Hedgehog signaling enhances delivery of 
chemotherapy in a mouse model of pancreatic cancer. Science, 2009. 324(5933): p. 
1457-61. 
6. 
Paproski, R.J., et al., The role of human nucleoside transporters in uptake of 3'-
deoxy-3'-fluorothymidine. Mol Pharmacol, 2008. 74(5): p. 1372-80. 
7. 
Abbruzzese, J.L., et al., A phase I clinical, plasma, and cellular pharmacology study 
of gemcitabine. J Clin Oncol, 1991. 9(3): p. 491-8. 
8. 
Gandhi, V., et al., Prolonged infusion of gemcitabine: clinical and pharmacodynamic 
studies during a phase I trial in relapsed acute myelogenous leukemia. J Clin Oncol, 
2002. 20(3): p. 665-73. 
9. 
Grunewald, R., et al., Gemcitabine in leukemia: a phase I clinical, plasma, and 
cellular pharmacology study. J Clin Oncol, 1992. 10(3): p. 406-13. 
10. 
Patel, S.R., et al., Phase II clinical investigation of gemcitabine in advanced soft 
tissue sarcomas and window evaluation of dose rate on gemcitabine triphosphate 
accumulation. J Clin Oncol, 2001. 19(15): p. 3483-9. 
11. 
Kroep, J.R., et al., Pretreatment deoxycytidine kinase levels predict in vivo 
gemcitabine sensitivity. Mol Cancer Ther, 2002. 1(6): p. 371-6. 
12. 
Bengala, C., et al., Prolonged fixed dose rate infusion of gemcitabine with 
autologous haemopoietic support in advanced pancreatic adenocarcinoma. Br J 
Cancer, 2005. 93(1): p. 35-40. 
13. 
Mackey, J.R., et al., Functional nucleoside transporters are required for gemcitabine 
influx and manifestation of toxicity in cancer cell lines. Cancer Res, 1998. 58(19): p. 
4349-57. 
14. 
Galmarini, C.M., et al., Potential mechanisms of resistance to cytarabine in AML 
patients. Leuk Res, 2002. 26(7): p. 621-9. 
15. 
Jansen, W.J., et al., The influence of BIBW22BS, a dipyridamole derivative, on the 
antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in 
human tumour xenografts. Eur J Cancer, 1995. 31A(13-14): p. 2313-9. 
16. 
Spratlin, J., et al., The absence of human equilibrative nucleoside transporter 1 is 
associated with reduced survival in patients with gemcitabine-treated pancreas 
adenocarcinoma. Clin Cancer Res, 2004. 10(20): p. 6956-61. 
17. 
Plunkett, W., P. Huang, and V. Gandhi, Preclinical characteristics of gemcitabine. 
Anticancer Drugs, 1995. 6 Suppl 6: p. 7-13. 

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18. 
Plunkett, W., et al., Gemcitabine: preclinical pharmacology and mechanisms of 
action. Semin Oncol, 1996. 23(5 Suppl 10): p. 3-15. 
19. 
Plunkett, W., et al., Gemcitabine: metabolism, mechanisms of action, and self-
potentiation. Semin Oncol, 1995. 22(4 Suppl 11): p. 3-10. 
20. 
Grunewald, R., et al., Saturation of 2',2'-difluorodeoxycytidine 5'-triphosphate 
accumulation by mononuclear cells during a phase I trial of gemcitabine. Cancer 
Chemother Pharmacol, 1991. 27(4): p. 258-62. 
21. 
Sigmond, J., et al., Gemcitabine uptake in glioblastoma multiforme: potential as a 
radiosensitizer. Ann Oncol, 2009. 20(1): p. 182-7. 
22. 
Pisters, P.W., et al., Effect of preoperative biliary decompression on 
pancreaticoduodenectomy-associated morbidity in 300 consecutive patients. Ann 
Surg, 2001. 234(1): p. 47-55. 
23. 
Raut, C.P., et al., Impact of resection status on pattern of failure and survival after 
pancreaticoduodenectomy for pancreatic adenocarcinoma. Ann Surg, 2007. 246(1): 
p. 52-60. 
24. 
Wang, L.Z., et al., An expedient assay for determination of gemcitabine and its 
metabolite in human plasma using isocratic ion-pair reversed-phase high-
performance liquid chromatography. Ther Drug Monit, 2003. 25(5): p. 552-7. 
25. 
Honeywell, R., et al., The determination of gemcitabine and 2'-deoxycytidine in 
human plasma and tissue by APCI tandem mass spectrometry. J Chromatogr B 
Analyt Technol Biomed Life Sci, 2007. 847(2): p. 142-52.  
26. 
Tempero et al., Randomized phase II comparison of dose-intense gemcitabine: 
thirty-minute infusion and fixed dose rate infusion in patients with pancreatic 
adenocarcinoma. J Clin Oncol  2003. 21(8): 3383-4. 
 

