Clinical trial NCT02209948
Conditions: Glioblastoma
Sponsor: Grupo Español de Investigación en Neurooncología
Start date: 2014-08-22; Completion date: 2019-06-14
Enrollment: 166 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to show if prolonging treatment with temozolomide to 12 cycles improve progression-free survival in patients with glioblastoma included in this study, randomized according to o6-methylguanine-DNA-methyltransferase (MGMT) methylation status and residual disease or not, to receive an additional 6 cycles of temozolomide.

Detailed description:

Arms: arm 1: Those patients will take 6 additional Temozolomide cycles arm 2: None
Interventions: intervention 1: Temozolomide
Intervention descriptions: intervention 1: None

Protocol documents:
Prot_001.pdf: 
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TITLE: Phase IIB, randomized, multicenter clinical trial, continuation or non-continuation with 6 cycles 
of temozolomide after the first 6 cycles of standard first-line treatment in patients with glioblastoma.  
PROTOCOL NUMBER OF THE sponsor: GEINO 01-14  
EUDRACT: 2014-000838-39  
VERSION: 1.2 of 8 September 2014  
SPONSOR: Spanish Group of Medical Neuro-Oncology - GEINO  
RESEARCH COORDINATOR:  
Dra. Carmen Balañá. Institut Catala Oncologia (ICO) Germans Trias i Pujol, Badalona 
Dra. Mª Angeles Vaz. Ramón y Cajal University Hospital, Madrid.  
The information included in this protocol is confidential. Publication is not permitted without the written consent of the researchers. This material may be used or 
disclosed by the study investigators and their associates since its use may be necessary for the development of the clinical study, as well as by the patients included in 
the study, the Public Health authorities and the ethical committees. 
 
 
 
 
 
 
 


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PROTOCOL SIGNATURE SHEET  
Protocol Code: GEINO 14-01 EudraCT  
No .: 2014-000838-39  
Version: 1.2 of September 8, 2014  
I have read this protocol and I agree to carry it out in accordance with the Good Practice Guidelines 
Clinic and with the Declaration of Helsinki.  
 Trial Coordinators 
 Dra. Carmen Balañà  
 Coordinating Firm 
 Dra. Mª Ángeles Vaz  
 Coordinating Firm 
 sponsor  
 Dr. Miguel Gil Gil  
 President of GEINO 
 
 
I have read this protocol and I agree to carry it out in accordance with the Good Clinical Practice 
Guidelines and the Declaration of Helsinki.  
 Principal Investigator:  
 Name:… ............................................ ... ..............  
 
 
 
 
 
 
 


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1. SUMMARY  
1.1. Trial 
Randomized phase II clinical trial with different types of adjuvant treatment duration after standard 
first-line treatment.  
1.2. Sponsor  
SPANISH GROUP FOR RESEARCH IN NEUROONCOLOGY - GEINO  
Headquarters: C / Velázquez nº7, 3 planta - 28001 Madrid  
Contact information:  
GEINO Technical Secretariat  
C / Secretary Coloma 64, esb B, entlo 5ª  
08024 Barcelona  
Tthe: 93.434.44.12  
Fax: 93.253.11.68  
Mail: secretaria@geino.es  
1.3. Title 
Phase IIB, randomized, multicenter continuation or non-continuation study with 6 cycles of 
temozolomide after the first 6 cycles of standard first-line treatment of glioblastoma.  
1.4 Sponsor code 
GEINO Code 14-01  
1.5. EudraCT number 
2014-000838-39  
1.6. Coordinators of the Clinical Trial
Dra. Carmen Balañà  
Medical Oncology Service.  
Institut Català Oncology Badalona  
Germans Trias i Pujol, Badalona.  
Crta. Canyet s / n 08916, Badalona  
1.7. Coordinator of the centralized study 
Dr. Cristina Carrato  
Pathological Anatomy Service  
Germans Trias i Pujol Hospital  
1.8. Participating centers and researchers 
Dra. Mª Ángeles Vaz  
Medical Oncology Service Ramón y Cajal University Hospital Crta.de Colmenar km 9,100 
28034 Madrid 
The Clinical Trial will be carried out through the participation of members of the Spanish Neuro-
Oncology Research Group (GEINO). A complete list of the researchers, centers and Ethics Committees is 
available as a separate document. 
1.9. Name of the Organization in charge of monitoring 
PHARMACEUTICAL MARKETING & CLINICAL RESEARCH - MFAR SL  
Calle Secretari Coloma 64-68, esc.B, entlo. 5th  
08024 - Barcelona  
Phone: +34 93 434 44 12  
Fax: +34 93 253 11 68  
e-mail: investigacion@mfar.net  
1.10. Study treatment 
Patients diagnosed with glioblastoma will be randomized after receiving 6 adjuvant cycles with 
temozolomide (standard treatment), to continue or not the treatment with temozolomide for 6 more 


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cycles.  
Treatment Groups:  
Experimental Arm: Temozolomide 150-200mg / m2 / dx 5 days every 28, for 6 
cycles. Control arm: No treatment. 
Presentation of the Drug: Temozolomide (Temodal®)  
 
. Pharmaceutical form: Hard capsules. Each capsule contains 5mg, 20mg and 100mg of temozolomide. 
The hard capsules have an opaque white body, an opaque green cap, and are imprinted with black ink. 
The cover is printed with "Temodal". 
. Route of administration: oral 
. ExcipientsCapsule: content: 132.8 mg anhydrous lactose, colloidal anhydrous silica, sodium starch 
glycolate, tartaric acid, stearic acid. Capsule bodies contain: gelatin, titanium dioxide (E 171), sodium 
lauryl sulfate, yellow iron oxide (E 172), red iron oxide (E 172) and are printed with black 
pharmaceutical ink, containing: rubber lacquer, propylene glycol, purified water , ammonium hydroxide, 
potassium hydroxide, black iron oxide (E 172). 
The medication used in the trial as the experimental arm will be the same as that used in the first 6 
adjuvant cycles, according to the usual practice of each center and will not be provided by the trial 
sponsor, since continuation up to 12 cycles is a standard procedure in most centers participating in the 
trial. 
1.11. Trial phase and design 
Phase IIB, multicenter, randomized clinical trial in which patients will be randomized after receiving 6 
adjuvant cycles with temozolomide (standard treatment) to continue or not treatment with 
temozolomide for 6 cycles.  
1.12. Study objective 
The main objective of the study is to detect differences in the probability of progression-free survival at 
6 months between patients with methylated or unmethylated MGMT, when receiving 6 additional cycles 
of temozolomide and stratifying for residual disease or not. 
1.13. Investigational disease 
Patients diagnosed with glioblastoma will be included.  
1.14. Primary endpoint 
The primary endpoint is to determine the differences between the two treatment groups will be 
progression-free survival at 6 months. This variable will be assessed in patients with glioblastoma who 
have already received 6 cycles of temozolomide (adjuvant) without progressing and who are randomized 
to continue with 6 additional cycles of temozolomide or to stop treatment from the date of 
randomization to the date of progression, defined according to RANO criteria. 
1.15. Sample Size 
A total of 160 patients with Glioblastoma will be included. Patients will be stratified by MGMT 
methylation status and presence of residual disease (visible on MRI) at enrollment. 
1.16. Duration of treatment 
Treatment of randomized patients in the experimental treatment arm will continue until the completion 
of the 6 additional cycles (12 cycles of adjuvant in total) or until disease progression, development of 
unacceptable toxicity, non-compliance, withdrawal of consent by part of patient or investigator's 
decision, whichever occurs first. 
1.17. Estimated study period 
3 years: 2 years for inclusion and one year for data analysis.  
Scheduled dates  
Expected start date: third quarter 2014  
Expected date of first inclusion: fourth quarter 2014  
Recruitment period: 24 months  
Expected end date: Second quarter 2017  
 
 
 
 


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2. INDEX  
1. SUMMARY 3  
2. INDEX 5  
3. GENERAL INFORMATION 7  
3.1. Type of clinical trial 7 
3.2. Description of the study drug 7 
3.3. sponsor Dating 7  
3.4. Name of the Organization in charge of monitoring 7 
3.5. Investigator General coordinator of the clinical study 7 
3.6. Coordinator of the centralized study 7 
3.7. Participating centers 8 
3.8. Estimated duration of the study 8 
4. OBJECTIVES OF THE TRIAL 8  
4.1. Background 8 
4.1.1 Epidemiology of glioblastoma 8  
4.2. Treatment of glioblastoma 8 
4.3. Justification 9 4.4. Objectives 10 
5. DESIGN AND TYPE OF CLINICAL TRIAL 10  
5.1. Development phase 10 
5.2. Control type 10 
5.3. Masking 10 
5.4. Study design 10 
5.4.1. Screeening of patients 10 
5.4.2. Centralized pathological review 11 
5.4.3. Inclusion of patients 11 
5.4.4. Translational substudy 12 
6. SELECTION OF PATIENTS 12  
6.1. Inclusion Criteria 12 
6.2. Exclusion criteria 13 
6.3. Diagnostic criteria for the pathologies in the study 13 
6.4. Inclusion 13 
6.5. Expected subjects and sample size calculation 14 
6.6. Patient withdrawal criteria 14 
6.6.1. Permanent interruption of study treatment 14 
6.6.2. Study output 14 
6.6.3. Procedures to follow when leaving the study 14 
6.7. Screening failures (Selection period) 15 
7. DESCRIPTION OF THE TREATMENT 15  
7.1. Treatment scheme 15 
7.2. Study medication 15 
7.3. Product accounting and adherence to treatment 15 
7.4. Concomitant medication 15 
7.5. Criteria for dose modification during the study 16 


