Clinical trial NCT00979810
Conditions: Brain Cancer Glioma
Keywords: PET scan CT scan 09-060 CNS
Sponsor: Memorial Sloan Kettering Cancer Center
Start date: 2009-09-16; Completion date: 2020-02-24
Enrollment: 11 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to evaluate high and low areas of growth, or proliferation, within the tumor. An imaging technique using a very small amount of a radioactive tracer called 18Ffluoro-deoxy-L-thymidine (18F-FLT) can detect areas of rapid growth within the tumor. This imaging technique is called a FLT PET imaging. This present study involves obtaining one scan using FLT PET imaging. The goal of this study is to investigate associations between the imaging findings showing differences in growth rate within the tumor and the biology of the tumor that is measured in the sampled tumor tissue. This information may be used in future brain tumor patients to determine the best combination of treatment for individual patients. These studies may also improve our understanding of the types of changes taking place in brain tumor tissue that could improve individual patient outcome. FLT is produced for human use by the MSKCC cyclotron facility under an investigational new drug (IND) approval issued by the US Food and Drug Administration (FDA). This means that FLT is produced under strict rules and regulations, is considered safe, and has been approved for use in humans for certain disease conditions. 18F-FLT has been used in several research studies to date at this institution.

Detailed description:

Arms: arm 1: This is a pilot study intended to collect preliminary data on 15 patients diagnosed with untreated high-grade glioma who are scheduled to undergo surgical resection.
Interventions: intervention 1: 18F-FLT PET Scan
Intervention descriptions: intervention 1: The patient will undergo MRI and 18F-FLT PET scans of the brain. A IV catheter will be placed in a superficial hand or arm vein for administration of 18F-FLT (approximately 370 MBq), prepared by the MSKCC Radiochemistry Core Facility. A second venous catheter will be placed in the opposite hand or arm for venous blood sampling. If a central venous catheter is present, it will be used for blood sampling or radiopharmaceutical administration, and only a single venous catheter will be placed. Sequential blood samples may be obtained following 18F-FLT infusion for assaying whole blood and plasma radioactivity. All catheters will be removed at the end of the day.

Protocol documents:
Prot_SAP_000.pdf: 
Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
 
 
IRB#: 09-060 A(4) 
 
Image-Guided Stereotactic Biopsy of High Grade Gliomas 
MSKCC NON-THERAPEUTIC/DIAGNOSTIC PROTOCOL 
 
Principal Investigator/Department: 
Michelle Bradbury MD, PhD 
Department of Radiology/ 
Neuroradiology Service 
Co-Principal 
Investigator(s)/Department: 
Cameron Brennan, MD 
Ingo Mellinghoff, MD 
Heiko Schöder, MD 
Department of Neurosurgery 
Department of Neurology 
Department of Radiology/ 
Nuclear Medicine Service 
Investigator(s)/Department: 
Steven Larson, MD 
John Humm, PhD 
Pat Zanzonico, PhD 
Philip H. Gutin, MD 
Eric Holland, MD PhD 
Vivianne Tabar, MD 
Andrew Lassman, MD 
Andrei Holodny, MD 
Sasan Karimi, MD 
Eric Lis, MD 
Jason Lewis, PhD 
Chaya Moskowitz, MD 
Marc Rosenblum, MD 
Department of Radiology/ 
Nuclear Medicine Service 
Department of Medical Physics 
Department of Medical Physics 
Department of Neurosurgery 
Department of Neurosurgery 
Department of Neurosurgery 
Department of Neurology 
Department of Radiology/ 
Neuroradiology Service 
Department of Radiology/ 
Neuroradiology Service 
Department or Radiology/ 
Neuroradiology Service 
Cyclotron and Radiochemistry Core 
Department of Epidemiology and 
Biostatistics 
Department of Pathology 
Consenting 
Professional(s)/Department: 
Cameron Brennan, MD 
Philip H. Gutin, MD 
Viviane Tabar, MD 
Eric Holland MD, PhD 
Andrew Lassman, MD 
Michelle Bradbury MD, PhD 
Department of Neurosurgery 
Department of Neurosurgery 
Department of Neurosurgery 
Department of Neurosurgery 
Department of Neurology 
Department of Radiology 
 
 
Please Note: A Consenting Professional must have completed the mandatory Human 
Subjects Education and Certification Program. 
 
 
 
 
 
 
 
 
 
 
 
Amended: 12/27/11 
Memorial Sloan-Kettering Cancer Center 
1275 York Ave. 
New York, NY 10021 


Memorial Sloan-Kettering Cancer Center 
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IRB#:09-060 A(4) 
 
 
Table of Contents 
MSKCC NON-THERAPEUTIC/DIAGNOSTIC PROTOCOL ..................................................... 1 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA ................................................................ 1 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS .......................................................................... 2 
3.0 
BACKGROUND AND RATIONALE ................................................................................... 2 
4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION ..................................................... 6 
4.2 
DESIGN .................................................................................................................. 6 
4.3 
INTERVENTION ................................................................................................... 9 
5.1 
CRITERIA FOR SUBJECT ELIGIBILITY ..................................................................... 12 
5.2 
SUBJECT INCLUSION CRITERIA .................................................................... 12 
5.3 
SUBJECT EXCLUSION CRITERIA ................................................................... 13 
6.0 
RECRUITMENT PLAN ................................................................................................... 13 
7.0 
ASSESSMENT/EVALUATION PLAN ........................................................................... 13 
8.0 
TOXICITIES/SIDE EFFECTS ......................................................................................... 13 
9.0 
PRIMARY OUTCOMES .................................................................................................. 13 
10.0 
CRITERIA FOR REMOVAL FROM STUDY ................................................................ 14 
11.0 
BIOSTATISTICS .............................................................................................................. 14 
12.1 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION 
PROCEDURES ............................................................................................................................. 17 
12.2 RESEARCH PARTICIPANT REGISTRATION ................................................... 17 
13.1 
DATA MANAGEMENT ISSUES ....................................................................................... 17 
13.2 
QUALITY ASSURANCE .................................................................................... 17 
13.3 
DATA AND SAFETY MONITORING ............................................................... 18 
14.1 
PROTECTION OF HUMAN SUBJECTS ........................................................................ 18 
14.2 
PRIVACY ............................................................................................................. 19 
14.3 SERIOUS ADVERSE EVENT (SAE) REPORTING .............................................. 19 
15.1 
INFORMED CONSENT PROCEDURES ........................................................................ 20 
15.2 
RESEARCH AUTHORIZATION ........................................................................ 20 
16.0 
REFERENCE(S) ............................................................................................................... 21 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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1.1 
PROTOCOL SUMMARY AND/OR SCHEMA 
 
This is a baseline feasibility study using fluorine-18-labeled fluorothymidine (18F-FLT), a proliferation 
tracer, and highly focal MR image-guided biopsy to evaluate whether characteristic metabolic/MR 
imaging findings can be linked to distinct molecular phenotypes and improve our understanding of the 
molecular and pathological heterogeneity within individual tumors. In addition, this study will assess 
the feasibility of voxel-wise estimation of proliferation and tracer delivery from overall FLT uptake 
assessments, as against traditional ROI-based pharmacokinetic approaches. The quantitative accuracy 
of these parametric images will be investigated through comparison of histologic determinations of 
tumor cell proliferation, CD31 expression, and gene expression differences, obtained at different biopsy 
locations, with the PET-MRI imaging features at those same locations. The results of these studies will 
lay the groundwork for larger prospective studies assessing 18F-FLT proliferative response in trials of 
targeted therapy, relating estimates of proliferation with prognosis, survival, and clinical outcome. 
 
