HER2 in Advanced NSCLC: an Observational, Prospective, Multi-centric Study Exploring HER2 Mutations Incidence and Therapeutic Management in Italy. The HEROS Study - GOIRC-01-2022
Conditions: NSCLC (Advanced Non-small Cell Lung Cancer), HER2
Sponsor: Gruppo Oncologico Italiano di Ricerca Clinica

HEROS Study - Protocol -  version 1.0 of 02 May 2024 
 
Pag. 1 of 27 
 
 
HER2 in advanced NSCLC: an Observational, prospective, multi-
centric Study exploring HER2 mutations incidence and therapeutic 
management in Italy. The HEROS study - GOIRC-01-2022 
 
Protocol code:  
 
 
 
GOIRC-01-2022 
 
 
 
 
Sponsor: 
Gruppo Oncologico Italiano di Ricerca Clinica 
(GOIRC)  
 
Coordinating Investigator:  
 
Marcello Tiseo 
 
Steering Committee:  
Marcello Tiseo, Sara Pilotto, Roberta Minari, 
Alessandro Leonetti, Michele Milella, Diana 
Giannarelli, 
Francesco 
Passiglia, 
Umberto 
Malapelle, Silvia Novello 
 
Coordinator Site:  
 UOC di Oncologia Medica, Azienda Ospedaliero-
Universitaria, Parma 
 
Protocol Version:   
 
 
1.0 of 02.05.2024 
 
 
 
 
 
Confidentiality Statement  
The information contained in this document, is the property of GOIRC, and therefore provided to you 
in confidence as an investigator, potential investigator or consultant, for review by you, your staff and 
an applicable Independent Ethics Committee. This information will not be disclosed to others without 
written authorization. 

 
 
 
 
HEROS Study - Protocol -  version 1.0 of 02 May 2024 
 
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STUDY STAFF  
COORDINATING INVESTIGATOR   
Prof. Marcello Tiseo 
Professore Associato e Direttore Scuola di Specializzazione in Oncologia Medica, Università di Parma 
SS di U.O. "Gestione attività ambulatoriali oncologiche complesse" 
U.O. Oncologia Medica 
Azienda Ospedaliero-Universitaria di Parma 
Via A. Gramsci, 14; 43126 Parma 
Tel: +39 0521702977; Fax: +39 0521995448 
E-mail: marcello.tiseo@unipr.it  
 
Co-COORDINATING INVESTIGATOR   
Prof.ssa Sara Pilotto 
Professore Associato in Oncologia Medica, Università di Verona 
U.O.C. Oncologia  
Azienda Ospedaliero Universitaria Integrata di Verona 
P.le L.A. Scuro, 10; 37134 Verona 
Tel: +39 0458028502 
E-mail: sara.pilotto@univr.it  
 
SCIENTIFIC COORDINATOR 
Dr. Alessandro Leonetti 
U.O. Oncologia Medica 
Azienda Ospedaliero-Universitaria di Parma 
Via A. Gramsci, 14; 43126 Parma 
Tel: +39 0521702316; Fax: +39 0521995448 
E-mail: aleonetti@ao.pr.it 
 
Co-SCIENTIFIC COORDINATOR 
Dr. Lorenzo Belluomini 
U.O.C. Oncologia  
Azienda Ospedaliero Universitaria Integrata di Verona 
P.le L.A. Scuro, 10; 37134 Verona 
Tel: +39 0458028502 
E-mail: lorenzo.belluomini@univr.it 
 
CLINICAL TRIAL OFFICE  
Dr.ssa Roberta Camisa 
Dr. Paola Onorini 
U.O. Oncologia Medica 
Azienda Ospedaliero-Universitaria di Parma 
Via A. Gramsci, 14; 43126 Parma 
Tel: +39 0521702682; Fax: +39 0521995448 

 
 
 
 
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E-mail: rcamisa@ao.pr.it; paola.onorini@ao.pr.it  
 
Dr.ssa Jessica Insolda 
U.O. Oncologia Medica 
Azienda Ospedaliero-Universitaria Integrata di Verona 
P.le L.A. Scuro 10, 37134, Verona 
jessica.insolda@univr.it 
Tel: + 39 0458123876 Borgo Trento 
Tel: + 39 0458128120 Borgo Roma 
 
 
LABORATORY 
Dr.ssa Roberta Minari 
U.O. Oncologia Medica 
Azienda Ospedaliero-Universitaria di Parma 
Via A. Gramsci, 14; 43126 Parma 
Tel: +39 0521702316; Fax: +39 0521995448 
E-mail: rominari@ao.pr.it  
 
Dr.ssa Anna Caliò 
Department of Diagnostic and Public Health 
Section of Pathology 
University of Verona, Italy 
Tel: 0458127458 
E-mail: anna.calio@univr.it 
 
CONTRACT RESEARCH ORGANIZATION (CRO) 
Dr.ssa Irina Mantovani 
VIS Ethic Research 
Via Lavezzola, 140 – 44123 Ferrara (FE) 
Email: i.mantovani@vis-er.com 
Tel: +39 344 011 9397 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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AMENDMENTS  
 
NUMBER 
VERSION 
DESCRIPTION  
APPROVAL 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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INVESTIGATOR AGREEMENT  
 
Study titol: “HER2 in advanced NSCLC: an Observational, prospective, multi-centric 
Study exploring HER2 mutations incidence and therapeutic management in Italy. The 
HEROS study”. 
 
Coordinating Investigator 
 
Prof. Marcello Tiseo 
 
Signature _______________________________________________________________  
 
Date _________ 
 
 
I have read the Protocol and I agree to conduct the study as detailed herein and in 
compliance with ICH Guidelines for Good Clinical Practice and applicable regulatory 
requirements.  
 
Principal Investigator 
 
Dr./Prof. ________________________________________________________________  
 
Signature _______________________________________________________________  
 
Date ________ 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
HEROS Study - Protocol -  version 1.0 of 02 May 2024 
 
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TABLE OF CONTENT 
1. PROTOCOL SYNOPSIS ............................................................................................... 8 
2. BACKGROUND AND RATIONALE ............................................................................. 11 
2.1 
Background ..................................................................................................... 11 
2.2 
Study rationale ................................................................................................ 12 
3 
STUDY OBJECTIVES ................................................................................................ 12 
3.1 
Primary objective ............................................................................................ 12 
3.2 
Secondary objectives ...................................................................................... 12 
3.3 
Exploratory objectives .................................................................................... 12 
4 
STUDY DESIGN ....................................................................................................... 13 
5 
STUDY POPULATION .............................................................................................. 13 
5.1 
Inclusion criteria ............................................................................................. 13 
6 
VARIABLES AND EPIDEMIOLOGICAL MEASUREMENTS ........................................... 14 
6.1 
Exposures ....................................................................................................... 14 
6.2 
Medical history and demographic data ........................................................... 14 
6.3 
Tumor molecular typing .................................................................................. 14 
7 
DESCRIPTION OF STUDY ASSESSMENTS ................................................................. 15 
7.1 
Data collection ................................................................................................ 15 
7.2 
Samples collection (only for Prospective Biomarker Analysis) ......................... 15 
7.3 
Storage, Re Use and Destruction of Biological Samples (only for Prospective 
Biomarker Analysis) .................................................................................................. 15 
7.4 
Radiological Assessments ............................................................................... 16 
7.5 
Molecular Analyses (only for Prospective Biomarker Analysis) ....................... 16 
7.6 
Schedule of activities for Prospective Biomarker Analysis ............................... 17 
8 
CRITERIA OF EVALUATION ...................................................................................... 17 
8.1 
Primary endpoints .......................................................................................... 18 
8.2 
Secondary endpoint ........................................................................................ 18 
8.3 
Exploratory endpoints ..................................................................................... 18 
8.3.1 Objective response ...................................................................................... 19 
8.3.2 Real World Objective Response Rate ........................................................... 19 
8.3.3 Disease Control Rate ................................................................................... 19 
8.3.4 Definition of disease progression ................................................................ 19 
8.3.5 Real World Progression-free survival ........................................................... 19 
8.3.6 Time to next treatment ............................................................................... 19 
8.3.7 Time to treatment discontinuation .............................................................. 20 
8.3.8 Overall survival ............................................................................................ 20 
9 
STUDY DISCONTINUATION ..................................................................................... 20 
10 
STATISTICAL CONSIDERATIONS ........................................................................... 20 

