HANSE - Holistic Implementation Study Assessing a Northern German Interdisciplinary Lung Cancer Screening Effort, Population-based Screening Study -Prospective, Randomized Comparator Controlled
Phase: NA
Conditions: Lung Cancer
Sponsor: Hannover Medical School

Observational Study Protocol - Amendment 
Study Code 
ESR-20-20770  
Version 
3.0 
Date 
13.12.2024 
 
 
 
Study amendment: 
HANSE - Holistic implementation study Assessing a Northern German 
interdisciplinary lung cancer Screening Effort 
Additional follow-up and implementation of newly available blood biomarkers to strengthen 
the results and refine the diagnostic workup of the prospective, randomized, and comparator-
controlled population-based screening study. 
Sponsor: 
Medizinische Hochschule Hannover  

Observational Study Protocol 
Study Code ESR-20-20770  
Version 13.12.2024 
Date 13.12.2024 
2 
TABLE OF CONTENTS 
PAGE 
 
TITLE PAGE ............................................................................................................................ 1 
TABLE OF CONTENTS ......................................................................................................... 2 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 4 
RESPONSIBLE PARTIES ...................................................................................................... 5 
PROTOCOL SYNOPSIS ......................................................................................................... 6 
AMENDMENT HISTORY .................................................................................................... 11 
MILESTONES ....................................................................................................................... 12 
1. 
BACKGROUND AND RATIONALE .................................................................. 13 
1.1 
Background ............................................................................................................ 13 
1.2 
Rationale ................................................................................................................ 14 
2. 
OBJECTIVES AND HYPOTHESES .................................................................... 15 
2.1 
Primary Objective(s) & Hypothesis(es) ................................................................. 15 
2.2 
Secondary Objective(s) & Hypothesis(es) (Optional) ........................................... 15 
2.3 
Exploratory Objective(s) & Hypothesis(es) (Optional) ......................................... 17 
3. 
METHODOLOGY ................................................................................................ 17 
3.1 
Study Design ± General Aspects ............................................................................ 17 
3.1.1 
Data Source(s) ........................................................................................................ 17 
3.2 
Study Population .................................................................................................... 17 
3.3 
Inclusion Criteria ................................................................................................... 17 
3.4 
Exclusion Criteria .................................................................................................. 17 
3.5 
Participant Follow-up (Optional) ........................................................................... 17 
4. 
VARIABLES AND EPIDEMIOLOGICAL MEASUREMENTS ........................ 18 
4.1 
Exposures ............................................................................................................... 18 
4.1.1 
Definition of Primary Drug Exposure (Optional) .................................................. 18 
4.1.2 
Definition of Comparison Drug Exposure (Optional) ........................................... 19 
4.2 
Outcomes ............................................................................................................... 19 
4.3 
Other Variables and Covariates ............................................................................. 20 
5. 
STATISTICAL ANALYSIS PLAN ...................................................................... 20 
5.1 
Statistical Methods ± General Aspects................................................................... 20 

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5.1.1 
Primary Objective(s):  Calculation of Epidemiological Measure(s) of 
Interest (e.g. descriptive statistics, hazard ratios, incidence rates, test/retest 
reliability) ............................................................................................................... 21 
5.1.2 
Secondary Objective(s):  Calculation of Epidemiological Measure(s) of 
Interest (e.g. hazard ratios, incidence rates, test/retest reliability) ......................... 21 
5.1.3 
Exploratory Objective(s):  Calculation of Epidemiological Measure(s) of 
Interest (e.g. hazard ratios, incidence rates, test/retest reliability) ......................... 22 
5.2 
Bias ........................................................................................................................ 23 
5.2.1 
Methods to Minimize Bias ..................................................................................... 23 
5.2.2 
Adjustment for Multiple Comparisons .................................................................. 23 
5.2.3 
Strengths and Limitations ...................................................................................... 23 
5.3 
Interim Analyses (Optional)................................................................................... 23 
5.4 
Sample Size and Power Calculations ..................................................................... 23 
6. 
STUDY CONDUCT AND REGULATORY DETAILS....................................... 25 
6.1 
Study Conduct ........................................................................................................ 25 
6.1.1 
Study Flow Chart and Plan .................................................................................... 25 
6.1.2 
Procedures .............................................................................................................. 26 
6.1.3 
Quality Control ...................................................................................................... 30 
6.2 
Protection of Human Subjects................................................................................ 31 
6.3 
Collection and Reporting of Adverse Events/Adverse Drug 
Reactions/Special Situations .................................................................................. 31 
7. 
LIST OF REFERENCES ....................................................................................... 32 
8. 
APPENDICES ....................................................................................................... 33 
9. 
ATTACHMENTS .................................................................................................. 34 
10. 
SIGNATURES ....................................................................................................... 35 
 

Observational Study Protocol 
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Abbreviation or 
special term 
Explanation 
CAT 
COPD assessment test 
CBC 
Complete blood-cell count 
CMP 
Comprehensive molecular profiling 
LC 
Lung cancer 
LDCT 
Low-dose computed tomography 
LungRADS 
Lung CT Screening Reporting & Data System 
MDT 
Multidisciplinary tumor board 
mMRC 
OR 
Modified medical research council 
Odds ratio 
PLCO 
Prostate, lung, colorectal, and ovarian cancer screening 
PPV 
Positive predictive value 
 
 
 
 

Observational Study Protocol 
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Version 13.12.2024 
Date 13.12.2024 
5 
RESPONSIBLE PARTIES 
Name 
Professional 
Title 
Role in Study Affiliation 
Email Address 
Jens Vogel- 
Claussen 
Prof. Dr. 
Principal 
Investigator 
Institute for Diagnostic and 
Interventional Radiology  
Biomedical Research in End-
stage and Obstructive Lung 
Disease Hannover (BREATH)  
German Center for Lung 
Research (DZL) 
vogel-
claussen.jens@mhha
nnover.de 
Sabine Bohnet 
Dr. 
Co- 
Investigator 
Universitätsklinikum 
Schleswig-Holstein - Campus 
Lübeck 
Airway Research Center 
North (ARCN) 
German Center for Lung 
Research (DZL) 
Sabine.Bohnet@uksh
.de 
Martin Reck 
Prof. Dr. 
Co- 
Investigator 
LungenClinic Grosshansdorf 
gGmbH 
Airway Research Center 
North (ARCN) 
German Center for Lung 
Research (DZL) 
m.reck@lungenclinic
.de 
 
 

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Version 13.12.2024 
Date 13.12.2024 
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PROTOCOL SYNOPSIS 
 
Study amendment: 
HANSE - Holistic implementation study Assessing a Northern German interdisciplinary 
lung cancer Screening Effort 
Additional follow-up and implementation of newly available blood biomarkers to strengthen 
the results and refine the diagnostic workup of the prospective, randomized, and comparator-
controlled population-based screening study. 
 
