RETROSPECTIVE EPIDEMIOLOGY STUDY OF ALK REARRANGEMENT IN NON-SMALL CELL LUNG CANCER PATIENTS IN THE MIDDLE EAST & NORTH AFRICA.
Conditions: Non-small Cell Lung Cancer
Sponsor: Pfizer

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 1 
 
 
 
 
RETROSPECTIVE NON-INTERVENTIONAL, NON-PFIZER PRODUCT STUDY PROTOCOL 
(NINPP Study) 
 
 
RETROSPECTIVE EPIDEMIOLOGY STUDY OF ALK REARRANGEMENT IN NON-
SMALL CELL LUNG CANCER PATIENTS IN THE MIDDLE EAST & NORTH AFRICA 
 
 
Compound Numbers: 
NA 
Compound Names: 
NA 
Phase:                                                               NA 
Study Number: 
A8081046 
           
Version and Date: 
FINAL VERSION 
                                                                           April 21st, 2014 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 2 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 3 
 
1. INTRODUCTION ............................................................................................................. 4 
1.1. 
Background & Rationale  ............................................................................................ 4 
2. STUDY OBJECTIVES AND ENDPOINTS ....................................................................... 6 
2.1. 
Objectives .................................................................................................................. 6 
2.2. 
Endpoints ................................................................................................................... 7 
2.2.1. 
Primary Endpoint .................................................................................................. 7 
2.2.2. 
Secondary Endpoints ............................................................................................ 7 
3. STUDY DESIGN .............................................................................................................. 7 
4. SUBJECT SELECTION ................................................................................................... 9 
4.1. 
Inclusion Criteria ........................................................................................................ 9 
4.2. 
Exclusion Criteria ....................................................................................................... 9 
5. STUDY TREATMENT AND DURATION ........................................................................ 10 
6. STUDY PROCEDURES ................................................................................................ 10 
7. DATA ANALYSIS/STATISTICAL METHODS ................................................................. 12 
7.1. 
Sample Size Considerations..................................................................................... 12 
7.2. 
Analysis Populations ................................................................................................ 13 
7.3. 
Statistical Methods ................................................................................................... 13 
7.4. 
Primary Analysis ....................................................................................................... 13 
7.5. 
Secondary Analysis  ................................................................................................. 13 
8. DATA COLLECTION ..................................................................................................... 14 
8.1. 
Primary Endpoint ...................................................................................................... 14 
8.2. 
Secondary Endpoints ............................................................................................... 14 
8.3. 
Record Retention ..................................................................................................... 14 
9. ADVERSE EVENT REPORTING .................................................................................. 15 
10. ETHICS ....................................................................................................................... 15 
10.1. 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) ................... 15 
10.2. 
Ethical Conduct of the Study…………………………………………………………….15 
10.3. 
Subject Information and Consent……………………………………………………….16 
11. PUBLICATION OF STUDY RESULTS .........................................................................16 
11.1. 
Communication of results by Pfizer ....................................................................... 16 
11.2. 
Publications by Investigators ................................................................................. 16 
11.3. 
Communication of Issues………………………………………………………………..17 
12. REFERENCES ............................................................................................................17 
        
Appendix A: Site Case Report Form………………………………….……………………….….18 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 4 
 
 
1. INTRODUCTION 
 
1.1. Background & Rationale  
 
Lung cancer is the most common cancer worldwide [1]. In 2008, an estimated 1.61 million 
people worldwide were diagnosed with pulmonary carcinoma, accounting for 13% of total 
cancer diagnoses [1]. Pulmonary carcinoma can be divided into two major histopathological 
groups: small-cell lung cancer (SCLC) and non-small-cell lung cancers (NSCLC) [2]. The latter 
group, NSCLC, comprises around 85% of lung cancers and represents a heterogeneous group 
of malignancies including adenocarcinoma, squamous cell carcinoma and large-cell 
carcinoma.  In small tissue sample when diagnostic features are not clear, cases may be 
classified as NSCLC, not otherwise specified (NOS) [2].  
 
Recently, a number of oncogenic drivers of NSCLC have been identified, such as activating 
mutations in the epidermal growth factor receptor (EGFR) gene, and translocations in the 
anaplastic lymphoma kinase (ALK) gene discovered in 2007 [3-5].  
 
