Assessment of MGMT Promoter Methylation and Clinical Benefit From Temozolomide-based Therapy in Ewing Sarcoma Patients
Conditions: Ewing Sarcoma Family of Tumors
Sponsor: Istituto Ortopedico Rizzoli

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Assessment of MGMT promoter methylation and clinical benefit from temozolomide-based therapy in 
Ewing sarcoma patients 
 
Versione 1.0 07Jan2014 
Coordinator: 
 
Emanuela Palmerini, MD  
Sezione  di Chemioterapia dei Tumori dell' Apparato Locomotore Istituti  
Istituto Ortopedico Rizzoli,  
Bologna, ITALY 
 
 
 
 
 
 
 
Contatti: 
Emanuela Palmerini, MD  
Sezione  di Chemioterapia dei Tumori dell' Apparato Locomotore Istituti  
Istituto Ortopedico Rizzoli,  
40136 Bologna, ITALY 
e-mail: emanuela.palmerini@ior.it 
Tel. +39/051 6366199 
FAX +39/051 6366277  
 

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INDEX 
 
Introduction 
 
 
 
 
 
 
 
 
 
pg. 3 
Bibliography 
 
 
 
 
 
 
 
 
 
pg. 7 
Study Objectives 
 
 
 
 
 
 
 
 
pg. 9 
Trial Design 
 
 
 
 
 
 
 
 
 
pg. 9 
Subject Selection 
 
 
 
 
 
 
 
 
pg. 10 
Enrollment  
 
 
 
 
 
 
 
 
 
pg. 10 
Sample size and Statistic 
 
 
 
 
 
 
 
pg. 11 
Ethic and logistical aspects 
 
 
 
 
 
 
 
pg. 11 
Table 1: RECIST 1.1 
 
 
 
 
 
 
 
 
pg. 12 
Table 2: PERCIST 
 
 
 
 
 
 
 
 
pg. 14 
 
 

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Introduction 
The current treatment modalities for Ewing sarcoma (EWS), including surgery, radiotherapy 
and chemotherapy, significantly improved the survival of these tumors, shifting the average 5-
years disease free survival rate from 10% to 65-75%. Nonetheless metastatic patients and 
patients with unfavorable prognostic factors (like poor pathological  response,  large tumor 
mass, multiple sites lesions) have poor survival. 
Patients who are metastatic at the time of diagnosis have an expected survival < 40%, while it 
is <20% in case of relapse. 
The standard treatment for Ewing sarcoma is based on the use of a doxorubicn, vincristin, 
ifosfamide, dactinomycin D, cyclofosfamide ad etoposide regimen with, in selected cases, high 
dose busulphan and melphalan with autologous peripheral blood stem cells rescue. 
In the last years, for patients with high risk Ewing’s sarcoma, the treatment with irinotecan 
and temozolomide has been introduced.  
The activity of irinotecan (a topoisomerasis I inhibitor) and temozolomide (alkylating agent ) 
has been study either in monotherapy and in combination, using different schedules and in 
combination with other drugs and target therapy. 
The experience in the use of temozolomide as single agent in is limited to 1 case report: 180 
or 215 mg/m2/die orally administered for 5 days in 7 pediatric patients with relapsed Ewing 
sarcoma. This study, with the compassionate use of temozolomide,  documented a 
progressive disease in all the treated patients, after 1 or 2 cycle of therapy. 
The actual available studies are related to patients treated with irinotecan as single agent 
continuously administered at low dosage, based on the pre-clinical results that demonstrated 
that this schedule has a minor toxicity compared to irinotecan given at high doses for a 
shorter time,  

