Global collaboRAtion on CIC-DUX4, BCOR-CCN3, High-grade Undifferentiated Round Cell Sarcoma (URCS) Project
Conditions: Undifferentiated Round Cell Sarcoma
Sponsor: Istituto Ortopedico Rizzoli

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
1 
 
 
 
 
“Global collaboRAtion on CIC-DUX4, BCOR-CCN3, high-grade Undifferentiated 
round cell sarcoma (URCS) project’ 
 
 
Study code GRACefUl 
Sponsor’s Name 
and Address: 
IRCCS Istituto Ortopedico Rizzoli 
Via di Barbiano 1/10 
40136 Bologna 
Italy 
Study 
Number/Version/
Date: 
Vers 3.0  
06 Feb 2023 
Coordinating 
Center: 
IRCCS Istituto Ortopedico Rizzoli 
SC Osteoncologia sarcomi dell’osso e dei tessuti molli e terapie innovative 
Via Pupilli 1 
40136 Bologna, Italy 
Coordinating 
Investigator and 
address: 
Emanuela Palmerini MD 
Phone: 051-6366.199 
Email: emanuela.palmerini@ior.it 
Methodology: Experimental study with biological material : case series review of clinical and 
histological data 
Type: Academic 
Founding: None 
 
 
 
 
 
 
 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
2 
 
                                                                   Protocol Signature 
 
Sponsor: IRCCS Istituto Ortopedico Rizzoli 40136 Bologna, Italy 
Study Title: “Global collaboRAtion on CIC-DUX4, BCOR-CCN3, high-grade Undifferentiated round 
cell sarcoma (URCS) project’ 
Study Acronym/Code: GRaCefUl 
Protocol Version and Date: Version 3.0 of   06 Feb 2023 
 
I read this protocol and I accept to conduct this trial in accordance with the protocol stipulations, 
GCP guidelines and the Declaration of Helsinki. 
 
 
Study global coordinator : 
 
Dott. ssa Emanuela Palmerini  
SC Osteoncologia sarcomi dell’osso e dei tessuti molli 
e terapie innovative  
IRCCS Istituto Ortopedico Rizzoli 
40136 Bologna, Italy 
 
 
Local Site Name:  IRCCS Istituto Ortopedico Rizzoli 
 
Local Site Principal Investigator Name: Dr. Emanuela Palmerini 
 
Local Site Principal Investigator Signature  
 
 
Signature Date  
 
Emanuela Palmerini 
 
06 Feb 2023 
 
 
 
 
 
 
 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
3 
 
 
TABLE of CONTENTS 
Background 
 
 
 
 
 
 
 
 
 
pag. 5 
Study Design  
 
 
 
 
 
 
                         pag.  5 
Objective of the study 
  
 
 
 
 
 
              pag. 5 
Population     
 
 
 
 
 
 
 
 
pag.  6 
Material and Methods   
 
 
 
 
 
 
 
pag.  6 
Statistics 
 
   
 
 
 
 
 
 
 
pag.  7 
Enrolment procedure   
 
 
 
 
 
 
            pag.  7 
Data Collection            
  
 
 
 
 
 
 
pag.  8 
Ethic and quality assurance   
 
 
 
 
 
 
pag.  8 
Informed Consent   
 
  
 
 
 
 
 
 
pag.  8  
 
Confidentiality    
pag.  9 
Publication of the results                                                                                     
pag.  9 
Sponsor Role and Responsibility                                                                                       pag.  10 
References 
                                                                                       
 
 
pag.  11 
 
 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
4 
 
BACKGROUND 
Undifferentiated round cell sarcomas (URCSs) represent a diagnostic challenge. URCSs cell 
histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may 
include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell 
osteosarcoma. Definitive classification usually requires detection of entity-specific mutations.  
Recently, the use of modern molecular techniques led to the description of a variety of new, rare, 
sarcoma subtypes, which have morphological appearance similar to Ewing sarcomas ("Ewing-like" 
sarcomas), but carry gene fusions different from that of Ewing sarcoma, with CIC-DUX4, and BCOR-
CCNB3 being the most frequent. Other non-Ewing round cells sarcomas are characterized by 
rearrangement of CIC (CIC-rearranged sarcomas) or BCOR (BCOR-rearranged sarcomas) with other 
genes, by Internal Tandem Duplication of BCOR or by other translocations (i.e.: CRTC1-SS18).  
The identification of these gene fusions suggests that other, yet to be identified, gene fusions 
could be associated with this type of tumour. Furthermore, novel gene fusions in Ewing-like" 
sarcomas have been reported recently, in case reports, including CIC-FOXO4 BCOR-MAML3 and 
ZC3H7B-BCOR. Identifying these genetically defined entities may contribute towards 
understanding the pathogenesis and the behaviour of these tumours. The optimal treatment for 
these ultra rare tumors is not known.  
We propose to collect all cases of URCSs treated within the referral centres for sarcoma, including 
Italian sarcoma group network (ISG), in order to reclassify and describe their treatment and 
outcome. 
 
