Response and Toxicity Prediction by Microbiome Analysis After (Concurrent) Chemo RT in Locally Advanced NSCLC Treated With IO (Durvalumab)
Conditions: Non Small Cell Lung Cancer Stage III, Immunotherapy
Sponsor: Leiden University Medical Center

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PREDICTION PROTOCOL 
Response and Toxicity Prediction by Microbiome analysis in locally advanced 
NSCLC treated with IO (durvalumab, MEDI4736) after Chemoradiotherapy 
 
 

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PROTOCOL TITLE: Response and Toxicity Prediction by Microbiome analysis in locally 
advanced NSCLC treated with IO (durvalumab) after chemoradiotherapy. 
Protocol ID 
NL76017.058.21 
Short title 
PREDICTION 
EudraCT number  
Not applicable 
Version 
 6.0 
Date:  
March 17, 2023 
Coordinating 
investigator/project leader 
Prof. Dr. P. Baas  
Leiden University Medical Center 
Department of Pulmonology 
Albinusdreef 2; 2333 ZA Leiden, NL 
Phone: +31 71 526 4438 
Principal investigator(s) /  
 
 
Sub investigators 
 
 
 
Multicenter research: per site 
 
Prof. Dr. P. Baas (LUMC) 
Drs. I. Smesseim (AVL/NKI) 
Dr. K. Cuppens (Jessa hospital) 
 
Drs. L.C. Steinbusch (co-PI) 
Prof. Dr.P.S. Hiemstra (co-PI)  
Prof. Dr. Ir B. Keijser (co-PI, TNO) 
Prof. Dr. E.F. Smit (co-PI) 
 
LUMC, Leiden 
 
AVL/NKI, Amsterdam 
 
Jessa Hospital, Hasselt, Belgium 
 
AZ Turnhout, Turnhout, Belgium 
 
AZ Delta, Roeselare, Belgium 
 
OLV Aalst, Aalst, Belgium 
 
 
Sponsor   
LUMC 
 
 
Subsidising party 
AstraZeneca 
Independent experts 
Dr. J. Stolk, pulmonologist 
Department of Pulmonology  
Leiden University Medical Center  
 

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Laboratory sites  
TNO (microbioom analysis 
LUMC Pulmonology (blood analysis) 
 
 
Pharmacy 
Not applicable 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department:  
 
Prof. dr. E.F. Smit, head of 
department of pulmonary medicine 
 
 
 
 
Coordinating 
Investigator/Project leader:  
 
 
Prof. dr. P. Baas 
department of pulmonary medicine 
 
 
 
 
Site Principal investigator 
 
 
Site: ……………………………. 
 
 
 
Name: 
 
……………………………………..… 
 
Department: 
 
……………………………………..… 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................10 
1.1 
Pre-clinical Trial Data .............................................................................................10 
2. 
OBJECTIVES ................................................................................................................12 
2.1 
Primary objective ....................................................................................................12 
2.2 
Secondary Objectives ............................................................................................12 
3. 
STUDY DESIGN ...........................................................................................................13 
4. 
STUDY POPULATION ..................................................................................................14 
4.1 
Population (base) ...................................................................................................14 
4.2 
Inclusion criteria .....................................................................................................14 
4.3 
Exclusion criteria ....................................................................................................15 
4.4 
Sample size calculation ..........................................................................................17 
5. 
METHODS ....................................................................................................................19 
5.1 
Study parameters/endpoints ...................................................................................19 
5.1.1 
Main study parameter/endpoint .......................................................................19 
5.1.2 
Secondary study parameters/endpoints ..........................................................19 
5.2 
Study procedures ...................................................................................................19 
5.3 
Withdrawal of individual subjects ............................................................................21 
5.4 
Replacement of individual subjects after withdrawal ...............................................21 
6. 
SAFETY REPORTING ..................................................................................................22 
6.1 
Temporary halt for reasons of subject safety ..........................................................22 
6.2 
AEs, SAEs and SUSARs ........................................................................................22 
6.2.1 
Adverse events (AEs) ......................................................................................22 
6.2.2 
Serious adverse events (SAEs) .......................................................................22 
6.3 
Follow-up of adverse events ...................................................................................23 
7. 
STATISTICAL ANALYSIS .............................................................................................24 
7.1 
Primary study parameter(s) ....................................................................................24 
7.2 
Secondary study parameter(s) ...............................................................................24 
8. 
ETHICAL CONSIDERATIONS ......................................................................................25 
8.1 
Regulation statement .............................................................................................25 
8.2 
Recruitment and consent........................................................................................25 
8.3 
Benefits and risks assessment, group relatedness .................................................25 
8.4 
Compensation for injury .........................................................................................25 
8.5 
Role of Astra Zeneca ..............................................................................................26 
9. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .............................27 
9.1 
Handling and storage of data and documents ........................................................27 
9.2 
Monitoring and Quality Assurance ..........................................................................27 
9.3 
Amendments ..........................................................................................................27 
9.4 
Annual progress report ...........................................................................................28 
9.5 
Temporary halt and (prematurely) end of study report ............................................28 
9.6 
Public disclosure and publication policy ..................................................................28 
9.7 
Synthesis ...............................................................................................................28 