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Memorial Sloan Kettering reported 3 Ewing’s Sarcoms cases, treated with irinotecan 20 
mg/m2 for 5 days, given for 2 weeks  (20 mg/m2 x 5 x 2) every 3 weeks.  All the patient had 
progressive disease (the time for the response evaluation is not reported). 
St Jude’s Hospital presented a Phase I dose finding study which included 2 patients with 
Ewing’s Sarcoma that were treated with irinotecan up to 45 mg/m2 for 5 days given for 2 
weeks.  One of the cases, reported a Partial Response (PR) that lasts for 6 cycles. Another St 
Jude’s study of 3 Ewing’s Patients treated with of irinotecan 5-20 mg/m2 for 5 days for 2 
weeks, and Gefitinib orally administered, documented 1 PR lasted for 4 cycles. 
The most interesting study on the use of irinitocan in metastatic Ewing’s Sarcomas as single 
agent, is the phase II window study, in  patients enrolled in EuroEWING99 protocol, and 
receiving ifosfamide 600/m2, single infusion, every 3 weeks. Five out of 22 patients included 
had a PR,  1 patient had  minimal response and 9 had SD. 
Combination studies were based on sinergic activity of irinotecan, when combined with 
temozolomide; this effect is dose dependent. 
Four irinotecan plus temozolomide in relapsed Ewing sarcoma studies were published, as 
reported in Table 1.  In all studies irinotecan was given in prolonged infusion, at a low dosage, 
with a 4-fold irinotecan dose difference, in a total number of 65 patients. 
In the first phase I study 14 patients were evaluable, reporting 1 CR, 3 PRs, and 4 SDs. 
In a retrospective series from MDACC in 25 patients, 7 CRs, 9 PRs, and  6 SDs were 
documented. The MSKCC reported 5 CRs, and 7 PRs, as “best responses”  out of 19 patients. 
The Nino Jesús Pediatric Hospital, in Madrid, documented 3 PRs and 2 SDs out of 7 patients. 
Combining these 4 series, the  65 included patients ha a response rate (RR) of 54% and a 
median time to progression (mTTP) ranging from 4.6 to 8.3 months. The progression free 

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survival was not reported. Three groups reported a total of 31 patients treated with 
vincristine in combination with temozolomide and irinotecan, with 4 differrent schedules, 
inlcluding irinotecan per os (see Table 1). 
The larger study is a polish retrospective series in 22 cases, reporting 5 CRs, 7 PRs and 3 SDs, 
for a RR of 55%. 
Oral irinotecan was employed at Cincinnati Children’s Hospital, in different trials. In the 1st 
study 1 unconfirmed CR, 1 PR and 1 SD were documented in 5 patients.  In the second, with 
the addition of bevacizumab 15 mg/kg on day 1, 2 Ewing cases were included, with 1 CR after 
6 cycles and 1 PR after 6 cycles. 
Table 1 
Authors 
TMZ (mg/m2) 
Irinotecan (mg/m2) 
Other agents 
Responses 
Wagner 
100 x 5 
IV 10-20 x 5 x 2 
None 
4 
Anderson 
100 x 5 
IV 10 x 5 
IV 10 x 5 x 2 
None 
16 
Casey 
100 x 5 
IV 20 x 5 x 2 
None 
12 
Hernandez-
Marques 
80-100 x 5 
IV 10-20 x 5 x 2 
None 
3 
Raciborska 
125 x 5 
IV 50 x 5 
Vincristine x 1 
12 
McKnall-Knapp 
100 x 5 
IV 20 x 5 x 2 
Vincristine x 1 
1 
Wagner 
100-150 x 5 
Orale 35-90 x 5 
Orale 35-90 x 5 x 2 
Vincristine x 1 
Vincristine x 2 
2 
Wagner 
150 x 5 
Orale 90 x 5 
Vincristine x 1 
Bevacizumab  
2 
 
Toxicity of irinotecan temozolomide combination is mainly hematological and gastrointestinal. Even in 
heavily pretreated patients, infrequent were G 3-4 thrombocytopenia and neutropenia. Grade 1-2 diarrhea 
was reported after about 50% of cycles, nonetheless in only 10% of patients reached grade 3-4, rarely 
requiring treatment discontinuation. 

