The Detection Of Circulating Tumor Cells (CTC) In Patients With Non-small Cell Lung Cancer (NSCLC) Undergoing Definitive Radiotherapy Or Chemoradiotherapy
Conditions: Non-small Cell Lung Cancer
Sponsor: Abramson Cancer Center at Penn Medicine

 
CTC's and NSCLC 
Version Date: 4/2/2021 
 
PROTOCOL 
UNIVERSITY OF PENNSYLVANIA 
IRB # 816263 
UPCC # 12512 
 
The Detection of Circulating Tumor Cells (CTC) in Patients with 
Non-small cell lung cancer (NSCLC) Undergoing Definitive 
Radiotherapy or Chemoradiotherapy 
 
 
 
 
 
Principal Investigator:  
Steven Feigenberg, MD 
 
 
 
 
University of Pennsylvania 
 
 
 
 
Perelman Center for Advanced Medicine 
 
 
 
 
Department of Radiation Oncology 
 
 
 
 
3400 Civic Center Blvd.  
 
 
 
 
TRC 2 West 
 
 
 
 
Philadelphia, PA 19104 
 
 
 
 
Phone  215-662-3998 
 
 
 
 
Email: Samuel.Swisher-McClure2@uphs.upenn.edu 
 
 
 
Sub-Investigators: 
 
Jay Dorsey, MD, PhD 
 
 
 
Gary Kao, MD, PhD 
 
 
 
Amit Maity, MD, PhD 
 
 
 
William Levin, MD 
 
 
 
Keith Cengel, MD, PhD 
 
 
 
Abigail Berman, MD 
Constantinos Koumenis, PhD 
 
 
Statistician: 
 
Rosemarie Mick, MS  
 
 
 
 
 
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authorized in writing by the study sponsor. 

 
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TABLE OF CONTENTS 
1 
ABSTRACT ........................................................................................................................... 4 
2 
BACKGROUND ................................................................................................................... 4 
3 
OBJECTIVE ......................................................................................................................... 7 
4 
SUBJECT POPULATION ................................................................................................... 8 
5 
STUDY PROCEDURES ...................................................................................................... 8 
6 
STATISTICAL CONSIDERATIONS .............................................................................. 10 
7 
REFERENCES .................................................................................................................... 13 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
. 
 
 
 
 
 
 
 
 
 
 

 
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LIST OF ABBREVIATIONS 
 
CACs  
cytogenetically abnormal cells 
CTC 
 
circulating tumor cells 
NSCLC 
non-small cell lung cancer  
PCR   
polymerase chain reaction 
WBC  
white blood cells 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1. ABSTRACT 
 
Non-small cell lung cancer (NSCLC) is the most common cause of cancer mortality in the United 
States with approximately 180,000 patients diagnosed each year.  Systemic disease and relapse 
after definitive therapy (surgery, chemotherapy and radiation) are common.  There is a need to 
develop new approaches for treating patients with NSCLC.  There is also a need to develop 
methods for predicting response to treatments, monitoring response to therapy, and early detection 
of relapse and/or systemic disease.  There is little known about the numbers, time course, and 
effect of therapy on CTCs in NSCLC patients.  The primary objective of this protocol is to describe 
the pattern of CTC detection in NSCLC patients undergoing definitive radiation therapy. 
Exploratory studies on the isolation of circulating tumor DNA (ctDNA) and its contributory value 
to the collection of circulating tumor cells will also be conducted.  Patients with NSCLC who 
undergo radiation as part of a definitive course of treatment will be enrolled. Blood collections 
will be obtained before, during, and after the completion of radiotherapy.  In patients who are also 
treated with the novel signal transduction inhibitor, nelfinavir, in addition to chemoradiotherapy, 
an additional blood sample will be obtained 4-6 days after starting nelfinavir and prior to starting 
chemoradiotherapy.  We will collect demographic and treatment data and explore & describe the 
pattern of CTC detection in all patients.  
 
