Sensitivity of PDL-1-analysis From Pleural Effusion in Non-small Cell Lung Cancer
Conditions: Lung Neoplasms
Sponsor: Wissenschaftliches Institut Bethanien e.V

 
 
 
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Sensitivity of PDL-1-analysis from pleural effusion in Non-
small cell lung cancer  
Sensitivität einer PDL-1 Analyse bei NSCLC mit Pleuraerguß 
 
 
Study Protocol 
 
 
Study Sites 
 
Wissenschaftliches Institut Bethanien für Pneumologie e.V. / Krankenhaus Bethanien gGmbH 
Klinik für Pneumologie und Allergologie 
Zentrum für Schlaf- und Beatmungsmedizin 
Aufderhöher Str. 169-175 
42699 Solingen 
Germany 
Prof. Dr. med. Winfried J. Randerath 
Dr. med. Lars Hagmeyer 
 
Klinik und Poliklinik für Herz- und Thoraxchirurgie der Uniklinik Köln 
Kerpener Str. 62 
50937 Köln 
 
PD Dr. med. Elmar Kuhn 
 
Protocol Version  
1.5 
 
Date  
 
 
04/Nov/2016 
 
 
 
Principal Investigator: 
 
 
 
Prof. Dr. med. Winfried J. Randerath 
Krankenhaus Bethanien gGmbH, Solingen 
 
 
_____________________ 
Signature 
 
 
_____________________ 
Date

Sensitivity of PDL-1-analysis from pleural effusion in Non-small cell lung cancer – Study Protocol Version 1.5 from 04/Nov/2016 
 
 
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1. Background / Rationale 
 
The programmed death receptor-1 (PD-1) physiologically acts as an immune checkpoint 
receptor, enabling self-tolerance by T-cells in normal tissue. Unbound PD-1 allows the 
normal immune response by T cells to occur. Binding of PD-1 to the ligands PD-L1 and 
PD-L2 suppresses the immune response. Abnormal PD-L1 expression on tumor cells 
leads to activation of PD-1 and suppression of cytotoxic T-cell activity. The T-cell 
tolerance allows the tumour cells to avoid recognition and elimination by the immune 
system 
(1). 
In 
September 
2014 
Pembrolizumab, 
a 
humanized 
monoclonal 
immunoglobulin G4 kappa antibody against PD-1, was approved in the United States of 
America for the treatment of incurable melanoma in patients with disease progression 
following first line treatment. Anti-PD-1 therapies may be a novel therapeutic option in the 
treatment strategy of advanced non-small cell lung cancer (NSCLC). Data from the 
KEYNOTE-001 study showed that NSCLC patients with detectable PD-L1 expression 
had a response under treatment with Pembrolizumab (2). Positive results could be 
demonstrated in treatment-naïve as well as in previously treated advanced NSCLC. 
There is probably a correlation between tumor PD-L1 expression and improved 
pembrolizumab antitumor activity (3). In March 2015 Nivolumab, a humanized 
monoclonal immunoglobulin G4 kappa antibody against PD-1, was approved in the 
United States of America for the treatment of incurable NSCLC. The approval was 
reached after the publication of the CHECKMATE-017 trial, where a benefit in overall 
survival of 3.2 months could be demonstrated in comparison to docetaxel in subjects 
after standard platinum-based chemotherapy with following tumor progression (4). In the 
CHECKMATE-063 trial under Nivolumab a response could be achieved in 15% of 
subjects after at  
least two lines of standard chemotherapy (5). In May 2015 a positive CHMP opinion was 
published by the EMA. 
Lung cancer is the second most common cancer and is the primary cause of cancer-
related death in both men and women in the United States (6). Currently, 80% of patients 
with lung cancer are given a diagnosis of primary NSCLC. Malignant pleural effusion 
(MPE) is a common complication of advanced lung cancer. The presence of MPE 
indicates a poorer prognosis for patients with lung cancer and impairs their quality of life. 
Pleural effusion is a convenient clinical sample with important clinical diagnostic sig-
nificance. It may be an alternative source supplying useful information about the 
neoplasm’s biology in terms of moleculargenetic and immunopathologic profile. 
 
2. Objectives 
 
This is a prospective diagnostic pilot study to create hypotheses regarding 
immunocytochemistry (ICC) PD-L1 analysis of pleural effusions in NSCLC patients as 
compared to the reference standard of PD-L1 immunohistochemistry (IHC). This 
comparison will be done to assess sensitivity and specificity of PD-L1 detection by ICC in 
pleural effusions. 
 
The main hypothesis is, that ICC analysis of PD-L1 from pleural effusion has a relevant 
diagnostic value with a specificity of ~90% as compared to the immunohistochemical 
reference standard. 
 