 
Background/Rationale: 
HANSE is an ongoing pilot lung cancer (LC) screening study in three specialized LC centers 
in northern Germany (NCT04913155). It was intended to provide evidence that a holistic and 
effective LC screening program can be implemented in Germany and that such a screening 
program can be integrated in the current infrastructure of certified lung cancer centers. For 
details, please find the initial study protocol in section 9 (Attachments). 
Participants between 55 and 79 years of age with a smoking history were eligible for low-dose 
computed tomography (LDCT) given that they met the inclusion criteria of the NELSON trial 
or had a PLCOM2012 ULVNVFRUH % (6 year risk).1,2 The primary objective of this study, 
which was recently met.4 , is to compare the efficiency of the PLCOM2012 and the NELSON 
inclusion criteria in identifying patients with LC3 by comparison of the positive predictive 
value (PPV) for LC detection with the two different inclusion methods (NELSON vs. 
PLCOM2012) after 2 rounds of LDCT screening (at baseline and after 1 year follow-up) 
Secondary endpoints include the proportion of individuals selected for screening, proportion 
of LC cases detected within the different cohorts after a 5-year follow-up, sensitivity and 
specificity after 5-year follow-up, rate of initiation of cardiovascular treatments, efficiency of 
nodule management algorithms, success and quality of the screening program, success of 
smoking cessation counselling, evaluation of blood-based biomarkers in positive LDCT cases, 
as well as analysis of the cost effectiveness.  
HANSE included 13,016 participants which were assessed for their individual lung cancer 
risk, of which 5,191 met the risk criteria of NELSON and/or PLCOM2012 and received a 
baseline LDCT and 4,356 participants received a 2nd round LDCT. In total, 111 LC cases were 
detected in both screening rounds (64 in round 1 and 47 in round 2), of which 108 LC cases 
were detected by the PLCOM2012  % risk score and 85 were detected by the NELSON 
criteria, showing a significantly higher LC detection rate of 97.3 % in PLCOM2012-selected 
when compared to 76.6 % in NELSON-selected participants (p<0.0001). Positive predictive 
values (PPV) were 2.59 % (PLCOM2012) and 2.17 % (NELSON), respectively.4  

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However, longer follow-up is needed to confirm these very promising results and to proof 
their validity considering also slowly growing lung cancer which might be overseen within 
only two LDCT rounds with an interval of 12 months and which could have an influence on 
the detection rate of the two risk scores. In the NELSON trial, the highest LC detection rate 
was found 3 years after the initial baseline LDCT. Therefore, this amendment is intended to 
include a third LDCT round in participants of the HANSE study fulfilling the NELSON 
and/or PLCOM2012  % risk criteria after a follow-up of approximately 4 years after the 
baseline screening round to further support the primary objective. 
Furthermore, recently novel blood biomarkers have been tested with promising performances 
in early-stage lung cancer detection.5±7 It is yet to be proven in how far such biomarkers can 
expand and refine the established risk criteria and/or the diagnostic workup within LDCT-
based LC screening programs to increase LC detection and to reduce the rate of false positive 
interventions. 
Integration of a certified smoking cessation program was already part of the initial HANSE 
protocol. Since intensified cessation programs comprising a combination of behavioural 
counselling and pharmacotherapy were shown to produce the highest success rates in smoking 
cessation8, effectiveness and safety of an intensified smoking cessation program is to be 
studied in comparison to the short smoking cessation counselling following the WHO 
recommendations. Participation is fully voluntary.  
Objectives and Hypotheses: 
The primary objective of the HANSE study remains unchanged and can be found in section 
2.1 of the initial study protocol (for details, see section 9. Attachments). However, the primary 
endpoint will be extended by a third LDCT screening round after follow-up of ~4 years to 
further strengthen the positive results of the HANSE study. 
In addition, this amendment is intended to include three additional secondary objectives: 
 
To proof the real-world practicability of the PLCOM2012 and NELSON risk scores by 
re-evaluating HANSE participants with an initially low risk score. Participants 
changing from initially low to high risk due to their increased age by ~4 years or by 
other changed variables will be invited to receive a baseline LDCT scan. 
 
To study the effectiveness of a guideline-based and intensified smoking cessation 
program in comparison to the already included short smoking cessation counselling 
with respect to the smoking cessation rates (participants being nicotine free).  
 
To proof if novel LC blood biomarkers can:  
o expand the risk assessment of LC screening. For this, HANSE participants with 
a low risk profile (after PLCOM2012 and NELSON risk re-evaluation) will be 
invited for blood sampling, and participants with positive LC biomarker 
findings will be invited to receive a LDCT scan.  

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o improve the diagnostic workup of positive LDCT findings within 
Multidisciplinary Tumor Boards (MDT). Therefore, participants will be 
randomized 1:1 and for half of the participants with positive LDCT findings, 
MDT discussions will be supplemented by blood biomarker results (biomarker 
reporting arm) whereas the other half will be discussed without these results 
(control arm). This randomization does not affect the initial study 
randomization (for details, see section 6.1.2). 
All participants with a positive LC blood biomarker test will receive a 2nd LDCT about 6 
months after the baseline CT of the HANSE 3rd round or baseline round respectively to 
confirm the negative diagnosis according to the mod. Lung RADS 1.1 score used in the 
HANSE study, except for participants with a positive lung cancer diagnosis on histology. 
All other objectives and endpoints of the HANSE study remain unchanged (for details, see the 
initial study protocol in section 9. Attachments). 
Methods: 
The principal methodology of the HANSE study remains unchanged. For better overview, 
only changes to the initial study protocol will be described in this amendment. 
Study design: 
Unchanged 
Data Source(s): 
In addition to the initially defined data sources, blood samples will be taken from all 
participants of the HANSE study (high risk and low risk participants) and blood biomarkers 
for early LC detection will be analyzed by a metabolomic blood test of 9 metabolites 
(BioMark Diagnostics, Richmond, Canada). 
Study Population: 
The general HANSE study population remains unchanged. However, the high risk population 
will be extended by initially low risk participants who become high risk by PLCOM2012 and 
NELSON re-evaluation. 
Exposure(s): 
Participants in the initial high risk group will undergo a third LDCT screening round after 
approximately 4 years after the baseline screening round. Participants either switching from 
the low to high risk group by PLCOM2012 and NELSON re-evaluation or who are tested 
positively for metabolic LC biomarkers will undergo a first LDCT screening round. To 
analyze the rate of false-positives, participants with positive LC biomarkers but without LC 
findings after the first LDCT will undergo a follow-up LDCT 6 months after baseline. 

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Outcome(s): 
Primary endpoint: 
The primary endpoint will be extended by a third LDCT screening round after follow-up of ~4 
years to proof and validate the positive results of the HANSE study considering slowly 
growing lung cancer. 
Additional secondary endpoints: 
 
LC detection rates (PPV) will be assessed for participants initially rated as low risk 
who become high risk after PLCOM2012 and NELSON re-evaluation after ~4 years of 
follow-up.  
 
Success of a guideline-based and intensified smoking cessation program will be 
analysed in comparison to the short smoking cessation counselling by:  
o Assessing the proportion of participants being nicotine-free (i.e. absolute 
abstinence from taking nicotine from any source) after 6 months (determination 
of cotinine in urine). 
o Assessing the proportion of participants being nicotine-free (i.e. absolute 
abstinence from taking nicotine from any source) after 1, 3, and 12 months 
(determination of cotinine in urine). 
o Assessing the proportion of participants being abstinent from combustible 
cigarettes, after 1, 3, 6, and 12 months 
o Survey of the Fagerström test, survey after craving, absence/sick days of the 
previous year as well as QoL survey 
o Assessment of the adverse events of the products as well as the withdrawal 
symptoms (no nausea, difficulty falling asleep and staying asleep, dry mouth, 
etc.) 
o Assessment of respiratory symptom burden (cough, perceived shortness of 
breath via mMRC or CAT) 
o Optional: Assessment / improvement of the function of the small airways or 
lung function 
o Optional: Assessment / improvement of central blood pressure measurements 
and arterial vascular stiffness 
 

Observational Study Protocol 
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 
LC detection rates (PPV) will be assessed after LDCT for low risk participants (after 
PLCOM2012 and NELSON re-evaluation) with positive LC biomarker findings 
(metabolomic blood test, BioMark Diagnostics, Richmond, Canada). 
 