STUDY DESIGN 
This is a multicenter retrospective and prospective study that will analyze all cases with a diagnosis 
of URCSs from 01 January 1983 and all new cases referring to each participant site. 
OBJECTIVE OF THE STUDY  
Primary Objective  
Correlate clinical and pathological variables with event-free survival (EFS) and overall-survival 
(OS) in URCSCs 
 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
5 
 
Secondary objective 
Molecular re-classification of URCSs based on presence of CIC-rearrangement, and BCOR-
CCNB3 fusion genes or of other genetic alterations. 
Exploratory objective 
1. In all the “true-negative” URCSs (unclassified URCS), genomic/genetic characteristics will 
be correlated with clinical outcome. 
2. In all URCS  samples with frozen tissue available, potential diagnostic and therapeutic 
target will be searched through RNA-seq 
 
 
POPULATION 
Inclusion criteria  
1. Patients with URCSs treated from 01 January 1983 and all new patients will be included. 
The study will include the following: 
 CIC –rearranged sarcomas   
 BCOR-CCNB3 fusion-positive sarcomas and sarcomas with BCOR genetic alterations 
 Undifferentiated round cell sarcoma with other genetic alterations 
 Undifferentiated round cell sarcomas with no evidence of genetic alterations 
(unclassified URCS) 
2. Clinical and outcome information available. 
3. Histological slides/formalin-fixed paraffin-embedded tissue tumor (FFPE) blocks from 
archive available to perform the histology analysis/frozen tissue representative of the 
tumor available.   
Exclusion criteria 
1) Diagnosis different from URCSs 
2) Patient with no clinical or outcome information available 
 
The study will include male and female patients without limit of age 
 
MATERIAL AND METHODS 
Patients with a morphological diagnosis of URCSs will be included and will be classified as  

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
6 
 
 CIC –rearranged sarcomas   
 BCOR-CCNB3 fusion-positive sarcomas and sarcomas with BCOR genetic alterations 
 Undifferentiated round cell sarcoma with other genetic alterations 
 Undifferentiated round cell sarcomas with no evidence of genetic alterations 
(unclassified URCS) 
 
Formalin-fixed paraffin embedded tissue and/or frozen tumoral tissue of URCSs cases negative for 
all Ewing’s sarcoma molecular fusions, will be analyzed for the presence of CIC and BCOR-CCNB3 
translocations fusion at each Institution. All these analyses will be performed by using real-time 
PCR, FISH analysis or NGS.  
Positive cases will be then re-classified accordingly, while negative cases will be defined as “true-
negative”. For patients defined as “true-negative, NGS or RNA-seq will be performed (if not 
already done), in order to detect the possible presence of other genetic alterations. 
Cases that remain negative for genetic alterations will be labelled as unclassified URCS. 
Treatment, outcome, and prognostic factors will be related with the molecular re-classification. 
Each participating center will have to send to the global study coordinator the pathology form 
correctly filled in. 
RNA-seq is an increasing used methodology to study either coding and non-coding RNA 
expression, for the study of the transcriptome based on next-generation sequencing (NGS). In all 
URCS  samples with frozen tissue available, differentially expressed genes, and potential diagnostic 
and therapeutic targets will be searched through RNA sequencing 
 
STATISTICS 
For survival analysis the following factors will be correlated with Overall Suvival (OS) and Event 
Free Survival (EFS): stage (localised vs metastatic), histology (CIC-DUX4, BCOR-CCNB3, other), age 
(pediatric < 18 years vs adult ≥ 18 years), gender, pathologic response (good vs poor) to 
neoadjuvant chemotherapy. 
EFS and OS will be estimated according to the Kaplan and Meier method with their respective 95% 
confidence intervals (CI) and calculated from the first day of chemotherapy administration to 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
7 
 
death or last follow-up visit. A Cox regression model will also be used to examine the effect of 
factors on OS and EFS. 
For retrospective analysis, it is expected to include about 200 patients. For prospective study, we 
expect to include 60 patients. 
 
ENROLLMENT PROCEDURE 
Patients considered eligible and who have provided a written informed consent will be 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.  
 
 
 
DATA COLLECTION 
Clinical data will be retrieved by reviewing clinical charts, radiology and pathology report by local 
investigators. 
A protocol-specific CRF reporting pathologic report at the time of study inclusion and the results of 
re-classification will be provided. 
A CRF is required and should be completed for each included subject. 
 
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  

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
8 
 
(ref: http://www.emea.eu.int/pdfs/human/ich/013595en.pdf).  
 
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 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.  
For retrospective analysis, due to the high incidence of mortality of the disease under 
investigation, it would be possible that some potential eligible subjects will be deceased.  
 