Please note that all patients entered on this protocol will undergo the standard of care for their brain 
tumor. The information obtained from the use of 18F-FLT PET scans will NOT be used to guide 
patients’ surgical planning on this protocol. Since FLT PET is considered investigational, subjects 
enrolled in this protocol will only undergo FLT PET scans under an FDA-approved institutional IND 
(#104742). 
 
Schema: 
a. Evaluation and consent of 15 patients with initial presentation of presumptive glioblastoma 
based on MRI appearance. 
b. Baseline pretreatment MRI studies following injection of gadolinium (GdDTPA) and pre- 
surgical dynamic 18F-FLT PET study with CT for attenuation correction. 
c. Planned tumor resection volume will be pre-determined by the operating neurosurgeon using 
anatomic MRI per routine, who will be blinded to the results of the preoperative PET study. 
d. Two neuroradiologists and a nuclear medicine attending, blinded to the surgical planning, will 
independently select “patterns of interest” or sites of heterogeneity within the tumor on co- 
registered PET-MRI scans for targeted biopsy. 
e. Surgical resection will take place per routine with frameless stereotactic tracking of resection 
location, updated by intraoperative MRI (iMRI,1.5T Siemens magnet). 
f. Histopathological and molecular characterization of the biopsied tumors: immunohistochemical 
staining for MIB-1 and gene expression profiling. 
g. Data analysis: physician interpretation of brain MRI and PET scans; volumetric region-of- 
interest (ROI) analysis for derivation of tumor and contralateral brain activity; 18F-FLT time 
activity curves (TACs) and steady-state SUVs; non-invasive determination of plasma TACs 
(venous and arterial input curves); voxel-based and ROI-based pharmacokinetic modeling. 


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2.0       OBJECTIVES AND SCIENTIFIC AIMS 
 
Primary Aim: To investigate relationships between voxel-based determinations of proliferation rate 
and observed MR imaging features (i.e., Ktrans or microvascular permeability; fBV, tissue fractional 
blood volume), as well as with spatially registered histologic measures of tumor cell proliferation 
(Ki67) and microvascular density (CD31) at corresponding locations. 
 
Secondary Aim #1: To attempt to corroborate voxel-based parameter estimates reflecting tumor cell 
proliferation with estimates derived using standard ROI-based pharmacokinetic modeling methods, for 
improving the characterization of high-grade gliomas using dynamic 18F-FLT PET-CT. 
Secondary Aim #2: To assess whether static measures of 18F-FLT uptake can adequately serve as non- 
invasive biomarkers of proliferative activity or whether parametric images, based on compartmental 
analyses of the FLT pharmacokinetics, are required by correlating findings of both approaches with 
regional histologic assays of tumor cell proliferation. 
 
Exploratory Aim: To evaluate whether differences in gene expression seen between areas of increased 
and decreased proliferative activity on parametric maps define consistent differential transcriptome 
signatures for comparison with known molecular subclasses of GBM and known pathways. 
 
3.1 
BACKGROUND AND RATIONALE 
 
3.2 Introduction 
3'-deoxy-3'-18F-fluorothymidine (FLT) is a PET radiotracer used to assess tumor cell proliferation1. 
Tumor-cell proliferation has been identified as an important surrogate marker of survival in brain tumor 
patients, as determined by staining of ex vivo biopsy specimens with proliferative markers such as Ki- 
672,3. Imaging with FLT takes advantage of the fact that pyrimidine nucleosides and several of their 
analogues are phosphorylated to the respective monophosphate (MP) by thymidine kinase 1 (TK-1) and 
are incorporated into DNA. FLT is also a substrate for TK-1, which has been demonstrated both in 
vitro4 and in vivo5,6. TK-1 activity is up-regulated in cells entering the S-phase, whereas the protein is 
nearly undetectable in growth arrested cells. TK-1 catalyzes the phosphorylation of FLT to FLT- 
monophosphate. Because it lacks a 3'-hydroxyl group, very little FLT is incorporated into DNA. Thus, 
in the amounts used for a radiotracer, it has no pharmacological properties and measures an early event 
in DNA synthesis rather than DNA incorporation. Intracellularly, phosphorylated FLT remains trapped. 
Nevertheless, many reports have shown a positive correlation between FLT uptake and S-phase fraction 
of cells in vitro and in vivo7. Its limited transport across the intact blood-brain barrier and the absence 
of proliferating cells in the normal adult brain result in low levels of 18F-FLT accumulation within the 
brain. Accordingly, FLT-PET has been used to assess tumor proliferative status in high-grade glioma 
patients using static tracer uptake estimates and/or assessing time-dependent kinetic changes in uptake 
following intravenous administration of 18F-FLT7-18. 
FLT has been synthesized by the MSKCC cyclotron facility for more than 2 years, and has been 
used in humans here and in several institutions in this country and Europe. For the purpose of this 
protocol, the synthesis will be done according to practice guidelines set forth in the NCI IND file. The 
primary source of the FLT will be the MSKCC cyclotron facility; however, alternative commercial 


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sources are available to ensure consistent supply of FLT. If a commercial supplier of FLT is used for a 
particular study, the supplier will be required to meet all of the acceptance criteria outlined in the FDA- 
approved MSKCC FLT IND (#104742) prior to administration. 
3.3 18F-FLT Tracer Kinetic Modeling: 
The utility of static (SUV) versus dynamic measures (i.e., k3) of 18F-FLT PET as an imaging 
biomarker represents different schools of thought, with examples of both found in the literature, and 
without a clear consensus offered7,19,20. Static measures of brain tumor 18F-FLT uptake at various times 
post-injection have been shown to correlate with histopathologic proliferation markers (Ki-67) in 
patient studies9,19. More recently, measured FLT SUV values were used to assess treatment response in 
recurrent malignant gliomas18. However, it is not clear what the FLT SUV measurements in such 
studies reflect, as both changes in tracer delivery and/or metabolic trapping will contribute to net tracer 
uptake. As an alternative approach, several previous clinical investigations have assessed uptake in 
brain tumors dynamically, generally using ROIs having maximum proliferative activity, in conjunction 
with two-tissue compartment (four-rate constant) models7,8,10,16, in order to estimate kinetic rate 
constants reflecting tracer transport and cellular proliferation. Both methods continue to be utilized for 
in vivo brain tumor applications, noting that recently recommended 18F-FLT data acquisition and 
analysis techniques have included static PET imaging at 40-60 min and corresponding SUV 
measurements of tumor activity concentration for brain tumors20. 
Accurate assessment of proliferation in brain by 18F-FLT PET requires analysis of uptake kinetics to 
extract separate rate constants for the initial transport (K1) and subsequent metabolic (trapping or 
phosphorylation) phases of 18F-FLT uptake (k3), the latter rate constant being directly proportional to 
the number and/or rate of proliferating cells within the tumor volume. This pharmacokinetic tracer 
uptake analysis requires knowledge of the fraction of 18F-FLT in the blood, given known variations in 
in-vivo blood clearance. In addition to direct arterial or arterialized venous blood sampling21, a number 
of alternative methods have been utilized22-28. Non-invasive ROI assessments of blood pools identified 
on PET images have also been successfully performed using the internal carotid arteries29-31 and venous 
sinuses32,33. However, the small size of these vessels (relative to the spatial resolution of PET 
tomographs) will require corrections for partial volume effects and spillover. The use of a venous, 
rather than an arterial, input function assumes that the tracer has a very low permeability, with no 
measurable extraction occurring in a first pass through the vascular bed34. Under these conditions, 
tracer concentrations in the large venous structures will be good approximations for those in the 
cerebral capillaries. 
 