LC detection rates (PPV) and application rates of invasive procedures will be assessed 
for positive LDCT findings which are discussed within MDT with respect to 
supplementation with blood biomarker results. 
Sample Size Estimations: 
Sample size estimation for the primary endpoint remains unchanged. However, expected LC 
cases in initially low risk participants who will receive baseline LDCT screening within this 
study amendment either by switching to high risk after PLCOM2012 and NELSON re-
evaluation or by positive blood biomarker results for early LC detection will be estimated in 
section 5.4 of this protocol amendment for LDCT justification. In addition, sample size 
estimation for the intensified smoking cessation program and power calculation of the new 
secondary endpoints can be found in section 5.4 of this protocol. 
Statistical Analysis: 
No major changes to initial protocol (for details, see section 9. Attachments). Only details 
concerning the new secondary endpoint to study the effectiveness of a guideline-based and 
intensified smoking cessation program in comparison to the already included standard 
smoking cessation counselling are included in section 5.1.2. 

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AMENDMENT HISTORY 
Date 
Section of study protocol 
Amendment or update 
Reason 
15.09.2020  
N/A 
Initial Protocol Version 1.0 
23.11.2020 Study objectives and 
power calculation of 
primary endpoint 
Amendment 
Protocol Version 2.0 
09.02.2021 Administrative changes: 
Recruitment methods, 
potential sampling bias and 
biomarkers 
Amendment 
Protocol Version 2.1 
13.12.2024 Addition of 3rd LDCT 
screening round (primary 
endpoint) and extension of 
secondary objectives 
Amendment 
Protocol Version 3.0 
 
 
 
 
 
 
 
 
 
 
 
 

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MILESTONES 
Milestone 
Planned date 
August 2020 
Final study protocol 
August 2020 
Contract Astra Zeneca 
August 2020 
Contract CRO 
August 2020 
Contract Coreline 
August 2020 
Contract truck/CT 
September 2020 
Ethics approval 
September 2020 
BfS application 
March 2021 
Study fully set-up 
July 2021 
Start of enrolment: First patient in 
August 2023 
Date of last data entry in database 
September 2023 
Date of Database Lock (Clean Database) 
December 2023 
Publication and Final report 
October 2024 
Final protocol amendment 
October 2024 
Contract AstraZeneca (amendment) 
December 2024 
Ethics submission (amendment) 
December 2024 
Final study setup (amendment) 
May 2025 
BfS approval (amendment) 
September 2025 
Start blood sampling low risk participants 
November 2025 
LDCT screening start (amendment) 
January 2027 
Date of last data entry in database 
February 2027 
Date of Database Lock (Clean Database) 
July 2027 
Publication and Final report 
 
 

Observational Study Protocol 
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1. 
BACKGROUND AND RATIONALE 
1.1 
Background 
HANSE is an ongoing pilot lung cancer (LC) screening study in three specialized LC centers 
in northern Germany (NCT04913155). It was intended to provide evidence that a holistic and 
effective LC screening program can be implemented in Germany and that such a screening 
program can be integrated in the current infrastructure of certified lung cancer centers. For 
details, please find the initial study protocol in section 9 (Attachments). 
Participants between 55 and 79 years of age who were current or former smokers and who met 
WKHLQFOXVLRQFULWHULDRIWKH1(/621WULDOVPRNLQJTXLWWLPH\HDUV!FLJDUHWWHVGD\
for >25 years or >10 cigarettes/day for >30 years) or a PLCOM2012 risk score of at least 1.58 % 
within 6 years were recruited from 3 certified lung cancer sites (Hannover, Großhansdorf, and 
Lübeck).1,2 Such high risk participants received two consecutive LDCT screening rounds: at 
baseline and after 12 months follow-up.3 The primary objective of this study, which was 
recently met.4 , is to compare the efficiency of the PLCOM2012 and the NELSON inclusion 
criteria in identifying patients with LC by a comparison of the positive predictive value (PPV) 
for LC detection with the two different risk selection methods (NELSON vs. PLCOM2012) after 
two rounds of LDCT screening For testing the null hypothesis of equal PPVs for LC detected 
in PLCOM2012-selected versus NELSON-selected individuals, the weighted generalized score 
statistic by Kosinski was used. Secondary endpoints include the proportion of individuals 
selected for screening, proportion of LC cases detected within the different cohorts after a 5-
year follow-up, sensitivity and specificity after 5-year follow-up, rate of initiation of 
cardiovascular treatments, efficiency of nodule management algorithms, success and quality 
of the screening program, success of smoking cessation counselling, evaluation of blood-
based biomarkers in positive LDCT cases (LungRADS 4A PET, 4B, 4X), as well as analysis 
of the cost effectiveness.  
HANSE included 13,016 participants which were assessed for their individual lung cancer 
risk, of which 5,191 met either one or both of the two risk criteria and received a baseline 
LDCT scan, and 4,356 participants received a 2nd round LDCT. In total, 111 LC cases were 
detected in both screening rounds (64 in round 1 and 47 in round 2), of which 108 LC cases 
were detected by the PLCOM2012 ULVNVFRUHDQGZHUHGHWHFWHGE\WKH1(/621
criteria, showing a significantly higher cancer detection rate of 97.3 % by PLCOM2012 in 
comparison to 76.6 % by the NELSON criteria (p<0.0001, see Figure 1). When the sample 
was supplemented with the low risk population, who did not meet the high risk criteria and 
consented to participate in the HANSE study (n=7,463), the calculated lung cancer detection 
rates (sensitivity) of the PLCOM2012 and the NELSON criteria were 77.1 % and 60.7 %, 
respectively. Accordingly, the HANSE study showed a 19.4 % relative increase of the PPV 
for LC detection in the PLCOM2012-selected group (PPV=108/4,167 [2.59 %] compared to the 
NELSON-selected group (PPV=85/3,916 [2.17 %], p=0.004). Hence, PLCOM2012 is reliable 
and more efficient than the NELSON criteria for selecting individuals to be enrolled into a LC 
screening program and should be used for identifying high risk individuals based on the 
results of the HANSE study. 

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Figure 1 Lung cancer detection within two LDCT screening rounds (baseline & after 12 months follow-up) 
of PLCOM2012- and NELSON-selected participants. 
1.2 
Rationale 
Despite the positive findings of a significant difference of LC detection between PLCOM2012 
and NELSON criteria within HANSE, longer follow-up is needed to confirm these results and 
to proof their validity considering slowly growing lung cancer which might be overseen 
within only two LDCT rounds with an interval of 12 months and which could have an 
influence on the detection rate of the two risk scores. For example, in the NELSON trial the 
highest LC detection rate was found 3 years after the initial baseline LDCT scan.1 Therefore, 
this amendment is intended to include a third LDCT round for high risk participants of the 
HANSE study fulfilling the NELSON and/or PLCOM2012 ULVNFULWHULDDIWHUDIROORZ-
up of approximately 4 years to further support the primary objective. 
Furthermore, recently novel blood biomarkers have been tested with promising performances 
in early-stage lung cancer detection.5±7 It is yet to be proven in how far such biomarkers can 
expand and refine the established risk criteria and/or the diagnostic workup within LDCT-
based LC screening programs to increase LC detection and to reduce the rate of false positive 
interventions. 
Integration of a certified smoking cessation program was already part of the initial HANSE 
protocol, according to the requirements defined by the Joint Statement of the German 
Radiological Society. For this, participants were informed about and encouraged to participate 