The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-
ALK) fusion gene has been identified as a potent oncogenic driver in non–small-cell lung 
cancer, in particular adenocarcinoma (ADC), & it represents a novel, promising molecular 
target for NSCLC treatments. [8] ALK alterations play a significant role in the pathogenesis of 
NSCLC, where it has been demonstrated to be a potent oncogenic driver, and accordingly, the 
ALK pathway represents a therapeutic target for novel NSCLC treatment [5, 6, 7] This fusion 
gene results from an inversion within the short arm of chromosome 2 (involving 2p21 and 
2p23), which leads to a fusion of the echinoderm microtubule-associated protein-like (EML4) 
gene with the anaplastic lymphoma kinase (ALK) gene[8] . This gene translocation activates 
ALK oncogene expression that induces cell transformation in vitro and in vivo [9].  
 
Treatment decisions by using biomarkers including ALK rearrangements and mutations in the 
EGFR gene have brought dramatic improvements in response and clinical benefit for NSCLC 
patients.  
 
In clinical studies, Crizotinib, an oral small-molecule tyrosine kinase inhibitor targeting ALK 
MET, and ROS1 tyrosine kinases [5,10,11], showed marked antitumor activity in patients with 
advanced ALK-positive NSCLC, with objective response rates of approximately 60% and a 
median progression-free survival of 8.1 months and 9.7 months, respectively  [12, 13].  In a 
recent comparative open label phase 3 study crizotinib proved superior to standard 
chemotherapy in patients with previously treated advanced NSCLC with ALK rearrangement, 
with median progression-free survival of 7.7 months in the  Crizotinib group  versus 3.0 months 
in the chemotherapy group (P<0.001), &  the response rates were 65% with crizotinib, as 
compared with 20% with chemotherapy (P<0.001). [14] 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 5 
 
Therefore, acquiring relevant biomarker information is essential in order to provide targeted 
and personalized treatment aimed at tumors harboring specific genetic abnormalities. In the 
era of personalized medicine, diagnostic strategies should be carefully considered in order to 
provide the necessary information to implement targeted therapy. Biopsy techniques, sample 
processing, histological diagnosis and biomarker testing should all be optimized. To achieve 
this goal requires adoption of a multidisciplinary approach in which interactions between the 
oncologist, molecular biologist, pathologist, pulmonologist, radiologist and/or other specialists 
are paramount. 
 
Development of robust and reliable laboratory tests for predictive biomarkers is essential to 
select appropriate patients for targeted therapy. A variety of methods have been adopted for 
the detection of ALK rearrangement, including fluorescent in situ hybridization (FISH), 
immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction (RT-PCR). 
[8] Currently, FISH analysis is the only FDA approved companion diagnostic test to detect ALK 
gene rearrangements.. However, the equipment required for FISH analysis is not always 
readily available in routine diagnostic laboratories. ALK rearrangement frequently involves 
short intrachromosomal inversion. [8]   The resulting subtle changes may be difficult to 
interpret by FISH analysis, and have led to false-negative results. [15, 16] IHC has been 
considered an alternative to FISH, which also can detect ALK rearrangements independent of 
the fusion partners. [8]    
 
In 2008, the highest incidence rates for lung cancer were seen in Central and Eastern Europe 
[1]. However, developing countries are now experiencing an increasing lung cancer burden 
[17]. Of note, lung cancer has the highest incidence rate of all cancers in 7 of 14 Arab 
countries and is the third most common cancer in Africa [1, 18]. 
 
Several studies confirmed the presence of EML4-ALK fusion in 2% to 7% of NSCLC patients. 
[19 - 24] In a meta-analysis of 14 publications, the ALK rearrangement has been observed in 
4.84% of cases of NSCLC (1). ALK rearrangements in NSCLC are mainly associated with 
adenocarcinomas. Patients affected are on average 10–15 years younger than patients 
lacking an ALK rearrangement and mostly have a history of never having smoked or of former 
light smoking (≤10 pack-years). ALK rearrangement, EGFR mutation, and KRAS mutation are 
generally (but not always) found to occur independently of one another and represent distinct 
molecular subsets [2,4,6]. However, little is known about the prevalence of EML4-ALK gene 
fusion in NSCLC patients in Africa Middle East countries and on their clinic-pathological 
parameters related to ALK fusion. 
 