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10. 
REFERENCES ..........................................................................................................29 
Appendix 1 ...........................................................................................................................32 
Appendix 2 ...........................................................................................................................33 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; 
in Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-
formulier) 
AE 
Adverse Event 
ALK 
anaplastic lymphoma kinase 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count  
aPPT 
activated partial thromboplastin time 
AST 
aspartate aminotransferase 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CRT 
chemoradiotherapy 
CRF 
case report form 
CT 
computed tomography 
CTLA-4 
cytotoxic T-lymphocyte-associated protein 4 
CV 
Curriculum Vitae 
CyTOF 
cytometry by time of flight 
DCR 
disease control rate 
EU 
European Union 
ECOG 
eastern cooperative oncology group 
EGFR 
epithelial growth factor receptor 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
GFR 
glomerular infiltration rate 
HCV 
hepatitis C virus 
IC 
ICI 
Informed Consent 
Immune Checkpoint Inhibitors 
INR 
international normalized ratio 
irAE 
immune-related adverse event 
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
NK 
natural killer 
NSCLC 
non-small cell lung cancer 
PFS 
progression free survival 
PD 
pharmacodynamic 
PD-L1 
programmed death-ligand 1 
PT 
prothrombin time 
RNA 
ribonucleic acid 
(S)AE 
(Serious) Adverse Event  
SOC 
standard of care 

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Sponsor The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
TPS 
tumor proportion score 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 
ULN 
upper limit of normal 
VOC 
Volatile Organic Compounds 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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SUMMARY 
 
Rationale:  
To investigate the role of the microbiome in response and toxicity prediction in patients with 
locally advanced NSCLC treated with durvalumab after concurrent or sequential 
chemoradiotherapy. 
 
Objective:  
The predictive value of the microbiome (throat swabs and stool samples) to identify patients 
who will relapse during standard durvalumab treatment after chemoradiotherapy at 6 months. 
Exploratory endpoints include the effects of antibiotic therapy before and during durvalumab 
treatment on toxicity and clinical outcome. 
 
Study design: 
Prospective observational multicenter study. 
 
Study population: 
Patients ≥ 18 years with non-small cell lung cancer who will start treatment with durvalumab 
after completion of chemoradiotherapy. 
 
Intervention: 
Not applicable 
 
Main study parameters/endpoints: 
To investigate if the microbiome (throat swabs and stool samples) can be used to identify 
patients who will relapse during durvalumab treatment after chemoradiotherapy at 6 months. 
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: 
Collection of throat swabs and stool samples before start of durvalumab treatment; sampling 
of blood. No benefit is foreseen in participation to this study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. INTRODUCTION AND RATIONALE 
The development of antibodies targeting the programmed death 1 (PD-1) axis and cytotoxic 
T-lymphocyte-associated protein 4 (CTLA-4) provided a major clinical improvement in 
treatment across tumor types. These drugs promote antitumor immunity by blocking 
inhibitory immune pathways, often resulting in tumor regression and improved survival. In 
advanced metastatic non–small-cell lung cancer (NSCLC) patient selection, based upon PD-
L1 tumor proportion score (TPS) and tumor mutational burden (TMB), moved PD-1 targeted 
immunotherapy from further lines to frontline therapy in monotherapy, bitherapy or combined 
with chemotherapy, showing a consistent more favorable response over conventional 
chemotherapy [1][2][3][4][5][6][7][8].  
 
Improved clinical outcomes after adjuvant treatment with durvalumab following 
chemoradiotherapy (CRT) for locally advanced NSCLC (PACIFIC trial program) [9][10][11], 
led to the rapid adoption of this treatment strategy as standard of care. However, despite the 
improved progression free survival and overall survival, recurrence rate remains high. 
Approximately 45% of patients will relapse within 1 year, despite adjuvant durvalumab 
therapy. To date no performant biomarker predicting treatment response or failure nor 
toxicity exists and the number of prospective studies addressing this issue is limited. Both 
PD-L1 TPS and TMB should be considered ‘enriching’ parameters improving response-
chances, but they are far from an ideal biomarker. Non-invasive biomarkers are essential in 
the future for better patient selection and therapy allocation. One of the potential non-
invasive biomarkers of interest is the microbiome.  
 
The microbiome gained a lot of interest as a potential biomarker the past years. Multiple 
studies demonstrated that the gut microbiota modulates the response to inhibitors of PD1-
PDL1 axis [12][13][14][15]. Overall, they indicate that a “healthier”, highly diverse microbiota 
and the presence of certain bacterial species favor the establishment of an anti-tumor 
immune response at baseline that may be enhanced by the anti-PD1 treatment with 
favorable clinical response. Alteration of microbiota balance by antibiotics treatment near the 
initiation of the therapy reduces its efficacy. 
 