2011-0703 
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Appendix 1 
 
Karnofsky Performance Status Scale: 
 
Description 
Status Score 
Normal, no evidence of disease 
100 
Able to perform normal activity with only minor symptoms 
90 
Normal activity with effort, some symptoms 
80 
Able to care for self but unable to do normal activities 
70 
Requires occasional assistance, cares for most needs 
60 
Requires considerable assistance 
50 
Disabled, requires special assistance 
40 
Severely disabled 
30 
Very sick, requires active supportive treatment 
20 
Moribund 
10 
 
 
 
 

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Appendix 2 
 
Reported Gemcitabine Toxicity (all of the listed toxicities were reported with prolonged 
treatment, high doses, and in multiple-cycle studies, thus not anticipated to any 
significant degree in our single-low-dose protocol): 
 
- Hematologic:  Myelosuppression is the dose-limiting toxicity with gemcitabine, but 
<1% of patients discontinued therapy for anemia, leukopenia, or thrombocytopenia.  
Red blood cell transfusions were required by 19% of patients.  The incidence of sepsis 
was less than 1%.  Petechiae or mild blood loss (hemorrhage), from any cause, were 
reported in 16% of patients; less than 1% of patients required platelet transfusions. 
 
- Gastrointestinal:  Nausea and vomiting were commonly reported (69%) but were 
usually mild to moderate.  Severe nausea and vomiting (WHO grade 3/4) occurred in 
<15% of patients with prolonged infusions and doses.  Diarrhea was reported by 19% 
of patients, and stomatitis by 11% of patients with prolonged infusions and doses. 
 
- Hepatic: Gemcitabine was associated with mild transient elevations of serum 
transaminases in approximately two-thirds of patients, but there was no evidence of 
increasing hepatic toxicity with either longer duration of exposure to gemcitabine or 
with greater total cumulative dose. 
 
- Renal: Mild proteinuria and hematuria were commonly reported.  Clinical findings 
consistent with the hemolytic-uremic syndrome (HUS) were reported in 0.25% of 
patients receiving gemcitabine in clinical trials.  Four patients developed HUS on 
prolonged gemcitabine therapy, two immediately post-therapy. Renal failure may not 
be reversible, even with discontinuation of therapy, and dialysis may be required. 
 
- Fever: The overall incidence of fever was 41%. This is in contrast to the incidence of 
infection (16%) and indicates that gemcitabine may cause fever in the absence of 
clinical infection. Fever was frequently associated with other flu-like symptoms and 
was usually mild and clinically manageable. 
 
- Rash: Rash was reported in 30% of patients.  The rash was typically a macular or 
finely granular maculopapular pruritic eruption of mild-to-moderate severity, involving 
the trunk and extremities with prolonged infusions and doses.  Pruritus was reported 
in 13% of patients. 
 
- Pulmonary:  Dyspnea was reported in 23% of patients, severe dyspnea in 3%.  
Dyspnea may be due to underlying disease, such as lung cancer (40% of study 
population) or pulmonary manifestations of other malignancies.  Dyspnea was 
occasionally accompanied by bronchospasm (<2% of patients).  Rare reports of 
parenchymal lung toxicity consistent with drug-induced pneumonitis have been 
associated with the use of gemcitabine. 
 
- Edema:  Peripheral edema (13%), and generalized edema (<1%) were reported.  
Less than 1% of patients discontinued due to edema. 
 