Immunohistochemical (IHC) analysis of FFPE tissue specimens remains a mainstay of routine 
pathology practice. The major advantage of this approach is an ability to assay for tumor-
specific antigen expression without loss of the cytologic and architectural features that 
distinguish normal from pathologic tissue (5). Cell Signaling Technologies and Ventana have 
developed the “VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody” which is 
intended for laboratory use in the detection of the anaplastic lymphoma kinase (ALK) protein in 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 6 
 
formalin-fixed, paraffin-embedded NSCLC tissue stained with  Ventana BenchMark XT, or 
BenchMark Gx immunohistochemical automated slide stainers.  The VENTANA ALK IHC 
assay is the only CE-marked IVD IHC test indicated as an aid in identifying patients eligible for 
treatment with XALKORI® (crizotinib).(25) 
 
There is almost complete lack of any epidemiologic data regarding the prevalence of ALK 
rearrangement in Middle East & North Africa NSCLC patients. Also the clinico-pathologic 
characteristics for patients with ALK-positive NSCLC in this region need to be investigated. 
 
Therefore the current study aims to estimate the prevalence of ALK rearrangement in the 
Middle East North Africa (MENA) population by using the Ventana ALK-IHC method for ALK 
protein detection in retrospective NSCLC clinical samples, & to evaluate the association of 
ALK rearrangement with clinical and pathological parameters of NSCLC patients in MENA. 
 
2. STUDY OBJECTIVES AND ENDPOINTS 
 
2.1. Objectives 
 
The primary objective of this retrospective epidemiology study is to conduct a large-scale study 
to estimate the prevalence of ALK rearrangement in a large cohort of approximately 700 
retained tissue samples of MENA NSCLC patients. The diagnostic testing of retained clinical 
tissue samples (tissue block) will be tested by using the Ventana IHC test for ALK protein 
detection.  
The secondary objectives are: 
- 
To evaluate the association of ALK rearrangement with demographic, clinical and 
pathological parameters in NSCLC patients with or without ALK rearrangement, if this 
data is available.  
- 
To assess the concordance between Vysis FISH and Ventana IHC methods for ALK 
rearrangement detection in a subset of tissue samples included. 
The study results will provide valuable information to understand the epidemiologic and clinical 
characteristics of ALK alterations in Africa Middle East NSCLC patients.  
 
 
 
 
 
 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 7 
 
2.2. Study Endpoints 
 
2.2.1. Primary Endpoint 
 
To estimate the prevalence of ALK rearrangement in the Middle East & North Africa NSCLC 
patients 
 
2.2.2. Secondary Endpoints 
 
Medical charts will be reviewed to gather data on the secondary endpoints described 
below if available. 
 
1- To evaluate the association of ALK rearrangement with demographic, clinical and 
pathological parameters in NSCLC patients  
 
2- To assess the concordance between Vysis FISH and Ventana IHC methods for ALK 
rearrangement detection in a subset of tissue samples (200-250) included in 2-3 centers. 
A Case Report Form (CRF) will be developed for the study with the above variables defined 
carefully to standardize data collection.   
 
3. STUDY DESIGN 
 
This is a retrospective, cross-sectional non-interventional epidemiology study to investigate the 
prevalence of ALK rearrangement in NSCLC patients in Middle East & North Africa. 
Approximately 700 retained tumor tissue specimens (tissue block) of patients previously 
diagnosed with NSCLC will be selected & subjected to  ALK immune-staining using Ventana 
anti-ALK (D5F3) Rabbit Monocolonal Primary Antibody combined with OptiView Benchmark 
System in 6-8 centers in 5-7 countries in the MENA region.  
 
The tissue samples of NSCLC cases will be retrieved from tissue banks of the molecular 
diagnostic units & pathology departments in these study centers. The histological diagnosis 
will be confirmed by the pathologists. The retained samples will then be tested by performing 
the Ventana ALK-IHC to assess the absence or presence of the ALK rearrangement by the 
detection of the ALK protein in formalin-fixed, paraffin-embedded NSCLC stored tissue 
samples using Ventana anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody (Roche 
Diagnostics GmbH) in the selected study centers.  
 
 
The results of ALK testing within this study population will then be used to establish the 
prevalence of ALK rearrangement in MENA NSCLC patients.  

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 8 
 
 
The patients’ characteristics, demographic, clinical and pathologic parameters will be 
obtained from their medical records & analyzed to determine any association with the 
presence of the EML4-ALK fusion gene in MENA NSCLC patients. 
 