1.1 Pre-clinical Trial Data 
In the PACIFIC study, 709 patients who were randomized to receive consolidation therapy 
(473 received durvalumab and 236 received placebo) were included. The median 
progression-free survival from randomization was 16.8 months ( [CI], 13.0 to 18.1) with 
durvalumab versus 5.6 months (95% CI, 4.6 to 7.8) with placebo. At 6 and 12 months there 
was a superior outcome for the durvalumab arm. At 6 months the percentage of patients who 
progressed was 32% versus 52% for placebo. At 12-month the progression-free survival rate 
was 55.9% versus 35.3%. The response rate was higher with durvalumab than with placebo 
(28.4% vs. 16.0%; P<0.001), and the median duration of response was longer (72.8% vs. 
46.8% of the patients had an ongoing response at 18 months). The median time to death or 
distant metastasis was longer with durvalumab than with placebo (23.2 months vs. 14.6 
months; P<0.001). Grade 3 or 4 adverse events occurred in 29.9% of the patients who 
received durvalumab and 26.1% of those who received placebo; the most common adverse 
event of grade 3 or 4 was pneumonia (4.4% and 3.8%, respectively). A total of 15.4% of 
patients in the durvalumab group and 9.8% of those in the placebo group discontinued the 
study drug because of adverse events. Clinical benefit however could be obtained without 
compromising patient reported outcomes [16]. 

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Microbiome data: An eloquent study involving mice subcutaneously injected with melanoma 
(B16) and colon carcinoma (MC38) cells pretreated with an antibiotic cocktail was among the 
first to show the relationship between the stool microbiome and response to immunotherapy 
[17]. Antibiotic-treated and germ-free mice showed significantly shorter survival and less 
tumor volume reduction with immunotherapy (through injections of CpG-oligonucleotides and 
anti-interleukin (IL)-10 antibodies), when compared to controls, and highlighted that 
commensal gut microbiota primed tumor-infiltrating myeloid-derived cells through Toll-like 
receptor 4 (TLR4) activation and produce cytokines such as tumor necrosis factor (TNF) 
critical to antitumor efficacy. Notably, administration of cultured Allstipes species (spp., A. 
shahii) or Lactobacillus spp. by gavage reconstituted or attenuated TNF-dependent tumor 
response to immunotherapy in antibiotic-treated mice, respectively. Numbers of Lactobacillus 
spp. recovered as early as 1 week after stopping antibiotics, but recovery of Allstipes and 
Ruminococcus spp. was delayed, taking up to 4 weeks after stopping antibiotics.  
Single cell analyses by mass cytometry: Systems immunology is a relatively new field that 
aims at better understanding the immune system as a whole, and at identifying cells, 
molecules and pathways involved in particular pathological settings [18] [19]. Systems 
biology approaches to immunology combine cutting-edge techniques, including single-cell 
transcriptomics and mass cytometry with bioinformatics, for the analysis of multidimensional 
data [20][21][22]. Mass cytometry, also called cytometry by time-of-flight (CyTOF), is a 
variation of flow cytometry, in which antibodies are labelled with heavy metal ion tags rather 
than fluorochromes, the readout being by time-of-flight mass spectrometry. This allows for 
the combination of many more antibody specificities in a single sample, without significant 
spillover between channels. The lack of any significant mass spectral overlap and 
autofluorescence background makes mass cytometry uniquely suited for complex high-
dimensional phenotypic and functional analysis at the single cell level. 
Despite the improvements in overall and progression free survival in patients treated with 
chemoradiotherapy followed by consolidation therapy with PD-L1 blockade (durvalumab), a 
significant number of patients suffer from recurrent disease within one year. 
To allow for a more adequate patient follow-up, identification of a biomarker predicting early 
recurrence or long-lasting disease control is required. The former group could be monitored 
more closely while the latter can suffice with less outpatient visits and less radiological 
evaluations. Ineffective therapies can be ceased earlier and allow a switch to a more 
effective regimen. This seems appropriate since assessment of tumor recurrence after CRT 
is more complicated due to the ample possibility of non-malignant changes in the lung 
(radiation pneumonitis or infection). So far, no biomarkers have been identified for this 
purpose. 
Recently, the microbiome has attracted attention for its ability to modify the immune system 
and thereby changing the immune surveillance in humans. This is considered to be of pivotal 
importance to sustain a long-lasting tumor control in different tumor types treated with 
immune checkpoint inhibitors. 
 
There is an urgent need to improve the survival in patients who progress early during 
adjuvant immunotherapy (IO) therapy after CRT for NSCLC. The standard of care (SOC) is 
based of clinical and radiological changes which can be difficult to interpret (see 4.2.1). The 
microbiome may be able to identify patients at risk for early relapse or development of 
toxicity. By identifying these patients, we might be able to improve the clinical outcome.  

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2. OBJECTIVES 
 
2.1 Primary objective 
Primary objective: To investigate the predictive value of the microbiome (throat swabs 
and stool samples) to identify patients who will relapse during durvalumab treatment after 
chemoradiotherapy at 6 months. 
Hypothesis: In approximately 32% of patients a tumor relapse will occur within 6 months 
after completion of chemoradiotherapy despite adjuvant durvalumab therapy. The 
microbiome plays a key role in the modulation of the immune system. Using the microbiome 
as non-invasive biomarker we will aim to identify those patients at risk of an early 
recurrence. 
 
2.2 Secondary Objectives 
1. To investigate the predictive value of the microbiome (throat swabs and stool samples) to 
identify patients that develop immune-related adverse event (irAE’s) during durvalumab 
treatment after chemoradiotherapy.  
 