3.4 Voxel-wise Pharmacokinetic Modeling of Dynamic 18F-FLT-PET Data: 
Pharmacokinetic modeling software for performing voxel-wise parameter estimates across the entire 
tumor volume has recently become available for multi-compartmental kinetic analyses.  BioGuide™ is 
a research software module that operates from within the Philips radiotherapy planning Pinnacle™ 
software. BioGuide™ includes a module that performs voxel-based pharmacokinetic modeling of 
dynamic PET image data as well as co-registered serial PET images. This kinetic modeling algorithm 
will compute voxel-wise parameter estimates of the compartmental model parameters (K1, k2, k3, and 
k4) defining tracer exchange between the blood and tumor tissue compartments, as well as Vb, the 
intravascular blood volume fraction, which will be estimated as a fifth parameter. 


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Voxulus, a Bio-Guide™ software program, is currently being used by the Department of Medical 
Physics for pharmacokinetic modeling of dynamically-acquired radiotracer (18F-FMiso) uptake on a 
voxel-by-voxel basis in both experimental models and head and neck cancer patients. BioGuide™ will 
be applied to brain 18F-FLT images using defined volume-of-interest measurements within the tumor 
and contralateral brain at the same location for generating voxel-wise parameter estimates. Parametric 
maps will then be determined using the estimated proliferation rate constant (k3) values that, for kinetic 
FLT PET data, are expected to reflect the spatial distribution of rates of cellular proliferation. 
 
3.5 Multimodal Image-Guided Surgical Targeting: 
With the exception of one recent investigation in which multiple targeted biopsy specimens were 
retrieved along a single biopsy tract17, image-based measurements of 18F-FLT uptake have not been 
made in the region of histologic sampling. Given the high degree of heterogeneity of glial tumors, it 
would be preferable to correlate several imaging measurements per patient with histologic analyses in 
the same areas of the tumor in order to detail relationships between tumor metabolism, physiology, and 
specific malignant features. Local relationships between histologic features and radiotracer uptake or 
anatomic/functional MR imaging findings will additionally influence image analysis and interpretation 
of gliomas. Multimodal image-guided stereotactic biopsies have been previously performed by co- 
registering functional MR and PET imaging studies17,35-37. By combining anatomic scans with 
metabolic and functional data from PET imaging, complementary information may be obtained that 
permits tumor heterogeneity and extension to be better defined, in addition to adding prognostic 
value37. These studies have sought to detail relationships between functional imaging parameters 
reflecting tumor vascularity (relative microvascular blood volume, rCBV) and tumor metabolism using 
11C-methionine or 18F-FDG, and their relationship to malignant features, MVD, or VEGF by 
histopathologic correlation. However, these correlations generally relied on representative tissue 
samples obtained from biopsy or resection, not in the region of image-based measurements35,37. Thus, 
local comparisons between imaging and tissue measurements could not be performed. Correlations 
between brain tumor 18F-FLT uptake and histopathologic proliferation markers have also been based on 
representative biopsy or resection specimens9. Neuronavigated surgery, however, facilitates increased 
precision of tissue sampling at locations that correspond with imaging measurements. 
 
3.6 Rationale 
Improved molecular imaging of brain tumors has been linked to several research priorities, identified 
in the report published by the NCI/NINDS Brain Tumor Progress Review Group (November 2000), 
including (1) a need for establishing noninvasive markers of early treatment response and (2) the need 
to understand treatment-relevant molecular alterations in relation to anatomic site and tumor 
heterogeneity. There has been limited advancement on both fronts, in large part due to the difficulty of 
systematically acquiring targeted biopsies and correlating imaging with tissue measures. The clinical 
relevance of this proposal is the demonstration of feasibility for (1) highly focal image-guided biopsy 
that can power a prospective study of FLT as a marker of prognosis or early response (2) identifying 
molecular correlates of imaging heterogeneity. 
Although a number of clinical FLT-PET investigations have assessed uptake in newly diagnosed or 
recurrent high-grade gliomas, using either static measures or dynamic approaches to estimate tracer 
kinetic parameters, these studies have not established FLT-PET as a prognostic or predictive marker. 


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This baseline study will serve to establish feasibility of performing analyses of prognostication post- 
hoc using the derived SUVs and/or rate constants reflecting proliferation/metabolic flux. If 
prognostication can be established, a larger patient cohort will be followed prospectively to assess 
clinical outcomes over longer time intervals. While baseline FLT-PET studies have not offered 
prognostic information, changes in FLT SUV values during treatment have been investigated as a 
predictor of long-term outcome. It is important to determine the component of FLT uptake 
measurements that may correlate with tumor cell proliferation (Ki-67), which is intriguing as  a 
potential biomarker, given that it was predictive of overall survival in bevacizumab- and irinotecan- 
treated recurrent gliomas as early as 1-2 wk after treatment. By establishing such response predictors 
and refining our ability to detect response to novel therapies in malignant brain tumor clinical trials, 
treatment protocols can be more efficiently optimized, expediting the evaluation of promising 
compounds in clinical trials for assessing patient outcomes. 
Tailoring targeted therapies based on an individual tumor’s unique molecular and biochemical 
signatures is an ultimate goal in managing brain tumor patients. Characterizing tumors at multiple sites, 
guided by heterogeneous metabolic/functional imaging findings, may capture variation in potential 
tissue biomarkers, such as EGFR amplification or MGMT methylation, which are frequently 
heterogeneously distributed. To meet this challenge, it will be necessary to determine clinically useful 
biomarkers with prognostic and predictive value to improve selection of specific therapeutics and to 
assess early response/progression for modifying treatment protocols. High throughput molecular 
analyses of surgically procured tissue have enhanced the characterization of gliomas, resulting in the 
definition of novel tumor sub-classes, better diagnostic accuracy, and improved prognostication. 
Prolonged progression-free and overall survival in glioblastoma patients treated with DNA alkylating 
agents, for instance, has been linked to MGMT hypermethylation in the majority of tumors. The results 
of such studies have suggested that gene expression profiling will impact future clinical practice in 
terms of cancer diagnosis, prognosis, and choice of optimal treatment. Under certain conditions, 
however, such as during the course of treatment, brain tumor biopsy may be impractical, and given that 
radiographic disease progression is often associated with morbid decline, there is a critical need to 
establish early and reliable non-invasive biomarkers of treatment response. 
This study will also lay the groundwork for establishing the interpretability of 18F-FLT heterogeneity 
within and across tumors on a voxel-wise basis. The feasibility of voxel-wise estimation of 
proliferation will be established, and validated against traditional ROI-based pharmacokinetic 
approaches. The results of these studies may inform the design of larger prospective studies assessing 
18F-FLT proliferative response in trial of targeted therapies, as well as determine the potential for FLT- 
PET to serve as a biomarker for proliferation at the tissue level. An FDA-approved IND has been 
issued for 18F-FLT (#104742) at this institution. 