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7.5.1. General toxicity and dose modifications 16Dose 
7.5.2adjustment criteria 16  
8. STUDY DETERMINATIONS AND PROCEDURES 17  
8.1. Determinations during the study 17 
8.2. Description of procedures 21 
8.3. Procedures prior to treatment 22 
8.4. Tests during treatment 23 
8.5. Safety visit 23 
8.6. After end of treatment 24 
8.7. Response evaluation 24 
8.7.1. Assessment of Clinical Response due to corticosteroids and neurological deterioration 
24 
8.7.2 Assessment of Radiological Response 25  
 
8.7.3. RANO response assessment 25 
9. ADVERSE EVENTS 26  
9.1. Definitions 26 
9.2 Notification and Documentation of Adverse Events 27  
9.3. Expedited notification of RAGI by thesponsor 28  
9.3.1 Deadlines for expedited notifications of RAGI to regulatory authorities 28  
9.3.2 Expedited notification of other relevant safety information 28 
9.3.3 Notification to Investigators 29 10. STATISTICAL CONSIDERATIONS 29  
10.1. Study variables 29 
10.1.1. Primary efficacy29 
endpoint10.1.2. Secondary variables 30 
10.2. Effectiveness evaluation 30 
10.3. Safety assessment 30 
10.4. Study populations 31 
10.5. Sample size and statistical analysis 31 
10.5.1. Sample size 31 
10.5.2. Statistical analysis 31 
11. ETHICAL AND LEGAL CONSIDERATIONS 32  
11.1. Ethics Committee 32 
11.2. Authorities 32 
11.3. Informed Consent 33 
11.4. Confidentiality 33 
11.5. Insurance 33 
11.6. Completion of the test 33 
11.7. Premature termination of the trial 33 
12. PRACTICAL CONSIDERATIONS 34  
12.1. Diagnostic criteria for the disease under study 34 
12.2. Investigator Responsibilities according to Good Clinical Practices 34 
12.3. Instructions for completing the electronic CRD 34 
12.4. Final Manuscript and Publications 34 
12.5. Monitoring 35 
12.6. Amendments to Protocol 35 
12.7. Data processing 36 
12.8. Documentation 36 
13. TRANSLATIONAL STUDY 36  
14. BIBLIOGRAPHY 37  
Annex I. KARNOFSKY INDEX 38  
Annex II. BARTHEL INDEX 39 
Annex III. MINIMENTAL TEST. MMS 41 


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Annex IV. RANO RESPONSE CRITERIA 42 
Annex V. ASSOCIATED MOLECULAR SUB-STUDY 43  
Annex VI. HELSINKI STATEMENT 44 
Annex VII. NCI COMMON TERMINOLOGY CRITERIA V.4.0 FOR ADVERSE EVENTS 45 
Annex VIII. TRIAL SCHEME 46 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3. GENERAL INFORMATION  
TITLE: Phase IIB, randomized, multicenter clinical trial of continuation or no continuation with 6 cycles 
of temozolomide after the first 6 cycles of standard first-line treatment in patients with glioblastoma.  
PROTOCOL Number .: GEINO 14-01 EudraCT  
EudraCT Number.: 2014-000838-39  
3.1. Type of clinical trial 
Phase IIB randomized, multicenter, continuation or non-continuation study with 6 cycles of 
temozolomide after the first 6 cycles of standard first-line treatment of glioblastoma.  
3.2. Description of the study drug 
Patients diagnosed with glioblastoma will be randomized after having received 6 adjuvant cycles with 
temozolomide (standard treatment) to continue or not the adjuvant treatment with 6 additional cycles.  
Treatment Groups:  
Experimental Arm: Temozolomide 150-200mg / m2 / dx 5 days every 28 for 6 cycles. 
Control arm: No treatment. 
Presentation of the Drug: Temozolomide (Temodal®). Pharmaceutical form: Hard capsules. 5mg, 20mg 
and 100mg capsules of temozolomide. 
The hard capsules have an opaque white body, an opaque green cap, and are imprinted with black ink. 
The cover is printed with "Temodal". 
3.3. Sponsor Data  
Spanish Group of Neurooncology Research - GEINO  
C / Velázquez nº7, 3 floor  
28001 Madrid  
Contact information:  
TechnicalGEINO  
SecretariatSecretari Coloma, 64-68, esc. B, entlo. 5th  
08024 Barcelona  
Phone: + 34 93 434 44 12  
Fax: + 34 93 253 11 68  
e-mail: secretaria@geino.es  
3.4. Name of the Organization in charge of monitoring 
PHARMACEUTICAL MARKETING & CLINICAL RESEARCH - MFAR SL  


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Calle Secretari Coloma 64-68, esc.B, entlo. 5th  
08024 - Barcelona  
Tel: +34 93 434 44 12  
Fax: +34 93 253 11 68  
e-mail: investigacion@mfar.net  
3.5. Investigator General Coordinator of the trial 
 Dr. Carmen Balañá (ICO Badalona) and Dra. Mª Angeles Vaz (H. Ramon y Cajal) will be the 
coordinating investigators of the trial, responsible for the preparation of analyyess, findings, conclusions 
and development of the final report.  
3.6. Coordinator of the centralized study 
Dra. Cristina Carrato - Dra. Carolina Sanz  
Pathological Anatomy Service  
H. Germans Trias i Pujol  
3.7. Participating Centers 
A complete list of participating centers available as a separate document.  
 
3.8. Estimated study duration 
3 years: 2 years for inclusion and one year for data analysis.  
Scheduled dates:  
Expected start date: third quarter 2014  
Expected date of first inclusion: fourth quarter 2014  
Recruitment period: 24 months  
Expected end date: first quarter 2017  
4. TRIAL OBJECTIVES  
4.1. Background 
4.1.1 Epidemiology of glioblastoma 
Gliomas are the most common tumors of the central nervous system (CNS) with an annual incidence of 
5.4 cases per 100,000 inhabitants. Of them, 54% are glioblastomas (GB) according to European 
statistics and our national registries. It is estimated that in Spain approximately 1,376 patients are 
diagnosed with this disease each year. The prognosis is inexorably poor with a median survival of 15-18 
months and only 10% of patients alive at 5 years. GB (WHO 2007 grade IV astrocytoma) is 
histologically characterized by presenting in addition to nuclear atypia and mitotic activity (traits that 
define grade II and III astrocytomas respectively), microvascular proliferation and / or necrosis. It is 
located preferentially in the cerebral hemispheres. Most arise 'de novo', without there being evidence of 
a previous precursor lesion, they are the so-called primary GBs. Secondary GBs develop from lower 
grade (II or III) astrocytomas. Although morphologically similar, these two groups have different clinical 
characteristics and a different molecular profile. Low-grade tumors that progress to high-grade often 
have mutations in IDH1 / 2 encoding isocitrate dehydrogenase enzymes. The IDH1 / 2 mutation in 
glioblastoma diagnosed de novo or primary confers a better prognosis. 
In GB, there is a prognostic classification, the Recursive partitioning analysis (RPA) described and 
validated (based on age, initial Minimental status, previous resection, and Karnofsky Index) that 
provides the prognosis of patients according to the benefit of treatment cancer and faciltes the 
comparison of results between studies. 
4.2. Treatment of glioblastoma 
The initial treatment is surgery that allows obtaining the histological diagnosis and achieves immediate 


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decompression of the brain. You must achieve maximum resection while maintaining the best 
neurological function that preserves the patient's quality of life. The age of the patient, and his general 
condition (KPS) are factors considered in the selection of surgical treatment. Post-operative irradiation 
has been the traditional treatment as it has been shown to increase survival from 6 to 8 months already 
in the first trials. 
The European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer 
Institute of Canada (NCIC) conducted a randomized phase III trial comparing standard radiotherapy 
(60Gy focal in 6 weeks) with the same combined treatment with temozolomide (TMZ) concomitant 
followed by 6 monthly cycles of adjuvant TMZ. 573 patients were included. The median survival was 
14.6 months with the combined treatment compared to 12.1 months in the control group. Two-year 
survival increased from 10.4% to 26.5% for the TMZ-treated group (([HR], 0.63, P <0.001). Toxicity 
was tolerable with 7% hematologic toxicity degrees III-IV in the concomitance phase and 14% in the 
adjuvant phase. 
 