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in smoking cessation programs comprising professionally guided group courses within short 
counselling sessions by trained staff. However, numerous studies have shown that a 
combination of behavioural counselling and pharmacotherapy produces the highest success 
rates in smoking cessation leading to implementation of respective treatment 
recommendations into the German S3 smoking cessation guidelines.8 Therefore, within this 
amendment effectiveness and safety of an intensified smoking cessation program consisting of 
behavioural counselling and pharmacotherapy is going to be studied in comparison to the 
short smoking cessation counselling following the W+2UHFRPPHQGDWLRQVRI5¶VDQG
$¶V 
2. 
OBJECTIVES AND HYPOTHESES 
Details of the initial study protocol can be found in section 9. Attachments. 
2.1 
Primary Objective(s) & Hypothesis(es) 
The primary objective of the HANSE study is to compare the efficiency of the PLCOM2012 
 % (6 year risk) risk score and the NELSON inclusion criteria in identifying participants 
with lung cancer in the age group 55-79 years, which remains unchanged from the initial 
protocol. However, the primary endpoint will be extended by a third LDCT screening round 
after follow-up of ~4 years to potentially further strengthen the positive results of the HANSE 
study. 
2.2 
Secondary Objective(s) & Hypothesis(es) (Optional) 
Secondary objectives will be extended by the following three objectives: 
 
To proof the real-world practicability of the PLCOM2012 risk score by re-evaluating 
HANSE participants with an initially low risk score who have not yet received a 
LDCT. Since the PLCOM2012 ULVNVFRUHLQFRUSRUDWHVDSHUVRQ¶VDJHORQJHUIROORZ-up 
will lead to an increased risk score crossing the threshold of 1.58% (6 year risk) for 
some participants. In addition, also other personal variables and the continued or 
changed smoking habits relevant for PLCOM2012 and NELSON risk calculation (i.e. 
smoking behaviour, BMI, comorbidities) can change over time and consequently can 
FKDQJHDSHUVRQ¶VULVNVFRUH)URPWKH+$16(GDWDDSSUR[LPDWHO\SDUWLFLSDQWV
are estimated to change from low to high risk. After proofing their actual PLCOM2012 
and Nelson risk scores on site, these participants will receive a baseline LDCT scan. 
 
To study the effectiveness of a guideline-based and intensified smoking cessation 
program in comparison to the already included standard smoking cessation counselling 
with respect to the smoking cessation rates (participants being nicotine free). For this, 
participants who change from low to high risk PLCOM2012 risk score will be 
randomized 1:1 to receive an intensified or a short smoking cessation counselling. The 
participation in smoking cessation programs is voluntary. This randomization does not 
affect the initial study randomization (for details, see section 6.1.2). 
 
To proof if novel LC blood biomarkers can:  

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o expand the risk assessment of LC screening. For this, HANSE participants with 
a low risk profile (not fulfilling the high risk criteria of NELSON and/or 
PLCOM2012) will be invited for blood sampling. Participants with positive LC 
biomarker findings will be invited to receive a LDCT scan, and the PPV will be 
compared to the NELSON and PLCOM2012 risk criteria. 
o improve the diagnostic workup of positive LDCT findings within 
Multidisciplinary Tumor Boards (MDT) in terms of detected LC cases and 
application of invasive procedures (biopsies or resections). Therefore, 
participants will be randomized 1:1 and for half of the participants with 
positive LDCT findings, MDT discussions will be supplemented by blood 
biomarker results (reporting arm) whereas the other half will be discussed 
without these results (control arm).  
 
All participants with a positive LC blood biomarker test will receive a 2nd LDCT about 6 
months after the baseline CT of the HANSE 3rd round or baseline round respectively to 
confirm the negative diagnosis according to the mod. Lung RADS 1.1 score used in the 
HANSE study, except for participants with a positive lung cancer diagnosis on histology. 
All high risk participants (by PLCOM2012, NELSON, and/or blood biomarker) receiving an 
LDCT with a positive LC blood test will be informed personally about their positive 
biomarker results by trained local staff after the date of their (potential) MDT, except for  
 low risk participants theoretically switching from low to high risk by an 
increase of their age by four years, who are then rated as low risk at the 
on-site physician PLCOM2012/NELSON risk confirmation and who are 
tested positively for LC blood biomarkers afterwards. Such patients will 
get a second LDCT screening invitation due to their biomarker test 
results and therefore can conclude that they are biomarker-positive. 
Consequently, such participants are excluded from the randomization 
for MDT reporting since blinding cannot be maintained. However, they 
will also be informed personally about their positive results by trained 
local staff after the date of their (potential) MDT.  
 
All (high and low risk) participants with negative LC blood test will be informed by mail 
about their negative blood test results also after the date of their (potential) MDT. 
 
 
All other objectives and endpoints of the HANSE study remain unchanged (for details, see the 
initial study protocol in section 9. Attachments). 

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2.3 
Exploratory Objective(s) & Hypothesis(es) (Optional) 
N/A 
3. 
METHODOLOGY 
The principal methodology of the HANSE study remains unchanged. For better overview, 
only changes to the initial study protocol will be described in this amendment. 
3.1 
Study Design ± General Aspects 
No changes to initial protocol (for details, see section 9. Attachments). 
3.1.1 
Data Source(s) 
In addition to the initially defined data sources, blood samples will be taken voluntarily from 
all participants of the HANSE study (high risk and low risk participants) and blood 
biomarkers for early LC detection will be analyzed by a metabolomic blood test of 9 
metabolites (BioMark Diagnostics, Richmond, Canada). 
In addition, urine sampling and questionnaire completion is planned for participants of the 
intensified and standard smoking cessation programs within additional study visits at 1, 3, 6, 
and 12 months after the initial LDCT scan. 
3.2 
Study Population 
The general HANSE study population remains unchanged. However, the high risk population 
will be extended by initially low risk participants who become high risk by PLCOM2012 or 
NELSON risk criteria re-evaluation. 
3.3 
Inclusion Criteria 
No changes to initial protocol (for details, see section 9. Attachments). 
3.4 
Exclusion Criteria 
No changes to initial protocol (for details, see section 9. Attachments). 
3.5 
Participant Follow-up (Optional) 
In the initial protocol, a regular follow-up of 1 year per patient, resembling two LDCT 
screening rounds, was planned with an additional follow-up of 5 years to assess LC 
development and of 10 years to assess cardiovascular and all-cause mortality. Within this 
amendment, the regular follow-up is to be expanded by a third LDCT screening round at 
approximately 4 years after the baseline LDCT screening.  
For confirmation of a false positive blood test, all participants with a positive LC blood 
biomarker test and negative LC diagnosis will receive a 2nd LDCT about 6 months after the 

