Analysis of the Molecular Profile of the Mixofibrosarcoma of the Extremities: Diagnostic and Prognostic Impact
Conditions: Sarcoma,Soft Tissue
Sponsor: Istituto Ortopedico Rizzoli



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TABLE of CONTENTS 
Background 
 
 
 
 
 
 
 
 
 
pg. 3 
Objective of the study 
 
 
 
 
 
 
 
pg.  3 
Study design   
 
 
 
 
 
 
 
 
pg.  3 
Population     
 
 
 
 
 
 
 
 
pg.  3 
Material and Methods   
 
 
 
 
 
 
 
pg.  4 
Statistics 
 
   
 
 
 
 
 
 
 
pg.  4 
Enrolment procedure   
 
 
 
 
 
 
 
pg.  4 
Data Collection            
  
 
 
 
 
 
 
pg.  4 
Informed Consent   
 
  
 
 
 
 
 
 
pg.  5 
Ethic and quality assurance   
 
 
 
 
 
 
pg.  5 
Confidentiality                                                                                       
 
 
pg.  6 
Publication of results                                                        
 
 
             pg.  6 
Sponsor Role and Responsibility                                                                                   pg.  6 
References 
                                                                                       
 
 
pg.  7 
 

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BACKGROUND 
Myxofibrosarcoma (MFS) represents one of the most common sarcomas in the adult / elderly age 
and its incidence is increasing due to the increase in the average age of the population; mainly 
affects the limbs and limbs, especially the lower limb. When compared to other histotypes, the 
typical feature of MFS is the tendency to a greater risk of local recurrence (RL) regardless of the 
degree of histological malignancy and surgical margins (60% vs. 25-30%)(1).  Such local aggression 
could be caused by the specific histological pattern of infiltrative growth. Local recurrence is also 
characterized by a variable, but high (15–38%) progression of degree, thus increasing the 
metastatic potential at the time of any local relapse (2).The use of local (radiotherapy) or systemic 
(chemotherapy) adjuvant therapies have not shown efficacy in terms of increasing the control of 
local and / or systemic diffusion. Despite this, the prognosis of MFS is relatively good with a 
metastasis rate of 20-25% and an estimated survival of approximately 75% at 5 years and 70% at 
10 years(3).Morphologically, it appears as a tumor with distinctive characteristics (association of 
hypocellular mixoid areas and hypercellular fibrous areas); however, it does not have a specific 
immunohistochemical pattern, so the histological diagnosis is mainly based on the hematoxylin-
eosin morphology (4). MFS is characterized by nonspecific cytogenetic aberrations and complex 
and unbalanced karyotypes, which increase with increasing degree, suggesting a multi-step tumor 
progression process due to the acquisition of genetic instability (5).Recently, Heitze et al. in a 
series of 25 cases of MFS (grade 1 and 3) they identified some genes with amplified expression 
including CCND1, CCNE1, EGFR, PTEN, RET (MET) and some deleted genes including TP53 and RB1. 
The mutation analysis also highlighted the presence of somatic mutations in TP53, PTEN, FGFR3, 
CDKN2A, and RB1 (6). 
However, for none of these alterations has a correlation been observed with the prognosis (in 
terms of overall or progression-free survival). On this basis, knowledge of genetic changes appears 
essential for the identification of potential biomarkers useful both at diagnostic / prognostic and 
therapeutic level. 
 
 
 
 
 
 

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OBJECTIVE OF THE STUDY  
The aim of the present study, in a serie of patients  with a diagnosis of  Mixofibrosarcoma treated 
at the Rizzoli Institute from 1 January 1993 up to 01 December 2017, is  to identify  patients with 
different risk classes in terms of disease-free survival (local and systemic recurrence) and total 
survival. The study will exam all the clinical, radiological, histological and immunohistochemical 
features of this tumour in all samples and  in a limited serie  of cases  the presence of mutation of  
50 genes cancer related.  
 
STUDY DESIGN 
To identify patients with different risk classes in terms of disease-free survival (local and systemic 
recurrence) we will review about 200 cases of mixofibrosarcoma of extremities treated at our 
institution. We will perform immunohistochemical and molecular analysis by NGS, Sanger 
sequencing and Digital PCR  considering 50 cancer-related genes. Survival analysis for disease free-
survival and overall survival was carried out using Kaplan-Meier curves and log-rank tests.  
 