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4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
 
4.2 
Design 
 
This is a two-year open-label pilot study at Memorial Sloan Kettering Cancer Center that will enroll 15 
patients with initial presentation of presumptive glioblastoma based on prior radiographic studies for 
18F-FLT PET imaging correlation with histologic and molecular markers. Standard baseline 
pretreatment MRI studies will be obtained following injection of gadolinium (GdDTPA) and a pre- 
surgical dynamic 18F-FLT PET study with CT for attenuation correction will be performed on Day 1. 
Surgical resection will be performed within a 7 day period of these imaging studies. The planned tumor 
resection volume will be pre-determined by the operating neurosurgeon on the basis of 
anatomic/functional MRI per routine, and blinded to the results of the preoperative PET study. Two 
neuroradiologists (Drs. Michelle Bradbury and Sasan Karimi) and a nuclear medicine attending (Dr. 
Heiko Schöder), blinded to the surgical planning, will independently identify “patterns of interest” or 
sites of heterogeneity within the tumor on co-registered PET-MRI scans for targeted biopsy. Fused 
images containing the marked biopsy sites will be uploaded to the BrainLAB VectorVision® 
neuronavigation system for viewing by the neurosurgeon in the operating room. Selected targeted sites 
will be marked as potential biopsy targets if they are: (1) contained with the pre-determined planned 
tumor resection volume, (2) over 1cm3, and (3) abnormal on intraoperative visualization. Surgical 
resection will take place per routine with frameless stereotactic tracking of resection location, updated 
by intraoperative MRI (iMRI, 1.5T Siemens magnet). As target sites are encountered, up to 1gm of 
tumor will be marked as a separate biopsy specimen and flash frozen in liquid nitrogen. Biopsy 
specimens will be taken only after adequate material has been acquired for routine pathology. Prior to 
histopathologic and molecular characterization, biopsy specimens will be inspected by the pathologist 
to insure adequacy of tumor tissue sampling and to definitively diagnose tumor. Histopathologic and 
molecular characterization of the biopsied tumors will be performed, specifically immunohistochemical 
staining for MIB-1, in addition to gene expression profiling. Data analysis will include physician 
interpretation of brain MRI and PET scans, volumetric region-of-interest (ROI) analyses of tumor and 
contralateral brain, generation of 18F-FLT time activity curves (TACs) and steady-state SUVs, non- 
invasive determination of plasma TACs (venous and arterial input curves), as well as voxel-based and 
ROI-based pharmacokinetic modeling. Periodic meetings will be held to discuss issues related to data 
acquisition, collection, and analysis, as well as to discuss the imaging and histopathology data. 
 
Primary Aim Study Design: Preoperative anatomical MRI images will be uploaded to the BrainLAB 
VectorVision® neuronavigation system, a frameless stereotaxy and image-guided surgical navigation 
system integrated with the Intraoperative MRI suite. Planned resection volume will be contoured by the 
neurosurgeon conventionally, based on the anatomic MRI. 18F-FLT PET-MRI studies will then be 
uploaded and registered by a radiologist. For each patient undergoing resection, up to four prescribed 
ROIs within the planned resection volume will be selected for potential targeted biopsy based on 
concordance/discordance or heterogeneity of PET and MRI imaging characteristics (i.e., increased 18F- 
FLT uptake/MRI-enhancing; increased 18F-FLT uptake/MRI-non-enhancing; low 18F-FLT uptake/MRI- 
enhancing; increased 18F-FLT uptake/MRI-nonenhancing). ROIs will be no smaller than a cubic 
centimeter. The operative plan, in terms of the tumor resection volume, will not be guided by decisions 
regarding sites of potential biopsy selected on the co-registered FLT PET-MRI study. It is expected that 


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the majority of target sites selected for each patient (up to 4) will be confined within the  area 
determined for resection; these tumors are usually large at presentation with heterogeneous 
enhancement and rim enhancement. Thus, it is expected that several areas selected by the 
neuroradiologists and nuclear medicine attending will be biopsied. Frameless stereotaxy will be utilized 
to estimate proximity to an ROI during resection. An intraoperative MRI will be obtained when one or 
more ROIs are accessible for biopsy. Multiple biopsies will then be acquired from the vicinity of the 
ROI with recording of the stereotactic locations of the sites and collection in separately numbered 
tubes. 
Brain shift and deformation will likely be present at the time of biopsy, shown by intraoperative 
MRI performed immediately before or after biopsy, limiting our ability to determine whether or not an 
ROI has been accurately sampled until after the procedure. Non-linear registration algorithms will be 
applied to correct for geometric distortions, and will be applied after maximizing global imaging 
registration using a linear registration algorithm. Non-linear registration methods will permit us to 
further adjust and refine post-hoc accuracy of tumor tissue sampling with regard to the preselected 
target ROIs, thereby enabling biopsied samples to be accepted or rejected. We have chosen to perform 
this compensation of brain shift post-hoc because it is procedurally time-consuming and might be 
subject to errors that could potentially misdirect the surgeon if used to directly guide biopsy. Instead, 
our method relies on the ability to take multiple biopsies from the vicinity of an ROI, annotating each 
with accurate localization based on intraoperative MRI. The accuracy of localization is about 1 cm3. 
Such piecewise acquisition of tissue is a routine approach for obtaining pathology and banking 
specimens at our institution. 
Post-hoc mapping of biopsy sites into presurgical image coordinates will determine which samples 
were in fact within the target ROI. The primary limitation of the approach is that it relies on the 
surgeon's estimation of where an ROI has shifted intraoperatively in order to direct biopsies: it is 
possible that some ROIs may be missed or deemed to be outside the resection volume and hence 
unavailable for biopsy. However, the minimum proposed ROI size of 1.0 cm3 is within the range of 
intraoperative accuracy routinely used by neurosurgeons during resection to avoid vascular and 
neuroanatomic structures. Therefore it is anticipated that at least one specimen be taken from the 
vicinity of the ROI, as judged by the surgeon; it will be established post-hoc as being accurately 
mapped to within an ROI. Acquiring tissue from surgically exposed tumor, as proposed here, avoids the 
significant risk of parenchymal bleeding from deep needle biopsy. If difficulties are encountered 
implementing the above plan, needle biopsy sampling through an initial minimal surgical exposure will 
be employed under a separate IRB-approved protocol. 
Biopsy specimens will be acquired for research purposes after pathologic specimens are submitted 
for the patient per routine by neurosurgery. A minimum of 1.0 g of biopsy tissue will be acquired per 
patient for research purposes, as the priority for the study is histologic validation. We will ensure that 
as much tissue as possible is sent for histology, aiming for roughly half of the specimens to be further 
processed as frozen sections for research purposes. 
Biopsy specimens will be frozen immediately in liquid nitrogen and stored at -80 °C until later use. 
All biopsy cores will be divided in half, with one half of the frozen tissue specimen sectioned for RNA 
and DNA expression analysis, and the remaining half processed as a formalin-fixed paraffin-embedded 
(FFPE) specimen for immunohistochemistry by fixing the tissue in 10% neutral buffered formalin for 2 
days,  followed  by paraffin  embedding.    Tissue  markers  for tumor cell  proliferation  (MIB-1)  and 


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microvessel density (CD31) will be applied to FFPE sections. Serial sections will be reviewed by an 
experienced neuropathologist. Ki-67 staining will be evaluated and a score designated indicating the 
percentage of positively stained tumor cells per quartile of tumor tissue. All cells with nuclei staining of 
any intensity will be defined as positive. The proliferative activity score, quantified as the percentage of 
MIB-1 stained nuclei per total nuclei in the sample, will be estimated from a representative slide 
selected by the neuropathologist. For MVD assessment using CD31, tumor sections will be scanned at 
low magnification (x40) to identify the region of the section with the highest microvessel density. This 
area will be counted at a magnification of x200 for CD31-positive expression, and the proportion of 
CD31-positive cells computed for all specimens. 
Histologic findings at a given biopsy site will be compared with ROI analyses at the same location 
on the co-registered PET-MRI studies. Pharmacokinetic modeling will be applied to these ROI 
measurements in order to generate parametric maps reflecting the proliferation rate constant, k3, and 
Ktrans (microvascular permeability). Estimated parameter values will be correlated with the presence of 
molecular markers (MIB-1, CD31), and compared with imaging features at the prospectively defined 
biopsy sites. 
 