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baseline CT of the HANSE 3rd round or baseline round respectively to confirm the negative 
diagnosis according to the mod. Lung RADS 1.1 score used in the HANSE study, except for 
participants with a positive lung cancer diagnosis on histology. If the LDCT report of this 6 
months LDCT is Lung RADS, 3, 4A, 4B or 4X they will be discussed in the MDT. 
For analysis of an intensified smoking cessation program, voluntary participants of the two 
smoking cessation programs will be invited to additional visits at 1, 3, 6, and 12 months after 
their LDCT scan for determination of cotinine in urine and for completion of questionnaires.  
4. 
VARIABLES AND EPIDEMIOLOGICAL MEASUREMENTS 
4.1 
Exposures  
In the initial study protocol, it was defined that recruited participants in the high risk group 
undergo 2 low-dose CT screening rounds (baseline and 1 year follow up). 
This amendment adds a third LDCT screening round to participants in the initial high risk 
group at ~4 years follow-up. In addition, participants of the initial low risk group can become 
high risk by PLCOM2012 and NELSON re-evaluation (i.e. due to an increase of their age by ~4 
years or changes in personal or behavioural variables). Such newly rated high risk participants 
will undergo a baseline LDCT screening round. Furthermore, all participants will be asked to 
provide a blood sample which will be analysed for biomarkers of early LC detection 
(metabolomic blood test, BioMark Diagnostics, Richmond, Canada). In addition to initially 
high risk participants and participants switching from low to high risk by PLCOM2012 and 
NELSON reassessment, also blood biomarker-positive participants will be eligible for 
baseline LDCT screening. MDT discussions of positive LDCT scans will be supplemented 
with the respective biomarker findings in half of the participants (1:1 randomization, for 
details, see section 6.1.2). To study the rate of false-positives, blood biomarker-positive low 
risk participants without detection of lung cancer will undergo a follow-up LDCT after 
approximately 6 months. 
Table 1 Blood biomarkers to be used in HANSE after study amendment. 
Biomarker 
Biomarker 
Type 
# of 
Biomarkers 
Sensitivity 
Specificity 
Area under 
the curve 
Metabolomic 
Blood Test 
(BioMark 
Diagnostics) 
Metabolites 
9 
0.93 
0.93 
0.93 
 
4.1.1 
Definition of Primary Drug Exposure (Optional) 
Participants who are current smokers and who change from low to high risk by PLCOM2012 or 
NELSON re-evaluation or by a positive blood test will be randomized 1:1 to receive an 
intensified smoking cessation program or a short standard smoking cessation counselling, 

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when they show-up for their baseline LDCT exam. The intensified smoking cessation program 
comprises behavioural counselling and pharmacotherapy with a partial agonist of nicotinic 
acetylcholine receptors in accordance with the guideline recommendations.8 The counselling 
intervention allows for an intensive examination of personal motives and barriers to smoking, 
while the medication support alleviates withdrawal symptoms and reduces the craving for 
nicotine. This dual approach significantly increases the chances of permanent smoking 
cessation. A partial agonist of nicotinic acetylcholine receptors, for example cytisine, will be 
given for 25 days, and dosage will be tapered over 12 weeks. Behavioural counselling is based 
on group-based tobacco cessation according to the 4+2 scheme in the sense of behavioural 
therapy.  
4.1.2 
Definition of Comparison Drug Exposure (Optional) 
N/A 
4.2 
Outcomes 
Primary endpoint: 
The primary endpoint will be extended by a third LDCT screening round after follow-up of ~ 
4 years to proof and validate the positive results of the HANSE study considering slowly 
growing lung cancer. 
Additional secondary endpoints: 
 
LC detection rates (PPV) will be assessed for participants initially rated as low risk 
who become high risk after PLCOM2012 and NELSON re-evaluation after ~4 years of 
follow-up. These LC detection rates will be compared with the rates of the initial high 
risk participants (NELSON and/or PLCOM2012) after the baseline LDCT screening 
round.  
 
Success of a guideline-based and intensified smoking cessation counselling will be 
analyzed in comparison to the standard smoking cessation counselling by:  
o Assessing the proportion of participants being nicotine-free (i.e. absolute 
abstinence from taking nicotine from any source) after 1, 3, 6, and 12 months 
(determination of cotinine in urine). 
o Assessing the proportion of participants being abstinent from combustible 
cigarettes, after 1, 3, 6, and 12 months 
o Survey of the Fagerström test, survey after craving, absence/sick days of the 
previous year as well as QoL survey 

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o Assessment of the adverse events of the products as well as the withdrawal 
symptoms (no nausea, difficulty falling asleep and staying asleep, dry mouth, 
etc.) 
o Assessment of respiratory symptom burden (cough, perceived shortness of 
breath via mMRC or CAT) 
o Optional: Assessment / improvement of the function of the small airways or 
lung function 
o Optional: Assessment / improvement of central blood pressure measurements 
and arterial vascular stiffness 
For this, participants who change from low to high risk by risk re-evaluation and who 
report to be current smokers will be randomized 1:1 to receive an intensified smoking 
cessation program or a short standard smoking cessation counselling. 
 
LC detection rates (PPV) will be assessed after LDCT for low risk participants (after 
PLCOM2012 and NELSON re-evaluation) with positive LC biomarker findings 
(metabolomic blood test, BioMark Diagnostics, Richmond, Canada) and compared to 
detection rates of the NELSON and PLCOM2012 risk criteria. 
 
LC detection rates (PPV) and application rates of invasive procedures will be assessed 
for positive LDCT findings which are discussed within MDT with respect to 
supplementation with blood biomarker results (metabolomic blood test, BioMark 
Diagnostics). Therefore, high risk participants who receive an additional LDCT (initial 
and by risk re-evaluation) will be randomized 1:1 to a biomarker-reporting arm, in 
which MDT discussions of positive LDCT findings will be supplemented with blood 
biomarker results, and a control arm without reporting of biomarker results to the 
MDT.  
 
 
4.3 
Other Variables and Covariates 
No changes to initial protocol (for details, see section 9. Attachments). 
5. 
STATISTICAL ANALYSIS PLAN 
5.1 
Statistical Methods ± General Aspects 
No major changes to initial protocol (for details, see section 9. Attachments). Only details 
concerning the new secondary endpoint to study the effectiveness of a guideline-based and 

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intensified smoking cessation program in comparison to the already included standard 
smoking cessation counselling are included in section 5.1.2. 
5.1.1 
Primary Objective(s):  Calculation of Epidemiological Measure(s) of Interest 
(e.g. descriptive statistics, hazard ratios, incidence rates, test/retest reliability) 
No changes to initial protocol (for details, see section 9. Attachments). 
5.1.2 
Secondary Objective(s):  Calculation of Epidemiological Measure(s) of Interest 
(e.g. hazard ratios, incidence rates, test/retest reliability) 
Secondary objectives:  
- 
to integrate blood biomarkers prospectively in a LDCT screening program to reduce 
the number of false positive invasive procedures (resections or biopsies). 
Hypotheses: Null hypothesis (H0): The positive predictive value (PPV) for the invasive 
procedures in the HANSE study in the MDT group with integrated blood biomarker (BioMark 
Diagnostics, Richmond, Canada) and for the MDT group without integrated blood biomarker 
(BioMark Diagnostics, Richmond, Canada) are equal. 
Alternative hypothesis (H1): The positive predictive value (PPV) for the invasive procedures 
in the HANSE study in the MDT group with integrated blood biomarker (BioMark 
Diagnostics, Richmond, Canada) and for the MDT group without integrated blood biomarker 
(BioMark Diagnostics, Richmond, Canada) are different. 
Statistical procedure: Kosinski test (weighted generalized score statistic)  
- 
to report the test performance of the blood test (BioMark Diagnostics, Richmond, 
Canada) in the low risk and the high risk populations  
Statistical procedure: ROC curve analysis, AUC values, sensitivity, specificity. 
Statistical significance of the effects of integrating blood biomarker tests into the MDT is 
determined using the p-values, at which Bonferroni correction for multiple testing will be 
applied to keep the overall alpha level < 0.05. If the p-value is below the limit yielded by the 
Bonferroni correction, the null hypothesis can be rejected and the alternative hypothesis that 
the PPV for invasive procedures is increased by integration of blood tests into the MDT can 
be accepted. 
 
The study compares an intensified smoking cessation program (including group therapy 
together with medication and non-medication support) with a standard smoking cessation 
counselling in terms of the success of nicotine abstinence. The main endpoint is the number of 
patients who are nicotine-free after 6 months. 