2. BACKGROUND 
  
NSCLC 
Non-small cell lung cancer (NSCLC) is the most common cause of cancer mortality in the United 
States with approximately 180,000 patients diagnosed each year.  Systemic disease and relapse 
after definitive therapy (surgery, chemotherapy and radiation) are common.  There is a need to 
develop new approaches for treating patients with NSCLC.  There is also a need to develop 
methods for predicting response to treatments, monitoring response to therapy, and early detection 
of relapse and/or systemic disease.  There is little known about the numbers, time course, and 
effect of therapy on CTCs in NSCLC patients (Katz et al., 2010)  
 
Current Methods of Circulating Tumor Cells Detection and Inherent Limitations 
The absolute number of CTCs that have been reported from cancer patients has varied greatly from 
one study to another (Katz et al., 2010). Technical constraints may lead to an underestimation of 
CTCs in some studies.  Tumor cells can adopt a wide range of shapes and sizes that may be 
indistinguishable from or overlap with normal cells, including white blood cells.   CTC-detecting 
techniques based on morphology or size alone may therefore fail to isolate CTCs that mimic 
normal cells in size or shape. Such tumor cells however can be uncloaked by staining for tumor-
specific antigens, including cell surface markers. Expression levels of such markers may, however, 
vary substantially between CTCs of different histologies or even possibly within the cells from a 
single patient.  The specific CTC analysis technique employed, how the patient-derived materials 
are handled and stored, and the thresholds set for detection not to mention the patient’s disease or 
treatment status may all potentially affect the sensitivity by which CTCs are detected. 
 
Size exclusion filtration is a common CTC detection technique based upon the observation that 
tumor cells are in general larger than non-cancerous circulating cells.  An advantage of this method 

 
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is that it may isolate CTCs of any histology, not just of epithelial origin, unlike other technologies 
currently being investigated and it uses polycarbonate membrane filters which are relatively 
inexpensive.  However, a disadvantage is that the trach-etching process that is employed to create 
the filter pores can occasionally create two pores that overlap, resulting in larger cavities that allow 
all cells to pass through, including those that otherwise would not be able to pass through a single 
pore.  This in turn leads to CTC loss and thus decreased sensitivity. 
 
Density Gradient Separation Techniques attempt to separate CTCs from peripheral blood cells 
based on their density.  The OncoQuick ® method (Greiner Bio-One, Frickenhausen, Germany) is 
one example of this technique.  This method relies on the fact that erythrocytes and neutrophils 
have greater densities than mononuclear cells and CTCs.  The method uses density gradient 
separation fluids such as Ficoll-Paque ®  or Percoll ® through which  erythrocytes and 
granulocytes will pellet while mononuclear cells and CTCs remain in the interphase layer above 
the fluid and below the plasma (Fizazi et al., 2007).    It is unclear, however, if all CTCs have the 
appropriate buoyant density for capture via density gradient separation methods. 
 
Other techniques have been described to distinguish CTCs from WBCs or to distinguish between 
subsets of CTCs expressing different biomarkers.  For example, laser scanning cytometry is a 
technique in which pre-stained cells are subject to laser light illumination and then scanned under 
microscopy to detect and count cells that stain brightly.   WBCs and CTCs are then differentially 
detected based on different degrees of light scatter, size, or other criteria (Pachmann et al., 2008).  
Polymerase chain reaction (PCR) is a sensitive method of detecting the expression of specific 
mRNA sequences, including those characteristic of CTCs.   However, there is often uncertainty 
regarding whether CTCs or WBCs or other sources are the origin of the detected mRNA, which 
thus confounds the analysis.  (Obermayr et al., 2010).  Another method that relies on antibody-
based detection of epithelial cell antigens expressed on the cell surface to identify CTCs is the 
EPISPOT (epithelial immunospot), in which live cells are incubated for 48-hours to ensure 
maximize cell surface protein expression, followed by the addition of primary anti-epithelial 
protein antibody and then a method to detect the binding of the primary antibody to identify CTCs  
(Alix-Panabières et al., 2007).  The CellSearch System (by Veridex LLC, so far the only method 
currently approved by the FDA for clinical assays) relies on ferrofluid particles coated with anti-
EpCAM (EpCAM = Epithelial cell adhesion molecule) antibodies to purify CTCs (Allard et al., 
2004).  The ferrofluid particles coated with anti-EpCAM antibodies bind to CTCs, and the entire 
complex is then removed via magnets.  There may, however, be cellular components (debris) such 
as membranes retaining EpCAM that can be found freely circulating.   Consequently, the 
magnetically captured material is further assessed using a panel of markers to confirm the presence 
of cells of tumor origin.   These secondary markers include the DNA stain 4,6-diamino-2-
phenylindole (DAPI), cytokeratins (CKs), and CD-45 (the latter two are detected via specific 
antibodies raised against the respective antigens).  The CellSearch System is heavily reliant on 
EpCAM expression.  Consequently there are many tumor types, especially those of non-epithelial 
origin, which would not be detectable via this system.  Furthermore, it remains possible that loss 
of EpCAM, or lowered levels of expression such as that due to mutation or downregulation 
(epithelial-to-mesenchymal transition), would likely impede the ability to isolate even cancer cells 
of epithelial origin.  The panel of secondary markers also requires regular validation and the 