 
 
 
 
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10.1 Statistical design and sample size ................................................................... 20 
10.2 Statistical analysis plan ................................................................................... 21 
10.2.1 Analysis populations’................................................................................ 21 
10.2.2 Statistical methods ................................................................................... 21 
10.2.3 Data recording and display ....................................................................... 22 
10.2.4 End of study ............................................................................................. 22 
11 
DATA MONITORING BOARD ................................................................................ 22 
12 
INVESTIGATOR AUTHORIZATION PROCEDURE .................................................... 23 
13 
PATIENT REGISTRATION PROCEDURE .................................................................. 23 
14 
FORMS AND PROCEDURES FOR COLLECTING DATA ............................................ 23 
15 
DATA QUALITY ASSURANCE ................................................................................ 23 
16 
ETHICAL CONSIDERATIONS ................................................................................. 24 
16.1 Patient protection ........................................................................................... 24 
16.2 Subject identification ...................................................................................... 24 
16.3 Informed consent............................................................................................ 24 
17 
ADMINISTRATIVE RESPONSABILITIES .................................................................. 25 
17.1 Overall study management ............................................................................. 25 
17.2 Clinical Trial Office .......................................................................................... 25 
17.3 CRO ................................................................................................................. 25 
18 
SPONSORSHIP .................................................................................................... 25 
19 
INSURANCE ........................................................................................................ 25 
20 
PUBLICATION POLICY.......................................................................................... 25 
21 
REFERENCES ....................................................................................................... 26 
 
 
 
 
 
 

 
 
 
 
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1. PROTOCOL SYNOPSIS 
 
Study Title 
HER2 in advanced NSCLC: an Observational, prospective, multi-centric Study 
exploring HER2 mutations incidence and therapeutic management in Italy. 
The HEROS study. 
Sponsor 
 
Version  
Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC) 
 
1.0 of 02.05.2024 
Rationale 
Considering the clinical and molecular heterogeneity of HER2-aberrant NSCLC, 
currently lacking real-world evidence about prevalence and specific treatment 
algorithms, the HEROS study aims to investigate the current diagnostic and 
therapeutic approach towards HER2-mutated NSCLC in the Italian clinical 
practice, with the final aim of enriching the current knowledge, as well as to 
provide exploratory data regarding outcome with different treatments, 
according to the specific type of HER2 mutation. 
Primary objective 
 
To describe the prevalence of HER2 mutations in advanced NSCLC in Italy. 
Secondary 
objective 
 
To describe the therapeutic management in clinical practice of HER2 mutated 
advanced NSCLC patients. 
Exploratory 
objectives 
• 
To describe potential correlations among baseline demographics, 
clinicopathological variables and HER2 mutations in advanced NSCLC. 
• 
To explore the clinical outcome according to the type of anti-HER2 
therapies applied in HER2 mutated advanced NSCLC patients. 
• 
To investigate potential correlations between PD-L1 expression and 
HER2 mutations. 
• 
To analyse the association between co-mutations and clinical outcome 
in HER2 mutated advanced NSCLC patients treated with anti-HER2 
agents. 
The subsequent exploratory objectives are only for a Prospective Biomarker 
Analysis that will be performed in a subset of centers (25 centers): 
• 
To analyse the association between FcγR polymorphisms (evaluated in 
blood and/or tissue) and clinical outcome in HER2 mutated advanced 
NSCLC patients treated with anti-HER2 agents. 
• 
To analyse the IHC expression of HSP90 on NSCLC tissue samples to 
test its potential impact as a prognostic/predictive factor during 
treatment with anti-HER2 agents. 
• 
To analyse the serum level of HSP90 and circulating level of HER2 
mutation in cell-free DNA as possible prognostic/predictive factors 
during treatment with anti-HER2 agents. 
Study design 
The HEROS study is an Italian observational multicenter prospective study 
aimed to investigate the current diagnostic and therapeutical approach 
towards HER2 mutated NSCLC in clinical practice. The enrolment will start in 

 
 
 
 
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September 2024 until September 2025. A 12-months follow-up window will 
be performed.  
The HEROS study will be conducted in partnership with ATLAS project: all 
patients prospectively enrolled in ATLAS project will be used to fulfil primary 
and secondary objectives. Information will be obtained by querying ATLAS 
database (59 centers) in aggregated manner. ATLAS project is a multi-centric 
retrospective/prospective Italian study aimed to describe prevalence of 
different oncogene alterations in all new diagnosed advanced NSCLC patients 
(5,000 patients/year). In particular, prevalence of HER2-mutated patients will 
be calculated on all patients enrolled in ATLAS project. Approximately 100 
HER2 mutated patients enrolled in ATLAS study will be studied for secondary 
and explorative objectives. 
Moreover, a subset of centers (25 centers) will enrol patients also for 
Prospective Biomarker Analysis in order to perform exploratory analysis on 
blood and tissue samples according to the availability of tissue specimens 
from the primary tumor or metastatic sites. 
All centers that will participate for Prospective Biomarker Analysis necessarily 
will be included into ATLAS project. 
Number 
of 
Patients 
 
The study will include three cohorts of patients:  
1. Cohort 1: Prevalence study population (5,000 patients from ATLAS 
project in 59 Italian centers) 
2. Cohort 2: HER2 mutations study population (100 patients from Cohort 
1) 
3. Cohort 3: Prospective Biomarker Analysis population (50 patients from 
Cohort 2 in 25 Italian centers) 
Inclusion Criteria 
Cohort 1 (Prevalence study population) 
1. Male or female, aged at least 18 years. 
2. Pathologically confirmed diagnosis of NSCLC from September 2024 to 
September 2025. 
3. Locally advanced (IIIC), not amenable to multimodal approach (chemo-
radiotherapy), or metastatic (IV) NSCLC according to TNM VIII edition. 
4. Enrolled in ATLAS project. 
Cohort 2 (HER2 mutations study population) 
5. Included in Cohort 1. 
6. Presence of HER2 mutation.  
7. Enrolled in ATLAS project. 
Cohort 3 (Prospective Biomarker Analysis population) 
8. Included in Cohort 2. 
9. Availability of tissue sample from the first 50 patients enrolled in 
cohort 2. 
10. Written informed consent (HEROS project) must be obtained before 
any study-related procedure. 
Study duration 
The duration of the study is expected to be a maximum of 24 months. The 
study recruitment period is expected to be approximately 12 months.  

 
 
 
 
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Primary Endpoint 
 
Percentage of advanced NSCLC with HER2 mutations in Italy clinical practice. 
Secondary 
Endpoints 
Percentage of type of therapies in advanced HER2 mutated NSCLC patients. 
Exploratory 
Endpoints 
• Percentage of clinicopathological variables (i.e. central nervous system 
metastases, ECOG PS, smoking status, etc) in advanced HER2 mutated 
NSCLC patients. 
• Real world objective response rate (RW-ORR), real world disease 
control rate (RW-DCR), real world progression-free survival (RW-PFS), 
time to next treatment (TTNT), time to treatment discontinuation (TTD) 
and overall survival (OS), according to the type of anti-HER2 therapies 
applied in advanced HER2 mutated NSCLC patients; 
• Percentage of PD-L1 expression among advanced HER2 mutated NSCLC 
patients; 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of 
anti-HER2 therapies according to the most represented co-mutations 
(e.g. TP53); 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of 
anti-HER2 therapies applied in advanced HER2 mutated NSCLC patients 
and the FcγR polymorphisms (evaluated in blood and/or tissue); 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of 
anti-HER2 therapies applied in advanced HER2 mutated NSCLC patients 
and the IHC tissue expression of HSP90; 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of 
anti-HER2 therapies applied in advanced HER2 mutated NSCLC patients 
and the serum level of HSP90 and circulating level of HER2 mutation in 
cell-free DNA. 
Statistical 
methods 
The sample size was estimated according to the available literature and to the 
patients’ population expected to fulfil the pre-specified inclusion criteria at 
the participating centers during the enrolment time. Considering the 
incidence of HER2 mutations in advanced NSCLC, the number of centers, trial 
duration, as well as the current diagnostic management of lung cancer, we 
plan to enroll from ATLAS project (approximately 5,000 advanced NSCLC 
patients per year) information regarding 100 HER2-mutated patients, of which 
50 patients (2-3 patients/center) will be enrolled also for Prospective 
Biomarker Analysis. Descriptive statistics will be performed to analyze the 
characteristics and outcomes of patients. 
 