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The Cincinnati study suggests that grade  ≥3  diarrhea  is more common when irinotecan is given at a dose   
> 10 mg/m2/die.  
Other side effects included nausea and vomiting, up to 15% of the patients, and 2 cases of grade 3-4 of 
hepatic toxicity. 
Temozolomide is an alkylating agent and recent studies demonstrated a key role of O6-methylguanine-DNA 
methyltransferase (MGMT) enzyme  in mechanisms of resistance to this class of drugs. 
.Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA repair gene by 
promoter methylation has been associated with longer survival in TMZ treated glioblastoma patients, and 
we believe that methylation studies could identify the most appropriate population to treat. 
No studies on MGMT methylation status have been reported in patients with Ewing sarcoma. 
Given these considerations and due to the demonstrated efficacy of irinotecan / temozolomide 
combination in Ewing, we propose to assess MGMT methylation status in metastastic  Ewing patients 
treated with temozoloide and irinotecan, in order to identify the most responsive patients. 
This project has been supported by University-Region Project, Alessandro Liberati (PRUA1GR-2013-
00000114), Principal Investigator: Dr. Emanuela Palmerini. 

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Bibliography 
 
 Kushner BH, Meyers PA.  How effective is dose-intensive/myeloablative therapy against Ewing's 
sarcoma/PNET metastatic to bone or bone marrow. The MSKCC experience and a literature review. J Clin 
Oncol, 2001 
 Bacci G, Ferrari S, Longhi A, Donati D, De Paolis M, Forni C, Versari M, Setola Therapy and survival after 
recurrence of Ewing's tumors: the Rizzoli experience in 195 patients treated with adjuvant and 
neoadjuvant chemotherapy from 1979 to     1997. Ann Oncol. 2003 Nov;14(11):1654-9. 
 Casey DA, Wexler LH, Merchant MS, Chou AJ, Merola PR, Price AP et al. Irinotecan and temozolomide for 
Ewing sarcoma: the MSKCC experience. Pediatr Blood Cancer. 2009  
 Wagner, L.M., et al., Temozolomide and intravenous irinotecan for treatment of advanced Ewing 
sarcoma. Pediatr Blood Cancer, 2007. 48(2): p. 132-9. 
 Anderson, P., et al., Novel bone cancer drugs: investigational agents and control paradigms for primary 
bone sarcomas (Ewing's sarcoma and osteosarcoma). Expert Opin Investig Drugs, 2008. 17(11): p. 1703-
15. 
 Casey, D.A., et al., Irinotecan and temozolomide for Ewing sarcoma: the Memorial Sloan-Kettering 
experience. Pediatr Blood Cancer, 2009. 53(6): p. 1029-34. 
 Hernández-Marqués, C., et al., Irinotecán y temozolomida en tumores sólidos pediátricos en recaída o 
refractarios. An Pediatr (Barc), 2013. 79(2): p. 68-74. 
 
 Raciborska, A., et al., Vincristine, irinotecan, and temozolomide in patients with relapsed and refractory 
Ewing sarcoma. Pediatr Blood Cancer, 2013. 60(10): p. 1621-5. 
 McNall-Knapp, R.Y., et al., Extended phase I evaluation of vincristine, irinotecan, temozolomide, and 
antibiotic in children with refractory solid tumors. Pediatr Blood Cancer, 2010. 54(7): p. 909-15. 
 Wagner, L.M., et al., Phase I trial of two schedules of vincristine, oral irinotecan, and temozolomide 
(VOIT) for children with relapsed or refractory solid tumors: a Children's Oncology Group phase I 
consortium study. Pediatr Blood Cancer, 2010. 54(4): p. 538-45. 