 
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appropriate negative and positive controls.  Commercial antibodies are often derived from animal 
sources, with attendant batch-to-batch variability in sensitivity and specificity. 
Relatively few studies have been published describing the detection of CTCs in NSCLC.  (Wu et 
al., 2009) described a technique using depletion of CD45 cells from blood with magnetic beads 
followed by CK18/19 staining of enriched cells.  Relatively few CTCs were detected although the 
number of CTCs correlated with stage. Katz reported on the development of a quantitative antigen-
independent fluorescent in situ hybridization-based (FISH) method for detecting cytogenetically 
abnormal cells (CACs) in the peripheral blood of NSCLC patients (Katz et al., 2010).  This FISH-
based assay detected losses and gains of selected chromosomal regions in the nuclei of cells 
regardless of their lineage.  These investigators detected mean number of CACs ranging from 7.23 
± 1.32/μL for deletions of 10q22.3/CEP10 to 45.52 ± 7.49/μL for deletions of 3p22.1/CEP3.   
 
Novel Methods of CTC Detection 
There is evidence that alternate techniques of CTC detection, and in particular detection of viable 
tumor cells which have lost epithelial cell markers, are possible. Kojima and colleagues have 
described one such method, in which a green fluorescent protein (GFP)-expressing attenuated 
adenovirus (OBP-401) is used to selectively label cells with high activity of telomerase. (Kojima 
et al., 2009).This method is independent of cell-surface markers and therefore is independent of 
epithelial to mesenchymal transformation. The Kojima method labels only those cells susceptible 
to adenovirus transduction and with high levels of telomerase activity. In the peripheral blood there 
exists a paucity of cells, other than CTC, in which adenovirus transduction may occur. 
Additionally, while it is established that telomerase is activated in the majority of human cancer 
cells, other circulating cells, including hematopoietic cells, show low telomerase activity.(Kojima 
et al., 2009). This combination of factors makes this an ideal vector for the labeling and detection 
of CTCs. 
 
To our knowledge a telomerase-based method of CTC detection has yet to be applied 
systematically to a NSCLC cancer population. Our group, however, has experience in measuring 
CTCs from lung tumors as part of the tissue collection/registry study in the Department of 
Radiation Oncology, thereby demonstrating the feasibility of this approach. 
 
Among 23 patients with non-small cell lung cancer (NSCLC) undergoing definitive 
chemoradiation the range of CTCs detected per ml of blood ranged from 0.57 to 570.7 cells (mean 
62.7, median 9.1, Standard Error 28.4). The rate of background, non-CTC cells, detected via the 
telomerase-based method in 4 normal, healthy controls ranged from 0.1 to 1.7 (mean 0.76, median 
0.72).  
 
Figure 2 shows the change in the number of CTCs detected among patients prior to and following 
definitive chemoradiation for NSCLC. Patient #1 decreased from 56.9 CTC/ml prior to treatment 
to 0.9 CTC/ml following treatment. Patient #2 decreased from 9.1 to 1.8 and patient #3 from 1.2 
to 0.2. Percent decreases ([pre-post/pre]*100) for Patients 1-3 were: 98%, 80% and 83%. 
 