In another minor randomized phase II study, the same benefit was repeated. The results after 5 years 
of follow-up again confirmed the survival benefit for the group of treated patients. with TMZ and the 
prognostic value of the RPA classification. No other study has improved the results. Concurrent and 
adjuvant treatment with 6 cycles of TMZ was established as standard after surgery in 2005. 
TMZ is a second generation derivative of imidazoltetrazinone, is spontaneously hydrolyzed under 
physiological conditions to the active metabolite and acts as a DNA methylating agent. It is administered 
orally and penetrates the blood-brain barrier (BBB). 
Due to its mechanism of action, it is known that cells that have the ability to repair methylated DNA to 
unmethylated DNA by, among other enzymes, 6-methylguanine DNA methyl-transferase (MGMT), they 
can overcome DNA damage and therefore not enter apoptosis . The enzyme is encoded by the MGMT 
gene. MGMT can be methylated in the GpG islets and not transcribed correctly, causing enzyme 
inactivity. MGMT methylation status is a predictive factor of response to TMZ. Methylated patients 
benefit the most from treatment, but it is not useful for first-line treatment decisions due to marginal 
benefit in unmethylated patients. 
4.3. Rationale 
Despite the lack of scientific evidence that prolongation of adjuvant therapy beyond 6 cycles benefits 
patients, the presence of residual disease before starting adjuvant in all patients, and an excellent 
tolerability profile of TMZ, it has led to the prolongation of adjuvant treatment beyond 6 cycles, 
sometimes even until disease progression. Prolongation of treatment beyond 6 cycles occurs basically 
due to 5 factors. 
1- The excellent tolerance profile of TMZ with apparently non-cumulative toxicity.  
2- Virtually all GB relapse after first-line treatment. Occasionally they can be re-operated or re-
irradiated in very specific cases, but there is no recognized and standardized second-line treatment of 
effectiveness. Bevacizumab is a promising drug, but it is not approved by the European Medicines 
Agency (although it is by the FDA) for the treatment of recurrent GB. In Spain, some patients can be 
treated in selected centers for compassionate use indication. Other drugs such as nitrosoureas may 
provide some clinical benefit even though they never demonstrated results of undeniable effectiveness 
before the appearance of TMZ. 
3- The concept of adjuvant in GB is not the same as in other diseases since the surgery is never radical 
and there is always residual disease after the intervention.  
4- It is difficult to evaluate the efficacy of the treatment, since magnetic resonance imaging (MRI) is not 
objective and reproducible to evaluate tumor and residual disease, although it is the recommended 
imaging technique. Contrast with gadolinium, it can assess tumor size (T1Gd sequences); infiltration 
and edema can be assessed on sequences (T2 and T2 / FLAIR), along with other imaging methodologies 
such as perfusion, diffusion, and spectroscopy. However, changes in gadolinium uptake are not 
pathognomonic for tumor growth but for increases in the permeability of the BBB that may be secondary 
to necrosis produced by chemoradiotherapy, changes in dexamethasone doses, etc. An event that 
occurs after treatment with irradiation (with or without TMZ) called pseudo-progression has been 
described, consisting of a false increase in the lesion in radiology and that can occur in up to 30% of 
patients who have been treated with TMZ and radiation, especially in the first 3 months after finishing 
treatment. These false images, sometimes accompanied by reversible neurological deterioration, can 
last for months and confuse the evaluation of viable disease. Pseudo-progression has been associated 
with longer survival and has been related to MGMT methylation, but can often lead to a premature and 
erroneous decision to discontinue TMZ treatment, thus preventing any potential therapeutic benefit. 
Since it is impossible to differentiate real residual disease from pseudo-progression, it is recommended 
to continue adjuvant TMZ at least 3 months before changing therapy and if the doubtful image persists, 
5- The selection of 6 cycles in the pivotal EORTC / NCIC trial was a random decision not based on 
previous experience.  


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The choice of 12 instead of 6 cycles of adjuvant TMZ is based on the opinion that possibly 6 cycles are 
not sufficient to control a disease in which tumor remains atthe end of the adjuvant. The American first-
line clinical studies have been designed with the administration of 12 cycles RTOG 0525, RTOG 0825 
and the American and Canadian treatment guidelines recommend continuing treatment with TMZ 
especially in caseof residual disease. This is not the case in Europe, where 6 cycles are recommended 
based on the evidence provided in the pivotal EORTC-NCI-C trial. However, treatment is not usually 
stopped after 6 cycles in most cases, in the absence of effective rescue treatments. 
A priori, the prolongation of treatment should benefit patients who present methylation of the MGMT 
gene, a predictor of benefit from TMZ treatment. 
In a study carried out in Spain and recently published in Clin Transl Oncol, it was observed that 80.5% 
of professionals continue treatment beyond 6 cycles: 44.4% only in case of residual disease, 27.8% 
always administer 12 cycles and 8.3% continue treatment until progression. The economic and 
demographic study estimated that this practice leads to spending on the National Health System of 1.5 
million euros per year. The majority of the survey participants considered that there was no evidence to 
continue or stop treatment and that it would be interesting to answer the question about the optimal 
duration of treatment in a clinical trial. For this reason, a grant was requested from the FIS Fund of the 
Carlos III Institute, which was granted to carry out the essay presented in this document. 
4.4.ORbjectives:  
Main Objective  
The main objective of the study is to detect differences in the probability of progression-free survival at 6 months 
between patients with methylated or unmethylated MGMT, when receiving 6 additional cycles of temozolomide and 
stratifying for residual disease or not. 
 
Secondary Objectives:  
Detect differences in:  
- Progression-free survival at 6 months for each of the stratification factors after inclusion in the study: Methylation 
status / residual disease  
- Progression-free survival (for all patients and by factors stratification) after inclusion in the study  
- Overall survival (for all patients and by stratification factors) after inclusion in the study  
- Differences in toxicity between both treatment arms 
- Study of markers / enzymes of resistance to temozolomide  
5. DESIGN AND TYPE OF CLINICAL TRIAL  
5.1. Development phase 
Phase IIB study, open, randomized and multicenter  
5.2.Control type  
lControl without placebo.  
5.3. Masking 
There will be no masking.  
5.4. Study design 
5.4.1. Screening of patients 
The signature of the informed consent of the patient for the clinical trial will be obtained before starting 
the selection period.  
Pre-screning may be performed in the 6th cycle of adjuvant standard treatment, but the patient will not 
be randomized until:  
 
 the result of the methylation status of the MGMT gene is available.  
 a baseline MRI has been performed on the patient in which it can be evaluated if there is residual tumor.  


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The different procedures carried out as part of the usual clinical management of the patient (for 
example, blood tests, imaging tests, etc.) and carried out before signing the informed consent can be 
used for selection or as baseline results as long as these tests have been carried out as specified in the 
protocol. 
Once the informed consent has been signed, a selection number will be assigned to each patient. Each 
center will receive a selection form within the Center's Investigator File in which predetermined selection 
numbers will be assigned. Said document must always remain in the study center under the custody of 
the research team. This selection number will identify patients through the necessary procedures to 
confirm the suitability of the first for the study (laboratory analysis, centralized imaging tests, central 
pathological review, etc.). An additional document will be added to the Investigator's Manual with 
detailed information on the identification procedures for the selection of patients. 
5.4.2. Centralized pathological review 
In the trial, two centralized reviews are carried out at the beginning of the study:  
1. To confirm the diagnosis of Glioblastoma: in all cases, a tumor block sample or at least 15 
unstained or stained histological slides will be sent with eosin / hematoxylin.  
2. For the determination of the methylation status of the MGMT gene: If the center of origin performs 
the determination of the MGMT methylation status locally, the result of the center will be accepted. If 
the center of origin does not make the determination of the MGMT, it will be carried out centrally at 
the Germans Trias i Pujol Hospital. It will therefore be necessary to send a tumor block. 
To participate in the study, the patient must sign the informed consent for participation in the clinical 
trial.  
There is associated a subsequent substudy for the determination of resistance proteins (MSH2, MSH6). 
To participate in this associated substudy (immunohistochemical study of resistance proteins), a tissue 
microarray (TMA) must be manufactured and for this it is necessary to have the tumor block to 
manufacture the TMA and subsequent immunohistochemical study. For the participation of the patient in 
the associated substudy, the patient must sign an informed consent different from that of their 
participation in the clinical trial. 
All samples will be sent by courier to the central laboratory. The response to the result of the review and 
the MGMT study will be obtained in a maximum of 7 days, at which point the patient can be 
randomized. All cases must be confirmed by the external reviewing pathologist, and although patients 
may be registered without waiting for the review report, they could be ineligible a posteriori, if the 
diagnosis is not confirmed. 
All excess slides will be returned to the center of origin together with a report after the central 
pathological review. The paraffin blocks will be returned after making the MA tissue. Subsequently, all 
remaining samples will also be returned after the completion of the project. 
No tumor sample can be mobilized from the hospital of origin until the patient has signed the informed 
consent. The centralized review for both pathological confirmation and the determination of MGMT will 
be carried out at: 
Dra. Cristina Carrato- Dra. Carolina Sanz  
Germans Trias i Pujol Hospital  
Pathological Anatomy Service  
Crta. Del Canyet s / n - Badalona  
To send the sample, please contact:  
MFAR, SL - TechnicalGEINO  
SecretariatSecretari Coloma, 64-68, esc. B, entlo. 5th  
08024 Barcelona  
Tthe: 93 434 44 12  
Fax: 93 253 11 68  
e-mail: investigacion@mfar.net - secretaria@geino.es 
 
 
No inclusion will be possible until:  
1- The center has the result of the methylation of the MGMT gene, either the local review or the 
centralized review of the tumor sample (The centralized review will take place in approximately 7 days).  
2- All patients must show before signing the IC (around the 6th adjuvant cycle) of an MRI in which it is 
demonstrated that there is no radiological progression. The presence of residual tumor or image 
compatible with residual tumor will be recorded on the patient record sheet for stratification in the 
assay. 