Sensitivity of PDL-1-analysis from pleural effusion in Non-small cell lung cancer – Study Protocol Version 1.5 from 04/Nov/2016 
 
 
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3. Outcome 
 
3.1. Primary Outcome Measures 
 
 
Number/prevalence of PD-L1-positive patients according to immunohistochemistry 
(IHC) of pleural biopsy 
 
Number/prevalence of PD-L1-positive patients according to immunocytochemistry 
(ICC) of pleural aspirate 
 
3.2. Secondary Endpoints 
 
 
IHC results of different bronchoscopic samples 
 
4. Study Population 
 
The population for selection of potential study participants is comprised of clinical routine 
patients presenting with (suspected malignant) pleural effusion. These patients must 
have an indication for pleural puncture. The cause and nature of the pleural effusion (e.g. 
presence of non-small cell lung cancer, NSCLC) at this time point will be unknown in 
many cases. To establish this, further routine diagnostic procedures are required. While 
patients will be included before the planned routine examinations and thus before 
presence of NSCLC can be confirmed, only patients with confirmed diagnosis of non-
small cell lung cancer will be included in the data analysis. 
 
A patient in whom suspicion of pleural manifestation of lung cancer is not confirmed, will 
be considered a screening failure and thus excluded from the study. 
 
4.1. 
Inclusion Criteria 
 
- 
Age ≥ 18 years 
- 
Presence of malignant pleural effusion with indication for pleural puncture and 
thoracoscopy 
- 
Confirmed diagnosis of non-small cell lung cancer according to ERS guidelines (to 
be confirmed after study inclusion when results from routine clinical examinations 
are available) 
- 
Written informed consent 
 
 
4.2. 
Exclusion Criteria 
 
- 
Age <18 years 
- 
Pregnancy and/or lactation 
- 
Acute and life-threatening illness (instable angina pectoris, acute pulmonary 
arterial embolism, myocardial infarction, etc.) 

Sensitivity of PDL-1-analysis from pleural effusion in Non-small cell lung cancer – Study Protocol Version 1.5 from 04/Nov/2016 
 
 
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- 
Any contraindication to undergo thoracoscopy (e.g. anticoagulation therapy which 
cannot be discontinued for the procedure) 
- 
Any medical, psychological or other condition impairing the patient’s ability to 
provide informed consent 
- 
Exclusion of non-small cell lung cancer as cause of pleural effusion after clinical 
routine examinations leads to study exclusion of patient 
 
5. Study Plan and Assessments 
 
5.1. Selection of Patients 
 
Patients presenting within clinical routine with pleural effusion and suspected or known 
underlying lung cancer and who satisfy the in- and exclusion criteria as far as they can be 
assessed at that time, are considered for study participation. 
 
These patients will be informed about all routine clinical examinations and assessments 
before informed consent will be obtained. However, informed consent for study 
participation will be obtained before (routine) pleural puncture to ensure that patients 
agree with the study-specific additional diagnostic analysis of the collected pleural 
effusion material. 
 
All invasive procedures described herein are part of the patient’s clinical routine treatment 
to achieve a clinical diagnosis. The routine examinations and study-associated data 
collection for each patient are outlined below. The individual patient’s study participation 
will be concluded, once all of these steps are completed. 
 
5.2. Anthropometry, Vital Signs, Medical History, Medication 
 
The following data will be collected for analysis within the study: 
 
1. Anthropometric data (age, gender, height, weight, BMI) 
2. Office blood pressure according to ESH/ESC guidelines 
3. Current medication 
4. Medical history incl. any oncological pre-medication 
5. Smoking history 
 
5.3. Pleural puncture (non-study specific routine examination) 
 
The pleural effusion will be treated and diagnosed by puncturing of the pleural cavity at 
the location of effusion. During this process the excess pleural fluid will be 
removed/collected and used for later analysis by an external pathology department. 
 
5.4. Thoracoscopic pleural biopsy (non-study specific routine examination) 
 
After pleural puncture, histological pleura samples will be obtained by pleural biopsy, 
primarily applying internistic thoracoscopy. If internistic thoracoscopy is not feasible (e.g. 
due to distinct intergrowth), the patient will undergo surgical thoracoscopy. In either case, 
at least five biopsies will be taken. 

Sensitivity of PDL-1-analysis from pleural effusion in Non-small cell lung cancer – Study Protocol Version 1.5 from 04/Nov/2016 
 
 
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5.5. Optional bronchoscopic lung biopsy (non-study specific routine 
examination) 
 
In some cases, suspicious tumor masses within the lung and/or intrathoracic lymph 
nodes are detected by preceding radiologic assessment and routinely sampled for further 
histological analyses. This applies in particular when no histological samples can be 
obtained from the pleura. In these cases, the corresponding results will also be 
considered for study-associated analysis. 
 
