An Observational Study of Local Relapse After High Grade Osteosarcoma
Conditions: Osteosarcoma
Sponsor: Istituto Ortopedico Rizzoli

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An observational study of local relapse after high grade osteosarcoma 
 
 
Study Code Osteo LR 
Sponsor’s Name IRCCS Istituto Ortopedico Rizzoli 
Via di Barbiano 1/10 
40136 Bologna, Italy 
Study 
Number/Version/Date: 
Vers. 1.0 
Jan 10th, 2022 
Coordinating Center and 
Principal Investigator: 
Osteoncology, Bone and Soft Tissue Sarcomas and 
Innovative Therapies Unit 
IRCCS Istituto Ortopedico Rizzoli  
Via pupilli 1 
40136 Bologna, Italy 
 
Dott.ssa Emanuela Palmerini 
Phone: 051-6366.199 
Email: emanuela.palmerini@ior.it 
Methodology: Prospective and retrospective study 
(multi-institutional cases series review of clinical data) 
  
Type: Academic/No profit 
 
Funding None 
Principal Investigator 
Signature 
I confirm that I’ve read this protocol and I accept to 
run the study in compliance with what is stated in the 
protocol and with the ICh-GCP and all applicable law 
 
Emanuela Palmerini M.D., Ph.D. 
 
Firma 
 
_________________________ 
 
 
 

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TABLE of CONTENTS 
Background  
 
 
 
 
 
 
 
 
pg. 3 
Objective of the study 
 
 
 
 
 
 
 
pg.  4 
Study design   
 
 
 
 
 
 
 
 
pg.  4 
Analysis and data collection    
 
 
 
 
 
 
pg.  4                                   
Statistics 
 
   
 
 
 
 
 
 
 
pg.  7 
Enrolment procedure 
  
 
 
 
 
 
 
pg.  7 
Data Collection           
 
  
 
 
 
 
 
pg.  8  
 
Ethic and quality assurance                                                                          pg.  8 
Informed Consent     
 
 
 
 
 
                     pg.  8 
General principles for HBM collection                                                          pg.  9 
Confidentiality                                                                                       
pg.  10 
Publication of results                                                        
 
 
pg.  10 
Sponsor Role and Responsibility                                                                  pg.  11               
References                                                                                        
pg.  12 
 
.   
 
 
 
 
 
 

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1. Background 
 
Treatment of osteosarcoma has evolved in the last 40 years: with the introduction of neo-
adjuvant chemotherapy, 5-year survival rates for patients affected by high-grade 
osteosarcoma have improved from 15% to more than 70%1-6.   
To date, many osteosarcomas are cured by multiagent chemotherapy and surgery, but a 
significant number still relapse: after treatment of the primary osteosarcoma, 40% to 50% 
of patients will develop local relapse (LR), distant metastases (DM) or both7-15.  
Few analyses in literature report on locally recurrent disease as it remains uncommon, 
with LR rate ranging from 4% to 10%10-17. Prognosis of patients experiencing LR is 
generally considered poor, however most data available do not provide sufficient 
details7,8,15. DM are such an overriding determinant of survival, and LR is so infrequent, 
that it is difficult to study local recurrence as an independent variable15-22.  
Also, in case of LR, while the importance of achieving a second surgical complete 
remission is well known (CR2), the role of second line chemotherapy is under discussion, 
with a mono-institutional experience showing no advantage in of chemotherapy after local 
relapse23. Several molecules and their receptors are known to be involved in migration and 
invasiveness of tumour cells including CXCL12 (SDF-1) which is the ligand of Receptor-4 
(CXCR4). The binding of CXCL12 to CXCR4 it is known to activate downstream cascades 
involving many signalling pathways, such as JAK/STAT, PI3K/Akt, MAPK, JNK which are 
key in cell migration. The expression of CXCR4 is low or absent in normal cells, while it is 
elevated in tumor cells. There are preclinical evidences that CXCR4 has an important role 
on tumor progression in sarcoma24-25.  Also, nuclear expression of CXCR4 and IGF-1R 
negatively influences survival in patients with localized soft tissue sarcoma26.  

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2. Objectives 
Aim of this study is to analyse the modalities of diagnosis (symptoms versus planned 
radiology) of LR for high-grade osteosarcoma patients, pattern of recurrence, treatment of 
LR and factors influencing post-LR survival (PLRS) on the role of second line 
chemotherapy, margins, Local relapse-free interval (LRFI) (“early LR” recurrences with an 
LRFI ranging between 1 and 24 months, and “late LR” as those having an LRFI longer 
than 24 months. 
 