Hypothesis; The microbiome plays a pivotal role in modulating host immunity and has 
been suggested to be a major determinant of immunotherapy-related toxicity. Using the 
microbiome as a non-invasive biomarker we will try to identify those patients at risk for 
developing irAE’s.  
 
2.  To determine disease control rate (DCR ie. stable disease, partial response, and 
complete response) and PFS at 6 months after completion of chemoradiotherapy and 
correlate this to the microbiome samples. 
 
Hypothesis: Durvalumab consolidation after chemoradiotherapy for locally advanced 
NSCLC was able to improve DCR and PFS. We will try to identify patients that benefit 
from durvalumab consolidation using the microbiome as a non-invasive biomarker. 
 
3. To investigate to what extend cytological characteristics of circulating immune cells 
obtained from responders and non-responders differ and to explore to what extend these 
differences relate to the microbiome.  
 
Hypothesis: The changes in microbiota composition of the stool and throat will correlate 
with the phenotype of immune cells observed by mass cytometry. 
 
 
 
 
 
 
 
 
 

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3. STUDY DESIGN 
This is a prospective observational multicenter study, during 2 years after first patient in, 126 
patients will be included. Patients be included from 2 centers in the Netherlands and 4 
centers in Belgium. 
 
 
 

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4. STUDY POPULATION 
 
4.1 Population (base)  
Patients ≥ 18 years with non-small cell lung cancer stage III who will be treated with 
durvalumab as per standard of care after completion of concurrent or sequential 
chemoradiotherapy. 
 
4.2 Inclusion criteria 
         In order to be eligible to participate in this study, a subject must meet all of the     
         following criteria: 
1. Stages IIIA, IIIB and IIIC (as per UICC 8th TNM edition) NSCLC (histologically or 
cytologically confirmed) who will be treated with durvalumab treatment after 
concurrent or sequential chemoradiotherapy according to local standards. Patients 
that received neoadjuvant/adjuvant chemotherapy for surgically treated stages I to 
III NSCLC are allowed as long as therapy was completed at least 6 months prior to 
the diagnosis of disease recurrence amenable for CRT and resolution of all 
treatment related toxicity ≤ grade 1. 
 
2. No signs of disease progression after chemoradiotherapy 
 
3.  18 years 
4. ECOG ≤ 1 
5. Must be willing to provide collected stool samples and allow to obtain a throat swab 
during the observation period. 
6. Demonstrate adequate organ function as defined in Table 1, all screening labs 
should be performed within 10 days of start durvalumab 
 
 

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Table 1: Adequate Organ Function Laboratory Values 
System 
Laboratory Value 
Hematological 
Absolute neutrophil count (ANC) 
≥1,500 /mcL 
Platelets 
≥100,000 / mcL 
Hemoglobin 
≥9 g/dL or ≥5.6 mmol/L  
Renal 
Serum creatinine OR 
Measured or calculated creatinine 
clearance* 
(GFR can also be used in place of 
creatinine or CrCl) 
≤1.5 X upper limit of normal (ULN) OR 
 
≥50 mL/min for subject with creatinine levels > 1.5 X 
institutional ULN 
Hepatic 
Serum total bilirubin 
≤ 1.5 X ULN OR 
 
Direct bilirubin ≤ ULN for subjects with total bilirubin 
levels > 1.5 ULN 
AST (SGOT) and ALT (SGPT) 
≤ 2.5 X ULN  OR 
≤ 5 X ULN for subjects with liver metastases 
Coagulation 
International Normalized Ratio (INR) 
or Prothrombin Time (PT) 
 
 
Activated Partial Thromboplastin 
Time (aPTT) 
≤1.5 X ULN unless subject is receiving anticoagulant 
therapy as long as PT or PTT is within therapeutic 
range of intended use of anticoagulants 
 
≤1.5 X ULN unless subject is receiving anticoagulant 
therapy as long as PT or PTT is within therapeutic 
range of intended use of anticoagulants 
*Creatinine clearance should be calculated per institutional standard. 
 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
 
1. Is currently participating in or has participated in a study of an investigational 
agent or using an investigational device within 4 weeks of the first dose of 
treatment. 
 
2. Has had prior monoclonal antibody therapy within 4 weeks prior to study Day 1 or 
who has not recovered (i.e., ≤ Grade 1 or at baseline) from adverse events due to 
agents administered more than 4 weeks earlier.  
 
3. Previous treatment with PD-1-PD-L1 axis inhibiting immunotherapy. 
 
4. Active or history of autoimmune disease or immune deficiency, including, but not 
limited to, myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus 
erythematosus, rheumatoid arthritis, inflammatory bowel disease, anti-
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Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis with 
the following exceptions: 
• 
Patients with a history of autoimmune-related hypothyroidism who are on 
thyroid-replacement hormone are eligible for the study. 
• 
Patients with controlled Type 1 diabetes mellitus who are on an insulin 
regimen are eligible for the study. 
• 
Skin disorders (such as vitiligo, psoriasis, or alopecia) not requiring 
systemic immunosuppressive treatment (including corticosteroids) are 
permitted to enrol. 
 