In summary: 
Prevalence study population: 5,000 patients 
HER2 mutations study population: 100 patients 
Prospective Biomarker Analysis population: 50 patients. 
 

 
 
 
 
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2. BACKGROUND AND RATIONALE  
 
 
 
 
 
 
 
2.1 
Background 
In recent years, HER2 alterations emerged as candidate actionable targets in advanced non-small-cell 
lung cancer (NSCLC). Although a clear identification and definition of HER2 alterations in lung cancer 
are still missing, three main subgroups were recognized: mutation, amplification, and protein 
overexpression. The assessment of HER2 alterations in NSCLC is performed by immunohistochemistry 
(IHC) for overexpression, fluorescent in situ hybridization (FISH), and next generation sequencing 
(NGS) for genetic alterations [1].  
The incidence of HER2 mutations in NSCLC is 2-4%, mainly insertions in exon 20-21 causing 
constitutive activation of the tyrosine kinase domain. The most common is duplication or insertion of 
the amino acids YVMA at codon 75 in exon 20, the A775_G776 ins YVMA, accounting for around 80%-
90% of all HER2 mutations [2]. HER2 overexpression is detected in 2-38% of cases, while amplification 
is observed in 2-5% [3]. Differently from breast and gastric cancer, the association between 
amplification and overexpression is controversial with no significant correlation between increased 
gene copy number and protein expression at IHC [4, 5]. As a potential explanation, the lack of 
standardized testing techniques and cut-offs for defining HER2 positivity represents a crucial issue [6]. 
In terms of clinical characteristics, patients harbouring HER2 alterations are more frequently women, 
young, never smokers, and mainly adenocarcinomas [7, 8].  
To date, phase III prospective randomized studies are missing. Available data exploring anti-HER2 
approaches in NSCLC derive mainly from retrospective studies and early phase prospective trials. 
HER2-mutated NSCLC harbour a worse prognosis with chemotherapy compared with wild-type tumors 
[9].  
Regarding tyrosine kinase inhibitors (TKI), afatinib showed modest activity in this setting (response 
rate, RR 13-19%, with heterogeneous outcomes according to the type of HER2 insertion) [10, 11]. 
Pyrotinib, an irreversible pan-HER receptor TKI, resulted in a RR of 53.3% with a median progression-
free survival (PFS) of 6.4 months [12]. Another irreversible pan-HER TKI, poziotinib, demonstrated in 
the phase II ZENITH20 trial, an ORR of 35.1% and a mPFS of 5.5 months in pre-treated patients 
affected by NSCLC harbouring HER2 exon 20 insertions. Use of poziotinib was limited by relevant 
toxicities (grade 3 diarrhea and rash in 26% and 30%, respectively), requiring a dose reduction in 78% 
of patients [13].  
Recently, the RAIN-701 trial suggested a promising activity (RR 22% and disease control rate, DCR 67%) 
of tarloxotinib, a hypoxia-activated prodrug of a pan-HER TKI, in HER2 exon 20 insertion mutations 
[14]. No significant clinical benefit was observed using lapatinib [15], dacomitinib [16], and neratinib 
[17]. Finally, TKIs were not active in HER2-amplified/-overexpressed NSCLC patients [18]. 
Trastuzumab in combination with chemotherapy (vinorelbine, docetaxel, or paclitaxel) was active in 
pre-treated HER2-mutant lung cancer patients [2, 19, 20]. Ado-trastuzumab emtansine (T-DM1), an 
antibody-drug conjugate targeting HER2, induced a RR of 44% in HER2-mutant lung cancer [21]. 
Moreover, T-DM1 demonstrated efficacy in patients with HER2 overexpression (IHC 3+), especially 
when concomitant HER2 mutations are present [22]. Based on the results of a promising phase 1 trial, 
trastuzumab deruxtecan, an antibody-drug conjugate composed of an anti-HER2 antibody and a 
topoisomerase I inhibitor payload, was recently evaluated in phase 2 DESTINY-Lung01 trial 
demonstrating a very promising clinical activity (RR 61.9%, DCR 90.5%, mPFS 14 months) in patients 
with HER2-mutated NSCLC [23].  

 
 
 
 
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Several monoclonal antibodies (MoAB), including rituximab in follicular lymphoma, cetuximab in colon 
cancer, and trastuzumab in breast cancer, demonstrated to induce antibody-dependent cell-mediated 
cytotoxicity (ADCC), based on the interaction of the Fc domain of the antibody with the Fc-γ receptors 
(FcγR) on the surface of the immune cells, such as natural killer (NK) and macrophages. Specific 
polymorphisms of FcγRIIA, homozygous for histidine expression at position 131, and of FcγRIIIA, 
homozygous for valine at position 158, are associated with an increased ADCC and with better clinical 
outcomes with trastuzumab in both metastatic and adjuvant HER2-positive breast cancer, when 
compared with other FcγR genotypes [24, 25].  
Another promising marker in this setting is represented by the evaluation of HSP90 expression levels 
in HER2 positive NSCLC since its role in regulation and stabilization of HER2 receptor is recognized in 
preclinical and clinical models [26–29]. In this regard, available data showed the co-targeting of HSP90 
and HER2 with HSP90 inhibitors (such as tanespimycin 17-AAG or ganetespib) and trastuzumab, in 
patients affected by trastuzumab-refractory HER2-amplified breast cancer, seemed to restore 
sensitivity to anti-HER2 MoAB, improving clinical outcomes [30, 31].   
Another field of interest is represented by the occurrence of co-mutations together with HER2 
alterations, which have been shown to impact patients’ prognosis and response to targeted 
treatments. In this regard, co-occurring TP53 mutation was found in 52-66% of HER2-mutated NSCLC 
[32, 33], and was associated with a worse prognosis [32]. In addition, TP53, as well as PI3K/AKT/mTOR 
co-mutations, were significantly correlated with worse clinical outcomes in HER2-mutated NSCLC 
patients treated with afatinib [34]. 
In conclusion, HER2 alterations, mainly mutations, recently emerged as a promising actionable target 
in lung cancer, deserving deepened investigations regarding diagnostic and therapeutical 
management, as well as the underlying mechanisms of activity/resistance of anti-HER2 agents. 
 
2.2 
Study rationale 
Considering the clinical and molecular heterogeneity of HER2-aberrant NSCLC, currently lacking 
specific treatment algorithms, the HEROS study aims to investigate the current diagnostic and 
therapeutic approach towards HER2-mutated NSCLC in the Italian clinical practice, with the final aim 
of enriching the current knowledge, as well as to provide exploratory data regarding outcome with 
different treatments (MoAb, TKI, chemotherapy), according to the specific type of HER2 mutations. 
 
3 
STUDY OBJECTIVES 
 
3.1 
Primary objective 
To describe the prevalence of HER2 mutations in advanced NSCLC. 
 
3.2 
Secondary objectives 
To describe the therapeutic management in clinical practice of HER2 mutated advanced NSCLC 
patients. 
 
3.3 
Exploratory objectives 
• 
To describe potential correlations among clinicopathological variables and HER2 mutations in 
advanced NSCLC. 