Secondary Aim #1 Study Design: The objective will be to investigate the feasibility of applying a 
novel imaging software platform to the measured dynamic brain PET imaging data. Voxel-based 
parameter estimates will be generated using ROIs selected for targeted biopsy and, in turn, utilized to 
construct parametric images (maps) reflecting the spatial distribution of the rate of 18F-FLT cellular 
trapping (or proliferation). These estimates will be compared with kinetic parameters determined using 
traditional ROI-based pharmacokinetic modeling methods. As input to the model, venous time-activity 
curves will be derived non-invasively at the level of the confluence of the sagittal, straight, and 
transverse sinuses, and corrected for hematocrit and partial volume effects. 
Venous input functions will be generated using previously described protocols32. We will use a 
summed image of the first 2 frames (1 min) of the study and place small ROIs (5 mm) on two to five 
successive planes in the image at the level of the confluence of the superior sagittal, transverse, and 
straight sinus, covering a total axial depth of 1.0-1.5 cm. The co-registered MRI and PET-CT will be 
used to confirm our interpretation of the vascular anatomy revealed by PET and to verify the position 
of the ROIs. In addition, threshold values of 50% of the peak activity in the sinuses will be used for 
further guiding the placement of the ROIs, with data for the ROIs on successive planes averaged. The 
resulting time course of the SUV will be automatically corrected for detection efficiency, scatter 
attenuation, and decay. In order to correct for partial volume averaging effects, the transverse 
dimension of the confluence of sinuses will be measured on axial T2-weighted imaging. Using the PET 
system recovery coefficient, derived from phantom studies as a function of the source dimension, a 
recovery coefficient for the confluence of sinuses will be determined. Each TAC data point will be 
divided by this recovery coefficient to yield the corrected TAC data. 
Venous input functions will be compared with non-invasively derived arterial input functions, the 
latter determined from the internal carotid arteries at the level of the skull base on PET after fusing with 
MR images for artery identification. The feasibility of using arterial structures at this level will be 
investigated using the same method described above for determining venous input functions. The 
agreement of kinetic parameter values calculated using both venous and arterial input functions will be 
examined. 


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A number of studies have compared rate constants estimated using non-invasive, image-based input 
functions versus time-activity curves (TACs) generated from sampled arteries or arterialized veins, and 
have found no statistically significant differences for rate constants obtained with these input 
functions32,39,40. More recently, image-derived input functions for transverse and cavernous sinuses 
have been used in conjunction with compartmental modeling to investigate the kinetic behavior of 
malignant brain tumors, with the resulting rate constants found to adequately describe 18F-FLT uptake 
characteristics7. Thus, as the collective results of these studies have demonstrated no statistically 
significant differences between blood samples and tomographically obtained input functions in terms of 
the kinetic parameters estimated, it is not essential that blood samples be acquired for determining an 
input function. However, up to 4 blood samples (approximately 3-milliliter per sample) may be taken 
post-injection, within the 1 hr imaging time period (i.e., roughly 15, 30, 45, and 60 min), for calibrating 
the corresponding image-based FLT TAC and/or improving pharmacokinetic parameter estimates, as 
well as for test consistency.). It should be noted that a range of post-injection time points (ultimately 
defining the blood TAC) can be selected for blood draws, as estimated pharmacokinetic parameters will 
not be critically dependent on the exact times chosen. This procedure will be performed in a number of 
patients to establish whether there is a relatively constant calibration factor or improved 
pharmacokinetic data that can be derived, which in turn would be applied to all the remaining patient 
data analyzed. 
 
Secondary Aim #2 Study Design: Voxel-wise parameter estimates derived in secondary aim #1 will be 
compared with static measures of 18F-FLT uptake using ROIs selected for targeted biopsy. To obtain 
static 18F-FLT images, data will be summed between 30 and 60 minutes post-injection. Estimated 
parameter values will be correlated with the proliferative activity score or the percentage of MIB-1 
stained nuclei per total nuclei in the sample as described under the Primary Aim Study Design. 
 
Exploratory Aim Study Design: Biopsy samples will be acquired from regions of increased/decreased 
proliferative activity (FLT uptake). Gene expression from microdissected portions will be assayed by 
Affymetrix U133Plus2 arrays. Differential gene expression signatures will be derived for each pair of 
samples in a resection, representing differences between FLT uptake values. Signatures will be 
compared across samples to attempt to characterize a core signature. Core signatures will be 
investigated by comparison to known transcriptome signatures of molecular subclasses of GBM as well 
as compared with other pathways and signatures. 
 
4.3 
Intervention 
 
PET scan protocol 
• Fifteen (15) patients with newly diagnosed or recurrent gliomas, deemed appropriate for this scan 
by the investigator on the basis of imaging studies and surgical management considerations, will 
undergo the FLT-PET study for purposes of collecting pilot data (in compliance with RDRC 
regulations). 
 
• All studies will be performed using a dedicated PET/CT, which integrates a dedicated PET scanner 
and a spiral CT with proprietary fusion software. Images are acquired with the patient in the fasted 


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state (about four to six hours), but water for hydration is allowed. A low dose spiral CT is performed 
first, followed by acquisition of PET images (emission images only), of the brain. Attenuation 
correction of the PET/CT images is performed using the CT data. PET, CT and fusion images are 
displayed on a standard workstation. 
 
• A IV catheter will be placed in a superficial hand or arm vein for administration of 18F-FLT 
(approximately 370 MBq), prepared by the MSKCC Radiochemistry Core Facility. A second venous 
catheter will be placed in the opposite hand or arm for venous blood sampling. If a central venous 
catheter is present, it will be used for blood sampling or radiopharmaceutical administration, and only 
a single venous catheter will be placed. Sequential blood samples may be obtained following 18F-FLT 
infusion for assaying whole blood and plasma radioactivity. All catheters will be removed at the end 
of the day. 
 
• Dynamic PET/CT images of the brain will be acquired for up to 1-hr on a dedicated PET/CT 
machine, with image acquisition starting immediately after injection using the following sequence: 6 
frames of 30sec, 7 frames of 1-minute, then 10 frames of 5-minutes. 
 
• The intravenous line will be removed at the end of the study. 
 
• Venous blood samples (up to four) may be drawn from a vein in the arm opposite to that used to 
inject FLT by research staff. A single sample (approximately 3 milliliters) will be acquired at the 
end of the study (i.e., 60 min) for calibrating the FLT TAC, with the option to perform additional 
blood sampling (i.e., 15, 30, 45 min post-injection) for improving estimates of pharmacokinetic 
parameters. 18F radioactivity in the 18F-FLT blood/plasma samples will be measured by research 
staff. 
• Dynamic FLT scans will be reconstructed by both filtered back projection and iterative 
reconstruction in order to check for quantitative accuracy at the smaller time segments immediately 
post injection. 
 
• 18F-FLT PET and CT examinations will be fused using a Vector-Vision® cranial navigation 
software (BrainLab AG, Heimstetten, Germany), after being transferred to a commercial 
workstation, BrainLab 1 (BrainLab AG). 
 
• The 18F-FLT PET-CT and baseline MRI studies will be uploaded onto BrainLab 1 and co-registered 
by the neuroradiologist using Vector-Vision® cranial navigation software. This will not be done in 
the operating room. The PET and MR image sets will be co-registered by applying the 3D rigid 
transformation (translations/rotations) to the PET image set, which aligns the CT image set of the 
PET-CT study and the MR image set. 
 