POPULATION 
Inclusion criteria 
1) Male and female patients treated at Rizzoli Institute from 01 Jan  1993 to 01 Dec 2017 
2) Diagnosis of mixofibrosarcoma of extremities  
3) Histological slides/formalin-fixed paraffin-embedded tissue tumor (FFPE) blocks from 
archive available to perform the histology analysis and molecular analysis 
4) Enough tumor biobanked material available for molecular analysis 
5) Presence of the biobank signed informed consent  
6) Written informed consent prior to any study-specific analysis and/or data collection  
 
Exclusion criteria 
1) Patients with histological diagnosis different from mixofibrosarcoma of extremities  
MATERIAL AND METHODS 
We will retrieve from the database of the Rizzoli institute all the cases with a histological diagnosis 
of mixofibrosarcoma of the extremities from 01 Jan 1993 to 01 Dec 2017  
We aspect to find approximately 200 cases.  
We will review all the medical records, radiological imaging, and histological data of these cases. 

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The immunohistochemical analysis of the principal alterated genes identified in this tumor by the  
literature will be performed. In a limited serie of cases, we will perform NGS analysis of specific 
hot spot mutations cancer-related in 50 genes. Results obtained will be validated by Sanger 
sequencing and Digital-PCR in all the selected cases.  
 
STATISTICS 
To the case series will be applied a descriptive statistic. 
 
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. 
 
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).  
 
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.  

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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.  
Due to the high incidence of mortality of the disease under investigation, it would be possible that 
some potential eligible subjects will be deceased.  
 
GENERAL PRINCIPLES FOR HUMAN BIOLOGICAL MATERIAL (HBM) COLLECTION 
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 and frozen tissue of tumor sample) are already stored in the 
archive of the Istituto Ortopedico Rizzoli, Pathology Department. 
The biological material will be used and stored according with the sample characteristic and 
applicable regulation. 
• The Istituto Ortopedico Rizzoli will have a designated person responsible for collection and 
will act as a communication point  
• The collected HBM should be documented, i.e. the amount remaining and its location. act 
as a communication point  
 
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 

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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 kept at all times 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. 
 
PUBBLICATION OF RESULTS  
The results from this study will be published or shown at scientific conferences.  
The final publication of the study results will be written by the Principal Investigator. 
 
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.  
 
REFERENCES 
1. Willems SM, Debiec-Rychter M, Szuhai K, Hogendoorn PC, Sciot R.  Local recurrence of 
myxofibrosarcoma is associated with increase in tumour grade and cytogenetic 
aberrations, suggesting a multistep tumour progression model. Mod Pathol. 2006. 19: 407-
416. 
2. Mentzel T, Calonje E, Wadden C, Camplejohn RS, Beham A, Smith MA, et al. 
Myxofibrosarcoma. Clinicopathologic analysis of 75 cases with emphasis on the low-grade 
variant. Am J Surg Pathol. 1996. 20: 391-405. 
3. Gronchi A, Lo Vullo S, Colombo C, Collini P, Stacchiotti S, Mariani L, et al.  Extremity soft 
tissue sarcoma in a series of patients treated at a single institution: local control directly 
impacts survival. Ann Surg. 2010. 251: 506-511 

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4. Kaya M, Wada T, Nagoya S, Sasaki M, Matsumura T, Yamaguchi T, et al.  MRI and 
histological evaluation of the infiltrative growth pattern of myxofibrosarcoma. Skeletal 
Radiol. 2008. 37: 1085-1090. 
5. Willems, S. M. et al. Cellular/intramuscular myxoma and grade I myxofibrosarcoma are 
characterized by distinct genetic alterations and specific composition of their extracellular 
matrix. J. Cell Mol. Med. 2009; 13, 1291–1301  
6. Heitzer E, Sunitsch S, Gilg MM, Lohberger B, Rinner B, Kashofer K, Stündl N, Ulz P, 
Szkandera J, Leithner A, Liegl-Atzwanger B. Expanded molecular profiling of 
myxofibrosarcoma reveals potentially actionable targets. Mod Pathol. 2017;30(12):1698-
1709.