CONFIDENTIALITY 
In order 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 will have 
to maintain at all times personal data confidentiality as specified in all the applicable regulations.  
All patients included in the study will be identified with a numeric code. Study Responsible and 
Collaborators of each laboratory and participating Institutions will have to keep a patients’ study 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
9 
 
inclusion registry containing the following personal data of each patient: name, surname, address 
and corresponding identification code used for this study. 
Study Responsible, Investigators and Collaborators of each laboratory and participating 
Institutions will guarantee that patients anonymity will be kept at all times and that their identity 
will be protected from access from unauthorized persons and Institutions.  
 
PUBBLICATION OF RESULTS  
The results from this study can be published or shown at scientific conferences. According to usual 
practice, this multicenter 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 will be 
submitted for review to all co-authors prior to submission.  
 
 
 
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. 
 

Protocol GRACefUl 
 
 
 
 
 
Version 3.0 06 Feb 2023 
10 
 
REFERENCES 
1. Gambarotti M, Benini S, Gamberi G, Cocchi S, Palmerini E, Sbaraglia M, Donati D, Picci P, 
Vanel D, Ferrari S, Righi A, Dei Tos AP. CIC-DUX4 fusion-positive round-cell sarcomas of soft 
tissue and bone: a single-institution morphological and molecular analysis of seven cases. 
Histopathology. 2016 Oct;69(4):624-34. 
2. Italiano A, Sung YS, Zhang L, Singer S, Maki RG, Coindre JM, Antonescu CR. High prevalence 
of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative 
undifferentiated small blue round cell sarcomas. Genes Chromosomes Cancer. 2012 
Mar;51(3):207-18. 
3. Antonescu C. Round cell sarcomas beyond Ewing: emerging entities. Histopathology. 2014 
Jan;64(1):26-37. 
4. Antonescu CR, Owosho AA, Zhang L, Chen S, Deniz K, Huryn JM, Kao YC, Huang SC, Singer S, 
Tap W, Schaefer IM, Fletcher CD. Sarcomas With CIC-rearrangements Are a Distinct 
Pathologic Entity With Aggressive Outcome: A Clinicopathologic and Molecular Study of 
115 Cases. Am J Surg Pathol. 2017 Jul;41(7):941-949. 
5. Peters TL, Kumar V, Polikepahad S, Lin FY, Sarabia SF, Liang Y, Wang WL, Lazar AJ, 
Doddapaneni H, Chao H, Muzny DM, Wheeler DA, Okcu MF, Plon SE, Hicks MJ, López-
Terrada D, Parsons DW, Roy A. BCOR-CCNB3 fusions are frequent in undifferentiated 
sarcomas of male children. Mod Pathol. 2015 Apr;28(4):575-86. 
6. Peters TL et al. BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male 
children. BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children. 
Mod Pathol. 2015 Apr;28(4):575-86. 
7. Cohen-Gogo S et al. Ewing-like sarcomas with BCOR-CCNB3 fusion transcript: A clinical, 
radiological and pathological retrospective study from the Société Française des Cancers de 
L'Enfant. Pediatr Blood Cancer. 2014 Dec;61(12):2191-8. 
8. Puls F et al. BCOR-CCNB3 (Ewing-like) sarcoma: a clinicopathologic analysis of 10 cases, in 
comparison with conventional Ewing sarcoma. Am J Surg Pathol. 2014 Oct;38(10):1307-18. 
9. Ludwig K, Alaggio R, Zin A, Peron M, Guzzardo V, Benini S, Righi A, Gambarotti M. BCOR-
CCNB3 Undifferentiated Sarcoma-Does Immunohistochemistry Help in the Identification? 
Pediatr Dev Pathol. 2017 Jul-Aug;20(4):321-329. 
10. Sugita S, Arai Y, Tonooka A, et al. A novel CIC-FOXO4 gene fusion in undifferentiated small 
round cell sarcoma: a genetically distinct variant of Ewing-like sarcoma. Am J Surg Pathol 
2014; 38: 1571-1576.  
11. Specht K, Zhang L, Sung YS, et al. Novel BCOR-MAML3 and ZC3H7B-BCOR Gene Fusions in 
Undifferentiated Small Blue Round Cell Sarcomas. Am J Surg Pathol 2016; 40: 433-442.  
12. Brohl AS, Solomon DA, Chang W, et al. The genomic landscape of the Ewing Sarcoma family 
of tumors reveals recurrent STAG2 mutation. PLoS Genet 2014; 10: e1004475.  
13. Heyer EE, Deveson IW, Wooi D, Selinger CI, Lyons RJ, Hayes VM, O'Toole SA, Ballinger ML, 
Gill D, Thomas DM, Mercer TR, Blackburn J. Diagnosis of fusion genes using targeted RNA 
sequencing. Nat Commun. 2019 Mar 27;10(1):1388. doi: 10.1038/s41467-019-09374-9. 
Erratum in: Nat Commun. 2020 Apr 8;11(1):1810. PMID: 30918253; PMCID: PMC6437215.