- Flu-like Symptoms: "Flu syndrome" was reported for 19% of patients.  Individual 
symptoms of fever, asthenia, anorexia, headache, cough, chills, and myalgia, were 

2011-0703 
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commonly reported.  Fever and asthenia were also reported frequently as isolated 
symptoms.  Insomnia, rhinitis, sweating, and malaise were reported infrequently.  Less 
than 1% of patients discontinued due to flu-like symptoms. 
 
- Infection:  Infections were reported for 16% of patients with prolonged infusions and 
doses.  Sepsis was rarely reported (<1%). 
 
- Alopecia: Hair loss, usually minimal, was reported by 15% of patients. 
 
- Neurotoxicity: There was a 10% incidence of mild parasthesias and a <1% rate of 
severe parasthesias. 
 
- Extravasations:  Injection-site-related events were reported for 4% of patients.  There 
were no reports of injection-site necrosis.  Gemcitabine is not a vesicant.  
 
- Allergic: Bronchospasm was reported for less than 2% of patients.  Anaphylactoid 
reaction has been reported rarely.  Gemcitabine should not be administered to patients 
with a known hypersensitivity to this drug. 
 
- Cardiovascular: 2% of patients discontinued therapy with gemcitabine due to 
cardiovascular events such as myocardial infarction, cerebrovascular accident, 
arrhythmia, and hypertension.  Many of these patients had a prior history of 
cardiovascular disease. 
 
 
 
 
 
 
 
 
 

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Appendix 3  
 
 
Intraoperative Schema:  
 
 
 
 
 
 
Timing of Blood Draws: 
 
 
 
 
Drug Dose 
 
Timing of Blood Draws (Gemcitabine infused at 10mg/m2/min) 
 
500mg/m2  
(Initial 5  patients) 
 
Pre-
incision 
0 min 
10 min 
20 min 
30 min 
40 min 
50 min 
(at completion) 
Approximately 
70 min* 
 
750 mg/m2 
(Subsequent 
patients) 
 
Pre-
incision 
0 min 
15 min 
30 min 
45 min 
60 min 
75 min 
(at completion) 
Approximately
95 min* 
 
*Last time point 20 minutes after Gemcitabine infusion completed. 
 
 
 
 

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Appendix 4 
 
Anticipated Perioperative Complications (as determined by previous institutional 
experience in pancreatic resection): 
 
 
Complication 
 
Anticipated Incidence 
Mortality 
1-2% 
All Major Complications 
30% 
       Anastomotic Leak/Fistula 
       10-20% 
       Abscess/Fluid Collection 
       10-15% 
       Pneumonia 
       5-10% 
       DVT 
       1-10% 
       GI Bleed 
       1-5% 
       Intra-abdominal Bleed 
       1-2% 
 
 
       VTE 
       1-2% 
       Sepsis 
       1-2% 
       Myocardial Infarction 
       1-2% 
       Reoperation 
       1-2% 
All Minor Complications 
70% 
       Atelectasis 
       40-60% 
       Pleural Effusion 
       20-40% 
       Delayed Gastric Emptying 
       15-20% 
       Wound Infection 
       5-10% 
       Cardiac Arrhythmia 
       5-10% 
       Ileus 
       5-10% 
       UTI 
       1-10% 
       Cellulitis 
       1-5% 
       Line Infection 
       1-5% 
       Chyle Leak 
       1-2% 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix 5 Photographs Depicting a Gross Pathologic Examination of the 
Pancreaticoduodenectomy Specimen: 
 
 
- Figure A: X = Adjacent Normal Tissue; Y = Superficial Carcinoma; Z = Deep 
Carcinoma. 
 
 
- Figure B: X = Adjacent Normal Tissue; Y = Superficial Carcinoma; Z = Deep 
Carcinoma. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix 6  
 
Assignment of Protocol Responsibilities 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix 7 
 
New York Heart Association (NYHA) Functional Classification: 
 
 
NYHA 
Class 
Symptoms 
I 
No symptoms and no limitation in ordinary physical activity, e.g. shortness of breath 
when walking, climbing stairs etc. 
II 
Mild symptoms (mild shortness of breath and/or angina) and slight limitation during 
ordinary activity. 
III 
Marked limitation in activity due to symptoms, even during less-than-ordinary activity, 
e.g. walking short distances (20–100 m). 
Comfortable only at rest. 
IV 
Severe limitations. Experiences symptoms even while at rest. Mostly bedbound 
patients.