 
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In summary, we have used novel methodology to detect CTCs in patients with NSCLC.  In this 
protocol, using this novel CTC detection assay, we will investigate the pattern of CTC detection 
in patients with NSCLC undergoing definitive radiotherapy as a part of their treatment.  We will 
systematically and prospectively collect demographic, treatment, and CTC data over a two-year 
period in each patient. These data will be exploratory and hypothesis-generating. 
 
Isolation of Circulating Tumor DNA (ctDNA) 
Circulating tumor DNA analysis may also be performed to provide additional correlated data. 
Following the isolation of the cells from the cell layer in the blood sample, the plasma from the 
rest of sample will be used to isolate circulating tumor DNA (ctDNA).  This DNA will then be 
amplified by PCR and analyzed for specific mutations associated with lung cancer (such as K-Ras, 
ALK, EGFR, etc), based on published methodologies (CAPP-Seq, Newman et al., Nature 
Medicine 20, 548–554, 2014).  
 
3. OBJECTIVE  
 
3.1 Primary Objectives 
1. To determine the intra- and inter-patient variability at baseline and test/re-test reliability of 
the method 
2. To describe the pattern of CTCs before, during and after definitive radiotherapy for each 
treatment stratum 
 
3.2 Secondary Objectives 
1. To explore the relationship between baseline CTC and metabolic tumor response 

 
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2. To determine the change in CTC during treatment (i.e., slope from baseline to week 5 
during radiotherapy) and metabolic tumor response 
3. To explore the changes in CTCs during treatment and changes after radiotherapy  
4. To determine the pattern of EGFR mutations in CTCs 
5. To determine the pattern of signaling pathway expression of relevant biomarkers of CTCs 
before, during and after definitive radiotherapy in patients receiving Nelfinavir 
6. To explore the contributory value of isolating circulating tumor DNA (ctDNA) in addition 
to the collection of CTCs. 
 
4. SUBJECT POPULATION 
 
We plan to enroll patients into seven distinct cohorts: 
1. Patients with early stage NSCLC undergoing stereotactic radiotherapy  
2. Patients with locally advanced NSCLC undergoing standard chemoradiotherapy  
3. Patients with stage I-III NSCLC undergoing, fractionated radiotherapy alone 
4. Patients with locally advanced NSCLC undergoing standard chemoradiotherapy with the 
novel signal transduction inhibitor, nelfianvir.  
5. Patients with resectable stage IIIa NSCLC undergoing pre-operative chemoradiotherapy 
6. Patients with suspected (no tissue diagnosis) early stage NSCLC undergoing stereotactic 
radiotherapy 
7. Patients with locally advanced NSCLC undergoing stereotactic radiotherapy followed by 
concurrent mediastinal chemoradiotherapy. 
Eligibility 
1. Patients with biopsy-proven (except for Cohort 6) NSCLC who are undergoing definitive 
radiotherapy as a part of their treatment regimen. 
2. Age 18 or older 
3. Signed informed consent. The consent process can be completed in person or via 
Telemedicine per University of Pennsylvania Health Systems telemedicine policy and 
procedures. 
4. Patients who are incapable of providing informed consent are excluded from participating 
in this study. 
 
5. STUDY PROCEDURES 
 
We will collect blood samples and determine CTC values in the seven cohorts of patients with 
NSCLC, as described below: 
Cohort 1:  Patients with early stage NSCLC undergoing stereotactic radiotherapy  
Cohort 2:  Patients with locally advanced NSCLC undergoing standard chemoradiotherapy  
Cohort 3:  Patients with stage I-III NSCLC undergoing, fractionated radiotherapy alone 
Cohort 4:  Patients with locally advanced NSCLC undergoing standard chemoradiotherapy with 
the signal transduction inhibitor, nelfinavir.  
Cohort 5:  Patients with resectable stage IIIa NSCLC undergoing pre-operative 
chemoradiotherapy* 
Cohort 6:  Patients with suspected (no tissue diagnosis) early stage NSCLC undergoing 
stereotactic radiotherapy 

 
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Cohort 7: Patients with locally advanced NSCLC undergoing stereotactic radiotherapy followed 
by concurrent mediastinal chemoradiotherapy. 
 
*Often a patient cannot be fully evaluated as a surgical candidate until the completion of 
chemoradiotherapy.  This means that the patients originally placed in cohort 2 can be moved to 
cohort 5 and vice versa upon the discretion of the PI. 
 