• The operating neurosurgeon will be blinded to the pre-operative PET study, and planned tumor 
resection volume will be pre-determined solely on the basis of anatomic MRI per routine. 
Preoperative  MRI  images  will  be  uploaded  to  the  BrainLAB  VectorVision®  neuronavigation 


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system, a frameless stereotaxy and image-guided surgical navigation system integrated with the 
Intraoperative MRI suite. 
 
• On co-registered PET-MRI scans and prior to surgical resection, up to four prescribed ROIs within 
the planned resection volume will be marked for potential targeted biopsy by two neuroradiologists 
and a nuclear medicine attending, blinded to the surgical planning. These ROIs will be selected 
based on heterogeneity of PET and MRI imaging characteristics (e.g., high 18F-FLT uptake/no 
enhancement; low 18F-FLT uptake/enhancement). Fused images containing the marked biopsy sites 
will be uploaded to the BrainLAB VectorVision® neuronavigation system for viewing by the 
neurosurgeon in the operating room. As noted above, however, the operative plan, in terms of the 
tumor resection volume, will not be guided by decisions regarding sites of potential biopsy selected 
on the co-registered FLT PET-MRI study. 
 
• ROI analyses for determination of tracer uptake measurements and the generation of time-activity 
curves for blood and tumor will be performed on an AW Workstation (GE Healthcare) that runs the 
Voxulus software 
module 
for 
voxel-based 
pharmacokinetic 
modeling. 
Voxel-based 
pharmacokinetic modeling, traditional ROI-based pharmacokinetic modeling (SAAM II program), 
and static uptake determinations will all be performed on this workstation. The information derived 
from these analyses will not be used for surgical planning or uploaded for use in the operating 
room. 
 
• Non-invasive input functions for blood will be obtained non-invasively using post-injection region- 
of-interest measurements of the superior sagittal sinus. These measurements will be compared with 
arterial blood activity-concentrations from an image containing the internal carotid artery, and will 
be calibrated using the measurement of a single intravenous blood sample acquired at one post- 
injection time point. If additional blood sampling (i.e., 15, 30, 45 min post-injection) has been 
acquired, these measurements will be compared with non-invasively derived values and used to 
improve estimates of pharmacokinetic parameters. 
 
• Surgical resection and tissue specimen acquisition will take place with frameless stereotactic 
tracking of resection location and updated by intraoperative MRI. 
 
• The radiation dose from the FLT studies was calculated based on published data. Given a realistic 
1- hour voiding interval, the target organ is the liver, receiving a dose of 0.168 rad/mCi, 
corresponding to 1.68 rad/10mCi. This is below the 5 rad single organ permissible dose. Total body 
dose is calculated at 0.046 rad/mCi, and 0.46 rad for a 10 mCi injection, the maximum dose that 
will be used (see Table below). With a 1 hour voiding interval, the dose to the urinary bladder wall 
will be 0.146 rad/mCi. Recent CT dosimetry literature quotes a dose of 0.9 rad/single organ from a 
typical CT scan, performed for the purpose of PET attenuation correction on a PET-CT scanner. 
The diagnostic protocol described here will not exceed the permissible limit of 5 rad/organ/single 
study and will also remain below the maximal permissible dose acceptable to the radioactive drug 
research. 


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• F18-Fluorothymidine (FLT) Patient Dosimetry 
Dosimetry for one PET FLT - CT scan 
 
 
Absorbed Dose 
 
 
 
One FLT PET-CT scan 
0.9 rad 3 + 
F18-FLT2 
  
10 mCi F18-FLT  
 
Target Organ 
rad/mCi 
rad/10 mCi 
rad 
Adrenals 
0.077 
0.766 
1.67 
Bone Surfaces 
0.058 
0.585 
1.48 
Brain 
0.013 
0.125 
1.03 
Breasts 
0.031 
0.310 
1.21 
Heart Wall 
0.062 
0.618 
1.52 
Kidneys 
0.132 
1.317 
2.22 
Large Intestine - Lower Wall 
0.048 
0.477 
1.38 
Large Intestine - Upper Wall 
0.046 
0.459 
1.36 
Liver 
0.168 
1.680 
2.58 
Lungs 
0.037 
0.374 
1.27 
Pancreas 
0.085 
0.851 
1.75 
Red Marrow 
0.089 
0.888 
1.79 
Small Intestine 
0.053 
0.525 
1.43 
Stomach Wall 
0.052 
0.522 
1.42 
Testes 
0.049 
0.488 
1.39 
Thyroid 
0.038 
0.385 
1.28 
0.110 
1.098 
2.00 
0.146 
1.463 
2.36 
0.220 
2.195 
3.10 
0.293 
2.927 
3.83 
 
5.1 
CRITERIA FOR SUBJECT ELIGIBILITY 
 
The patient population will be comprised of patients with newly-diagnosed or recurrent high grade 
gliomas. 
 
5.2 
Subject Inclusion Criteria 
 
5.1.1. Age > 18 years old. 
 
5.1.2. Radiographic appearance of a lesion presumed to be high-grade glioma. 
1 FLT PET-CT scans 
Urinary Bladder Wall 
45-min Voiding Interval 
1-hr Voiding Interval 
1.5-hr Voiding Interval 
2-hr Voiding Interval 


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5.1.3. Planned surgical resection. 
 
5.2 
Subject Exclusion Criteria 
 
5.2.1. Pregnant (confirmed by serum b-HCG in women of reproductive age) or breast feeding 
 
5.2.2  Patients with other active malignancies or prior treatment for non-CNS malignancies. 
 
6.0 
RECRUITMENT PLAN 
 
Patients will be evaluated by attending physicians from the Departments of Neurosurgery and 
Radiology entered onto the study if they are appropriate candidates. The attending physician will 
obtain informed consent from the eligible patient. 
 
7.0 
ASSESSMENT/EVALUATION PLAN 
 
Patient assessment and evaluation will be based on region-of-interest (ROI) measurements of tumor, 
non-tumor-bearing brain, the dural venous sinus/arterial blood pool, as well as additional data from 
whole blood/plasma radioactivity measurements. These assessments will be used to obtain static and 
dynamic 18F-FLT uptake measurements, namely SUV values and estimated kinetic rate constants 
(including proliferation) using both voxel-wise and ROI-based tracer kinetic modeling approaches. 
SUV values and rate constants will be compared with molecular markers (percentage of Ki-67 and 
CD31 expressing cells, differential gene expression signatures) determined at corresponding sites of 
biopsy. This evaluation is detailed in Section 4.0. 
 
8.0 
TOXICITIES/SIDE EFFECTS 
 
The radiotracer, 18F-FLT, has been synthesized by the MSKCC cyclotron facility for about two 
years, and has been used in humans in this and other institutions in this country and Europe. The 
primary source of the FLT will be the MSKCC cyclotron facility; however, alternative commercial 
sources are available to ensure consistent supply of FLT. If a commercial supplier of FLT is used for a 
particular study, the supplier will be required to meet all of the acceptance criteria outlined in the FDA- 
approved MSKCC FLT IND (#104742) prior to administration. 18F-FLT synthesized at MSKCC has 
been tested for toxicity and pyrogenicity. The product synthesized at MSKCC has met all requirements. 
No side effects are expected as a result of this study. However, in the unlikely event that an adverse 
reaction to the radiopharmaceutical occurs, the results must be documented and reported by the 
Principal Investigator to the Institutional Review Board Chairman and IND Committee. 
 