 
 
 
 
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• 
To explore the clinical outcome according to the type of anti-HER2 therapies applied in HER2 
mutated advanced NSCLC patients. 
• 
To investigate potential correlations between PD-L1 expression and HER2 mutations. 
• 
To analyse the association between co-mutations and clinical outcome in HER2 mutated 
advanced NSCLC patients treated with anti-HER2 agents. 
 
The subsequent exploratory objectives are only for the prospective biological analysis that will be 
performed in a subset of centers (25 centers): 
• 
To analyse the association between FcγR polymorphisms (evaluated in blood and/or tissue) 
and clinical outcome in HER2 mutated advanced NSCLC patients treated with anti-HER2 agents. 
• 
To analyse the IHC expression of HSP90 on NSCLC tissue samples to test its potential impact as 
a prognostic/predictive factor during treatment with anti-HER2 agents. 
• 
To analyse the serum level of HSP90 and circulating level of HER2 mutation in cell-free DNA as 
possible prognostic/predictive factors during treatment with anti-HER2 agents. 
 
4 
STUDY DESIGN 
 
The HEROS study is an Italian observational multicenter prospective study aimed to investigate the 
current diagnostic and therapeutical approach towards HER2-mutated NSCLC in clinical practice.  
The enrolment will last a year, approximately from September 2024 until September 2025. A minimum 
of 12-months follow-up window will be performed following the prospective phase.  
The HEROS study will be conducted in partnership with ATLAS project: all patients prospectively 
enrolled in ALTAS project will be used to fulfil primary and secondary objectives. Information will be 
obtained by querying ATLAS database (59 centers) in aggregated manner. 
A subset of centers (25 centers) will enrol patients also for the Prospective Biomarker Analysis, that 
will be performed on blood and tissue samples according to the availability of tissue specimens from 
the primary tumor or metastatic site. 
 
The study will include three cohorts of patients:  
• Cohort 1: Prevalence study population (5,000 patients) 
• Cohort 2: HER2 mutations study population (100 patients) 
• Cohort 3: Prospective Biomarker Analysis population (50 patients). 
 
5 
STUDY POPULATION 
 
5.1 
Inclusion criteria 
Cohort 1 (Prevalence study population) 
Patients enrolled in ATLAS project that met all the following criteria will be included into the study for 
the primary objective analysis: 
1. Male or female, aged at least 18 years 
2. Pathologically confirmed diagnosis of NSCLC during Heros enrolment period, approximately 
from September 2024 to September 2025 
3. Locally advanced (IIIC), not amenable to multimodal approach (chemo-radiotherapy), or 
metastatic (IV) NSCLC according to TNM VIII edition 

 
 
 
 
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4. Enrolled in ATLAS project 
 
Cohort 2 (HER2 mutations study population) 
5. Included in Cohort 1 
6. Presence of HER2 mutation 
7. Enrolled in ATLAS project 
 
Cohort 3 (Prospective Biomarker Analysis population) 
8. Included in Cohort 2 
9. Availability of tissue sample from the first 50 patients enrolled in cohort 2 
10. Written informed consent (HEROS project) must be obtained before any study-related 
procedure 
 
 
6 
VARIABLES AND EPIDEMIOLOGICAL MEASUREMENTS 
 
6.1 
Exposures 
Data on previous/ongoing anticancer treatment will be collected. Patients will receive anticancer 
treatments as per clinical practice, consisting of chemotherapy, TKIs, immunotherapy or anti-HER2 
agents (i.e., trastuzumab, T-DM1, trastuzumab deruxtecan). Available clinical data about medical 
history, demographic data, tumor molecular typing, treatment compliance and treatment activity for 
patients in cohorts 1, 2 and 3 will be collected from the ATLAS eCRF. Biological exploratory data of 
cohort 3 patients will be collected in a dedicated database, filled out with the results of the biomarker 
analyses. 
 
6.2 
Medical history and demographic data 
Medical history includes: 
Clinically significant diseases (active or resolved) 
Cancer history, including prior cancer therapies and procedures 
Prior surgeries 
Reproductive status 
Smoking history 
ECOG PS 
Presence/absence of central nervous system metastases 
 
Demographic data includes age, sex and self-reported ethnicity. 
The initial cancer diagnosis and the current cancer stage at the time of screening, along with tumor 
histology and all sites of disease, should be recorded per The American Joint Committee on Cancer 
(AJCC), 8th edition (http://www.cancerstaging.com). Any previously identified mutations and the dates 
of identification, must be recorded. 
 
6.3 
Tumor molecular typing 
Data regarding molecular typing of the tumor obtained before enrolment as per clinical practice will 
be collected and will include EGFR, ALK, ROS1 and PD-L1 status. When available, all additional 

 
 
 
 
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molecular alterations will be recorded (i.e., KRAS, RET, BRAF, MET, NTRK). If NGS analysis is available, 
all the molecular alterations will be reported.  
 
Regarding HER2 mutation history, any previously identified mutation, the dates and methods of 
identification, must be recorded at screening. 
 
7 
DESCRIPTION OF STUDY ASSESSMENTS 
 
Cohort 1 (Prevalence study population) 
 
Patients must be enrolled in ATLAS project.  
Data collected for ATLAS project compliant with HEROS project will be collected from ATLAS database. 
 
Cohort 2 (HER2 mutations study population) 
Patients must be enrolled in ATLAS project. 
Data collected for ATLAS project compliant with HEROS project will be collected from ATLAS database. 
 
Cohort 3 (Prospective biological analysis) 
Written informed consent for participation in the HEROS study must be obtained before collecting 
biological samples. Informed Consent Forms for enrolled patients and for patients who are not 
subsequently included will be maintained at the study site.  
Flow-chart of scheduled study assessments is provided in Section 6.6. All assessments must be 
performed and documented for each patient. 
 
7.1 
Data collection 
During the study, clinical data of patients will be collected (See Section 5). Data will be stored for 15 
years at the Medical Oncology Unit of University Hospital of Parma, in a dedicated password-
protected storage folder on a PC in charge of the aforementioned operating unit, under the 
responsibility of Prof. Marcello Tiseo. 
 
7.2 
Samples collection (only for Prospective Biomarker Analysis) 
Patients who will start anticancer treatment will undergo a basal blood sample collection, 
concurrently with the planned routinary blood tests according to clinical practice, that should be 
performed no more than 7 days before starting anticancer treatment, whatever it is. During the 
routine blood test, an extra 20 ml of blood will be drawn for study purposes. 
Tissue samples already collected as per clinical practice will be reviewed. 
 
7.3 
Storage, Re Use and Destruction of Biological Samples (only for Prospective Biomarker 
Analysis) 
Tissue samples will be labelled at the recruiting center, then delivered to the Department of 
Diagnostics and Public Health, University of Verona, P. le L.A. Scuro 10, 37134; Verona (Italy) where 
they will be stored and processed. The tissue samples will be stored for 5 years so that the planned 
evaluations can be repeated or completed. Liquid biopsies will be labelled at the recruiting center, 
then delivered to the Laboratory of Medical Oncology Unit of University Hospital of Parma, where 
they will be stored and processed. 

 
 
 
 
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The data collected during the study will be stored at the Laboratory of Medical Oncology Unit of 
University Hospital of Parma, under the responsibility of Prof. Marcello Tiseo. The processes adopted 
for the coding and storage of samples for molecular analysis are important to maintain subject 
confidentiality. Each sample will be pseudonymized so that it will be impossible to identify the patient 
but related information will remain available and will be used for future studies. In this regard, future 
studies will always concern the same pathology and/or drug of interest of this research, and they will 
be submitted to the ethics committee for approval.  
Samples will be destroyed or returned to the competent Institution, when feasible, after 5 years to the 
end of the study.at the end of the study. The results of any further analyses will be reported either in 
the Clinical Study Report itself or as an addendum, or separately in a scientific report or publication. 
 