Blood Collection Procedure  
 
The procedure of blood collection for each patient will be as follows: up to 5 mL of blood will be 
drawn into a waste tube (to avoid collecting skin material and potential contaminating skin stem 
cells.) The waste tube will either be discarded or used to investigate other laboratory methods 
involving CTC virus transduction.  This could lead to the use of a more effective virus for CTC 
analysis or the need for smaller subject blood samples in the future. Subsequently 10 mL of blood 
will be collected into heparinized tubes, and immediately placed on ice in a prepared plastic bag 
and transported to the laboratory for processing within approximately one hour.  A 10 mL sample 
may also be collected (typically in an EDTA tube) for the isolation of ctDNA.  These samples will 
be collected via venipuncture or by utilizing a patient’s previously place central/peripheral line 
being used for other purposes (i.e. chemotherapy).     
 
The total volume of blood obtained with each collection would therefore ideally be at least 25 cc.  
Blood collections will, be obtained up to 2 times before the start of radiation, during weeks 1-2, 
weeks 3-4, and weeks 5-6 of radiotherapy or as clinically indicated.  Following the completion of 
radiotherapy blood draws will occur at approximately:  1 month, 3 months, 6 months, 12 months, 
18 months and 24 months.  In patients who are also treated with nelfinavir an additional blood 
sample will be obtained 4-6 days after starting nelfinavir and prior to starting chemoradiotherapy.   
Blood collections are performed exclusively for research purposes. 
 
Ideal collection time points generally include:  
 
 A maximum of two pre-radiotherapy samples (ideally within approximately 30 days of each 
other) prior to the first fraction of radiation.  
 Approximately one to two weeks after the initiation of radiation therapy (for cohort 7 this will 
be obtained during the course of stereotactic radiotherapy) 
 Approximately three to four weeks after the initiation of radiation therapy (this collection is 
omitted for cohort 1 and 6 because the treatment course of those cohorts is generally shorter 
than four weeks, for cohort 7 this will be obtained approximately three to four weeks after the 
initiation of concurrent mediastinal chemoradiotherapy) 
 Approximately five to six weeks after the initiation of radiation therapy (this collection is 
omitted for cohort 1 and 6 because the treatment course of those cohorts is generally shorter 
than four weeks.  This collection may also be omitted for other cohorts if the investigator 
decides to use a hypofractionated treatment plan, for cohort 7 this will be obtained 
approximately five to six weeks after the initiation of concurrent mediastinal 
chemoradiotherapy). 
 Approximately one month  after the completion of radiation 

 
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 Approximately three months  after the completion of radiation 
 Approximately six months after the completion of radiation 
 Approximately twelve months after the completion of radiation 
 Approximately eighteen months after the completion of radiation 
 Approximately twenty-four months after the completion of radiation 
 
Due to unanticipated side-effects of treatment, scheduling issues or logistical difficulties, the 
above schedule will be modified at the discretion of the PI.  If a sample is unable to be obtained 
at a specified time point it will be collected at the next opportunity. Every attempt will be made 
to collect samples at normally scheduled standard of care appointments.  In cases where this is 
not possible and the participant is willing, a sample may be obtained at a research visit only. 
 
*cohort 5 patients who require follow up radiotherapy or chemoradiotherapy will have an 
additional CTC sample obtained prior to  follow-up radiotherapy (approximately 7 days from the 
initiation of radiation) and then will follow the schedule of cohort 2 thereafter. 
 
For patients on Nelfinavir, the two pre-radiotherapy collection samples will consist of: 
(1) before start of Nelfinavir and before initiation of radiotherapy 
(2) after start of Nelfinavir but before initiation of radiotherapy 
 
An additional blood sample may be requested from the patient if he/she develops clinical or 
biopsy-proven recurrent disease or progression of disease at any time along with continued 
collection of samples at the same time points already determined per protocol.  
 
*Adverse events will not be followed or reported for this protocol. 
 
Off Study Criteria  
Patients will be taken off the study for 1) patient refusal; 2) extraordinary medical circumstances 
or 3) at the discretion of the PI. 
 