5. Subjects with a condition requiring systemic treatment with either corticosteroids 
(> 10 mg daily prednisone equivalents) or other immunosuppressive medications 
within 14 days of start of Durvalumab. Inhaled or topical steroids, and adrenal 
replacement doses > 10 mg daily prednisone equivalents are permitted in the 
absence of active autoimmune disease. 
 
6. Subjects who have undergone organ transplant or allogeneic stem cell 
transplantation. 
 
7. Active malignancy or a prior malignancy within the past 3 years, with the following 
exceptions:  
• 
Patients with completely resected basal cell carcinoma, cutaneous 
squamous cell carcinoma, cervical carcinoma in-situ, breast carcinoma in-
situ, and patients with isolated elevation in prostate-specific antigen or low 
risk prostate cancer (managed with watchful waiting) are allowed. 
• 
Patients who underwent mediastinal radiotherapy in the past are not 
allowed.  
8. Subjects with known chronic infections/infectious disorders (eg. Clostridium colitis) 
 
9. Have known but untreated driver mutations of the EGFR gene or ALK or ROS1 
translocation. 
 
10. Has evidence of symptomatic interstitial lung disease or an active, non-infectious 
pneumonitis. 
 
11. Has an active chronic infection requiring systemic therapy. 
 
12. Has a history or current evidence of any condition, therapy, or laboratory 
abnormality that might confound the results of the trial, interfere with the subject’s 
participation for the full duration of the trial, or is not in the best interest of the 
subject to participate, in the opinion of the treating investigator.   
 
13. Has known psychiatric or substance abuse disorders that would interfere with 
cooperation with the requirements of the trial. 
 
14. Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies) 
 

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15. Has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV 
RNA [qualitative] is detected). 
 
16. Has received a live vaccine within 30 days prior to the first dose of trial treatment.  
 
4.4 Sample size calculation 
The primary goal of the study is identify patients, that develop disease progression or 
relapse (PD) within 6 months during adjuvant durvalumab after chemoradiotherapy, by 
the composition of the gut and throat microbiome as measured by the alpha and beta 
components (see 7.1). These are defined as a group of certain species and the distance 
(overlap) of the cultured species.  Since there are no well-defined data for the microbiome 
available and a search for the predictive value of any one of the thousand different 
bacteria species is in vain, we summarize the data using two diversity measures; the 
alpha and beta component according to the Shannon-Weaver index [23]. 
 
Formal power analysis and sample size calculation is difficult to perform within these 
premisses. Sample size is based on data from previous studies. These studies examined 
the microbiota composition in adults with different cancer types. Some studies were 
explorative of nature and correlated with OS or PFS. These studies were summarized in 
Table 2 (adapted from [24]). 
 
 
The purpose of our study is to train a classifier that will be validated in a subsequent study 
if it is promising enough. We will measure the predictive ability of the classifier on the 
basis of sensitivity and specificity, using cross-validation. 
We expect at 6 months to identify 41 patients with progressive disease and 85 with non-
progressive disease. We consider this study positive when the cross-validated sensitivity 
and specificity both lie entirely above 70% (one-sided 95% confidence intervals around 
both). With above numbers, this is achieved when minimally 29 out of 41 PD-patients and 
minimally 60 out of 85 non-PD patients are correctly identified, corresponding to an 
observed sensitivity of at least 82% and an observed specificity of at least 79% in our 
study population.  
 
 

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Table 2 
 
Metabolites 
N 
Disease 
ICIs 
Effects 
Reference 
SCFAs 
- 
Fecal acetate (high) 
- 
Fecal proprionate (high) 
- 
Fecal butyrate (high) 
- 
Fecal Valeric Acid (high) 
- 
Plasme isovaleric acid (high) 
 
52 
Solid Cancers 
Anti-PD-1 
Longer PFS 
[25] 
SCFAs 
- 
Fecal proprionate (high) 
- 
Fecal butyrate (high) 
Fecal Lysine (high) 
Plasme Nicotinic acid (high) 
 
11 
NSCLC 
Anti-PD-1 
Longer 
Response 
[26] 
SCFAs 
- 
Fecal butyrate (high) 
 
40 
Melanoma 
Prostate Cancer 
Anti-CTLA-
4 
Shorter PFS 
[27] 
SCFAs 
- 
Plasma proprionate (high) 
 
45 
Melanoma 
Anti-CTLA-
4 
Shorter PFS 
[27] 
Serum kynurenine/tryptophan 
ratio (high) 
 
106 Melanoma 
Renal cell carcinoma 
Anti-PD-1 
Shorter OS 
[28] 
3-Hydrozyanthranilic Acid 
(low) 
 
19 
NSCLC 
Anti-PD-1 
Longer PFS 
[29] 
Fecal 2-pentanone (high) 
Fecal Tridecane (high) 
 
11 
NSCLC 
Anti-PD-1 
Early 
progression 
[26] 
Serum indoleamine-2,3-
diozygenase 
 
23 
NSCLC 
Anti-PD1 
ICI resistance 
[30] 
 
 

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5. METHODS 
 
5.1 Study parameters/endpoints 
 
5.1.1 Main study parameter/endpoint 
Progression of disease defined by iRECIST criteria on regular CT scan at 6 months 
following chemoradiotherapy. 
 