7.4 
Radiological Assessments 
All eligible patients who will receive anticancer treatment as per clinical practice will undergo a tumor 
assessment as per clinical practice. Radiological assessments during treatment will be performed 
according to clinical practice. 
Measurable and non-measurable disease must be documented at screening prior to start of 
treatment. Screening assessment should include: 
 
• CT scan (with contrast) of the chest, abdomen and pelvis; MRI of the chest, abdomen, and pelvis 
with a non-contrast CT scan of the chest may be used in patients for whom CT scans with contrast 
are contraindicated (i.e., patients with contrast allergy or impaired renal clearance); 
• Brain CT (with contrast) or MRI scan should be performed at baseline to evaluate CNS metastases in 
all patients.  
 
7.5 
Molecular Analyses (only for Prospective Biomarker Analysis) 
Laboratory of Medical Oncology Unit of University Hospital of Parma will coordinate the collection and 
processing of blood samples.  
Blood samples will be collected from all patients enrolled in the Prospective Biomarker Analysis and 
will be sent to the central laboratory. Each center will process the blood sample in order to obtain an 
aliquot of blood for FcγR analysis, serum sample for quantification of circulating HsP90 levels, and 
plasma samples for cell-free DNA (cfDNA) extraction for HER2 analysis. Genomic DNA (gDNA) and 
cfDNA will be extracted with proper kit from blood and plasma samples, respectively. Where blood 
sample is not available, gDNA will be extracted from normal tissue micro-dissected from tissue biopsy. 
The analysis of FcγR will be performed by a quantitative PCR (qPCR) and specific probes for FcγRIIA 
and FcγRIIIA polymorphisms (rs1801274 and rs396991, Applied Biosystems) following the 
manufacturer's instructions.  
Hsp90 levels will be quantified with ELISA kit (Thermofisher) following the manufacturer's instructions.  
The analysis of HER2 on cfDNA will be performed by a digital droplet PCR (ddPCR) and specific ddPCR 
Mutation Assay (BioRad®, Hercules, CA, USA) following the manufacturer's instructions.  
 
Laboratory of Medical Oncology Unit of University Hospital of Verona will coordinate the collection 
and processing of tissue samples.  
Tissue from biopsy obtained at the time of diagnosis must be submitted in order to perform IHC of 
HSP90 and when feasible, NGS analysis to study co-mutations.  

 
 
 
 
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Immunostaining for HSP90 (clone EPR16621-67, dilution 1:1500, Abcam) will be performed in biopsy 
and surgical NSCLC specimens. All samples will be processed using a sensitive ‘Bond Polymer Refine’ 
detection system in an automated Bond immunohistochemistry instrument (Leica Biosystems). 
Labelling for the marker will be recorded as the percentage of positive cells and intensity of the 
staining. 
Genomic profiling will be performed using the CORE NGS panel developed by the International Cancer 
Genome Consortium that encompasses mutations, copy number alterations, and structural variants in 
a panel of 174 clinically actionable genes, genomic signatures, regulatory elements, immunological 
features, sample integrity/contamination, tumor mutational burden, microsatellite instability, and 
relevant fusion genes. 
 
7.6 
Schedule of activities for Prospective Biomarker Analysis  
Study procedures 
Enrollment phase 
(12 months) 
Follow-up 
(min 12 months) 
Signed Informed Consent Form(s)a 
X 
 
Review of eligibility criteria 
X 
 
Medical history, including cancer and demographic 
informationb 
X 
 
Data on treatment compliance and administration 
X 
X 
Blood samples for gDNA, serum, and cfDNA analysisc 
X 
 
Collection of tumor tissued 
X  
 
IHC analysis 
X 
 
NGS analysis 
X 
 
Tumor assessmente 
X 
 
Survival statusf 
 
X 
aWritten informed consent is required for performing any study-specific tests or procedures. Patient may allow for the 
collection and testing of archival or fresh tumor tissue by signing the main consent form. 
bCancer history includes stage, date of diagnosis and prior antitumor treatment. Demographic information includes sex, 
age and self-reported race/ethnicity. 
cPatients who will receive anticancer treatment will undergo a basal blood sample collection, to be performed no more 
than 7 days before starting treatment. 
d Tissue samples already collected as per clinical practice will be reviewed. 
eAll eligible patients who will receive anticancer treatment will undergo a tumor assessment as per clinical practice. 
Radiological assessments during treatment will be performed according to clinical practice. 
fSurvival status information will be collected every 3 months via telephone calls, patient medical records, and/or clinic 
visits. All patients will be followed for survival unless the patient withdrawn consent; this request must be documented 
in the source documents and signed by the investigator. 
 
 
8 
CRITERIA OF EVALUATION 
 
A summary of objectives and corresponding endpoints is provided below.   
 
Primary Objective 
Primary Endpoint 
Study cohort 
To describe the prevalence of HER2 mutations 
in advanced NSCLC. 
Percentage of advanced NSCLC with HER2 
mutations in Italy clinical practice  
Cohort 1 
Secondary Objectives 
Secondary Endpoints 
 
To describe the therapeutic management in 
Percentage of type of therapies in advanced 
Cohort 2 

 
 
 
 
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clinical practice of HER2 mutated advanced 
NSCLC patients. 
HER2 mutated NSCLC patients. 
Exploratory Objectives 
Exploratory Endpoints 
 
To describe potential correlations among 
clinicopathological 
variables 
and 
HER2 
mutations in advanced NSCLC. 
Percentage of clinicopathological variables (i.e. 
central nervous system metastases, ECOG PS, 
smoking status, etc) in advanced HER2 
mutated NSCLC patients. 
Cohort 2 
To explore the clinical outcome according to 
the type of anti-HER2 therapies applied in 
HER2 mutated advanced NSCLC patients. 
RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS 
according to the type of anti-HER2 therapies 
applied in advanced HER2 mutated NSCLC 
patients. 
Cohort 2 
To investigate potential correlations between 
PD-L1 expression and HER2 mutations. 
Percentage 
of 
PD-L1 
expression 
among 
advanced HER2 mutated NSCLC patients. 
Cohort 2 
To analyse the association between co-
mutations and clinical outcome in HER2 
mutated advanced NSCLC patients treated 
with anti-HER2 agents. 
RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS 
according to the type of anti-HER2 therapies 
according to co-mutations. 
Cohort 2 
To analyse the association between FcγR 
polymorphisms (evaluated in blood and/or 
tissue) and clinical outcome in HER2 mutated 
advanced NSCLC patients treated with anti-
HER2 agents. 
RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS 
according to the type of anti-HER2 therapies 
applied in advanced HER2 mutated NSCLC 
patients 
and 
the 
FcγR 
polymorphisms 
(evaluated in blood and/or tissue). 
Cohort 3 
To analyse the IHC expression of HSP90 on 
NSCLC tissue samples to test its potential 
impact as a prognostic/predictive factor during 
treatment with anti-HER2 agents. 
RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS 
according to the type of anti-HER2 therapies 
applied in advanced HER2 mutated NSCLC 
patients and the IHC tissue expression of 
HSP90. 
Cohort 3 
To analyse the serum level of HSP90 and 
circulating level of HER2 mutation in cell-free 
DNA as possible prognostic/predictive factors 
during treatment with anti-HER2 agents. 
RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS 
according to the type of anti-HER2 therapies 
applied in advanced HER2 mutated NSCLC 
patients and the serum level of HSP90 and 
circulating level of HER2 mutation in cell-free 
DNA. 
Cohort 3 
 
8.1 
Primary endpoints 
The primary endpoint is the percentage of advanced NSCLC with HER2 mutations in Italy clinical 
practice. The prevalence will be calculated by dividing the number of advanced NSCLC with HER2 
mutations enrolled in the ATLAS study by the number of advanced NSCLC patients who underwent 
NGS at the Centers participating to ATLAS project. 
 
8.2 
Secondary endpoint 
The secondary endpoint is the percentage of type of therapies performed in advanced HER2 mutated 
NSCLC patients as per clinical practice. 
 