9.0 
PRIMARY OUTCOMES 
 
Primary study measurements will be FLT uptake (SUV values); estimated rate constants, including 
that reflecting proliferation, derived from voxel-wise and ROI- based tracer kinetic modeling; and 
molecular markers (percentage of Ki-67 and CD31 expressing cells, differential gene expression 
signatures). 


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10.1 
CRITERIA FOR REMOVAL FROM STUDY 
 
10.1.1. Patients will not be removed from the protocol unless they request so. 
 
10.1.2. If at any time, the patient develops unacceptable protocol-associated toxicity, he/she will be 
removed from study. 
 
10.1.3. If at any time the patient is found to be ineligible for the protocol, as designated in the section 
on Criteria for Patient/Subject Eligibility (i.e., a change in diagnosis), the patient will be removed from 
the study. 
 
11.0     BIOSTATISTICS 
 
This is a pilot study intended to collect preliminary data on 15 patients diagnosed with untreated 
high-grade glioma who are scheduled to undergo surgical resection. 
 
 
Primary Aim: To investigate relationships between voxel-based determinations of proliferation rate 
and observed MR imaging features, as well as with spatially registered histologic measures of tumor 
cell proliferation (Ki67) and microvascular density (CD31) at corresponding locations. 
 
Statistical Methodology for Primary Aim: In this aim, we will first explore the relationships 
between 18F-FLT PET parameters and MR imaging features (Ktrans or microvascular permeability). 
The 18F-FLT PET parameters: K1 (tracer delivery rate constant), k2 (rate constant reflecting flow 
from the tumor interstitial compartment back to blood), k3 (rate constant reflecting proliferation), 
and k4 (rate constant reflecting flow from tumor cells back into the tumor interstitial space) will be 
compared to Ktrans on MR imaging. We will also explore relationships between Ktrans, each of the 
18F-FLT PET parameters, and the histological markers Ki67 and CD31. All of the parameters and 
markers are measured on a continuous scale. Initially graphical methods such as pair-wise scatter 
plots will be used to assess the relationships. The relationships between each pair of variables will be 
more formally evaluated using methods that account for correlated data (there may be up to four 
observations per patient) such as random effects regression models or generalized estimating 
equations. 
 
It is unlikely but possible that it may be determined at surgery that a patient does not have a high- 
grade glioma but instead has a metastasis or PCNSL. The likelihood that this will occur will be 
infrequent, if at all, given the constellation of distinctive imaging features that generally serve to 
differentiate these tumor types. If it does occur, however, we will continue accrual until 15 high 
grade glioma patients have been enrolled. In this case, two sets of analysis for the primary aim and 
all other aims will occur, one including all enrolled patients and one including only patients with 
high-grade gliomas. Patients found to have a metastasis or PCNSL may not yield different values 
than patients with glioma so the decision to include them in the analysis may not be unreasonable. 


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However, we will descriptively assess any differences in results between the two sets of analysis as 
much as the data permits if this situation arises. 
 
Secondary Aim #1: To attempt to corroborate voxel-based parameter estimates reflecting tumor cell 
proliferation with estimates derived using standard ROI-based pharmacokinetic modeling methods, 
for improving the characterization of high-grade gliomas using dynamic 18F-FLT PET-CT. 
 
Statistical Methodology for Secondary Aim #1: To corroborate the voxel-based method with the 
ROI-based approach, pharmacokinetic modeling will be assessed by comparing the estimated 
parameter values for the entire intra-tumor ROIs to the average of the voxel-by-voxel parameter 
values among the voxels comprising the same ROIs. This analysis will focus on the k3 value. For 
each ROI, we would like to see the estimated k3 value derived from the entire ROI to be within 25% 
of the average k3 value determined for the multiple voxels. The ratio of two k3 values will be 
computed for this purpose. As an additional analysis, we will calculate the concordance correlation 
coefficient (CCC) to evaluate the agreement between the ROI-based and the average voxel-based 
parameter values. A modified version of the CCC that allows for clustered data will adjust for the 
fact that there may be multiple observations per patient contributing to the estimates38. 
Secondary Aim #2: To assess whether static measures of 18F-FLT uptake can adequately serve as 
non-invasive biomarkers of proliferative activity or whether parametric images, based on 
compartmental analyses of the FLT pharmacokinetics, are required by correlating findings of both 
approaches with regional histologic assays of tumor cell proliferation. 
 
Statistical Methodology for Secondary Aim #2: Using the same methods described for the primary 
arm, the relationship between Ki67 and the static measure of 18F-FLT (the SUV), and the parametric 
measure (k3) will be explored. The strength of the relationship between these two parameters and 
Ki67 will be compared descriptively. 
 
Exploratory Aim: To evaluate whether differences in gene expression seen between areas of 
increased and decreased proliferative activity on parametric maps define consistent differential 
transcriptome signatures for comparison with known molecular subclasses of GBM and known 
pathways. 
 
Statistical Methodology for Exploratory Aim: 
 
ACGH: Array CGH data will be analyzed through the software pipeline developed within MSKCC's 
Cancer Genome Characterization Center within The Cancer Genome Atlas Project. This includes 
extensive QC annotation, normalization and artifact reduction, and segmentation analysis. 
 
Expression analysis for gene signatures: Expression array data will be normalized by RMA and 
differential expression of paired samples determined by Significance Analysis of Microarrays 
(SAM). Methods of analysis cross-sample will aim at determining core signature genes which are 
commonly differentially expressed in relation to imaging features. These methods will depend on the 


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number of samples from each tumor that represent differential features. For larger sample sets, 
simple Venn intersection may reveal core signature genes. Sparser samplings of feature contrasts 
will be analyzed by pairwise Gene Set Enrichment to identify a subset of mutually expressed 
signature genes. 
 
Analysis of gene signatures: Gene signatures representing contrasting imaging features will be 
compared with known signatures representing previously described molecular subclasses of GBM, 
including the "Proneural", "Proliferative" and "Mesenchymal" classes, as well as molecular 
subclasses already emergent in the analysis of 206 GBM samples in The Cancer Genome Atlas pilot 
project to date. Signatures will be compared by GSEA and gene ontology. GSEA will also be used to 
analyze feature-derived signatures in an exploratory process to characterize possible enrichment for 
altered expression within canonical pathways. The Connectivity Map offers another potentially 
valuable resource for mining feature-derived signatures, and can be approached with specific 
hypothesis: that differential proliferation and permeability, as image-derived features, may be 
associated with transcriptome evidence of PI3 kinase signaling or VEGF signaling, both of which 
are well represented in the database. Analysis will include exclusion of gene subsets such as cell 
cycle members whose differential regulation is likely to correlate with simple histologic measures of 
proliferation. 
 
For the exploratory aim, we can evaluate the power we will have to detect a difference in gene 
expression levels between areas of high and low tracer uptake (or signal intensity) within an 
individual. With 15 subjects contributing a sample from both a high and low uptake area, we expect 
to be able to detect a twofold difference in base 2 log intensity levels with a Type I error of 0.001 
and a Type II error of 0.05 if the standard deviation for the expression levels is 0.75 (using base 2 
logarithms) or less. A standard deviation of 0.75 is within the range of what we expect to see41. 
 
Sample size justification: 
For this pilot study, we expect there to be 15 eligible patients per year, and we will aim to reasonably 
accrue 8 of these patients per year for two years. 
 