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5.4.3. Inclusion of patients 
After confirmation that the patient is a candidate to be included in this study (result of MGMT 
methylation, MRI without progression, and fulfillment of the inclusion / exclusion criteria), patients will 
be randomized, assigning them centrally a study number to each subject. 
The patient enrollment procedure is described below:  
1. Complete and sign the patient enrollment form (registration forms must be signed by a 
clinician (PI or co-PI) identified in the signature list and in the registry delegation of 
responsibilities).  
2. Send the completed and signed form to the CRO:  
MARKETING FARMACÉUTICO & INVESTIGACIÓN CLÍNICA, SL 
FAX (+ 34 93 253 11 68) or e-mail to investigacion@mfar.net  
3. The CRO will conduct the patient inclusion process.  
4. Respond to the center, sending the confirmation form of the patient's randomization both by 
fax and by e-mail. Confirmation of patient randomization will contain the patient's study number. 
5. Likewise, the treatment arm to which it has been randomized (experimental or control) will be 
communicated.  
6. In no case will treatment be started without knowing the result of the randomization.  
The patient's study number will identify subjects throughout their participation in this study. 
An additional document will be added to the Center's Investigator File with detailed information on 
patient inclusion procedures.  
5.4.4. Translational substudy 
In the event that the patient has given their informed consent to participate in the translational sub-
study, the tumor samples sent for the centralized review and determination of the MGMT methylation 
status will be used to prepare a tissue matrix for subsequent immunohistochemical studies of IDH1 and 
TMZ resistance or sensitivity protein (MSH6, MSH2), and future studies that may arise as a result of the 
assay results. 
All samples will be coded prior to shipment.  
An additional document will be added to the Center's Archive with detailed information on the collection 
of the samples and the procedure for sending them.  
6. SELECTION OF PATIENTS  
Glioblastoma patients who meet all eligibility criteria will be included. 6.1. Inclusion 
criteria 
A subject will be considered eligible for inclusion if they meet each and every one of the following 
criteria:  
1. Ability to understand and sign the informed consent document.  
2. Age ≥ 18 years.  
3. Patients with glioblastoma according to WHO classification (glioblastoma) who have received 
chemo-radiotherapy and chemotherapy based on temozolomide (Stupp scheme) and have 
completed 6 cycles of adjuvant temozolomide (with or without bevacizumab) in the context of 
standard treatment without having presented disease progression. 
4. Availability of tumor tissue from the first surgery to perform the centralized histological review, to 
determine the methylation of the MGMT gene if it has not been performed in the center of origin 
(if it had been performed in the center of origin, it will be accepted the result of the center). 
5. Stable doses of dexamethasone on inclusion, never higher than the dose of corticosteroids 
received in cycle 6 of adjuvant.  
6. Karnofsky index ≥ 60%  
7. All patients must show absence of disease progression on brain MRI as defined in the RANO 
Criteria prior to randomization. 


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8. Baseline MRI of the study carried out a maximum of 6 weeks prior to inclusion, in which no 
progression was observed and allowed the administration of the 6th cycle of care. NMR 
performed after the 6th cycle of adjuvant is also acceptable as long as no progression is 
observed. 
9. Adequate marrow reserve: hematocrit≥29%, leukocytes> 3,000 / mcl, ANC ≥ 1,500 cells / ul, platelets ≥ 100,000 
cells / ul.  
10. Creatinine <1.5 times the upper limit of normal for the testing laboratory.  
11. Serum bilirubin <1.5 / ULN, SGOT and SGPT <2.5 times the upper limit of normal for the 
testing laboratory. Serum alkaline phosphatases <3 / ULN. 
12. Effective contraceptive method for patients and their partners.  
6.2. Exclusion criteria 
Subjects who meet any of the following criteria should not be included in the study:  
1. Less than 5 years of any previous infiltrating neoplasm. Carcinoma in situ of the cervix or 
cutaneous basal cell carcinoma are accepted. 
2. Concomitant treatment with other investigational agents (except concomitant bevacizumab).  
3. Presence of any clinically significant gastrointestinal abnormality that may affect the intake, 
transit, or absorption of the study drug, such as the inability to take oral tablet medication.  
4. Presence of any psychiatric or cognitive disorder that limits the understanding or signing of the 
informed consent and / or jeopardizes compliance with the requirements of this protocol. 
5. Concurrent disease that prevents the continuation of treatment with temozolomide. 6. 
Presence of leptomeningeal spread. 
7. Pregnant or lactating women.  
8. HIV positive patients on combined antiretroviral treatment.  
6.3. Diagnostic criteria for the study pathologies 
Patients diagnosed with Glioblastoma who have received standard treatment with irradiation, 
temozolomide and subsequent treatment with 6 adjuvant cycles (with or without bevacizumab) and who 
have not presented progression will be selected, in whom continued treatment with adjuvant 
temozolomide is considered until 12 cycles. 
Patients will be randomized at that time to continue or stop treatment with temozolomide. For this, they 
must have an MRI before randomization in which it is demonstrated that there is no disease progression 
and they have the result of the methylation of the MGMT gene. 
 
6.4. Inclusion 
The CRO MFAR SL should be contacted for the inclusion of patients.  
Pre-screning can be performed from the beginning of cycle 6, if the patient is clinically stable and by 
MRI. (An MRI that has been carried out before said cycle will be accepted to sign IC and start the 
inclusion procedures but it should not exceed 6 weeks). 
The patient will not be randomized until:  
1. the MGMT methylation result is available  
2. Baseline MRI confirms previous disease stability.  
3. the patient must remain clinically stable without increasing the dose of dexamethasone. 
Dexamethasone doses should be equal to or less than the dose received during cycle 6 of the 
adjuvant. 
 
Once the patient meets the requirements described above, the correctly completed inclusion sheet will 
be sent by fax and from MFAR SL a fax will be returned to the Center confirming the inclusion and the 
assigned treatment arm.  
Contact information: 
PHARMACEUTICAL MARKETING & CLINICAL INVESTIGATION - MFAR SL  
C / Secretari Coloma 64-68, esc.B, entlo. 5th  


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08024 - Barcelona  
Tthe: +34 93 434 44 12  
Fax: +34 93 253 11 68  
e-mail: investigacion@mfar.net  
6.5. Expected subjects and sample size calculation 
A total of 160 patients diagnosed with Glioblastoma will be included. Patients will be stratified by MGMT 
gene methylation status and presence of residual disease (visible on MRI) at the time of enrollment. 
6.6. Patient withdrawal criteria 
6.6.1. Permanent interruption of study treatment 
Patients will receive the treatment described above until one of the following occurs:  
● Completion of treatment according to protocol.  
● Termination by decision of the Principal Investigator.  
● Unacceptable toxicity or adverse events that could render the administration of treatment an unacceptable risk in the 
investigator's discretion. In case of unacceptable toxicity, patients will be followed until toxicity is resolved. 
● Progression of the disease by RANO criteria.  
● Withdrawal of consent by the patient.  
● The researcher or the Sponsor considers the patient not complying with the requirements of the study. ● The study 
ends or is closed early. 
● Major protocol deviations:  
o Non-compliance with the inclusion / exclusion criteria.  
 
o Inability to perform the full tumor evaluations required in the protocol.  
The reason for discontinuation of treatment should be clearly reflected in the case registration forms 
(CRD).  
6.6.2. Study output 
Patients will be encouraged to continue in the study; however, they can voluntarily withdraw their 
consent at any time. On the other hand, the investigator may withdraw a patient from the study at his 
discretion. Finally, the Sponsor can suspend the study if necessary. 
The reasons for the early withdrawal of a patient from the study should be documented in the CRD as 
follows:  
- The study is closed / finished.  
- The patient abandons the follow-up.  
- Investigator's decision.  
- The patient withdraws consent.  
- Major protocol violations.  
- Death.  
The CRD will detail the date of departure from the study and the cause of it. In the case of death, a 
death certificate should be obtained (if possible) with the cause of death assessed and documented. 
Patients who leave the study cannot be included again, whatever the reason for their withdrawal. 
6.6.3. Procedures to follow when leaving the study 
For all patients who leave the study, the investigator will follow the following procedures:  
a) In the event of the end of treatment, the follow-up visits specified in this protocol will be carried out. 
b) In case of abandonment of the treatment before its completion, the subsequent treatment and 
subsequent follow-up will be determined until death, guaranteeing the patient medical assistance 
according to the usual clinical practice.  
6.7. Screening failures (Selection period) 


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A selection failure is considered to be any patient who has signed the informed consent but withdraws it 
before inclusion. All potential subjects that are evaluated for inclusion in the study that are screening 
failures will be registered in the Identification list / Patient Selection Registry but will not be included in 
the study database. The reasons for exclusion of these patients who do not enter the trial must be 
recorded in the aforementioned forms. All patients who have been randomized will be registered and 
documented even if they do not start continuation treatment with temozolomide. These will be analyzed 
by intention to treat. 
7. DESCRIPTION OF THE TREATMENT  
7.1. Treatment scheme 
Patients will be randomized into the study (depending on the status of MGMT gene methylation and the 
presence or absence of residual disease on MRI) into one of the following treatment groups: 
EXPERIMENTAL GROUP: Temozolomide dose 150/200 mg / m2 for 5 days every 28 days for 6 cycles 
(total 12 cycles of adjuvant temozolomide). 
CONTROL GROUP: No treatment (total 6 cycles of adjuvant treatment).  
 