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 Wagner, L., et al., Pilot study of vincristine, oral irinotecan, and temozolomide (VOIT regimen) combined 
with bevacizumab in pediatric patients with recurrent solid tumors or brain tumors. Pediatr Blood Cancer, 
2013. 60(9): p. 1447-51. 
 Mixon, B.A., et al., Vincristine, irinotecan, and temozolomide for treatment of relapsed alveolar 
rhabdomyosarcoma. J Pediatr Hematol Oncol, 2013. 35(4): p. e163-6. 
 Wagner, L.M., et al., Phase I trial of temozolomide and protracted irinotecan in pediatric patients with 
refractory solid tumors. Clin Cancer Res, 2004. 10(3): p. 840-8. 
 Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and 
benefit from temozolomide in glioblastoma. N Engl J Med. 2005 

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Study Objectives 
- Primary Endpoint  :  
Assessment of MGMT promoter methylation and clinical benefit from temozolomide-based therapy in 
Ewing sarcoma patients 
 
- Secondary Endpoint:     
Correlation of the MGMT promoter methylation to the activity of the temozolomide + irinotecan 
combination  
 
Trial Design 
This is an observational retrospective and prospective study on the use of temozolomide and 
irinotecan in patients treated at SSD Chemioterapia of Istituto Ortopedico Rizzoli (Bologna Italy) 
and in other Italian and European Hospitals 
 The MGMT methylation status will be correlated with the disease clinical data and with the disease 
response in term of RECIST 1.1 response (Table 2). If available the metabolic response will be 
evaluated with PET TB (Table 3) 
The MGMG methylation analysis will be done in collaboration with the following laboratories 
- 
Research Unit "Molecular Characterization of Sarcoma",  
- 
CRS Development of  Biomolecular Therapies Experimental Oncology Lab,   
The test will be performed extracting from fresh tumor sample or from FFPE (based on the 
availability) , the DNA on the basis of the standard protocols. The MGMT methylation will be 
evaluated by the EpiTect Bisulfite analysis according the following steps: 
 
Target DNA will be incubated at high temperature and low pH with sodium bisulfite. 
Bisulfite conversion, transforms cytosine residues into uracil residues but leaves 5-
methylcytosine residues unchanged 
 
The bisulfite converted DNA,  will be then transferred into EpiTect spin column, when it 
will be eluted and purified 
 
 
The purified converted DNA, will be amplified by a DNA methylation-specific 
polymerase chain  reaction (MSP) with primers specific for MGMT. 

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 
After amplification the  MSP-PCR products will be electrophoresed on a 2% agarose gel 
and will visualized with UV light . 
All the patient’s data will collected in anonymized way and will be managed and retained 
by the SSD Chemioterapy ward of  Istituto Ortopedico Rizzoli Clinical Trial Office. 
The complete list of the collected data is reported into the Case Report Form (pg 12) 
Subject Selection 
 
Inclusion Criteria 
 
- Histological confirmed diagnosis of Ewing’s Sarcoma 
- Chemotherapic treatment with temozolomide and irinotecan 
- Written informed consent prior to any study related activities 
 
Exclusion Criteria 
 
-Lack of written informed consent for the study 
-Any situation that could interfere with the complete data collection of the clinical data related to the 
temozolomide and irinotecan treatment 
 
Enrollment 
 
The expected duration of this study is 36 months. An extension of this period could be evaluated if 
a correlation between the MGMT methylation status and temozolomide/irinotecan activity, will be 
observed and thus a sample size increase will be required. 
The study will be terminated if the first consecutives 15 patients will not present any alteration of 
the MGMT methylation status.  
Sample Size and Statistic 
 
On the basis of the incidence of the disease, the expected number of analysis will be 15/yr for the 
retrospective and 9/yr for the retrospective part of the study.  
The MGMT methylation status will be evaluated on 42 samples. 
Due to the lack of literature data it is not possible to predict the incidence of the expected MGMT 
methilation status in the study sample. 

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If the first consecutives 15 patients will not present any alteration of the MGMT methylation status, 
the study will be early terminated. 
If alteration of the MGMT methylation will be observed the data will be analyzed (-square test) so 
to correlate the presence and the absence of methylation with the radiological response variables 
based on RECIST 1.1 criteria (Complete Response –CR-, Partial Response – PR- and Stable Disease –
SD-). 
If this analysis will result in a possible correlation between the MGMT methylation status and the 
radiological response, a new prospective study will be developed on the basis of the results of this  
research. 
Due to the observational design of the study the sample size is not pre-determined and only the 
duration of the study can be estimated. 
 