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Hypotheses: Null hypothesis (H0): The success rates (nicotine abstinence) between the brief 
intervention and the group therapy are equal. 
Alternative hypothesis (H1): The success rates between the brief intervention and the group 
therapy differ. 
Statistical procedure: As this is a binary dependent variable (nicotine abstinence: yes/no), a 
logistic regression or a chi-square test is suitable. In this case, logistic regression is 
recommended, as additional covariates can be included in the model. 
Procedure for the statistical analysis:  
In the first step, the data are cleaned and variables are created which, among other things, code 
nicotine abstinence after 6 months (1 = nicotine-free, 0 = not nicotine-free) and the 
intervention groups (0 = standard smoking cessation, 1 = intensified smoking cessation). 
The distribution of the characteristics (age, gender, nicotine abstinence, previous lung 
diseases, CT findings) is calculated in the two groups.  
In the third step of the analysis, a logistic regression is performed to evaluate the effect of 
intensified smoking cessation compared to standard smoking cessation, with the intervention 
group included as a predictor variable and other variables as covariates to control for. The 
estimated coefficients, odds ratios (ORs) and confidence intervals (CIs) are documented as 
results. 
In the fourth step, the statistical significance of the effects of the intervention group is 
determined using the p-values, at which Bonferroni correction for multiple testing will be 
applied to keep the overall alpha level < 0.05. If the p-value is below the limit yielded by the 
Bonferroni correction, the null hypothesis can be rejected and the alternative hypothesis that 
the group therapy is significantly more effective than the brief intervention can be accepted.  
In the fifth step, a sensitivity analysis is carried out to check the robustness of the results. The 
possible influencing factors (age, gender, etc.) are taken into account and interaction effects 
are examined. Secondary parameters will be analysed by similar analysis models if possible. 
Safety parameters will be analysed by descriptive statistics only. 
The results are interpreted in the context of the efficiency of the forms of intervention and 
possible limitations of the study are discussed. 
5.1.3 
Exploratory Objective(s):  Calculation of Epidemiological Measure(s) of 
Interest (e.g. hazard ratios, incidence rates, test/retest reliability) 
No changes to initial protocol (for details, see section 9. Attachments). 

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5.2 
Bias 
5.2.1 
Methods to Minimize Bias 
No changes to initial protocol (for details, see section 9. Attachments). 
5.2.2 
Adjustment for Multiple Comparisons 
 
Bonferroni correction for multiple testing will be applied to keep the family wise error rate of 
the secondary endpoints < 0.05 when testing for statistical significance of the effects of 
integrating blood biomarker tests into the MDT as well as of the effect of an intensified 
smoking cessation program, respectively. If the p-values are below the limit yielded by the 
Bonferroni correction, the null hypotheses can be rejected and the alternative hypotheses can 
be accepted. 
5.2.3 
Strengths and Limitations 
The general strengths and limitations of the HANSE study remain unchanged (for details, see 
section 9. Attachments). However, this protocol amendment possesses the great potential of 
reducing two important limitations of the initial study protocol: 
I) 
LC detection rates within the low risk cohort, which were up to now only estimated 
on the basis of the respective PLCOM2012 score, can now be proven - at least for 
participants changing from low to high risk by PLCOM2012 and NELSON re-
evaluation or by positive blood biomarker results.  
II) 
The relatively short time horizon of the two screening rounds within the initial 
study protocol will be broadened to ~4 years, including a third screening round. 
This will allow for confirmation of the significant difference in terms of LC 
detection between the PLCOM2012  % (6 years) risk score and the NELSON 
criteria, also considering slowly growing LC cases which might affect LC 
detection rates. 
5.3 
Interim Analyses (Optional) 
Unchanged from initial study protocol 
5.4 
Sample Size and Power Calculations 
Expected LC cases in initially low risk participants who will receive LDCT screening within 
this study amendment either by switching to high risk after risk re-evaluation or by positive 
blood biomarker results for early LC detection will be estimated for LDCT justification. 
Low Æ high risk participants by PLCOM2012 re-evaluation 
Approximately 800 initially low risk participants are estimated to become high risk by 
PLCOM2012 re-HYDOXDWLRQ % after 6 years). However, correct high risk-status will be 

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assessed before LDCT performance by qualified medical staff. During the baseline LDCT 
screening of the HANSE study within initially high risk participants, the median PLCOM2012 
score was 2.47 % and 64 LC cases were detected within 5.191 LDCT scans. Conservatively 
assuming a median PLCOM2012 score of 1.60 % for participants changing from low to high 
risk, a total of 6 LC cases can be expected to be found within this population (X = 64 LC cases 
/ 2.47 % / 5.191 LDCT scans * 800 LDCT scans * 1.60 % = 6 LC cases). 
Low risk participants with positive blood biomarker findings 
7,464 participants initially did not meet the high risk criteria and consented to participate in 
the HANSE study. Their median PLCOM2012 was 0.45 %, wherefore 17 LC cases can be 
deduced to be detectable within LDCT screening (X = 64 LC cases / 2.47 % / 5.191 LDCT 
scans * 7,464 LDCT scans * 0.45 % = 17 LC cases). Minus ~800 participants and 6 LC cases 
changing to high risk after PLCOM2012 re-evaluation, approximately 6,600 low risk 
participants will be eligible for blood sampling, and 11 LC cases can be estimated to be 
detectable by LDCT within these participants. Taking a sensitivity and specificity of 0.93 each 
as a basis for the metabolomic blood test (BioMark Diagnostics, Richmond, Canada) used for 
testing the low risk group (for details, see Table 1 in section 4.1), it can be estimated that 
approximately 461 low risk participants will be tested positively for LC blood biomarkers and 
will receive a LDCT scan. 
 
Lung cancer 
No lung cancer 
Biomarker-positive 
True positive (A) 
False positive (B) 
Biomarker-negative 
False negative (C) 
True negative (D) 
x Sensitivity = 0.93 = A / (A+C) 
x Specificity = 0.93 = D / (B + D) 
x A + B + C + D = 6,600 participants 
x A = 11 detectable LC cases 
x C = A / 0.93 ± A = 11 / 0.93 ± 11 = 1 case 
x D = 0.93 * (B + D) = 0.93 * (6,600 ± 11 ± 1) = 6,127 cases 
x B = 6,600 ± 11 ± 1 ± 6,127 = 461 cases 
Supplementation of MDT discussions with blood biomarker results 
During LDCT rounds one and two, 236/9,547 (2.47 %) LDCT scans revealed a positive result 
(LungRADS 4A PET, 4B, or 4X) and were discussed within MDT. Within these 236 cases, 93 
LC cases were found, revealing a PPV of 39.4 % (93/236). Subsequently, 184 invasive 
procedures (biopsies or resections) were performed in which 115 malignancies were found, 
revealing a PPV of 62.5 % and a rate of false positive invasive procedures of 37.5 %. Taking 
the rate of 2.47 % positive LDCT results per screening round as a basis and assuming 
approximately 5,261 LDCT scans to be performed in high risk participants within this 
amendment (~4,000 initially high risk + ~800 becoming high risk after PLCOM2012 and 
NELSON re-evaluation + ~461 blood biomarker-positive participants), approximately 130 