8.3 
 Exploratory endpoints 
• Percentage of clinicopathological variables (i.e. central nervous system metastases, 
ECOG PS, smoking status, etc) in advanced HER2 mutated NSCLC patients. 
• Real world objective response rate (RW-ORR), real world disease control rate (RW-
DCR), real world progression-free survival (RW-PFS), time to next treatment (TTNT), 

 
 
 
 
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time to treatment discontinuation (TTD) and overall survival (OS), according to the 
type of anti-HER2 therapies applied in advanced HER2 mutated NSCLC patients; 
• Percentage of PD-L1 expression among advanced HER2 mutated NSCLC patients; 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of anti-HER2 
therapies according to co-mutations; 
The subsequent exploratory endpoints are only for Prospective Biological Analysis: 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of anti-HER2 
therapies applied in advanced HER2 mutated NSCLC patients and the FcγR 
polymorphisms (evaluated in blood and/or tissue); 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of anti-HER2 
therapies applied in advanced HER2 mutated NSCLC patients and the IHC tissue 
expression of HSP90; 
• RW-ORR, RW-DCR, RW-PFS, TTNT, TTD and OS according to the type of anti-HER2 
therapies applied in advanced HER2 mutated NSCLC patients and the serum level of 
HSP90 and circulating level of HER2 mutation in cell-free DNA. 
 
8.3.1 Objective response 
Objective Response (OR) is defined as a complete response (CR) or a partial response (PR) at the 
radiological evaluation, based on the Investigator’s assessment, and will be measured according to 
standard RECIST criteria v1.1. 
 
8.3.2 Real World Objective Response Rate 
Real World Objective Response rate (RW-ORR) is defined as the proportion of patients in which an 
objective response will be observed. 
 
8.3.3 Disease Control Rate 
Real World Disease Control Rate (DCR) is defined as the proportion of patients who achieved a 
complete response (CR) or a partial response (PR) or a stability of disease (SD) at the radiological 
evaluation, based on the Investigator’s assessment. 
 
8.3.4 Definition of disease progression 
Disease progression will be defined as any unequivocal progression of disease according to RECIST 
criteria. 
 
8.3.5 Real World Progression-free survival 
Real World Progression-free survival (RW-PFS) is calculated as the interval from the date of treatment 
start to either the date of disease progression or the date of death, whichever occurs first and 
whatever the cause. The date of first documented radiographic progression as assessed by RECIST1.1, 
if available, will be used as the date of event. Patients alive with no evidence of disease progression at 
the time of their last visit are censored at the time of the last examination. 
 
8.3.6 Time to next treatment 

 
 
 
 
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Time to next treatment (TTNT) is calculated as the interval from the date of treatment start to the 
date of commencement of the next line of therapy, if applicable. 
 
8.3.7 Time to treatment discontinuation 
Time to treatment discontinuation (TTD) is calculated as the interval from the date of treatment start 
to the date of treatment end, whatever the cause. Patients alive with treatment ongoing at the time 
of their last visit are censored at the time of the last examination. 
 
8.3.8 Overall survival 
Overall survival (OS) is defined as the time from the date of treatment start to death, whatever the 
cause. The follow-up of patients still alive will be censored at the moment of last visit/contact. 
 
 
9 
STUDY DISCONTINUATION 
 
The Investigator has the right to discontinue a patient from study or withdraw a patient from the 
study at any time. In addition, patients have the right to voluntarily discontinue study or withdraw 
from the study at any time for any reason. Discontinued patients will be not replaced. Reasons for 
withdrawal from the study may include but are not limited to the following: 
 
• Patient withdrawal of consent at any time 
• Investigator determines it is in the best of interest of the patient 
 
Every effort should be made to obtain information on patients who withdraw from the study. The 
primary reason for withdrawal from the study should be documented on the appropriate CRF. 
However, patients will not be followed for any reason after consent has been withdrawn. 
 
10 STATISTICAL CONSIDERATIONS  
 
10.1 Statistical design and sample size 
 
The sample size was estimated according to the patients’ population expected to fulfil the pre-
specified inclusion criteria at the participating centers during the enrolment time. Considering the 
incidence of HER2 mutations in advanced NSCLC, the number of centers, trial duration, as well as the 
current diagnostic management of lung cancer, we plan to collect, from ATLAS project (approximately 
5,000 advanced NSCLC patients per year), information regarding 100 patients and to enroll in HEROS 
study 50 patients (2-3 patients/center) for biological exploratory analysis. 
Descriptive statistics will be performed to analyze the prevalence of HER2 mutations and the 
prevalence of characteristics of patients in the overall registered population. Correlations with 
treatment outcomes will be analyzed.    
Assuming from literature that prevalence of HER2 mutation is 2% we plan to consider as denominator 
of this rate about 5,000 NSCLC patients. This sample size will allow to estimate this prevalence with a 
95% confidence interval based on the exact binomial calculation ranging from 1.6% to 2.4%. 
   

 
 
 
 
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10.2 Statistical analysis plan 
 
10.2.1 Analysis populations’ 
 
Registered population: registered population will include all patients who were enrolled into the 
ATLAS study. 
Study population: patients fulfilling selection criteria 
 
 
10.2.2 Statistical methods  
 
Primary endpoint:  the prevalence will be calculated by dividing the number of advanced NSCLC with 
HER2 mutations enrolled in the study by the number of advanced NSCLC patients who underwent NGS 
at the selected Centers in the study period participating to ATLAS project reported with the exact 95% 
confidence interval. 
 
Secondary endpoint: distribution of type of therapies performed in advanced NSCLC patients HER2 
mutated as per clinical practice calculated as absolute counts and percentages on the study 
population 
 
Exploratory endpoints: 
 
Clinical-pathological characteristics of HER2 mutated NSCLC patients 
They will be summarized using absolute counts and percentages if referred to categorical items or by 
calculating mean and standard deviations as well as median, interquartile range, minimum and 
maximum for quantitative variables. 
 
Real World Objective response rate 
The RW-ORR will be reported including the exact 95% CI. Patients that for any reason will not be 
evaluated for the objective response will be considered as non-responder for this analysis and 
included in the denominator. 
 
Real World Disease control rate 
The RW-DCR will be reported including the exact 95% CI. Patients that for any reason will not be 
evaluated for the objective response will be considered as non-responder for this analysis and 
included in the denominator. 
 
Real world progression-free survival, time to next treatment, time to treatment discontinuation and 
overall survival 
The distributions of RW-PFS, TTNT, TTD and OS probabilities and the estimates of the corresponding 
median times will be obtained by the Kaplan Meier technique. The 95% CI for the median times will be 
calculated using the Brookmeyer and Crowley method. Estimates of the PFS and OS rates at a fixed 
time point (6-months and 1-year) will be obtained using the Kaplan Meier technique and 95% CI will 
be calculated by the Greenwood’s formula for standard deviation. Patients who will not experience 
the events will be censored.  
 

 
 
 
 
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Percentage of PD-L1 expression among advanced NSCLC patients HER2 mutated 
PD-L1 expression will be summarized as a quantitative item and categorized according to the usual 
cut-points (0, 1%, 50%) 
 
Allelic frequency (AF) of HER2 mutation in the blood  
Allelic frequency of HER2 mutation in cfDNA will be calculated with fractional abundance method by 
QuantaSoft (BioRad).  
 
FcγR polymorphisms 
Differences in characteristics among the genotypes will be assessed using Fisher exact test for 
categorical data. Genotypes will be analyzed separately and as combined allelic groups. 
 
HSP90 IHC expression 
Chi-square test or Fisher’s exact test will be performed to analyze the relationship between HSP90 
status and patients’ characteristics. 
 
 
10.2.3 Data recording and display  
Frequency tables will be tabulated for all categorical variables by the levels of the variables as they 
appear on the ATLAS database eCRF (with percentages). Categories with a text field specification will 
be tabulated as categories and then supplemented by a listing with the following information for the 
patients fulfilling the condition for the specification (patient id, institution, value of the item and text 
field contents).  
Other continuous variables (for example age, dose) are presented using the median and range 
(minimum, maximum), mean and standard deviation.  
Continuous data may also be presented in categories (for example, age may also be grouped in 
decades).  
Continuous variables for which a coding system exists (such as for laboratory data) will be recoded 
into categories.  
Laboratory data derived from exploratory analyses will not be recorded in the ATLAS eCRF, but 
collected in a dedicated separate database under the supervision of the Coordinating Investigator. 
 