Table 1.  Outline of Study Blood Collection 
 
 
Pre-Treatment 
On Treatment 
Follow-up (by month) 
Cohort 
#1 
#2* 
1/2 wks 3/4 wks 
5/6wks 
1 
3 
6 
12 
18 
24 
1 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
6 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
7 
X 
X 
X** 
X*** 
X*** 
X 
X 
X 
X 
X 
X 
*In cases where it is not possible to obtain two pre-treatment samples due to logistical considerations and planned 
treatment start date only one pre-treatment sample will be obtained. 
**For cohort 7 this sample will be obtained during the course of stereotactic radiotherapy. 

 
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*** For cohort 7 these samples will be obtained approximately three to four weeks and five to six weeks after the 
initiation of concurrent mediastinal chemoradiotherapy. 
 
6. STATISTICAL CONSIDERATIONS 
 
6.1 Design: 
This is a pilot study to establish that circulating tumor cells (CTC) can be detected in blood samples 
of patients with NSCLC undergoing definitive radiotherapy. CTCs will be detected by a method 
which uses a telomerase-specific replication-selective adenovirus expressing the molecular 
marker, GFP. Patients will be assigned to one of seven strata, as defined by their treatment. A total 
of 280 patients will be enrolled, 10 in cohort 4, 60 per stratum in cohorts 1,2, and 6 and 30 per 
stratum in cohorts  3, 5, 7.  
 
Treatment Stratum 
1: Early stage NSCLC undergoing stereotactic body radiotherapy  
2. Locally advanced NSCLC undergoing standard chemoradiotherapy 
3. Stage I to III NSCLC undergoing radiotherapy alone 
4. Locally advanced NSCLC undergoing Nelfinavir + standard chemoradiotherapy 
5. Stage IIIa NSCLC undergoing pre-operative chemoradiotherapy 
6. Non-biopsy proven early stage NSCLC undergoing stereotactic body radiotherapy 
7. Locally advanced NSCLC undergoing stereotactic radiotherapy followed by 
concurrent mediastinal chemoradiotherapy. 
 
 
Up to two blood samples will be obtained at baseline (ideally within approximately 30 days of 
each other). Thereafter, samples will be obtained at approximately weeks 1/2, 3/4 and 5/6 during 
radiotherapy and at approximately 1, 3, 6, 12, 18 and 24 months after completion of radiotherapy. 
The sampling schedule varies slightly for patients who undergo SBRT or surgery or who receive 
Nelfinavir. EGFR mutations and signaling pathway expression of relevant biomarkers detected in 
CTCs will be measured at baseline, at week 5/6 during radiotherapy and at 3 and 6 months after 
completion of radiotherapy.  Metabolic tumor response will be assessed by PET/CT when possible 
if performed as standard of care at 3 months after completion of radiotherapy and if clinically 
indicated. 
6.2 Objectives:  
 
6.2.1 The primary objectives are to determine: 
1.   intra- and inter-patient variability at baseline and test/re-test reliability of the method 
2.   the pattern of CTCs before, during and after definitive radiotherapy for each treatment stratum 
 
6.2.2 Secondary objectives are to describe the relationship between: 
3.  baseline CTC and metabolic tumor response 
4. changes in CTC counts during treatment (i.e., slope from baseline to week 5/6 during 
radiotherapy) and metabolic tumor response 
5.  changes in CTC counts during treatment and changes after radiotherapy 
6. CTCs and the isolation of ctDNA 

 
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And the pattern: 
7.  of EGFR mutations in the detected CTCs 
8. of genetic mutations and other biomarkers in the detected CTCs before, during and after 
definitive radiotherapy 
9.  in patients receiving Nelfinavir 
 
6.3 Preliminary data: 
CTCs were measured in 23 NSCLC patients undergoing definitive therapy. Substantial inter-
patient variation was observed, with a coefficient of variation of 212%.  Thus estimation of intra-
patient variation and inter-patient variation at baseline in a prospectively identified patient cohort 
will be extremely important, in order to identify credible treatment-related changes in CTCs during 
and after radiotherapy. 
Mean+SE 
Median 
Standard Deviation 
Minimum 
Maximum 
Coefficient of variation 
62.7+28.4 
9.1 
133.2 
0.57 
570.7 
212% 
 