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positive LDCT results and 51 LC cases can be estimated to be detected within this screening 
round. 1:1 randomization of the high risk participants aims at equal distribution of these cases 
to MDT blood biomarker supplementation (biomarker reporting group, n = 65) and normal 
MDT discussion (control group, n = 65). If one estimates, that the PPV can be increased from 
62.5% to 90 % by the MDT supplementation with blood biomarker results, a total of 128 
positive LDCT cases randomized in two groups of 64 are sufficient to achieve a power of 80% 
and an alpha level of 0.025 if the PPV of the blood biomarker is 86%. 
Intensified smoking cessation program 
To calculate the number of cases comparing two types of intervention ± standard smoking 
cessation (group B) and intensified smoking cessation (group A) ± the following steps were 
carried out: 
Selection of statistical assumptions: 
x Effect size (odds ratio, OR): For group A versus group B, the OR is 1.77. 
x Effect size (odds ratio, OR): Within group A with or without drug support with an 
(odds ratio, OR): For the group with drug support versus group B, the OR is 3..5. 
x Two-sided significance threshold (alpha): Typically 0.025. 
x Power (1 - beta): Typically 0.8 (80%). 
x Ratio of group sizes: Assume that the group sizes are equal. 
Baseline frequency of the event: 
The baseline frequency of the event (nicotine abstinence) in Group B was estimated from the 
literature in order to accurately perform the case number calculation. For a hypothetical 
baseline frequency, 6% is used for group B.8 
For pooled group therapy, a pooled odds ratio is calculated to capture the benefit of total 
group therapy (with or without medication) over standard smoking cessation. 
A pooled odds ratio that takes into account the effect of group therapy (both with and without 
medication support) is used to calculate the number of cases. To do this, we consider the 
combined effect of group therapy. Taking into account a dropout rate of 25%, this results in a 
required total number of participants of 260, with 130 participants per group (brief 
intervention versus total group therapy). 
6. 
STUDY CONDUCT AND REGULATORY DETAILS 
6.1 
Study Conduct 
6.1.1 
Study Flow Chart and Plan 
No changes to initial protocol (for details, see section 9. Attachments). 

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6.1.2 
Procedures 
General details of the initial protocol remain unchanged and only details differing from the 
initial protocol will be described (for details, see section 9. Attachments). A flow chart of the 
planned procedures within this amendment is shown in Figure 2. 
Only participants who already gave their informed consent to participate in the HANSE study 
will be considered to take part in this study amendment. For this, participants will be informed 
via letter and/or email about the content and purpose of the study amendment and invited to 
participate. Before participants will be contacted, the PLCOM2012 risk score will be re-
evaluated for low-risk participants by increasing their initial age by 4 years. Participants 
UHYHDOLQJDULVNVFRUH % (6 years) will be handled as high-risk participants and invited 
for LDCT screening.  
/RZULVNSDUWLFLSDQWVQRWFURVVLQJWKHULVNVFRUH % (6 years) after age-corrected 
PLCOM2012 re-evaluation will be informed and invited for blood sampling. On site, PLCOM2012 
and NELSON will be re-assessed and participants revealing a PLCOM2012 ULVNVFRUH % 
(6 years) or fulfilling the NELSON criteria will be handled as high-risk participants and 
invited for LDCT screening. Blood sampling will take part during two months prior to the 
start of the third LDCT round, and blood samples will be shipped to BioMark Diagnostics 
(Richmond, Canada) for early LC detection via the metabolic blood test. Participants with 
positive test results will be invited for LDCT screening, together with the total high-risk 
cohort (initially high-risk + high-risk by age-corrected PLCOM2012 re-evaluation and by on-site 
PLCOM2012 and NELSON re-assessment). All participants with positive LDCT results 
scheduled for MDT conference will be randomized 1:1 to a biomarker reporting group or a 
control group. MDT discussions of positive LDCT findings within the reporting group will be 
supplemented with the respective biomarker findings, whereas MDT discussions of the 
control group will take place without biomarker results.  
All participants with a positive LC blood biomarker test will receive a 2nd LDCT about 6 
months after the baseline CT of the HANSE 3rd round or baseline round respectively to 
confirm the negative diagnosis according to the mod. Lung RADS 1.1 score used in the 
HANSE study, except for participants with a positive lung cancer diagnosis on histology. 
All high-risk participants receiving an LDCT with a positive LC blood test will be informed 
personally about their positive results by trained local staff after the date of their (potential) 
MDT, except for  
 low risk participants theoretically switching from low to high risk by an 
increase of their age by four years, who are then rated as low risk at the 
on-site physician PLCOM2012/NELSON risk confirmation and who are 
tested positively for LC blood biomarkers afterwards. Such patients will 
get a second LDCT screening invitation due to their biomarker test 
results and therefore can conclude that they are biomarker-positive. 
Consequently, such participants are excluded from the randomization 
for MDT reporting since blinding can not be maintained. However, they 

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will also be informed personally about their positive results by trained 
local staff after the date of their (potential) MDT.  
 
All (high- and low-risk) participants with negative LC blood test will be informed by mail 
about their negative blood test results also after the date of their (potential) MDT. 
 
Participants eligible for LDCT (low risk participants with positive biomarker test results as 
well as the total high-risk cohort) will be invited for LDCT screening. All study participants 
are required to sign informed consent. All participants except for the original high-risk cohort, 
which received at least one baseline MDCT (n=5191), will be assessed for their final LDCT 
eligibility (check for the initially defined inclusion and exclusion criteria, for details, see 
sections 3.3 and 3.4 within the initial study protocol which can be found on section 9. 
Attachments) at the study sites by qualified medical staff prior to the LDCT scan. Participants 
of the total high-risk cohort will answer standardized questionnaires as previously used for the 
baseline and one year LDCT screening rounds. In addition, these participants will be asked to 
provide blood samples for biomarker analyses of early LC detection (metabolomic blood test, 
BioMark Diagnostics, Richmond, Canada) and will be randomized 1:1 to a biomarker 
reporting group or a control group, if they have a positive LDCT exam (LungRADS 4A PET, 
4B or 4X). MDT discussions of positive LDCT findings within the reporting group will be 
supplemented with the respective biomarker findings, whereas MDT discussions of the 
control group will take place without biomarker results.  
LDCT performance tests and workup will be conducted as described within the initial study 
protocol, except for MDT discussions of positive LDCT findings, which will be supplemented 
with the biomarker results in half of the participants.  
In addition, participants who are current smokers and who change from low to high risk by 
PLCOM2012 or NELSON re-evaluation or by a positive blood test will be randomized 1:1 to 
receive an intensified smoking cessation program or a standard smoking cessation counselling 
IROORZLQJWKH:+2UHFRPPHQGDWLRQVRI5¶VDQG$¶V, when they show-up for their 
baseline LDCT exam. The intensified smoking cessation program comprises behavioural 
counselling and pharmacotherapy with a partial agonist of nicotinic acetylcholine receptors in 
accordance with the guideline recommendations.8 During the standard smoking cessation 
counselling, participants are informed about and encouraged to participate in smoking 
cessation programs comprising professionally guided group courses within short counselling 
sessions by trained staff. Participation is fully voluntary.  