The dose that the patient will receive in the adjuvant cycles before starting the trial will be maintained 
in such a way that the continuation dose may range between 125mg / m2 / dx 5 days and 200mg / m2 
/ dx 5 days. 
The initiation of treatment should take place a maximum of 2 weeks after the end of cycle 6 of 
adjuvant, that is, a maximum of 6 weeks is allowed after administration of day 1 of cycle 6 of adjuvant. 
6 cycles will be administered, after checking the blood count and other toxicity. The 7th cycle dose level, 
(1 additional trial cycle) once the patient is randomized to the continuation treatment group will be the 
same dose that was previously received in the 6th cycle prior to entering the trial. We establish a dose 0 
equivalent to the last dose received in cycle 6. 
Randomized patients in the control arm will be visited with the same frequency as if they were treated 
with the 6 additional cycles of temozolomide.  
See treatment scheme in Annex VIII.  
7.2. Study medication 
Temozolomide:  
The temozolomide dose is adjusted to the patient's body surface area and the total dose can be rounded 
to the nearest ten (10 mg).  
It is administered in a single oral dose at least 2 hours before or after the intake of any food, the first 5 
days of the 28-day cycle. An antiemetic should be prescribed one hour before taking it at least 5 days 
and exactly follow the pattern that was followed for the first off-trial cycles. 
For all phases, lost doses of the drug due to dose adequacy due to toxicity should not be recovered.  
7.3. Product accounting and adherence to treatment 
In accordance with local legislation, the researcher will state in the clinical history that the patient has 
taken the medication as prescribed for each new cycle prescribed in the active treatment arm. The dose 
will be recorded and if there has been a loss of any of them and the reason. 
7.4. Concomitant medication 
All study subjects will be asked for a complete list of the drugs they have taken during the 4 weeks prior 
to the screening period, including both prescription and non-prescription drugs. The investigator must 
be informed of any new drug administered to the patient from the screening visit to the post-treatment 
follow-up visit. All medications taken by the patient during the study will be recorded in the electronic 
CRD (eCRD), where the indication, dose and administration data of the new drug will be detailed. 
Use with caution  
Growth factors can be used, at the discretion of the investigator, to induce neutrophil count elevations 
when febrile neutropenia is present, but not prophylactically, in order to deliver temozolomide on 
schedule. 
7.5. Criteria for dose modification during the study 


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7.5.1. General toxicity and dose modifications 
Toxicity will be recorded according to the NCIC version 4.0 criteria (Annex VII). Adverse effects will be 
meticulously recorded and reported according to European and Spanish laws. 
7.5.2 Dose adjustment criteria  
This phase is considered care management but some recommendations are detailed to homogenize the 
treatment of patients.  
 
The dose that the patient will receive in the adjuvant cycles before starting the trial will be maintained 
in such a way that the continuation dose may range between 125mg / m2 / dx 5 days and 200mg / m2 
/ dx 5 days. 6 cycles will be administered, after checking the blood count and other toxicity. The 7th 
cycle dose level, once the patient has been randomized to the continuation treatment group, will be the 
same dose as previously received in the 6th cycle prior to entering the trial. We establish a dose of 0 
equivalent to the last dose received in cycle 6. 
A dose reduction level will be allowed for subsequent cycles in case of toxicity down to a minimum of 
125mg / m2 / dx 5 days. 
 
Dose level  
cycle 1 (7th) 
Dose mg / m2 / 
dx 5 days 
Notes 
0  
150-200 mg / m2 
Same dose as in cycle 6 before inclusion in the trial. 
-1  
125 mg / m2 
If the patient has to lower the dose due to toxicity 
according to the monthly hematological controls to 
a level lower than this, they will exit the study due 
to toxicity 
 
 
Table of dose modification for subsequent cycles:  
Neutrophils  
109 / l 
Platelets  
109 / l 
Tox no  
hematological  
Dose 
≥1500  
≥100  
≤ grade 2 *  
Continue treatment at the prescribed doses 
in cycle and subsequent 
≥500 <1500  
≥25 <100  
Grade 3-4 
Any of them:  
Treatment delay up to a maximum of 2 
weeks until normal figures are obtained. 
Continue treatment with reduction of a dose 
level in subsequent cycles. 
If there is no recovery at 2 weeks end of 
adjuvant treatment. 
<500  
<25 
GRADE 3-4 
not recovered 
in 2 weeks 
Any of them:  
Definitive stop of adjuvant TMZ.  
Control hemograms every 2-3 days until 
hematological recovery to Grade 3 (platelets ≥ 
25x109/ l, neutrophils≥0.5x109/ l) and continue 
evaluation schedule 
 
 
* except alopecia, nausea and vomiting  


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8. DETERMINATIONS OF THE STUDY AND PROCEDURES  
8.1. Determinations during the study 
Screening tests for inclusion of patients in the study should be performed days before the first dose of 
study drug.  
 
The table summarizes the procedures and determinations to be made throughout the trial and its 
schedule, in the two trial arms. 
All data referring to the patients entering the study will be collected in the specific data collection 
notebook for this clinical trial.  
 
 
1- EXPERIMENTAL ARM  
DETERMINATION  
PATIENTS IN ACTIVE TTO 
CYCLE 6  
ASSISTA
NCE  
In the 
previ
ous 
28 
days 
During treatment 
ADJUVANCE PHASE cycles / 28 d 
CYCLE 1 (7)  
CYCLE 2 
(8)  
CYCLE 3 
 (9) 
CYCLE 4  
(10) 
CYCLE 5 
(eleven) 
CYCLE 6 
(12) 
Informed consent  
X 
 
 
 
 
 
 
AP review  
X 
 
 
 
 
 
 
MGMT study  
X 
 
 
 
 
 
 
Randomization  
X 
 
 
 
 
 
 
NMR (1)  
X  
 
 
X  
 
 
X 
Anamnesis  
X 
 
 
 
 
 
 
Physical exploration  
X  
X  
X  
X  
X  
X  
X 
CORTICOIDS (DOSE / DRUG)  
X (2)  
X  
X  
X  
X  
X  
X 
ANTICOMICIAL  
(DOSE / DRUG) 
X  
X  
X  
X  
X  
X  
X 
KPS  
X  
X  
X  
X  
X  
X  
X 
TMZ  
 
X  
X  
X  
X  
X  
X 
Toxicity  
 
X  
X  
X  
X  
X  
X 
Clinical Analysis  
X  
X  
X  
X  
X  
X  
X 
Concomitant medication  
X  
X  
X  
X  
X  
X  
X 
I. BARTEL  
X  
 
 
X 
 
 
 


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MMS  
X  
 
 
X 
 
 
 
RX THORAX (3)  
X 
 
 
 
 
 
 
ECG (4)  
X 
 
 
 
 
 
 
Patient survival 
 
 
 
 
 
 
 
 
(1) MRI: Baseline MRI will be done around cycle 6 (2 weeks before or 3 weeks after) and will be taken as baseline. 
In case of neurological deterioration, the MRI and response assessment will be advanced. 
(2) Corticosteroids: the baseline corticosteroid dose should not be higher than the corticosteroid dose received 
during cycle 6 of the adjuvant.  
(3/4) Chest X-ray and ECG will only be performed if clinically indicated.  
 