6.4 Endpoints:  
1. Circulating tumor cells will be detected by imaging for green fluorescent protein (GFP) which 
measures cells which high levels of telomerase activity after adenovirus transduction. CTCs are 
measured in units of number of GFP-positive cells /mL.  
2. Metabolic tumor response will be determined by PET/CT (when performed as clinically 
indicated)  at 3 months post-treatment and dichotomized as responder (CR/PR) or nonresponder 
(<PR). 
3. Change in CTC will be measured by fold change (post/pre) and percent change from baseline 
(i.e., [pre-post)/pre]*100). 
4. EGFR mutations will be detected by IF staining with EGFR mutation-specific antibodies. 
5.  The presence or expression of other biomarkers and pathways involved in tumor biology will 
be assessed via such methods as RT-PCR for genetic mutations. 
6.    In patients treated with Nelfinavir, the levels of and changes in signaling pathways and other 
relevant biomarkers will also be assessed. 
 
6.5 Plans for Data Analysis:  
Descriptive statistics will support hypothesis generation and the design of adequately powered 
future studies. Most analyses will be conducted separately for each treatment stratum.  
 
Descriptive statistics/Graphs: Baseline CTC will be characterized by descriptive statistics (e.g., 
mean, median, SD, range, coefficient of variation) and reliability analysis (see below description). 
At each time point during and after radiotherapy, the CTC mean+SE and the fold change mean+SE 
(to account for each patient's baseline) will be calculated. Plots of mean and fold change over time 
will be generated. Test/re-test reliability analysis. Since repeated observations of CTC are obtained 
at baseline, a plot of Visit 1 and Visit 2 values will be constructed, Pearson correlation and linear 
regression (i.e., test of slope = 1) will be initial analyses to characterize overall test/re-test 
reliability for the study population, pooling over the 7 strata. Test-re/test reliability will be further 
evaluated by the within-patient coefficient of variation. Defining real change. To determine 
whether fold changes observed during and after treatment are beyond random variation, we will 
compute the ratio of Visit 1 to Visit 2 for each patient and then compute the standard deviation of 

 
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the distribution of the ratios. Values of fold change in CTC during and after treatment which fall 
outside the interval [1.0-2SD, 1.0+2SD] will be considered credible changes.  
 
Repeated measures ANOVA and linear models: Because we hypothesize CTCs will decrease 
during radiotherapy, changes over time will be characterized by modeling slopes. Log 
transformation and piecewise linear models may be employed, as needed. Repeated measures 
ANOVA will be used to compare mean CTC values at each time point during and after 
radiotherapy to mean CTC at baseline, although these exploratory analyses may be underpowered. 
Group comparisons: At 3 months post-treatment, metabolic response will be determined. 
Descriptive statistics of baseline CTC will be generated for responders and nonresponders within 
each treatment stratum. Comparison of responders to nonresponders will be conducted by pooling 
over the 7 strata. The affect of Nelfinavir in combination with chemoradiotherapy will be evaluated 
by mixed effects models which test the fixed effect of stratum (#2 vs #4) while modeling the time 
course of CTC values. Similarly, the affect of standard radiotherapy versus stereotactic 
radiotherapy will be evaluated by mixed effects models which test the fixed effect of stratum (#1 
vs Stage I patients in #3). Correlations: Relationships between CTC and biomarker expression or 
presence of genetic mutations will be evaluated at fixed time points, by scatter plots and 
correlations. Time courses of CTC, EGFR mutations and biomarkers will also be jointly plotted to 
investigate temporal relationships.  
 
Sample Size:  A total of 10 subjects will be enrolled in cohort 4, 30 subjects will be enrolled into 
cohorts 3, 5, & 7 and 60 subjects will be enrolled into cohorts 1, 2, & 6. Any subject who does not 
complete a full course of radiotherapy will be removed from the study and replaced. 
 
Study Duration:  
The study will enroll a total of 280 subjects. This study will likely complete enrollment in 48 
months. 
 
7. REFERENCES 
Alix-Panabières, C., Vendrell, J.-P., Pellé, O., Rebillard, X., Riethdorf, S., Müller, V., Fabbro, 
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