5.1.2 Secondary study parameters/endpoints 
Changes in circulating immune cells. 
 
Occurrence of immunotherapy-related toxicity within 12 months following 
chemoradiotherapy. 
 
 
5.2 Study procedures 
Details regarding laboratory procedures/assessments to be performed in this trial are 
according to the local policies.  
 
Stool samples and a throat swipe will be obtained using the recommended procedure 
developed by the TNO. See Appendix 1. 
 
The CyTOF analysis will be performed on the blood drawn at baseline and in week 4. 
The total amount of blood to be drawn/collected for correlative studies over the course of 
the trial is 2 x 20 ml. The sampling will be performed only in the LUMC and AVL/NKI.See 
Appendix 2. 
 
Pulmonary function test: Patients will perform hospital-based lung function 
measurements in accordance with the ERS/ATS criteria; Forced vital capacity (FVC), 
Forced Expiratory Volume in 1 second (FEV1) and diffusion capacity of the lung for 
carbon monoxide (DLCOc) will be measured.  
 
See table 3 on the next page for an overview of all procedures, both Standard of Care and 
study-specific. 
 
 
 

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Table 3: Schedule of assesments 
 
 
Only those items with an * are considered not the SoC procedures 
Item # is only applicable for the Dutch centers 
 
 
Screening 
Phase 
During IO treatment 
(weeks) 
 
Main Study 
Screening 
(Baseline) 
 4 
12 
24 
36 
52 
 
Informed Consent 
x 
 
 
 
 
 
Inclusion/Exclusion Criteria 
x 
 
 
 
 
 
Smoking status (active/recent vs 
hardly/never) 
x 
 
 
 
 
 
Demographics and Medical History 
x 
 
 
 
 
 
Antibiotics use review  
x 
x 
x 
x 
x 
x 
Progression Status 
 
 
x 
x 
x 
x 
 
Review Adverse Events 
x 
x 
 
 
 
 
Review immune related toxicity 
 
x 
x 
x 
x 
x 
Physical Examination 
x 
 
 
x 
 
x 
Vital Signs and Weight 
x 
 
 
x 
 
x 
ECOG Performance Status 
x 
 
 
x 
 
x 
 
Tumor Imaging (CT scan within +/- 2 
wks) 
x 
 
x 
x 
x 
x 
Stool sampling* 
x 
 
 
 
 
 
Throat swabs* 
x 
 
 
 
 
 
Blood Samples (CyTOF) * # 
x 
x 
 
 
 
 
Pulmonary Function test  
(FVC, FEV1, F/V, TLCO) 
x 
 
 
x 
 
x 

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5.3 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for 
urgent medical reasons. 
 
5.4 Replacement of individual subjects after withdrawal 
Patients will be replaced when withdrawn from the study. Due to the low burden of this 
study, it is not expected that patients will withdraw.  
 
 
 
 
5.5 
Premature termination of the study 
In case of slow accrual (<25 patients  entered in the first year) the study can be terminated 
prematurely. In this scenario, the METC will be notified within 15 days. 
 
 
 
 

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6. SAFETY REPORTING 
 
6.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety. The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed. 
 
6.2 AEs, SAEs and SUSARs 
6.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to the trial procedure. Only 
adverse events related to the collection of stool sampling, throat swabs and blood 
sampling will be recorded for this study. 
 
6.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
In case a subject is considered to be harmed by the collection samples of stool or 
blood the PI will be notified and discussed if the study must be adjusted or ended. 
 
Only study related (S)AE’s will be reported. This implies that all (S)AE’s related to 
participation in this study protocol, meaning from stool sampling, throat swabs and 
blood sampling will be reported. The local investigator will report all SAEs to the 
sponsor without undue delay after obtaining knowledge of the events. 
 
Immune related toxicity (so called immune related adverse events or irAEs) will be 
managed according to the local standard practice and recorded in the patient file.   
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 7 days of first knowledge for 
SAEs that result in death or are life threatening followed by a period of maximum of 8 
days to complete the initial preliminary report. All other SAEs will be reported within a 
period of maximum 15 days after the sponsor has first knowledge of the serious 
adverse events. 

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6.3 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study, as defined in the protocol. 
 
 
 
 

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7. STATISTICAL ANALYSIS 
        
SPSS (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 25.0. Armonk, 
NY: IBM Corp.) and MatLab (2017B, MathWorks, Natick, MA) will be used for data analysis. 
Descriptive statistics will expressed as mean ± SD if data are normally distributed and as 
median (interquartile range) if data are non-normally distributed. Between-group 
comparisons will be carried out using Mann–Whitney U tests, two-sample unpaired t-tests or 
chi-square tests. Within group comparisons will be carried out with paired T-test or in case of 
non-normal distribution with a Wilcoxon Signed Rank Test. Associations will be analyzed by 
(multiple) regression analysis. 
 