2- CONTROL ARM 
 
DETERMINATION  
PATIENTS WITHOUT 
TTO 
CYCLE 6TH ASSISTANCE  
In the previous 28 days 
Every 28 days 
for 6 months 
after 
randomizatio
n 
Every 12 weeks 
after 6 months 
after 
randomization 
Informed consent  
X 
 
 
AP review  
X 
 
 
MGMT study  
X 
 
 
Randomization  
X 
 
 
 
NMR (1)  
X  
X 
X 
Anamnesis  
X  
X 
 
Physical exploration  
X  
X 
X 
CORTICOIDS (dose / drug)  
X  
X 
X 
Anticomicials (dose / drug)  
X  
X 
X 
KPS  
X  
X 
X 
Toxicity  
 
X 
X 
Clinical Analysis  
X  
X 
X 


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Concomitant medication  
X  
X 
X 
I. BARTEL (1)  
X  
X 
X 
MMS (1)  
X  
X 
X 
RX THORAX (2)  
X 
 
 
ECG (2)  
X 
 
 
Patient survival 
 
 
 
(1) MRI, I. Barthel and MMS will be performed according to the usual clinical practice, every 12 weeks. In case of 
neurological deterioration, MRI and Barthel response assessment will be carried out 
(2) The performance of chest X-rays and ECG will only be performed if clinically indicated 
 
 
8.2. Description of procedures 
1. Informed consent: Each patient must sign written consent prior to undergoing the specific 
evaluations of the study and prior to the start of treatment. The patient must sign 2 separate informed 
consents, one for the clinical trial and the other for the biological samples. 
2. Tumor analysis: After obtaining the informed consent of the tumor sample, the tumor tissue from 
the original diagnostic biopsy or a recently obtained biopsy will be sent to the centralized laboratory, for 
confirmation of the diagnosis. The tumor block should be sent if it is necessary to determine the 
methylation status of the MGMT gene and to participate in the study of resistance proteins. In general, 
paraffin blocks are preferred for diagnostic confirmation, but 15 unstained slides are also acceptable. 
Details of suitable material for molecular analysis and shipping instructions will be provided in the study 
sample management guide. These determinations will be made in approximately 7 days. 
* If the center has the result of the methylation status of the MGMT gene carried out in the center itself, 
it will not be necessary to perform the centralized review. The methylation result report should be 
submitted along with the tumor sample for centralized diagnostic review 
3. Inclusion / Exclusion Criteria: They must be reviewed in detail for each patient prior to carrying 
out the specific evaluations of the study and prior to the start of treatment.  
4. Anamnesis: it will be carried out in the baseline period (in the 2 weeks prior to inclusion) general 
anamnesis with evaluation of Minimental Test (MMS), Barthel index and Kasnofsky index (KPS), 
existence of neurological deficit and severity. Details of coexisting diseases, oncological history 
(including smoking and history of weight loss in the last 6 months), description of first-line treatment 
with radiation dose and number of cycles of temozolomide and the concomitance or not with 
bevacizumab. 
5. Complete physical examination:  
5.1 Pre-treatment (Screening): Physical examination and vital signs (measurements of resting 
pulse, blood pressure, respiratory rate, temperature). Functional status will be assessed using 
the Karnofsky scale, Annex I of this protocol. Neurological and functional deterioration will be 
assessed using the MMS and IB, annexes II and III of this protocol. Doses of corticosteroids 
(dose / drug) and anticonvulsants (dose / drug) will be recorded 
5.2 During treatment and follow-up visits: Physical examination, functional status, KPS, and 
neurological deterioration at each treatment visit (on day 1 of each cycle prior to dispensing 


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treatment), and at all follow-up visits. Control of the drug, dose of corticosteroids and 
anticonvulsants. 
The IB and MMS will be carried out in the visit of cycle 1, in each control visit in which MRI is 
performed and in the safety visit once the patient has finished the treatment.  
6. Concomitant medication: All drugs taken in the last 4 weeks prior to randomization and 
concomitantly during the study, will be recorded in the clinical history and in the data collection 
notebook with the indication, information on the dose and the dates of administration. Especially the 
doses of corticosteroids and anticonvulsants 
7. Security Assessment: Safety assessments and adverse events include tumor-related, treatment-
related, and unrelated signs and symptoms. Adverse events will be documented and recorded in the 
CRD upon notification by patients. Specific questions will be asked about adverse events. The reporting 
period for non-serious AA ends 30 days after the last study treatment dose or subsequent initiation of 
antineoplastic therapy, whichever is earlier; the reporting period for severe AA ends 30 days after the 
last study treatment dose regardless of the initiation of any subsequent antineoplastic therapy. Adverse 
events will be followed up at the post-treatment follow-up visit at least 28 (and no more than 35) days 
after the end of treatment or until all drug-related toxicities have resolved or are considered. 
irreversible, whatever happens later. Classification of toxicity will be based on the National Cancer 
Institute Common Terminology Criteria for Adverse Events, version 4.0 (NCI CTCAE v4.0). 
8. Clinical analysis: should be performed on day 1 of each treatment cycle with a window of up to 72 
hours prior to the scheduled visit.  
8.1 Hemogram: includes complete count of the 3 series: red, white (differential count) and 
platelet count.  
 
8.2 Biochemistry: includes glucose, sodium, potassium, creatinine, AST, ALT, alkaline 
phosphatase, total bilirubin, and gamma glutamyltransferase.  
8.3 Pregnancy test (for women of childbearing potential): blood or urine pregnancy test 
(minimum sensitivity of 25 IU / L or equivalent units of beta human chorionic gonadotropin [β-
HCG]) during the 7 days prior to inclusion. Subsequently, the blood pregnancy test is only 
necessary if it is clinically indicated or if it is required by routine clinical practice at the center. 
8.4 Coagulation: coagulation studies will be performed if clinically indicated and will include: 
prothrombin time (PT), partial thromboplastin time TTP, and fibrinogen.  
9. Electrocardiogram (ECG): It will be performed on ECG if clinically indicated.  
10. Chest X-ray: A chest X-ray will be performed if it is clinically indicated.  
11. Radiological images: Baseline and follow-up NMR will consist minimally of T1, T1Gadolinium and 
FLAIR sequences in the axial planes, to allow the application of the RANO criteria. It is recommended to 
use the same device for each patient throughout the study. 
12. Therapeutic compliance: Any missed or missed dose due to vomiting should be indicated in the 
patient's medical record and CRDs, in the same way as all administered doses. 
13. Dispensing medication: Temozolomide will be dispensed on day 1 to 5 of each 28-day treatment 
cycle in the active treatment group.  
14. Dose of corticosteroids: Record in the patient's clinical history and in the CRD the doses of 
corticosteroids that the patient has received in cycle 6 of the adjuvant, as well as the basal dose prior to 
inclusion. At each visit, the dose of corticosteroids that the patient receives at that time should also be 
recorded in the clinical history and in the CRD. 
15. Follow-up: All patients will be followed up to determine subsequent antineoplastic treatments and 
survival, regardless of the reasons for withdrawal from study treatment. Information regarding post-
progression treatments should include the list of subsequent treatments received as well as whether the 
patient has been re-operated or received re-irradiation. All this will be noted in the clinical history and in 
the CRD. 
8.3. Pre-treatment procedures 
Before carrying out any specific study procedure, informed consent of the trial must be obtained, except 
for MRI, which allows the patient to be offered the study and which is considered healthcare but will be 
considered baseline.  


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Procedures that are part of routine treatment are not considered study specific procedures. Procedures 
that are part of regular attendance can be used as screening procedures to determine eligibility. Before 
initiating study treatment, all subjects will be evaluated for eligibility. The selection process begins the 
day the subject signs the informed consent approved by the CEIC and continues until the randomization 
of the patient in the trial and subsequent start of the study treatment. In this study, only eligible 
subjects will receive the study treatment. 
All subjects must have completed the following procedures in ≤ 28 days (unless otherwise specified) 
prior to the start of study treatment:  
● Review of the inclusion and exclusion criteria.  
● Medical and medication history  
● The paraffin-embedded tumor block must be identified, prepared, and sent to the central 
laboratory for molecular analysis at MGMT. The sample can be sent at any time prior to inclusion, 
even if the patient is still receiving first-line treatment, as long as the specific informed consent for 
sample submission is signed. 
● Physical examination, blood pressure, respiratory rate, temperature, weight and height. ● KPS 
(Karnofsky performance status) functional status (≤ 14 days before study inclusion). 
● Laboratory tests (≤ 14 days before study inclusion) and biochemical tests. ● Serum or urine 
pregnancy test for women of childbearing age (≤72 hours before the start of study treatment).  
● ECG and chest X-ray if clinically indicated.  
● IB and MMS.  
● Cranial MRI that will be taken as a baseline examination in terms of response evaluation (its 
performance is admitted in a period ≤ 6 weeks, that is, an MRI prior to the 6th cycle of 15 days, to 
verify the clinical stability of the disease.) The response it should always be assessed with the same 
equipment as the initial examination. In the event that the dose of corticosteroids must be increased 
before the start of treatment, or the patient suffers neurological deterioration during the screening 
and randomization period (between the 6th cycle and randomization), the patient will not be 
randomized and will be considered failure screening. For this reason, the dose of corticosteroids on 
the day of inclusion may never be lower than that on the day of randomization or initiation of 
treatment. 
Treatment should begin as soon as possible to allow a 4-week period between Cycle 6 (care) and Cycle 
7 (study), although a 2-week delay will be allowed.  
8.4. Tests during treatment 
- Experimental arm  
Patients will be visited every 4 weeks (28-day cycles) for 6 treatment cycles, and at each visit:  
● Physical examination: weight  
● KPS  
● Analytical and biochemical tests  
● Therapeutic compliance  
● Dose of corticosteroids and anticonvulsants  
● Toxicity registry  
If the patient needs to increase the dose of corticosteroids or neurologically worsens, it is advisable to 
repeat the initial radiology to rule out progression. The dose of corticosteroids on the day of IC inclusion 
or signature may never be higher than the dose on the day of randomization (to rule out progressions in 
that period). 
Cycle 3 and 6: additionally it will be carried out:  
● Response evaluation by RANO criteria on brain lesions visualized in MRI.  
● I. Barthel and MMS.  
MRI imaging tests, the Barthel Index and MMS will be performed every 12 weeks until disease 
progression; responses to treatment (PR, CR) must be confirmed at 4 weeks using the same method, if 
the patient abandons treatment for any reason before progression, MRIs should continue to be 
performed every 12 weeks as part of the follow-up until progression . 
- Control arm  
Patients will be visited every 4 weeks for 6 months after enrollment in the trial and at each visit they 
will:  
● Physical examination: weight  
● KPS  
● Analytical and biochemical tests  
● Therapeutic compliance  
● Dose of corticosteroids and anticonvulsants  