Primary Objective 
- 
post-LR survival (PLRS)  
 
Secondary Objectives 
- 
analyse the modalities of diagnosis of LR in high-grade osteosarcoma 
- 
pattern of recurrence 
- 
 treatment of LR  
- 
Prognostic factors for PLRS (second line chemotherapy, surgical margins, surgical 
complete remission after LR (CR2), type of surgery) 
Exploratory Objectives  
Assessment of CXCR4 and CXCL12 (SDF-1) expression classified as nuclear, 
cytoplasmatic, and both in primary tumor, LR and DM 
- 
Comparison of CXCR4 and CXCL12 (SDF-1) expression in the LR samples 
between the groups of patients with isolated LR and those presenting with LR and 
DM 
- 
Comparison of CXCR4 and CXCL12 (SDF-1) expression on primary tumor in 3 
group of patients: 
- 1- patients with non LR, nor DM (archival series from Rizzoli database) 
- 2- patients with isolated LR 
- 3- patients with LR + DM 
 
3. STUDY DESIGN 
This is a multicenter retrospective and prospective study that will analyze all cases with of 

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diagnosis of LR in high-grade osteosarcoma from 01 January 1980 and all new cases 
referring to each participant site. 
4. Prospective observational analysis and data collection 
Clinical data will be retrieved by patient charts.  
A protocol-specific CRF reporting the results of the review will be provided. 
 Data from all patients diagnosed with OS will be prospectively entered in this platform. 
Each patient will be provided with an informational leaflet for his/her general practitioner 
and asked to sign an informed consent prior to data entry. 
The following information will be recorded in the database:  
 Demographics – patient name, DOB, gender, date of first consultation, data of 
diagnosis  
 Data on primary tumor – size, site, evidence of multifocality  
 Data on histological diagnosis – morphology (classical type, proximal type, mixed), 
mitotic count, depth  
 Data on staging at diagnosis – evidence of lymph-nodal and/or distant metastases  
 Data on treatment of primary tumor – surgery, radiotherapy, chemotherapy  
 Data on recurrence – type of recurrence, site, date  
 Data on treatment of local relapse – surgery, radiotherapy, chemotherapy  
 Data on treatment of metastatic disease – pulmonary metastasectomy, radiotherapy  
 Data on chemotherapy for metastatic disease – line, regimen, number of cycles, 
best response, duration of response  
 Patient status at last follow-up (dead of disease, DOD, alive with disease, AWD, 
nonevidence of disease, NED) 
 
Optional sub-study 
For both prospective and retrospective analysis, in all cases with formalin - fixed paraffin 

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embedded 
(FFPE) 
tissue 
available, 
and 
if 
funding 
might 
be 
secured, 
an 
immunohistochemistry analysis to assess the expression of CXCR4 and CXCL12 (SDF- in 
LR, DM and primary tumor) will be assessed both in the nucleus and cytoplasm, as 
described [10]. The optional exploratory revision will start only if funding might be secured.   
 
4.1 Study population 
We plan to include approximately 50 patients in 36 months. 
Inclusion criteria 
- 
Patients who experienced LR as first event of recurrence after treatment for localized 
high-grade osteosarcoma of bone, including cases with synchronous lung 
metastases 
- 
Adequate patient compliance to follow-up visits 
- 
Written informed consent prior to any study-specific analysis and/or data collection  
- 
No age limits 
Exclusion criteria  
- 
Cases of LR demonstrated after > 3 months after metastatic disease  
- 
Impossibility to ensure adequate compliance  
 
5. Retrospective observational analysis and data collection 
Together with the prospective observational study, a retrospective analysis including all the 
cases with a histological diagnosis of localized high-grade osteosarcoma who experienced 
LR as first event of recurrence after treatment from 01 Jan 1980 to 31 Dec 2021 will be 
performed. 
The data will be extracted retrospectively from prospectively maintained institutional 
database and will be checked through a revision of patient records.  
 
The following data will be collected: 
 demographics (gender, date of birth, age at diagnosis),  

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 disease characteristics (morphological subtype, dimension, primary site),  
 treatment received (date of surgery, surgical margins, radiation therapy, timing of 
radiation therapy, chemotherapy, timing of chemotherapy, type of regimen used, 
best response according to RECIST 1.1)  
 date of LR, modality of LR diagnosis, clinical suspect or imaging, presence, or 
absence of DM at LR diagnosis, type of treatment after LR, date of surgical 
complete remission after LR (CR2), surgical margins 
 outcome (recurrence type and timing, patient status at last follow-up) 
 
5.1 Study population 
Through the collaboration of the joining institutions, we plan to include approximately 100 
patients 
 Inclusion criteria 
- 
Male and female patients treated for localized high-grade osteosarcoma from 01 Jan 
1980 to 31 Dec 2021.  
- 
Clinical Information available 
- 
No age limits 
Exclusion criteria  
- 
Cases of LR demonstrated after > 3 months after metastatic disease  
- 
Evidence of metastatic disease at presentation  
 
 
 
6. Statistics 
To the case series will be applied a descriptive statistic. 
For the exploratory objectives, positive and negative expression of CXCR4 and CXCL12 
(nuclear and cytoplasmatic) will be correlated with ES and OS. 
 