The samples & data collected will be coded by ‘Study Subject ID’ to protect patient 
confidentiality. No identifiable personal data will be stored. 
The medical records of included NSCLC patients’ tissue samples will be 
reviewed to obtain the following information if available: 
 
- 
Age 
- 
Gender (male / female) 
- 
Race 
- 
Nationality 
- 
Occupation 
- 
Medical history of prior lung disease, e.g. TB, COPD, etc. 
- 
Smoking history (never, current, or ex-smoker /  Age at starting smoking / Number of 
cigarettes/day, years of cigarette smoking / If ex-smoker, years since quitting smoking) 
- 
Specimen types (Surgically resected, Biopsy, Unknown) 
- 
Primary cancer diagnosis (Date of cancer diagnosis, duration since diagnosis, & age at 
diagnosis) 
- 
Date when the first sign/symptom of lung cancer appeared 
- 
Source of tissue sample 
- 
Tumor histologic types  (Adenocarcinoma, SCC, LCC, NOS) 
- 
Tumor stages 
- 
Treatment type (Surgery with or without chemotherapy, Chemotherapy and other, 
Unknown)  
- 
Line of therapy (First-line, ≥ Second-line, Unknown) 
- 
Response to treatment. 
- 
Presence of other biomarkers like EGFR & KRAS (Wild type, Mutant, Unknown) 
- 
Date of disease relapse/recurrence 
- 
Date of death 
- 
Last date of follow-up and vital status 
- 
Cause of death 
 
The assessment of concordance of the results between FISH & IHC will be done in 2-3 
centers. Results of FISH testing will be collected & recorded, if the retained tissue 
samples had been previously tested. If not, the Vysis FISH test will be performed, to 
assess the concordance between the results obtained using Vysis Break-apart FISH  & 
Ventana IHC testing methods for ALK rearrangement detection. 
 
 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 9 
 
 
 
4. SUBJECT SELECTION 
 
4.1. Inclusion Criteria 
 
Since this is a retrospective epidemiology study, no patients will be enrolled. Tissue samples 
of non-squamous NSCLC cases less than 5 years old from each of the centers will be 
selected. Cases will be eligible for inclusion in this study if all of the following criteria are met: 
1. Histological confirmation of nonsquamous NSCLC, with any TNM stage. 
2. Available and sufficient tissue sample for ALK testing 
3. Tissue samples are less than 5 years old 
4. Routinely processed formalin-fixed, paraffin-embedded tissue samples only (see 
exclusion criteria pertaining to tissue samples). 
5. Histological sections mounted on glass slides must not be older than 3 months 
6. Age > 18 years 
7. Any ECOG Performance status  
8. Still alive, or death confirmed before inclusion, or is unknown 
9. Disease diagnosis and/or treatment in one of the centers, in the last 5 years, assigned 
to participate in the study; 
10. Written informed consent for general investigational testing was previously obtained, or 
specifically obtained for this retrospective epidemiology study, or having a documented 
waiver for the Informed consent document use, as required by local regulatory 
authorities, &/or Research Ethics committee/Institutional Review Board. 
 
4.2. 
Exclusion Criteria  
 
Cases with any of the following exclusion criteria are not eligible: 
 
1- Tumor tissue samples older than 5 year period or samples not properly stored. 
2- Tumor tissue samples fixed by using AFA, B5, Bouin’s, 95% ETOH, & alcohol fixatives. 
3- Under-fixed tissue samples (i.e. < 6 hrs)  
4- Tumor tissue samples that have been subject to any decalcification processes. 
5- Recycled paraffin-embedded tissue samples. 
6- Cut slides stored longer than 3 months. 
7- Insufficient tissue samples with less tumor cells & high amount of necrosis. 
 
 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 10 
 
 
 
5. STUDY TREATMENT AND DURATION (Not Applicable) 
 
This is a study that does not required administration of study drug, placebo, or any study 
specific therapeutic intervention. 
 
6. STUDY PROCEDURES 
 
No patients will be enrolled or be directly involved. This study involves laboratory testing of 
retained tissue specimens, of retrospectively diagnosed cases with NSCLC, & reviewing of their 
medical records and therefore will not require enrollment of subjects for prospective 
surveillance. Prior to initiation of the study, Pfizer and the study team will be responsible for 
identifying and selecting sites (hospitals/institutes) suitable for performing the study tests & data 
collection. Sites will be selected so that they are as representative as possible of the general 
population within a country. Site selection also will be based on practical factors such as the 
availability of tissue banks, availability of Benchmark GX or XT system, & on its qualification to 
perform the Ventana ALK-IHC test. 
 
Retrospectively collected NSCLC formalin-fixed, paraffin-embedded tissue (FFPET) tumor 
tissue specimens will be identified & retrieved from the tissue banks of the study centers.  
 
Cases which will meet the inclusion/exclusion criteria will be selected for analysis for ALK 
rearrangements using Ventana ALK-IHC testing method. 
 