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● Toxicity registry  
If the patient needs to increase the dose of corticosteroids or neurologically worsens, it is advisable to 
repeat the initial radiology to rule out progression. The dose of corticosteroids on the day of IC inclusion 
or signature may never be higher than the dose on the day of randomization (to rule out progressions in 
that period). 
Months 3 and 6: additionally it will be carried out:  
● Response evaluation by RANO criteria on brain lesions visualized in MRI.  
● I. Barthel and MMS.  
MRI imaging tests, Barthel Index and MMS will be performed every 12 weeks until disease progression; 
responses to treatment (PR, CR) should be confirmed at 4 weeks using the same method. 
 
8.5. Security visit 
The safety visit will be carried out in patients treated in the experimental arm 4 weeks after the end of 
treatment (whatever the cause of termination),  
● Physical examination, and vital signs (measurements of resting pulse, blood pressure, respiratory 
rate, temperature, weight and height).  
● Functional status: KPS  
● Analytical and biochemistry  
● Toxicity registry  
MRI imaging tests will be performed every 12 weeks until disease progression. Since progression-free 
survival is the main objective of the study, it is essential to rule out progression on MRI. 
8.6. After end of treatment 
- End of treatment without progression:  
Once the treatment for any cause other than disease progression has been completed in the 
experimental arm or after 6 months from inclusion in the control arm, the following procedures will be 
performed every 12 weeks until disease progression: 
● Physical examination, and vital signs (measurements of resting pulse, blood pressure, respiratory rate, 
temperature).  
● KPS  
● Barthel index  
● Minimental Test  
● Analytical tests:  
● Analytical and biochemistry  
● DXM and anti-seizure doses  
● NMR  
- After progression of the disease:  
Once the patient progresses, follow-up for overall survival until death every 12 weeks, can be both visits 
to the oncology service and telephone calls. The patient's current status and registration of new cancer 
treatments will be determined (surgery, radiotherapy, chemotherapy and type, or / and bevacizumab, 
clinical trial) 
The date of death will be verified to determine survival.  
8.7. Response evaluation 
The RANO criteria will be applied to evaluate the response and define the progression. It will be carried 
out in each center based on the radiological results, the clinical picture of the patient and the doses of 
corticosteroids required. It will be based on the RANO response criteria. 
The progression is defined as:  
1. Radiological worsening (increased contrast uptake area or appearance of new lesions). 2. 
significant deterioration in FLAIR together with irreversible neurological deterioration 
3. irreversible neurological deterioration even in the absence of radiological deterioration.  
4. continuous increase in corticosteroids (> 2 weeks) to prevent neurological deterioration without 
reducing the dose.  
8.7.1. Assessment of Clinical Response by corticosteroids and neurological deterioration 


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The criteria modified by the RANO Committee are based on radiological response, neurological 
symptoms, and corticosteroid doses.  
Patients should be maintained with the minimum dose of corticosteroids they require throughout the 
treatment (the dose that keeps them neurologically stable).  
If a dose increase is required due to an event (eg fever, seizure with post-critical deficit, etc.), the 
doses must be returned to the previous dose. If continuous dose increases are required for more than 2 
weeks, the patient should be considered as in clinical progression. 
In the progression due to sudden neurological deterioration, a radiological examination is 
recommended. If it cannot be performed, progression of brain disease will be considered. 
 
It is important in patient evaluation visits to report in detail the dose of corticosteroids that the patient 
receives and the stability or not of the neurological symptoms, in addition to the results of the 
radiological examination (MRI).  
8.7.2 Assessment of Radiological Response  
A baseline MRI will be performed, in cycle 3 and every 12 weeks until progression.  
The size of the baseline lesion will be evaluated at the beginning of treatment using the product of the 2 
largest perpendicular diameters or the sum of the products of the measurable lesions in the axial plane 
and will be recorded as a baseline measurement. The same measurements will be taken for reference. 
In case of neurological deterioration or the appearance of new neurological symptoms, the MRI will be 
carried out to verify the radiological progression 
In the assessment of the response, contrast uptake and hyperintensity in FLAIR will be analyzed 
separately. 
Gadolinium T1 Sequence Evaluation  
- It will not by itself be the one that will define the criteria for partial response, stability and 
progression, as it must be accompanied by the absence of deterioration in the Flair sequences.  
- Measurement of the component that is enhanced after contrast in axial sections.  
- The maximum diameter in axial section and its maximum perpendicular in the same cut will be sought.  
- In successive controls the same methodology will be carried out. It will not necessarily correspond to 
the same cut / diameter made in the previous study, since the lesions often do not expand 
symmetrically but rather by a margin, which is not always the same. 
- If the lesion consists of several separate uptake nodules but within the hyperintense area in FLAIR, it 
will be measured as if it were a single nodule.  
- This value will be noted in the CRD.  
- The appearance of new enhancement foci will be noted in continuity or at a distance.  
Evaluation in T2 FLAIR sequences  
Given the difficulty of quantification, a subjective measurement based on the experience of the 
radiologist will be made with the following criteria:  
+2.  
The lesion presents a clearly greater extension (at first glance) than the immediately 
previous study. From experience in other studies, the clear visual difference represents an 
increase of more than 20%. 
+1.  
The lesion has a greater extension than the previous study but must be evaluated in 
several slices to reach this conclusion. 
0.  
No changes to the FLAIR extension. 
-1.  
Extension reduction compared to the previous study 
-2.  
Obvious reduction. 
 
 
The appearance of new hyperintense foci in FLAIR other than the initial lesion will be noted. These foci 
may correspond to new infiltrative areas or changes related to treatment. The opinion of the radiologist 


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will be indicated in FLAIR NOTES. 
8.7.3. RANO response rating 
In the present study it is important to determine the date of progression since the response to 
treatment will not be considered a study objective.  
It is recommended to use the following monitoring table to standardize data collection:  
 
 
BASAL NMR  
CONTROL 1  
CONTROL 2 
DATE 
 
 
 
T1GD  
T target 
PRODUCT 
DIAMETERS INJURY 
1,2,3 ... 
PRODUCT 
DIAMETERS INJURY 
1,2, 
ETC 
New spotlights  
 
IF NOT  
IF NOT 
Sum  
products 
 
MM2  
MM2  
MM2 
FLAIR 
 
VISUAL EVALUATION 
EQUAL (0)  
+1, +2 
EQUAL (0)  
+1, +2 
NEW SPOTLIGHTS   
IF NOT  
IF NOT 
DXM DOSE  
BASAL DOSE  
DOSE  
DOSE 
CLINIC  
BASAL CLINIC  
Stable / Better / Worse  
Stable / Better / Worse 
REPLY  
 
EE / RP / RC / P  
EE / RP / RC / P 
 
 
9. ADVERSE EVENTS  
The ICH GCP requires that both Investigators and Promoters follow a specific procedure when reporting 
adverse effects or reactions within a clinical trial.  
Any event related to drug toxicity, illnesses that debut during the study, or exacerbations of pre-
existing illnesses must be reported.  
Furthermore, clinically relevant changes on physical examination and abnormal parameters found on 
complementary examinations (eg, radiography, ECG) should also be reported as AE. 
The criteria to classify a result found in a complementary examination as abnormal and therefore be 
classified as AE are:  
● that the result of the complementary examination is associated with clinically relevant symptoms, and / or  
● that the result of the complementary examination implies a change in the dose of study drug or a study exit, or 
implies adding another drug for its control or another type of therapy and / or  
● that the result of the complementary examination implies an outcome that is included in the definition of AAG, 
and / or  
● that the result of the complementary examination is considered as an AE by the investigator. 9.1. 
Definitions 
The ICH PCB definitions apply to this protocol.  
Adverse event (AA) 


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Any detrimental health incidence in a patient or clinical trial subject treated with a drug, even if it does 
not necessarily have a causal relationship with said treatment.  
Adverse reaction (AR)  
An AR is any unintended and harmful reaction to an investigational drug, regardless of the dose 
administered.  
Serious Adverse Event (AAG) and Serious Adverse Reaction (RAG) 
Any adverse event or adverse reaction that, at any dose:  
● produces death (be fatal),  
● threatens the patient's life,  
● make hospitalization necessary or its prolongation,  
● produces persistent or significant disability or incapacity,  
● is a congenital anomaly or birth defect, or  
 