 
10.2.4 End of study 
The final analysis will occur at the earliest when the last patient with HER2 mutation has been 
followed up in the study for at least 12 months. Prior to the final analysis, the database will be 
fully clean and frozen. 
 
11 DATA MONITORING BOARD 
 
A Board formed by the Coordinating and Co-Coordinating Investigator, with the Scientific Coordinators 
and Clinical Trial Office will monitor the recruitment and the data quality at least twice a year. Arising 
problems will be discussed with the Coordinating and Co-Coordinating Investigator, with the Scientific 
Coordinators who will respond appropriately. Relevant information will be included in the study status 

 
 
 
 
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reports. No results will be presented at investigators meetings before the trial is closed to recruitment 
and data are mature for the analysis of the primary endpoint, unless recommended otherwise by the 
Board. 
 
12 INVESTIGATOR AUTHORIZATION PROCEDURE  
 
Investigators will be authorized to register patients in this study only when all regulatory requirements 
are being fulfilled and a site initiation visit is performed.  
 
 
13 PATIENT REGISTRATION PROCEDURE 
 
This is a multicenter observational study. All patients must be registered in the ATLAS eCRF at the time 
of study enrolment. Patient registration will only be accepted from authorized investigators, who will 
receive a personal user ID to access the registration system. 
Registration will be centralized and performed online. At the end of the procedure, a patient 
sequential identification number will be allocated to the patients. The sequential identification 
number attributed to the patient at the end of the registration procedure identifies the patient. 
All patients in the Cohort 3 must be registered in the HEROS CRF at the time of study enrolment. They 
will maintain the same ATLAS ID.  
 
 
14 FORMS AND PROCEDURES FOR COLLECTING DATA 
 
The primary study database for data analysis to assess the primary and secondary objectives of the 
study is the ATLAS case report form. All data concerning the collection and analysis of biological 
samples will be recorded in the HEROS paper CRFs.   
The HEROS CRFs pertaing the results of blood and tissue analysis will be completed, dated and signed 
by the Investigator of the centralized laboratory. 
Source documents (paper or electronic) are those in which patient data are recorded and documented 
for the first time. They include, but are not limited to, hospital records, clinical and office charts, 
laboratory notes, memoranda, evaluation checklists, pharmacy dispensing records, recorded data 
from automated instruments, copies of transcriptions that are certified after verification as being 
accurate and complete, X-rays, patient files, and records kept at pharmacies, laboratories and medico-
technical departments involved in a clinical study. 
 
15 DATA QUALITY ASSURANCE 
 
Computerized and manual consistency checks will be performed on newly entered forms; queries will 
be issued in case of inconsistencies. Consistent forms will be validated by the Clinical Trial Office to be 
entered on the master database. Inconsistent forms will be kept "pending" until resolution of the 
inconsistencies. 
Site visits will be conducted by the Sponsor or an authorized representative for inspection of study 
data, patients' medical records and eCRFs. The investigator will permit national and local health 

 
 
 
 
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authorities, Sponsor monitors, representatives and collaborators and the IRBs/ECs to inspect facilities 
and records relevant to this study. 
 
 
16 ETHICAL CONSIDERATIONS 
 
16.1 Patient protection 
The responsible investigator will ensure that this study is conducted in agreement with the Declaration 
of Helsinki. The protocol has been written, and the study will be conducted according to the 
International Conference on Harmonisation (ICH) Harmonized Tripartite Guideline for Good Clinical 
Practice (GCP).  
This protocol, the Informed Consent Forms, any information to be given to the patient, and relevant 
supporting information must be submitted to the IRB/EC and reviewed and approved by the IRB/EC 
before the study is initiated. In addition, any patient recruitment materials must be approved by the 
IRB/EC. Any protocol amendments will be prepared by the Sponsor. Protocol amendments will be 
submitted to the IRB/EC in accordance with local regulatory requirements. 
 
16.2 Subject identification 
The name of the patient will not be asked for nor recorded in the study Data Base. Patient enrolled in 
Heros study will keep the identification number assigned for ATLAS study. This number will identify the 
patient and must be included on all case report forms. A subject identification list will be stored at the 
selected Centers.  
 
16.3 Informed consent 
All patients will be informed of the aims and procedures of the study. They will be informed as to the 
strict confidentiality of their patient data, but that their medical records may be reviewed for study 
purposes by authorized individuals other than their treating physician. Being an observational study, 
there are no possible hazards to which the patients will be exposed, except for incidental genetic 
findings which could potentially reveal a hereditary predisposition to other malignancies, with 
potential risk for the patient’s relatives. Only in this last case, the patient will be informed and sent to 
genetic counselling, if the patient has given the consent to be informed on incidental genetic findings. 
Otherwise, considering the non-interventional nature of the study, results will not be returned.  
The informed consent form is part of the documents to be submitted to the ethic committee for 
approval. For deceased or untraceable patients for whom it is not possible to obtain the informed 
consent form, every effort would be made to reach the patient and obtain his consent, otherwise the 
General Data Protection Regulation will be consulted. 
It will be emphasized that the participation is voluntary and that the patient is allowed to refuse 
further participation in the protocol whenever he/she wants. This will not prejudice the patient’s 
subsequent care. Documented informed consent must be obtained for all alive patients. This must be 
done in accordance with the national and local regulatory requirements. 
For European Union member states, the informed consent procedure must conform to the ICH 
guidelines on Good Clinical Practice. This implies that “the written informed consent form should be 
signed and personally dated by the patient or by the patient’s legally acceptable representative”. All 
the personal data will be collected, stored and used according to the General Data Protection 
Regulation n. 679/2016, the Legislative Decree n. 196/2003 (Code on personal data protection) and 

 
 
 
 
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Pag. 25 of 27 
Decision of Guarantee for privacy protection n. 146/2019. 
 
17 ADMINISTRATIVE RESPONSABILITIES  
 
17.1 Overall study management 
 
The Coordinating and Co-Coordinating Investigator, with the Scientific Coordinators (in cooperation 
with the Clinical Trial Office) will be responsible for writing the protocol, reviewing all case report 
forms and documenting his/her review on evaluation forms, discussing the contents of the reports 
with the Clinical Trial Office and for publishing the study results. The Coordinating and Co-
Coordinating Investigator with the Scientific Coordinators will also generally be responsible for 
answering all clinical questions concerning eligibility, treatment, and the evaluation of the patients. 
 
17.2 Clinical Trial Office 
The Clinical Trial Office will be responsible for reviewing the protocol, collecting case report forms, 
controlling the quality of the reported data, and generating reports and analyses in cooperation with 
the Coordinating and Co-Coordinating Investigator and Scientific Coordinators. All methodological 
questions should be addressed to the Clinical Trial Office. 
Clinical Trial Office will supervise CRO’s activities. 
 
17.3 
CRO 
The CRO will be responsible of ethical submissions and site activation and site close out activities. 
 
18 SPONSORSHIP 
This study is sponsored by GOIRC (Gruppo Oncologico Italiano di Ricerca Clinica). The protocol will be 
supported by partial economic support from Astra-Zeneca.  
 
19 INSURANCE 
A study insurance is not necessary considering that the patients enrolled receive standard treatment 
according to clinical practice.  
 
20 PUBLICATION POLICY 
The final publication of the study results will be written by the GOIRC on the basis of the final analysis 
performed at the Clinical Trial Office. After revision by the Clinical Trial Office and other co-authors the 
manuscript will be sent to a major scientific journal. Authors of the manuscript will include at least the 
Study Staff, the members of the writing committee, the investigators who have included more than 
10% of the eligible patients in the trial (by order of inclusion). 
The manuscript will include an appropriate acknowledgment section, mentioning all investigators who 
have contributed to the trial, the data center staff involved in the study, as well as supporting bodies. 
All publications including data from the present trial will be submitted for review to the Clinical Trial 
Office and to all co-authors prior to submission. The Study Staff must approve all publications, 
abstracts and presentations of data pertaining to patients included in this study. 
 