7.  Enrolment procedures 
Patients considered eligible and who have provided a written informed consent will be 

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included into the study and their material will be used for the purpose of this research 
according to the protocol and to the informed consent provided. 
For the retrospective analysis, the investigator will extract from Institute database the list of 
potential eligible patients for whom the tumor material is available. After the verification of 
the presence of the study enrollment criteria, the patient will be included in the study.  
Due to the high incidence of mortality of the disease under investigation, it would be 
possible that some eligible subjects will be deceased 
 
8. Data Collection 
Clinical data will be retrieved by patient charts. 
A protocol-specific CRF reporting the results of the review will be provided. 
A CRF is required and should be completed for each included subject. 
 
9. Ethics and quality assurance  
The clinical trial protocol and its documents will be sent before initiating the study to the 
competent Authorities and Ethics Committees of each participating country for its approval. 
The responsible investigator will ensure that this study is conducted in agreement with 
either the most updated Declaration of Helsinki and all the international and local laws that 
apply to clinical trials and to patient protection.  
The protocol has been written, and the study will be conducted according to the principles 
of the ICH Harmonized Tripartite Guideline for Good Clinical Practice  
(ref: http://www.emea.eu.int/pdfs/human/ich/013595en.pdf).  
 
10. Informed consent 
All Patients will be informed, by the investigator, of the aims of the study, the possible risks 
and benefits that will derive from the study participation.  
The Investigator must clearly inform that the patient is free to refuse participation in the 

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study and that can withdraw consent at any time and for any reason.  
They will be informed as to the strict confidentiality of their patient data, but that their 
medical records may be reviewed for trial purposes by authorized individuals other than 
their treating physician.  
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".   
The Investigator must also sign the Informed Consent form and will keep the original at the 
site and a copy of the original must be handed to the patient.  
The competent ethics committee for each Institution participating to the study must 
validate local informed consent documents before the study can be opened. It will be 
emphasized that the participation is voluntary and that the patient is allowed to refuse 
further participation in the study whenever he/she wants. This will not prejudice the 
patient's subsequent care.  
 
11. General principles for human biological material (HBM) collection  
In all cases with formalin - fixed paraffin embedded (FFPE) tissue available, and if funding 
accessible, human biological material (HBM) collection involves the collection and storage 
of biological material, residual biological material, or derivatives in compliance with ethical 
and technical requirements.  
Biological material (FFPE blocks of tumor sample) are stored in the sites’ local archives. 
The biological material will be used and stored according with the sample characteristic 
and applicable regulation. 
 Each institute will have a designated person responsible for collection and will act 
as a communication point  

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 The collected HBM should be documented, i.e. the amount remaining and its 
location act as a communication point. 
12. Confidentiality  
To ensure confidentiality of clinical trial data as disposed the national and European 
applicable regulation, data will be only accessible for the trial Sponsor and its designees, 
for monitoring/auditing procedures, the Investigator and collaborators, the Ethics 
Committee of each corresponding site and the Health Authority. 
Investigator and the Institution will allow access to data and source documentation for 
monitoring, auditing, Ethic Committee revision and inspections of Health Authority, but 
maintaining at all times subject personal data confidentiality as specified in the “Directive 
95/46/EC of the European Parliament and of the Council of 24 October 1995”.  
 The Investigator must guarantee that patient anonymity is always kept and their identity 
must be protected from unauthorized persons and institutions.  
All patients included in the study will be identified with a numeric code, so that no 
identifiable personal data will be collected (pseudo anonymization) 
The Investigator must have and conserve a patients’ inclusion registry where it figures the 
personal data of the patient: name, surname, address and corresponding identification 
code into the study, this register will be kept on the Investigator File. 
 
13. Publication of results  
The results from this study can be published or shown at scientific conferences. According 
to usual practice, this multicentred study will be published as a whole, and not with the 
data obtained separately from each of the sites participants. It is expected that other 
articles are published about the exploratory aspects of this trial once the main data has 
been published. The final publication of the trial results will be written by the Coordinator 
Investigator. 
All publications (papers, abstracts, presentations…) including data from the present trial 

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will be submitted for review to all co-authors prior to submission.  
 
14. Sponsor role and responsibility  
The sponsor is the sole owner of the data and is responsible of all the clinical trial activities 
from study design, development, data collection, management, analysis, interpretation of 
data, writing and the decision to submit the report for publication written by the Principal 
Investigator.  
 
 

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