Local research ethical approvals will be in place or will be applied for, & appropriate 
consenting or waiver documentation will be obtained.  
 
Implementation of the ALK IHC procedure and validation on ALK positive slide (known ALK-
positive NSCLC case; human appendix tissue; Ventana 2 in 1 control cell lines slide). 
 
Online ALK IHC procedure and interpretation training through Webinar 
 
Ventana ALK-IHC testing method will be performed on the eligible NSCLC formalin-fixed, 
paraffin-embedded tissue (FFPET) tumor tissue specimens. 
 
The medical records of included NSCLC patients’ tissue samples will be reviewed to 
obtain data on their demographic, clinical and pathologic parameters. 
 
In 2-3 of the study centers, ALK break apart FISH results are either already available or will be 
done after IHC has been performed, in order to assess the concordance between Vysis FISH 
and Ventana IHC in this subset of tissue samples. 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 11 
 
Clinical & histopathological characteristics of ALK positive cases will be compared to the 
clinical characteristics of al ALK negative cases.  
 
The CRFs will be used to record all information required by the study for collection of primary 
endpoint data & for collection of the secondary endpoints. Upon receipt and compilation of the 
analytic datasets collected from each site, the data analyses described in the analysis plan will 
be performed. To complete the study, a final study report will be developed. 
 
 
- 
Immunohistochemistry (IHC): 
Routinely processed, formalin-fixed, paraffin-embedded tissues are suitable for IHC testing 
with VENTANA anti-ALK (D5F3) primary antibody. 
VENTANA anti-ALK (D5F3) primary antibody has been developed for use on VENTANA 
BenchMark XT and BenchMark GX automated slide stainers in combination with Rabbit 
Monoclonal Negative Control Ig, OptiView DAB IHC Detection Kit, and OptiView Amplification 
Kit and accessories. 
 
Sections should be cut approximately 4 μm thick and mounted on positively-charged glass 
slides. Slides should be stained promptly, as antigenicity of cut tissue sections diminishes 
over time and is compromised within 3 months after cutting. For reading of the slides, the 
Ventana kit guidance of the manufacturer should be followed.  (Please refer to VENTANA 
anti-ALK (D5F3) Scoring Interpretation Guide and Performance Characteristics using the 
package insert as a reference). 
Scoring Criteria for Determination of ALK status in NSCLC: 
Clinical Interpretation 
Staining Description 
Positive for ALK 
Presence of strong granular cytoplasmic staining in tumor 
cells (any percentage of positive tumor cells). Known 
staining elements should be excluded, including: 
• 
light cytoplasmic stippling in alveolar 
macrophages, 
• 
cells of neural origin (nerve and ganglion cells), 
• 
glandular epithelial staining, and 
• 
cells within lymphocytic infiltrate. 
Negative for ALK 
Absence of strong granular cytoplasmic staining in tumor 
cells.  
 
 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 12 
 
 
- 
Fluorescent In Situ Hybridization: 
 
FISH analysis may also be performed on the FFPE tumor tissues using a break apart probe 
specific to the ALK locus (Vysis LSI ALK Dual Color, break apart rearrangement probe; Abbott 
Molecular) according to the manufacturer’s instruction. In brief, 4-μm-thick sections are 
deparaffinized, dehydrated, immersed with Vysis pretreatment Solution (Abbott Molecular) at 
80°C for 15 minutes, and treated with Protease Solution (Abbott Molecular) at 37°C for 20 
minutes. Dual probe hybridization will be performed using the LSI ALK dual-color probe, which 
hybridizes to the 2p23 locus with Spectrum Orange and Spectrum Green on either side of the 
ALK gene breakpoint.  
 
ALK FISH will be considered positive when more than 15% of 50 or more analyzed cells 
showed splitting of the fluorescent probes flanking the ALK locus.  
 
For assay procedure, interpretation & result reporting, & other information, please refer for 
Vysis ALK Break Apart FISH Probe Kit document. 
 
 
 
7. DATA ANALYSIS/STATISTICAL METHODS 
 
This study involves the analysis of the test results performed on retrospectively collected 
NSCLC FFPE tissue specimens stored in tissue banks. The required study data will be 
collected via retrospective review of subject charts and/or hospital records for subjects’ clinical 
& histo-pathological characteristics. The data analyzed in this study will be documented in a 
non-interventional statistical analysis plan, which will be developed by the study team and 
maintained by Pfizer. This document may modify the plans outlined in the protocol; however, 
any major modifications will be reflected in a protocol amendment. 
 