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Figure 2 Procedural flow chart of the study amendment 
Visit type 1: risk assessment and blood sampling of low risk participants 
After invitation, low risk participants willing to provide a blood sample will be able to 
schedule an appointment at their preferred study site. On site, participants will be informed by 
qualified medical staff and their written consent will be obtained. AIWHUZDUGVSDUWLFLSDQWV¶
variables will be collected to re-assess their PLCOM2012 and NELSON score. Participants 
revealing a PLCOM2012 DULVNVFRUH % (6 years) or positive NELSON score will be 
handled as high risk participants and can schedule an appointment for LDCT screening (visit 
type 2). Blood samples for biomarker analysis of early LC detection will be obtained from all 
low risk participants, and blood samples will be shipped to BioMark Diagnostics (Richmond, 
Canada) for early LC detection via the metabolic blood test. After receiving the test results, 
low risk participants with a negative blood test will be informed about their test results via 
letter and/or email. Low risk participants with a positive blood test will be informed 
personally about their positive results by trained local staff after the date of their (potential) 
MDT. As they need to be informed that they have been reassessed to be in the high-risk group 
to be able to schedule an LDCT exam (visit type 2), they will be told initially that a 
reassessment of their general personal data at their initial visit has scored them into the high-

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risk group in order to keep them blinded for potential randomisation for the MDT if they have 
a positive LDCT.. 
All participants will: 
- 
Be questioned whether they receive treatment for any cardiovascular conditions.  
- 
Be assessed of current COPD treatment medication. 
- 
%HDVNHGWRDQVZHUDµFRQVHTXHQFHVRIVFUHHQLQJ¶VXUYH\ 
 
Visit type 2: LDCT of high risk participants. 
All high risk participants (either by initial scoring or by PLCOM2012 and NELSON re-
assessment or by positive blood test) will be invited to schedule an appointment for LDCT 
screening. In case of initially scored high risk participants, LDCT of this amendment reflects a 
third screening round, whereas in case of participants changing from low to high risk during 
conduct of this amendment LDCT reflects a first screening round. 
On site, all high risk participants will be informed by qualified medical staff and their written 
consent will be obtained. Variables will be collected to confirm the high risk status of the 
high-risk subgroup with estimated PLCOM2012 age adapted (4y) risk score based on the two 
risk scoring models (NELSON/PLCO) and the final LDCT eligibility (check for the initially 
defined inclusion and exclusion criteria, for details, see sections 3.3 and 3.4 within the initial 
study protocol which can be found on section 9. Attachments) will be assessed by qualified 
medical staff prior to the LDCT scan.  
As described in the initial study protocol, all participants will: 
- 
Be questioned whether they receive treatment for any cardiovascular conditions or 
whether they initiated treatment for any cardiovascular conditions during the study  
- 
Be assessed for their smoking status 
- 
Be assessed of current COPD treatment medication and treatment initiation during 
the study 
- 
%HDVNHGWRDQVZHUDµFRQVHTXHQFHVRIVFUHHQLQJ¶VXUYH\ 
prior to undergoing LDCT. Lung LDCT assessment will be conducted as described in the 
initial study protocol and randomization to the four reporting groups will be maintained for 
participants initially scored as high risk. Importantly, all high risk participants (initial or by 
risk re-evaluation) will be randomized after receiving the initial LDCT scan if their LDCT 
result is positive (Lung RADS 4A PET, 4B, 4X). Randomization will not affect the procedure 
and the schedule of the cancer screening process but only determine the allocation of the 
participants to a biomarker reporting group, in which MDT discussions are supplemented by 
biomarker results, or a control group. Randomization ratio of 1:1 will provide equally sized 
groups of patients in both groups using the actual PLCOM2012 score (by reassessment) as 
stratification factor. Patients being rated as low risk by PLCOM2012 and NELSON 
reassessment during visit type 2 and who will be tested positively for LC blood biomarkers 

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afterwards will be excluded from this randomization since they can deduce their positive 
blood biomarker findings from the procedural steps and become unblinded. This 
randomization is independent of and will not affect the initial reporting group randomization. 
Differing from the initial study protocol, participants changing from low to high risk will not 
be randomized into a reporting group, and coronary calcium score and emphysema score will 
always be reported in this group. Instead, participants of this group who reported to be current 
smokers will be randomized to an intensified smoking cessation program and a control group 
with standard smoking cessation counselling. Randomization ratio of 1:1 will provide equally 
sized groups of patients in both groups including age (5 year groups) and sex stratification. 
Visit type 3: follow-up LDCT schedule 
In accordance with the initial study protocol, follow-up LDCT scans will be scheduled 
according to the LDCT findings (LungRADS categories). In case of LungRADS 3, an 
additional LDCT will be performed after ~6 months. In case of LungRADS 4A 3months, an 
additional LDCT will be performed after ~3 months.  
All participants with a positive LC blood biomarker test will receive a 2nd LDCT about 6 
months after the baseline CT of the HANSE 3rd round or baseline round respectively to 
confirm the negative diagnosis according to the mod. Lung RADS 1.1 score used in the 
HANSE study, except for participants with a positive lung cancer diagnosis on histology. In 
case the first LDCT scan of a blood biomarker-positive participant reveals LungRADS 3, the 
regular ~6 months follow-up LDCT will be the same as the blood biomarker-based follow-up. 
All participants with positive blood biomarker results who are scored intermediate 
(LungRADS 3 or 4A 3months) or positive (LungRADS 4A PET, 4B, 4X) at their 6 months 
follow-up LDCT will be discussed in the MDT. Such patients will, however, not be 
randomized since their positive blood biomarker status is known at that time point. 
 
Visit type 4: additional smoking cessation visits 
In addition to behavioural counselling and pharmacotherapy (intensified program) and 
information about voluntary group courses (standard program), participants of both smoking 
cessation programs will be invited for additional visits at 1, 3, 6, and 12 months after the 
initial LDCT scan. Within these visits, urine will be analysed for cotinine as a measure of 
nicotine consumption and questionnaires (Fagerström test, craving survey, absence/sick days 
of the previous year, as well as QoL survey) will be completed by the participants. 
6.1.3 
Quality Control 
No changes to initial protocol (for details, see section 9. Attachments). 

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6.2 
Protection of Human Subjects 
General details of the initial protocol remain unchanged (for details, see section 9. 
Attachments). 
However, since the procedures described within this protocol amendment are not part of the 
initial protocol and hence not described within the initial patient information sheets and the 
informed consent forms, these documents will be adapted respectively, participants will be 
newly informed, and the written informed consent (re-consent) will be obtained before 
conduct of any of the procedures described within this protocol amendment. 
6.3 
Collection and Reporting of Adverse Events/Adverse Drug 
Reactions/Special Situations 
No changes to initial protocol (for details, see section 9. Attachments). 

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7. 
LIST OF REFERENCES 
1. de Koning, H. J. et al. Reduced Lung-Cancer Mortality with Volume CT Screening in a 
Randomized Trial. N. Engl. J. Med. 382, 503±513 (2020). 
2. Tammemägi, M. C. et al. Selection criteria for lung-cancer screening. N. Engl. J. Med. 368, 
728±736 (2013). 
3. Vogel-Claussen, J. et al. Design and Rationale of the HANSE Study: A Holistic German 
Lung Cancer Screening Trial Using Low-Dose Computed Tomography. ROFO. Fortschr. 
Geb. Rontgenstr. Nuklearmed. 194, 1333±1345 (2022). 
4. Vogel-Claussen, J., Bollmann, B.-A. & May, K. WCLC 2024 - MA18.05. Effectiveness of 
NELSON vs PLCOm2012 Lung Cancer Screening Eligibility Criteria: Final Analysis of 
the Prospective German HANSE Study. (2024). 
5. Dama, E. et al. Biomarkers and Lung Cancer Early Detection: State of the Art. Cancers 13, 
3919 (2021). 
6. Kenaan, N. et al. Advances in early detection of non-small cell lung cancer: A 
comprehensive review. Cancer Med. 13, e70156 (2024). 
7. Bibikova, M. & Fan, J. Liquid biopsy for early detection of lung cancer. Chin. Med. J. 
Pulm. Crit. Care Med. 1, 200±206 (2023). 
8. S3-Leitlinie Rauchen und Tabakabhängigkeit: Screening, Diagnostik und Behandlung 
Version 3.1 - Januar 2021 AWMF-Register Nr. 076-006. 
 
 

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8. 
APPENDICES 
No changes to initial protocol (for details, see section 9. Attachments). 
 

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9. 
ATTACHMENTS 
KB-Observational+ES
CR+Protocol+HANSE+