 
7.1 Primary study parameter(s) 
The primary goal of the study is to identify patients that develop disease progression or 
relapse (PD) within 6 months during adjuvant durvalumab treatment by the composition of 
the gut and throat microbiome as measured by the alpha and beta components.  
Microbiota composition is expressed in a table of sequence reads, clustered at single 
nucleotide variants, each assigned to microbial taxa at various taxonomic levels. This will 
be used to establish possible differences in terms of  (1) microbial (alpha) diversity, using 
the Shannon index and/or Simpson-index, (2) compositional differences (beta diversity), 
such as the Bray Curtis and/or UniFrac distances between samples, and (3) differential 
abundances of specific microbial taxa within the microbial community. Data might be 
further evaluated using principle component analysis and/or principal coordinate analysis. 
 
7.2 Secondary study parameter(s)  
Explorative analysis for relationships between immune-related toxicity (irAEs) during 
durvalumab treatment after chemoradiotherapy and composition of the microbiome (throat 
swabs and stool samples) will be done by (multiple) regression analysis. CT scans, lung 
function and other relevant clinical data will be used to identify irAE’s. 
 
Disease control rate (DCR ie. stable disease, partial response, and complete response) 
will be determined by CT scans and PFS at 6 months after completion of 
chemoradiotherapy. This will be correlated to the microbiome samples using multiple 
regression analysis. 
 
In addition, CyTOF analyses will be tested for their usefulness in predicting these 
progressors. These are of pure experimental value. 
 
 
 

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8. ETHICAL CONSIDERATIONS 
 
8.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(version 2013). We consider this study to be inside the boundaries of the Medical 
Research Involving Human Subjects Act (WMO). The coordinator will implement the 
research project in full respect of (inter-)national legal and ethical codes of practice. All 
study documents, including the full protocols and informed consent forms and any 
additional materials will be submitted to the relevant national authorities. Studies will 
commence upon approval.  
 
All information used in this study will be collected for routine clinical practice and will be 
handled, accessed and used according to the current regulatory framework, including the 
AVG. Appropriate mechanisms for data handling are installed for this study, and all 
investigators are notified of this and followed up during the research project.  
 
8.2 Recruitment and consent 
Eligible patients visiting the outpatient clinic will be asked whether they are interested to 
participate in this study according to standards of GCP, and if so they will receive verbal 
and written information. Recruitment procedures will be conducted by the investigator or 
the treating physician on the outpatient clinic in the different participating hospitals. 
Enrolment of the patients will be done according to principles stated above and the 
inclusion and exclusion criteria. Informed consent is required, in which the purpose of the 
research is explained, what the role of the subjects will be, and how the trial will be 
performed.  
 
Subjects will receive the Patient Informed Form and will be able to ask any questions 
when they do not understand the content. They will have enough time to consider their 
participation in the study.  
When informed consent is received the research nurse, investigator or treating physician 
can sign the patient informed consent. Participating in this clinical trial is voluntary and 
subjects can withdraw from participation for with any reason at any time. 
 
8.3 Benefits and risks assessment, group relatedness 
Participation to this study is not considered to bring any benefit to the patient. Since most 
of the analysis are coinciding with regular follow-up the negative impact on the subject is 
minimal. 
Study burden for the patient is relatively small. The study procedures are stool sampling, 
throat swabs and blood sampling. These procedures are of minimal invasiveness. 
 
8.4 Compensation for injury 
Participation in this study is expected to be without risk because the minimal invasiveness 
of the study procedures. In addition, during the study there will be no (change in) medical 
intervention. Therefore the investigator has been granted dispensation for insurance by 
the METC-LDD (Medical Ethical Committee Leiden Den Haag Delft). 
 

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8.5 Role of Astra Zeneca 
The company Astra Zeneca has provided the LUMC a grant for the execution of the 
translational research and financial support for personnel involved in the study. 
 
 
 

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9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
9.1 Handling and storage of data and documents 
The handling of personal data complies with the GDPR or General Data Protection 
Regulation (Dutch: Algemene Verordening Gegevensbescherming, AVG) as in effect 
from May 25th 2018. GDPR is an EU wide agreement which covers all member states. 
 
All obtained material and data from patients will be handled confidentially per site. 
Characteristics and clinical results from participants will be collected, labelled with a 
measurement ID and stored in an encoded database. All data will be inserted into a 
database management system (eCRF) for maintenance and use of clinical data 
management (Castor). Each subject will be assigned a subject number at the time the 
informed consent is signed. Data will be encrypted. No characteristics that can be traced 
to an individual (no dates, only age) are stored or shared between the study sites.   
 
A subject identification code list is made where the code is linked to the participant’s 
hospital number. This list is stored securely per study site and is only accessible for local 
clinical investigators and members of the study team throughout the study.  
 
Data will be accessible for the principal investigator, members of the study team, 
monitor and IGJ Following closure of the study. 
All study records will be maintained for 15 years in a safe and secure location, as local 
regulations require.The investigator will retain all patient information for a period of 15 
years after study completion. They will be securely stored per study site as local 
regulations require. 
 
The patients can revoke their permission for participating in this study at any moment. 
Performed sampling and measurements will be destructed and not been used for 
research. If the sampling and measurements were already analysed before revoked 
permission they will still be used for this study. 
 
The controller of the collected data is the principal investigator in this study. In case 
patients have either questions or complaints about protection of their privacy they can 
contact our data protection officer via privacy@lumc.nl. Information about this study, but 
no information about the patients, is stored at www.clinicaltrialsregister.eu 
 
9.2 Monitoring and Quality Assurance  
Monitoring in all sites in the Netherlands will be executed by (internal) monitors of the 
LUMC according to the monitor plan. 
 