 
7.1 Sample Size Considerations 
 
In the event that no sites can provide access to an electronic database of hospital discharge 
records, a quasi-random sample of records from the combined discharge populations at the 
selected sites will be selected (as described in Section 2.2.1) and conducted initially as a pilot, 
no minimum sample will be determined prior to this manual review, but as many charts as 
possible will be reviewed to maximize precision for the incidence estimates. 
 
Approximately 700 patients will be recruited for the proposed study. Assuming that the 
expected prevalence is no greater than 10%, a sample size of 700 patients previously 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 13 
 
diagnosed with NSCLC permits the estimation of the percent of patients with EML4-ALK fusion 
to within + or – 2.1% with 95% confidence; ie, the half-width of the 95% confidence interval will 
be < 2.2%. For example, if the prevalence is 5%, the half-width of the 95% confidence interval 
is + or – 1.6%. 
 
7.2 Analysis Populations 
 
For the analysis of ALK incidence, the number of incident cases will be collected directly 
from centers participating in the study 
 
Epidemiology data will be extracted by testing of retained clinical samples by using the 
Ventana ALK-IHC diagnostic method to assess the absence or presence of the ALK 
rearrangement by the detection of the ALK protein in formalin-fixed, paraffin-embedded 
NSCLC stored tissue samples. The results of ALK rearrangement within this study population 
will then be used to establish the prevalence of ALK rearrangement in MENA NSCLC patients.  
 
The Full Analysis Set (FAS) is defined as all included cases which meet the selection criteria in 
Section 4.  
 
7.3 Statistical Methods 
 
Analyses will be primarily descriptive in nature. Binary data will be summarized using the 
percent of subjects with the event and a 95% confidence interval. Continuous data will be 
reported using n, mean, standard deviation, median, and range; a 95% confidence interval for 
the mean will also be computed. No interim analyses are planned.  
 
7.4 Primary Analysis 
 
The prevalence of ALK rearrangement is the primary endpoint of the study, and it will be 
calculated as the 100 x number of cases with ALK rearrangement divided by the total number of 
cases which met the inclusion & exclusion criteria, and 95% confidence interval will be 
presented along with the estimated prevalence. 
 
7.5 Secondary Analysis  
 
Univariate comparison between the proportion of subjects with ALK rearrangement within 
categories of demographic, clinical and pathological parameters, will be summarized using p-
values of chi-square tests, odds ratios and 95% confidence intervals (CIs) for the odds-ratios. A 
multiple regression analysis will be performed using the presence of ALK rearrangement as the 
dependent variable. Independent variables to be included in the model are the ones above 
mentioned. A stepwise method will be used for the selection of independent variables. Odds 
ratios and 95% confidence intervals (CIs) for the odds-ratios will be presented for the variables 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 14 
 
selected for the final model. 
 
The concordance between Vysis ALK-FISH and Ventana ALK IHC tests results will be 
evaluated by assessment of positive, negative and overall percent agreement between tests 
and associated 95% CIs. 
 
Full details of the planned statistical analyses will be given in a statistical analysis plan. 
 
8. DATA COLLECTION 
 
8.1. Primary Endpoint: 
 
This study involves laboratory testing of retained tissue specimens, of retrospectively diagnosed 
cases with NSCLC, which meet the inclusion criteria of study. Epidemiology data will be collected 
by performing the testing on retained clinical samples by using the Ventana ALK-IHC 
diagnostic method to assess the absence or presence of the ALK rearrangement. The result 
of each test will be recorded as positive or negative for ALK rearrangement.  
 
 
The data obtained within this study population will then be used to establish the prevalence of 
ALK rearrangement in MENA NSCLC patients.  
 
8.2. Secondary Endpoints 
 
The manner in which data will be collected to evaluate the clinic-pathological characteristics 
associated with of ALK rearrangement will depend on the data availability and technical 
capabilities of each participating hospital/center. Ideally, the selected hospitals /centers will 
have an electronic database of the patients and the corresponding stored tissue samples. In this 
case medical records can be retrospectively analyzed locally to obtain the required information. 
For centers without electronic database, a manual review of their medical records will be 
necessary to collect data. 
 
In these 2-3 centers, if Vysis ALK-FISH was previously performed on this tissue sample & its 
result was reported in the patient’s medical record, so this result will be obtained, recorded, & 
be compared with ALK-IHC test result. 
 
Vysis ALK-FISH test will be performed if not previously done, & its result will be recorded. 
 