 
5.6. Immunocytochemistry (ICC) and Immunohistochemistry (IHC) 
 
The pleural fluid obtained by pleural puncture will be in part used for standard clinical 
routine analysis and another part will be subject to study-specific PD-L1 analysis. The 
same applies to the histological samples obtained by thoracoscopy and potential 
bronchoscopy. These analyses will reveal nature and cause of the pleural effusion. In 
case the corresponding results confirm an NSCLC diagnosis, all results from ICC and 
IHC will be collected for analysis within the study. Should the results rule out NSCLC, the 
patient will be considered a screening failure and excluded from the study. 
 
For PD-L1 analysis in both ICC and IHC, the PD-L1 IHC 22C3 pharmDx Kit (Dako) will be 
used. 
 
5.7. Documentation of Adverse Events 
 
All adverse events occurring during each patient’s actual study participation will be 
assessed for diagnostic procedure relatedness. Procedure-related adverse events will be 
documented for later study-specific analysis. 
 
 
 
 

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6. Statistical Analysis 
 
6.1. Primary Analysis 
 
The main hypothesis is, that ICC analysis of PD-L1 from pleural effusion has a relevant 
diagnostic value with a specificity of ~90% as compared to the immunohistochemical 
reference standard. To test this hypothesis, the number of PD-L1 positive and negative 
detections for each sample type will be documented and used for calculation of sensitivity 
and specificity. Furthermore, the frequency with 95% confidence interval of PD-L1 
positive patients according to ICC of pleural fluid will be calculated. 
 
6.2. Secondary Analyses 
 
Secondary analyses comprise intra-individual comparative descriptive analyses of all 
pathological results to assess correlation between ICC and IHC results.   
 
7. Sample Size Calculation 
 
According to previous experience concerning the value of ICC in lung cancer diagnosis, 
sensitivity/specificity of this method is expected to be lower as compared to IHC. While 
exact figures for PD-L1 detection (ICC and IHC) are not available we anticipate a 
specificity of approx.. 90% of PD-L1 detection via ICC as compared to IHC. Previous 
application of the PD-L1 antibody used for detection in histological samples revealed a 
prevalence of approx. 25% in PD-L1 positive NSCLC patients (Garon et al. 2015). 
Buderer (1996) described a method for sample size calculation for sensitivity and 
specificity incorporating the prevalence of disease. We used this method and specified 
the required parameters as followed: 
 
Sample size (n) was calculated according to the formula: 
 
𝑛=
𝑍𝛼/2
2 𝑆𝑃(1 −𝑆𝑃)
𝑊2
(1 −𝑃)
 
where 
 
Z/2  
z value from standard normal distribution with =0.05 
SP  
specified specificity (90%) 
W 
width of 95% confidence interval (10%) 
P 
prevalence of disease (25%) 
 
This resulted in a number of 46 patients, which was amended by a drop-out rate of 
approx. 10% to give a total patient number of 
 
n=50 patients  
 
which should at least allow for a valuable qualitative assessment of the feasibility of PD-
L1 diagnostic based on ICC and provide a basis for possible future studies. 
 
 
 

Sensitivity of PDL-1-analysis from pleural effusion in Non-small cell lung cancer – Study Protocol Version 1.5 from 04/Nov/2016 
 
 
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8. References 
 
1. Poole RM, Pembrolizumab: first global approval, Drugs. 2014 Oct;74(16):1973-81 
2. Rizvi NA et al., Safety and clinical activity of MK-2475 as initial therapy in patients with 
advanced non-small cell lung cancer (NSCLC) [Abstract No. 8007]; 50th Annual Meeting 
of the American Society of Clinical Oncology.2014; Chicago, IL  
3. Garon EB et al., Antitumor Activity of Pembrolizumab (Pembro; MK-3475) and 
Correlation With Programmed Death Ligand 1 (PD-L1) Expression in a Pooled Analysis 
of Patients With Advanced NSCLC ; ESMO 2014 
4. Brahmer J, Reckamp KL, Baas P et al, N Engl J Med 2015, Jul 9; 373(2):123-35  
5. Rizvi NA, Mazieres J, Planchard D et al, Lancet Oncol 2015 Mar; 16(3): 257-65 
6. Siegel R , Naishadham D , Jemal A . Cancer statistics, 2013 . CA Cancer J Clin . 2013 ; 
63 ( 1 ): 11 – 30 
7. Garon EB et al., Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J 
Med. 2015 May 21;372(21):2018-28. 
8. Buderer NM, Statistical methodology: I. Incorporating the prevalence of disease into the 
sample size calculation for sensitivity and specificity. Acad Emerg Med. 1996 
Sep;3(9):895-900. 
 
 
9. Abbreviations 
 
ICC 
Immunocytochemistry 
IHC 
Immunohistochemistry 
PD-L1 
Programmed death-ligand 1 
ERS 
European Respiratory Society 
ESH 
European Society of Hypertension 
ESC 
European Society of Cardiology 
BMI 
Body-Mass Index 
NSCLC 
Non-small cell lung cancer