9.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. All amendments will be notified to the METC that gave 
a favourable opinion. 
All substantial amendments will be notified to the METC and to the competent authority. 
 

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A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to 
affect to a significant degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
9.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 
the trial, serious adverse events/ serious adverse reactions, other problems, and 
amendments.  
 
9.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit.  
 
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action.  
    
In case the study is ended prematurely, the sponsor will notify the accredited METC 
within 15 days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC.  
 
9.6 Public disclosure and publication policy 
The study will be presented at major Lung Cancer meetings and is planned to be 
published in a major journal  (e.g. J Thoracic Oncology, Lancet Oncology) within 6 months 
after full analysis of the data in agreement with the CCMO publication policy. 
 
9.7 Synthesis 
The study is considered as minimal risk and therefore a DSMB is not necessary. The 
monitor coordinator of the LUMC will be informed about the study procedures and will 
define the intensity of monitoring in this study. In the unlikely event a potential harmful 
finding occurs, the patient will be duly notified. 
 
 
 

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Appendix 1 
 
Collection of microbiome samples 
 
Stool samples will be collected at baseline. Participants will receive a collection kit 
containing two sample tubes (cylindrical containers with a spatula in the lid) and gloves. They 
will be instructed on using these tubes. The tubes need to be stored in the refrigerator (at +/- 
4 ⁰C) in order to conserve the volatile SCFA’s and calprotectin,Times of collection will be 
noted. These stool samples will then be collected by the investigator during the clinical visit. 
Stool samples can be no more than 24 hours old.  
After arrival at the clinical research unit, the research personnel will immediately prepare and 
store one sample (meant for analysis of the gut microbiome) at -80 ⁰C. The cooled sample 
will be homogenized and transferred to multiple smaller cups, which will be stored at -20 ⁰C. 
The -80 ⁰C sample will be used for 16S-rRNA metagenomic sequencing of microbial content 
by TNO. The -20⁰C sample will be stored for metabolic biomarker analysis (e.g. SCFA).    
 
Throat swabs will be collected at baseline. 
Mucosal throat swab samples will be collected by trained andqualified personel. Swabs will 
be collected using the Copan eNAT swabs, comprising of a sterile swab and a sample tube. 
The sterile swab will be applied by gently stroking 5 times over the pharyngeal region. 
Strokes will be made in an anterior-posterior direction. 
The study of throat and gut microbiota composition will be performed by (TNO, Zeist & 
ACTA, University of Amsterdam, Netherlands). 16S rRNA sequencing will be first performed 
to determine differences in bacterial phyla in responders compared with non-responders and 
the data will be used to study the microbiota composition in correlation with the phenotype of 
DCs observed by mass cytometry. 
 
 

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Appendix 2 
 
CYToF analysis 
T cell response against commensal bacteria: The commensal-specific T cell response will be 
analyzed in order to determine whether it is linked with anti-PD-L1 efficacy and/or with DC 
function [18] [19] . T- lymphocytes from peripheral blood and tumor lysate will be expanded in 
order to obtain enough cells for the analyses. An analysis of the commensal-specific T cell 
response will be performed in collaboration within LUMC, and functional assays will be 
performed as described above. 
 
Immunophenotyping of peripheral blood from non-progressors and progressors by mass 
cytometry before and 4 weeks after the onset of the treatment, by using two panels (with 42 
metal-labeled antibodies and 6 barcode options based on palladium metals), in order to 
identify circulating biomarkers as described in Exploratory Objective 2.  
Using Panel 1, markers related to tumor-cell killing of CD8+ T cells and NK cells will be used. 
Panel 2 will allow us to investigate activation and tolerogenic markers in DC subsets (e.g. 
pDCs, cDC1, cDC2) and monocytes.  
 
Procedure: cells will be incubated with metal-labeled antibodies and barcoded-anti-beta2M 
antibodies, followed by fixation and acquisition on a Helios mass cytometer. Fcs files will be 
normalized using the included reference beads, concatenated and debarcoded. Then, viable 
immune cells will be pre-grated and exported as fcs files using Flowjo X (Treestar), followed 
by clustering and further analysis using the in-house developed software Cytosplore 
(www.cytosplore.org) and the R-package Cytofast [22] (Figure 1).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Pipeline for data analysis of airway tissue samples. The fcs files generated by mass cytometry are 
compiled and clustered together in Cytosplore. In the first level of hierarchical clustering, the cell lineages can be 
identified, including T cell, B cell, NK cell, dendritic cell, monocyte and granulocyte populations. In the second 
level of clustering, clusters within cell lineages are identified (example here: CD4+ T cells). The accompanying 
heatmap shows the marker expression profile of each cluster in the cell lineage (here: CD4+ T cells). This is used 
to identify particular subsets in the cell lineage of interest. The fcs files of the clustered cells are then further 
analyzed in R with the Cytofast package. The abundance of each cluster per group can be represented in a 
quantitative bar graph. Statistical comparison is performed with ‘global testing’ to highlight significant changes in 
cluster abundance between groups.