 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 15 
 
 
8.3. Record Retention 
 
To enable evaluations and/or audits from regulatory authorities or Pfizer, the principal 
investigator from the study team will keep records, including any electronic extracts of 
administrative hospital data, original signed informed consent forms, copies of all CRFs, 
serious adverse event forms, source documents, and detailed records of treatment disposition, 
and adequate documentation of relevant correspondence (e.g., letters, meeting minutes, 
telephone calls reports). The records should be retained by the investigator for a period 
according to local regulations, or as specified in the clinical study agreement, whichever is 
longer. 
 
9. ADVERSE EVENT REPORTING 
 
Should evidence emerge from study documentation of the occurrence of an adverse 
reaction due to a Pfizer product, such information must be reported within 24 hours to the 
local Pfizer Drug Safety Unit. 
 
10. ETHICS 
 
10.1. Institutional Review Board (IRB)/Independent Ethics Committee (IEC) 
 
Because this study is a retrospective analysis of preexisting, anonymous, and de- identified 
data, an informed consent waiver will be sought by an Independent Ethics Committee or 
Institutional Review Board (IEC/IRB). 
 
Depending on the level of risk and nature of the research, a study may be ruled as exempt from 
IRB review by an IRB chair or designated IRB member. Studies that are not exempt must be 
approved either by an IRB chair or designated IRB member (if the study   qualifies for 
expedited review) or by a full IRB committee. Since de-identified, non- interventional patient 
data will be collected retrospectively for the proposed study, an exemption from the 
participating IRB committee will be sought. It is the responsibility of the investigator to obtain 
the study protocol & other relevant documentations, if applicable, from the IRB/IEC. All 
correspondence with the IRB/IEC should be retained in the Investigator File. Copies of 
IRB/IEC approvals should be forwarded to Pfizer. 
 
10.2. Ethical Conduct of the Study 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 16 
 
This study will be conducted in accordance with legal and regulatory requirements, as well as 
with scientific purpose, value and rigor and follow generally accepted research practices such 
as Good Pharmacoepidemiology Practices (GPP) issued by the International Society for 
Pharmacoepidemiology (ISPE), the International Society for Pharmacoeconomics and 
Outcomes Research (ISPOR) guidances, Pharmaceutical Research and Manufacturers 
Association (PhRMA) guidelines and similar. 
 
10.3. Subject Information and Consent 
 
All parties will ensure protection of subject personal data and will not include subject names on 
any sponsor forms, reports, publications, or in any other disclosures, except where required by 
laws. In the case of data transfer, Pfizer will maintain high standards of confidentiality and 
protection of subject personal data. Because all data will be de- identified, non-interventional, 
and retrospective in nature, no personal information on subjects will be collected or transmitted 
to either the study investigator or to Pfizer. 
 
11. PUBLICATION OF STUDY RESULTS 
 
11.1. Communication of results by Pfizer 
 
This study is a non interventional epidemiology study and as such does not require 
disclosure as per Pfizer SOP CT28. 
 
11.2. Publications by Investigators 
 
The selected study investigator or contracted research team will provide manuscripts, 
abstracts, or the full text of any other intended disclosure (e.g., poster presentation invited 
speaker or guest lecturer presentation) to Pfizer at least 30 days before they are submitted for 
publication or otherwise disclosed. If any patent action is required to protect intellectual 
property rights, the investigator agrees to delay the disclosure for a period not to exceed an 
additional 60 days. The investigator will, on request, remove any previously undisclosed 
confidential information (other than the study results themselves) before disclosure. For all 
publications relating to this study, the investigators will comply with recognized ethical 
standards concerning publications and authorship, including Section II - “Ethical 
Considerations in the Conduct and Reporting of Research” of the Uniform Requirements for 
Manuscripts Submitted to Biomedical Journals, http://www.icmje.org/index.html#authorship, 
established by the International Committee of Medical Journal Editors. 
 
 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 17 
 
11.3. 
Communication of Issues: 
The investigator will inform Pfizer Immediately of any serious breached of this NI study 
protocol that the investigator becomes aware of. 
 
12. REFERENCES 
 
1- Ferlay J, Shin HR, Bray F, et al. GLOBOCAN 2008 v1.2, Cancer Incidence and 
Mortality Worldwide: IARC CancerBase No. 10 [Internet] Lyon, France: International 
Agency for Research on Cancer. http://globocan.iarc.fr. 2010. 
 