 
 
 
 
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21 REFERENCES 
 
1. Jebbink M, de Langen AJ, Boelens MC, et al: The force of HER2 – A druggable target in NSCLC? 
Cancer Treat Rev 86, 2020 
2. Arcila ME, Chaft JE, Nafa K, et al: Prevalence, clinicopathologic associations, and molecular 
spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas. Clin Cancer Res 18, 
2012 
3. Zhao J, Xia Y: Targeting HER2 Alterations in Non–Small-Cell Lung Cancer: A Comprehensive Review. 
JCO Precis Oncol, 2020 
4. Hirsch FR, Varella-Garcia M, Franklin WA, et al: Evaluation of HER-2/neu gene amplification and 
protein expression in non-small cell lung carcinomas. Br J Cancer 86, 2002 
5. Nakamura H, Saji H, Ogata A, et al: Correlation between encoded protein overexpression and copy 
number of the HER2 gene with survival in non-small cell lung cancer. Int J Cancer 103, 2003 
6. Hotta K, Yanai H, Ohashi K, et al: Pilot evaluation of a HER2 testing in non-small-cell lung cancer. J 
Clin Pathol 73, 2020 
7. Mazières J, Peters S, Lepage B, et al: Lung cancer that harbors an HER2 Mutation: Epidemiologic 
characteristics and therapeutic perspectives. J Clin Oncol 31, 2013 
8. Pillai RN, Behera M, Berry LD, et al: HER2 mutations in lung adenocarcinomas: A report from the 
Lung Cancer Mutation Consortium. Cancer 123, 2017 
9. Wang Y, Zhang S, Wu F, et al: Outcomes of Pemetrexed-based chemotherapies in HER2-mutant lung 
cancers. BMC Cancer 18, 2018 
10. Lai WV, Lebas L, Barnes TA, et al: Afatinib in patients with metastatic or recurrent HER2-mutant 
lung cancers: a retrospective international multicentre study. Eur J Cancer 109, 2019 
11. Peters S, Curioni-Fontecedro A, Nechushtan H, et al: Activity of Afatinib in Heavily Pretreated 
Patients With ERBB2 Mutation–Positive Advanced NSCLC: Findings From a Global Named Patient Use 
Program. J Thorac Oncol 13, 2018 
12. Wang Y, Jiang T, Qin Z, et al: HER2 exon 20 insertions in non-small-cell lung cancer are sensitive to 
the irreversible pan-HER receptor tyrosine kinase inhibitor pyrotinib. Ann Oncol 30, 2019 
13. Socinski MA, Cornelissen R, Garassino MC, et al: LBA60 ZENITH20, a multinational, multi-cohort 
phase II study of poziotinib in NSCLC patients with EGFR or HER2 exon 20 insertion mutations. Ann 
Oncol 31, 2020 
14. Liu SV, Villaruz LC, Lee VHF, et al: LBA61 First analysis of RAIN-701: Study of tarloxotinib in patients 
with non-small cell lung cancer (NSCLC) EGFR Exon 20 insertion, HER2-activating mutations & other 
solid tumours with NRG1/ERBB gene fusions. Ann Oncol 31, 2020 
15. Ross HJ, Blumenschein GR, Aisner J, et al: Randomized phase II multicenter trial of two schedules 
of lapatinib as first- or second-line monotherapy in patients with advanced or metastatic non-small 
cell lung cancer. Clin Cancer Res 16, 2010 
16. Kris MG, Camidge DR, Giaccone G, et al: Targeting HER2 aberrations as actionable drivers in lung 
cancers: Phase II trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with HER2-
mutant or amplified tumors. Ann Oncol 26, 2015 
17. Hyman DM, Piha-Paul SA, Won H, et al: HER kinase inhibition in patients with HER2-and HER3-
mutant cancers. Nature 554, 2018 
18. Hainsworth JD, Meric-Bernstam F, Swanton C, et al: Targeted therapy for advanced solid tumors on 
the basis of molecular profiles: Results from mypathway, an open-label, phase IIA multiple basket 
study, in Journal of Clinical Oncology. 2018 

 
 
 
 
HEROS Study - Protocol -  version 1.0 of 02 May 2024 
 
Pag. 27 of 27 
19. Mazéres J, Barlesi F, Filleron T, et al: Lung cancer patients with HER2 mutations treated with 
chemotherapy and HER2-targeted drugs: Results from the European EUHER2 cohort. Ann Oncol 27, 
2016 
20. Tomizawa K, Suda K, Onozato R, et al: Prognostic and predictive implications of HER2/ERBB2/neu 
gene mutations in lung cancers. Lung Cancer 74, 2011 
21. Li BT, Shen R, Buonocore D, et al: Ado-trastuzumab emtansine for patients with HER2-mutant lung 
cancers: Results from a phase II basket trial, in Journal of Clinical Oncology. 2018 
22. Peters S, Stahel R, Bubendorf L, et al: Trastuzumab emtansine (T-DM1) in patients with previously 
treated HER2-overexpressing metastatic non–small cell lung cancer: Efficacy, safety, and biomarkers. 
Clin Cancer Res 25, 2019 
23. Smit EF, Nakagawa K, Nagasaka M, et al: Trastuzumab deruxtecan (T-DXd; DS-8201) in patients with 
HER2-mutated metastatic non-small cell lung cancer (NSCLC): Interim results of DESTINY-Lung01. J Clin 
Oncol 38, 2020 
24. Gavin PG, Song N, Rim Kim S, et al: Association of polymorphisms in FCGR2A and FCGR3A with 
degree of trastuzumab benefit in the adjuvant treatment of ERBB2/HER2-positive breast cancer 
analysis of the NSABP B-31 trial. JAMA Oncol 3, 2017 
25. Musolino A, Naldi N, Bortesi B, et al: Immunoglobulin g fragment c receptor polymorphisms and 
clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast 
cancer. J Clin Oncol 26, 2008 
26. Falcone I, Carbognin L, Ciuffreda L, et al: PO-502 A potential role for HSP90 in HER2-driven breast 
cancer (BC). ESMO Open 3, 2018 
27. Macario AJL, de Macario EC: Sick Chaperones, Cellular Stress, and Disease. N Engl J Med 353, 2005 
28. Neckers L, Workman P: Hsp90 molecular chaperone inhibitors: Are we there yet? Clin Cancer Res 
18, 2012 
29. Whitesell L, Lindquist SL: HSP90 and the chaperoning of cancer. Nat Rev Cancer 5, 2005 
30. Garcia-Carbonero R, Carnero A, Paz-Ares L: Inhibition of HSP90 molecular chaperones: Moving into 
the clinic. Lancet Oncol 14, 2013 
31. Modi S, Stopeck A, Linden H, et al: HSP90 inhibition is effective in breast cancer: A phase II trial of 
tanespimycin (17-AAG) plus trastuzumab in patients with HER2-positive metastatic breast cancer 
progressing on trastuzumab. Clin Cancer Res 17, 2011 
32. Yuan B, Zhao J, Zhou C, et al: Co-Occurring Alterations of ERBB2 Exon 20 Insertion in Non-Small Cell 
Lung Cancer (NSCLC) and the Potential Indicator of Response to Afatinib. Front Oncol 10, 2020 
33. Liu Z, Wu L, Cao J, et al: Clinical characterization of ERBB2 exon 20 insertions and heterogeneity of 
outcomes responding to afatinib in Chinese lung cancer patients. Onco Targets Ther 11, 2018 
34. Fang W, Zhao S, Liang Y, et al:  Mutation Variants and Co-Mutations as Genomic Modifiers of 
Response to Afatinib in HER2 -Mutant Lung Adenocarcinoma. Oncologist 25, 2020