Ethic and logistical aspects  
 
This project has been awarded and received the financial support from “Bando per la ricerca Regione-
Università, Alessandro Liberati”. 
All the financial aspect of this research will be covered by “Progetto di ricerca Regione-Università, 
Alessandro Liberati (Progetto PRUA1GR-2013-00000114)”, Principal Investigator: Dr. Emanuela Palmerini. 
 

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Tabella 1: RECIST 1.1 
Target 
Non-target 
New 
Overall Response 
CR 
CR 
No 
CR 
CR 
Non-CR / non-PD 
No 
PR 
CR 
NE 
No 
PR 
PR 
Non-PD or NE 
No 
PR 
SD 
Non-PD or NE 
No 
SD 
Not all evaluated 
Non-PD 
No 
NE 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
Response criteria 
 
Evaluation of target lesions 
Complete Response (CR): Disappearance of all target lesions. 
Any pathological lymph nodes (whether target or non-target) must have reduction in short axis 
to <10 mm. 
Partial Response (PR): At least a 30% decrease in the sum of diameters of target lesions, 
taking as reference the baseline sum diameters.  
Progressive Disease (PD): At least a 20% increase in the sum of diameters of target lesions, 
taking as reference the smallest sum on study (this includes the baseline sum if that is the 
smallest on study). In addition to the relative  increase of 20%, the sum must also 
demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new 
lesions is also considered progression). 
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD, taking as reference the smallest sumdiameterswhile on study. 
 
Evaluation of non-target lesions 
While some non-target lesions may actually be measurable, they need not bemeasured and 
instead should be assessed only qualitatively at the time points specified in the protocol. 
Complete Response (CR): Disappearance of all non-target lesions and normalisation of tumour 
marker level. All lymph nodes must be non-pathological in size (<10mm short axis). 
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of 
tumour marker level above the normal limits. 
Progressive Disease (PD): Unequivocal progression of existing non-target lesions.  
 

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Reference: 
E.A. Eisenhauera,, P. Therasseb, J. Bogaertsc, L.H. et al. New response evaluation criteria in 
solid tumours: Revised RECIST guideline (version 1.1) EUROPEAN JOURNAL OF CANCER 45 ( 2 
0 0 9 ) 2 2 8 –2 4 7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 2: PERCIST: Positron Emission Tomography Response Criteria in 
Solid Tumors  
Response category Criteria 
Complete metabolic 
response 
Normalization of all lesions (target and nontarget) to SUL less 
than mean liver SUL and equal to normal surrounding tissue SUL 
Verification with follow-up study in 1 month if anatomic criteria 
indicate disease progression 
Partial metabolic 
response 
> 30% decrease in SUL peak; minimum 0.8 unit decrease* 
Verification with follow-up study if anatomic criteria indicate 
disease progression 
Progressive 
metabolic disease 
> 30% increase in SUL peak; minimum 0.8 unit increase in SUL 
peak* 
> 75% increase in TLG of the 5 most active lesions 
Visible increase in extent of FDG uptake 
New lesions 
Verification with follow-up study if anatomic criteria indicate 
complete or partial response 
Stable metabolic 
disease 
Does not meet other criteria 
Primary outcome determination is measured on the single most active lesion on each scan (not 
necessarily the same lesion).  
Secondary outcome determination is the summed activity of up to 5 most intense lesions (no more than 
2 lesions per organ). 
 
Abbreviazioni: SUL, standardized uptake value using lean body mass; TLG, total lesion glycolysis. 
 
Reference: 
Wahl RL, Jacene H, Kasamon Y, Lodge MA. From RECIST to PERCIST: Evolving Considerations for PET 
response criteria in solid tumors. J Nucl Med. 2009;50(Suppl 1):122S-150S