2- Gadgeel SM, Ramalingam SS, Kalemkerian GP. Treatment of lung cancer. Radiol Clin 
North Am 2012; 50:961-74. 
 
3- Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth 
factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N 
Engl J Med 2004; 350:2129-39. 
 
4- Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to 
gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. 
PLoS Med 2005; 2:e73. 
 
5- Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-
small-cell lung cancer. N Engl J Med 2010; 363:1693-703. 
 
6- Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion 
gene in non-small-cell lung cancer.Nature 2007;448:561–566. 
 
7- Soda M, Takada S, Takeuchi K, et al. A mouse model for EML4-ALKpositive lung 
cancer. Proc Natl Acad Sci USA 2008;105:19893–19897. 
 
8- Ka-Fai To, MBChB, Joanna HM Tong, et al. Detection of ALK Rearrangement by 
Immunohistochemistry in Lung Adenocarcinoma and the Identification of a Novel 
EML4-ALK Variant. J Thorac Oncol. 2013;8: 883-891. 
 
9- Horn L, Pao W. EML4-ALK: Honing in on a new target in non-small-cell lung cancer. J 
Clin Oncol. 27:4232-4235, 2009. 
10- Ou SH, Kwak EL, Siwak-Tapp C, et al. Activity of crizotinib (PF02341066), a dual 
mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) 
inhibitor, in a non-small cell lung cancer patient with de novo MET amplification. J 
Thorac Oncol 2011;6:942-6. 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 18 
 
11-  Bergethon K, Shaw AT, Ou S-HI, et al. ROS1 rearrangements define a unique 
molecular class of lung cancers. J Clin Oncol 2012;30:863-70. 
12-  Camidge DR, Bang YJ, Kwak EL, et al.Activity and safety of crizotinib in patients with 
ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet 
Oncol 2012;13:1011-9. 
13- Kim D-W, Ahn M-J, Shi Y, et al. Results of a global phase II study with Crizotinib in 
advanced ALK-positive non-small cell lung cancer (NSCLC). Presented at the Annual 
Meeting of the American Society of Clinical Oncology, Chicago, June 1–5, 2012. 
abstract. 
 
14-  Alice T. Shaw, Dong-Wan Kim, Kazuhiko Nakagawa, et al. Crizotinib versus 
Chemotherapy in Advanced ALK-Positive Lung Cancer N Engl J Med. June 2013.1-10 
 
15- Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features 
characterize ALK-rearranged lung adenocarcinoma in the western population. Clin 
Cancer Res 2009;15:5216–5223. 
 
16-  Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows 
for the routine detection of ALK rearranged lung adenocarcinomas by standard 
immunohistochemistry. Clin Cancer Res 2010;16:1561–1571. 
 
17- Proctor, R.N., Tobacco and the global lung cancer epidemic. Nat Rev Cancer, 2001. 
1(1): p. 82-6. 
 
18- Salim, E.I., et al., Cancer epidemiology and control in the arab world - past, present 
and future. Asian Pac J Cancer Prev, 2009. 10(1): p. 3-16. 
 
19-  Choi YL, Takeuchi K, Soda M, et al. Identification of novel isoforms of the EML4-ALK 
transforming gene in non-small cell lung cancer. Cancer Res 2008;68:4971–4976. 
 
20- Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological 
characteristics in a subset of lung cancers. J Thorac Oncol 2008;3:13–17. 
 
21- Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare 
acquired event. Neoplasia 2008;10:298–302. 
 
22- Shinmura K, Kageyama S, Tao H, et al. EML4-ALK fusion transcripts, but no NPM-, 
TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung 
carcinomas. Lung Cancer 2008;61:163–169. 
 
23- Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell 
lung cancer and non-tumor lung tissues. Am J Pathol 2009;174:661–670. 

RETROSPECTIVE NON-INTERVENTIONAL NON-PFIZR PRODUCT STUDY PROTOCOL 
Final Protocol, April 21, 2014 
Study Number: A8081046 
 
 
Page 19 
 
 
24- Wong DW, Leung EL, So KK, et al.; University of Hong Kong Lung Cancer Study 
Group. The EML4-ALK fusion gene is involved in various histologic types of lung 
cancers from nonsmokers with wild-type EGFR and KRAS. Cancer 2009;115:1723–
1733. 
 
25- Ventana; Roche Diagnostics International LTD.New Companion Diagnostic Assays. 
October 2012

NON-INTERVENTIONAL, NON-PFIZR PRODUCT STUDY PROTOCOL  
Final Protocol, December 10, 2013  
 Study Number: A8081046  
 
Page 20