All analyses are purely exploratory and intended to generate data for future studies. We do not plan 
to conduct any rigorous hypothesis testing. To explore what we might be able to say using this 
limited sample size, we find that with data on 15 patients we expect to be able to detect a moderately 
high correlation coefficient of 0.60 with 80% power using a two-sided 0.05 level test42. This 
calculation assumes that each patient contributes only one observation, but in our case, we expect an 
average of two observations for each patient (up to four samples per patient) which may increase our 
power to draw any conclusions. Simulation studies assuming an average of two observations per 
patient indicate that we can estimate the concordance correlation coefficient to within (half-width of 
95% confidence interval) ±0.27 for a CCC equal to 0.8, ±0.20 for a CCC equal to 0.9, and ±0.05 for 
a CCC equal to 0.99. 


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12.0 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 
 
12.1   Research Participant Registration 
 
Confirm eligibility as defined in the section entitled Criteria for Patient/Subject Eligibility. 
 
Obtain informed consent, by following procedures defined in section entitled Informed Consent 
Procedures. 
 
During the registration process registering individuals will be required to complete a protocol 
specific Eligibility Checklist. 
 
All participants must be registered through the Protocol Participant Registration (PPR) Office at 
Memorial Sloan-Kettering Cancer Center. PPR is available Monday through Friday from 
8:30am – 5:30pm at 646-735-8000. The PPR fax numbers are (646) 735-0008 and (646) 735- 
0003. Registrations can be phoned in or faxed. The completed signature page of the written 
consent/verbal script and a completed Eligibility Checklist must be faxed to PPR. 
 
 
13.1 
DATA MANAGEMENT ISSUES 
A Research Study Assistant (RSA) will be assigned to the study. The responsibilities of the RSA 
include project compliance, data collection, abstraction and entry, data reporting, regulatory 
monitoring, problem resolution and prioritization, and coordinating the activities of the protocol 
study team. 
 
The data collected for this study will be entered into a secure database. Source documentation will 
be available to support the computerized patient record. All research material from this study will be 
handled with the same confidentiality as the patient’s other medical data. 
 
 
13.2 
Quality Assurance 
 
Eligibility of patients will be verified with the principal investigator. Only the designated 
investigators can obtain informed consent. 
 
There are several different mechanisms by which clinical trials are monitored for data, safety and 
quality. There are institutional processes in place for quality assurance (e.g., protocol monitoring, 
compliance and data verification audits, therapeutic response, and staff education on clinical 
research QA) and departmental procedures for quality control, plus there are two institutional 
committees that are responsible for monitoring the activities of our clinical trials programs. The 
committees: Data and Safety Monitoring Committee (DSMC) for Phase I and II clinical trials, and 
the Data and Safety Monitoring Board (DSMB) for Phase III clinical trials, report to the Center’s 
Research Council and Institutional Review Board. 


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During the protocol development and review process, each protocol will be assessed for the level of 
risk and the degree of required monitoring. Every type of protocol (e.g., NIH sponsored, in-house 
sponsored, industrial sponsored, NCI cooperative group, etc.) is reviewed and monitoring procedures 
are established at the time of protocol activation. 
 
13.3 
Data and Safety Monitoring 
 
The Data and Safety Monitoring (DSM) Plans at Memorial Sloan-Kettering Cancer Center were 
approved by the National Cancer Institute in September 2001. The plans address the new policies set 
forth by the NCI in the document entitled “Policy of the National Cancer Institute for Data and 
Safety Monitoring of Clinical Trials” which can be found at 
http://cancertrials.nci.nih.gov/researchers/dsm/index.html. The DSM Plans at MSKCC were 
established and are monitored by the Office of Clinical Research. The MSKCC Data and Safety 
Monitoring Plans can be found on the MSKCC Intranet at: http://mskweb2.mskcc.org/irb/index.htm. 
 
 
14.1 
PROTECTION OF HUMAN SUBJECTS 
 
 
There are no foreseen additional risks to the patients from this study. 
 
Risks of Study Participation: Patients in this study will be receiving current standard of care for their 
specific disease site. 
 
Financial costs/burdens: 
Because all diagnostic and therapeutic interventions except for the FLT-PET scans are part of the 
current routine care of patients/subjects eligible for this study and that a research grant will cover the 
cost of the FLT-PET scans, there are no additional financial cost or burden to the patient beyond the 
charges routinely incurred as part of standard medical care. 
 
Patient Confidentiality: 
Patient/subject privacy and confidentiality will be maintained according to MSKCC guidelines and all 
data derived from this study will be kept in a secure database. All data and results will be anonymously 
reported with regard to individual subjects. 
 
Voluntary nature of the study: 
Subjects will be made aware of the voluntary nature of the study as part of the informed consent 
process. They will be allowed to withdraw participation at any time without the risk of alteration in the 
quality of their medical care. 


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14.2 
Privacy 
 
 
MSKCC’s Privacy Office may allow the use and disclosure of protected health information 
pursuant to a completed and signed Research Authorization form.  The use and disclosure of 
protected health information will be limited to the individuals described in the Research 
Authorization form.  A Research Authorization form must be completed by the Principal 
Investigator and approved by the IRB and Privacy Board. 
 
14.3 Serious Adverse Event (SAE) Reporting 
 
Any SAE must be reported to the IRB/PB as soon as possible but no later than 5 calendar days. 
The IRB/PB requires a Clinical Research Database (CRDB) SAE report be submitted 
electronically to the SAE Office at sae@mskcc.org containing the following information: 
Fields populated from the CRDB: 
• Subject’s name (generate the report with only initials if it will be sent outside of 
MSKCC) 
• Medical record number 
• Disease/histology (if applicable) 
• Protocol number and title 
 
Data needing to be entered: 
• The date the adverse event occurred 
• The adverse event 
• Relationship of the adverse event to the treatment (drug, device, or intervention) 
• If the AE was expected 
• The severity of the AE 
• The intervention 
• Detailed text that includes the following information: 
o A explanation of how the AE was handled 
o A description of the subject's condition 
o Indication if the subject remains on the study 
o If an amendment will need to be made to the protocol and/or consent form 
The PI’s signature and the date it was signed are required on the completed report. 
For IND/IDE protocols: 
The CRDB AE report should be completed as above and the FDA assigned IND/IDE number 
written at the top of the report. If appropriate, the report will be forwarded to the FDA by the 
SAE staff through the IND Office. 


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15.0     INFORMED CONSENT PROCEDURES 
 
Before protocol-specified procedures are carried out, consenting professionals will explain full 
details of the protocol and study procedures as well as the risks involved to participants prior to 
their inclusion in the study. Participants will also be informed that they are free to withdraw 
from the study at any time. All participants must sign an IRB/PB-approved consent form 
indicating their consent to participate. This consent form meets the requirements of the Code of 
Federal Regulations and the Institutional Review Board/Privacy Board of this Center. The 
consent form will include the following: 
 
1. 
The nature and objectives, potential risks and benefits of the intended study. 
2. 
The length of study and the likely follow-up required. 
3. 
Alternatives to the proposed study. (This will include available standard and 
investigational therapies. In addition, patients will be offered an option of supportive care for 
therapeutic studies.) 
4. 
The name of the investigator(s) responsible for the protocol. 
5. 
The right of the participant to accept or refuse study interventions/interactions and to 
withdraw from participation at any time. 
 
Before any protocol-specific procedures can be carried out, the consenting professional will 
fully explain the aspects of patient privacy concerning research specific  information.  In 
addition to signing the IRB Informed Consent, all patients must agree to the Research 
Authorization component of the informed consent form. 
 
Each participant and consenting professional will sign the consent form. The participant must 
receive         a         copy         of         the         signed         informed         consent         form.. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 09-060 A(4) 
Amended: 12/27/11 
- 21 - 
 
 
 
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Memorial Sloan-Kettering Cancer Center 
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Amended: 12/27/11 
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IRB#: 09-060 A(4) 
 
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