● is clinically significant  
Clinical and scientific judgment should be exercised when deciding whether it is appropriate to make an 
urgent notification of clinically significant events that may not be life threatening, or that do not require 
hospitalization, but that could put the patient at risk or require intervention. to prevent one of the 
outcomes detailed above. 
Likewise, all suspicions of transmission of an infectious agent through a drug will be reported as serious.  
Suspected Serious and Unexpected Adverse Reaction (RAGI) 
It is a serious adverse reaction that is "unexpected" eg. A serious adverse reaction, the nature and 
severity of which is not consistent with the information on the pharmaceutical product in question, which 
appears in the summary of product characteristics (or in the investigator's manual). 
The reference document to establish the “expectability” of adverse events for Temozolomide will be the 
latest version of the Product Data Sheet available on the EMEA website.  
Life threatening event 
Any event in which the patient is at risk of death at the time of the event; it does not refer to an event 
that hypothetically could have caused death if it had been more severe. 
Hospitalization / prolongation of hospitalization  
Any event that requires hospitalization (or prolongation of hospitalization) and that occurs or worsens 
during a patient's participation in a clinical study should be reported as an SAA. Prolongation of 
hospitalization is defined as any extension of a patient's hospitalization beyond the anticipated / 
required stay at the initial admission, in the opinion of the investigator or the physician responsible for 
the patient. 
The following cases of hospitalization do not meet the AAG reporting criteria:  
a) Reasons described in the protocol (for example, administration of the drug, complementary tests 
required by the protocol). Hospitalizations or extensions of hospitalization for a complication in the 
administration of treatment or procedures will be reported as SAG. 
b) Hospitalizations or extensions of hospitalization for technical, practical or social reasons, in the 
absence of an AA. 
c) Pre-scheduled hospitalizations (that is, scheduled prior to study entry). Any surgical intervention or 
procedure scheduled prior to study entry must be documented in the CRD. 
Unexpected Adverse Reaction (RAI)  
Any adverse reaction whose nature, intensity or consequences does not correspond to the reference 
information for the medicine (for example, the investigator's manual in the case of an investigational 
medicine not authorized for marketing, or the technical sheet of the product in the case of an authorized 
medicine). 
The reference document to establish the “expectability” of adverse events for temozolomide will be the 
latest version of the Product Data Sheet available on the EMEA website.  
Associated with the use of the drug 
An AA is considered associated with the use of the investigational drug if the causation assessment is 
related to any of the investigational drugs or is unknown according to the definitions below. 
Assessment of causality.  


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The investigator must provide an assessment of the causality of each investigational drug (including 
combination products and comparators) according to the following criteria:  
AND There is a reasonable possibility that the study drug (s) caused the serious adverse event. 
N There is no reasonable possibility that the study drug (s) caused the serious adverse event and 
other causes are more likely.  
UK Unknown. It should only be used in special situations where the investigator has insufficient 
information (for example, the patient has not been treated at his center), and if none of the above 
options can be used. 
 
9.2 Notification and Documentation of Adverse Events  
The sponsor will collect AA from the signing of the patient's informed consent until 30 days after the 
administration of the last dose of study treatment.  
All AAs must be registered in the source document and in the CRD using medical terminology. 
Investigators should assess the severity (grade) of the event according to NCI-CTC V 4.0, assign a 
relationship with each of the trial drugs, and seek and obtain adequate information to determine the 
outcome and to assess whether it meets the criteria for its classification as AAG requiring immediate 
communication. The researcher must provide any information requested by the promoter, in addition to 
that collected in the CRD. 
All SAAs (as defined above) that occur during the clinical trial or within 30 days of the last dose of study 
medication, regardless of the suspected relationship with the study treatment, must be reported by the 
investigator. In addition, any SAAs that occur as a result of specific diagnostic procedures or 
interventions in this protocol must also be reported. Beyond this time period, only SAEs suspected of 
being related to the study drug should be reported. 
All AEs that are suspected of being related to study treatment should be followed after the time of 
treatment suspension until the event or its sequelae have resolved or stabilized at a level acceptable to 
the Principal Investigator, the Lead Investigator of the Trial. and / or the Promoter.  
The research team must notify the sponsor of all pregnancies of patients or patient pairs that occurred 
during clinical studies within 24 hours. from your knowledge. The outcome of the pregnancy must also 
be communicated within 24 hours of its knowledge. 
The cause of death of a deceased patient in a clinical study, regardless of whether it is an expected 
event or associated with the investigational agent, is considered an AGA and should therefore be 
reported using the AGA Document. If the autopsy report is available, it should be sent to the sponsor 
identified exclusively with the patient's inclusion number. 
 
All serious adverse events must be reported by fax within 24 hours. to MFAR, SL 
Fax: +34 93 253 11 68  
e-mail: investigacion@mfar.net 
 
The AAG report must contain a complete written summary detailing the important aspects of the 
corresponding adverse events. Where appropriate, information about relevant medical records and 
autopsy reports should be included. The tracking information must be sent to MFAR, SL within 24 hours. 
All AEs that are suspected of being related to study treatment should be followed after the time of 
treatment suspension until the event or its sequelae have resolved or stabilized at a level acceptable to 
the Principal Investigator, the Lead Investigator of the Trial. and / or the Promoter. 
9.3. Expedited notification of RAGI by the Promoter 
The promoter will assume the responsibility of the adequate communication of the RAGI to the 
regulatory authorities. The procedure indicated in the most up-to-date version of the document 
“Detailed guidance on the collection, verification and presentation of adverse reaction reports arising 
from clinical trials on medicinal products for human use”, available on the EMEA web portal, will be 
followed. 
All suspected serious and unexpected adverse reactions (RAGI) will be reported in accordance with 
current regulations on clinical trials in Europe, to the Competent Authorities, to the CEICs, and to 
researchers, in the terms and in the manner established in the “Detailed guidance on the collection, 
verification and presentation of adverse reaction reports arising from clinical trials on medicinal products 
for human use ”and following the local regulations that apply. 
9.3.1 RAGI expedited notification deadlines to regulatory authorities  


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The promoter will notify any individual case of suspicion of RAGI within a maximum period of 15 
calendar days after having knowledge of the suspected adverse reaction. When the suspicion of RAGI 
has caused the death of the subject or put his life in danger, the promoter will send the information 
within a maximum period of 7 calendar days from the moment the promoter becomes aware of the 
case. Said information must be completed, if possible, within the following eight days. This information 
should include an assessment of the significance and implication of the findings, including relevant prior 
experience with the same or similar medications. 
9.3.2 Expedited notification of other relevant safety information  
The sponsor must notify (as soon as possible and no later than 15 days) all information that could 
modify the risk / benefit ratio of the investigational drug, or determine changes in its administration 
schedule or in the conduct of the trial, for example: 
● A qualitative change or increase in the percentage of occurrence of the expected SARs, which is considered 
clinically important.  
● RAGIs that occur after the completion of a clinical trial and that are notified by the investigator to the sponsor.  
New events related to the conduct of the trial or development of the investigational medicinal product 
and likely to affect the safety of the subjects, such as: 
● Serious adverse events that may be associated with the trial procedures and may modify the conduct of the trial.  
● A significant risk to subjects such as the lack of efficacy of an investigational drug used to treat a life-threatening 
disease.  
● Important new safety findings from new animal studies (such as carcinogenicity).  
● Any premature termination or temporary stoppage of a clinical trial with the same investigational drug for safety 
reasons, conducted in another country and by the same promoter.  
● RAGs related only to a non-investigational drug that are considered relevant as they are not subject to the 
general rules for expedited notification of individual RAGI cases.  
In addition, if additional relevant information is obtained, it will be notified as soon as possible. 
9.3.3 Notification to Investigators  
The Sponsor will communicate to the investigators any information that may affect the safety of the 
trial subjects as soon as possible.  
In addition, the sponsor will inform the researchers of the safety aspects that affect the performance of 
the clinical trial or the development of the product, including the interruption of the development 
program or modifications to the protocol related to safety. 
Notification procedure:  
1. The researcher must fill in the AAG form (or the consulting doctor, named in the list of signatures and 
in the responsibility registration form, which is in charge of the patient), indicating the degree, causality 
and expectancy of the event according to as stated above. In the absence of the responsible 
investigator, a member of the center's trial team will fill out the form and sign it. Subsequently, the 
responsible researcher must check the AAG form, make the appropriate changes, sign it, forward it by 
fax to MFAR SL (tel: 93 434 44 12 - fax: 93 253 11 68, mail: investigacion@mfar.net ) as soon as 
possible. This initial report will be followed by a detailed, written report if appropriate. 
2. Monitoring- Patients should be followed until clinical recovery is complete and laboratory results have 
returned to normal or baseline, or the event has stabilized. Follow-up should continue until completion 
of protocol treatment if necessary. Tracking information should be recorded on an additional AAG form, 
checking the tracking box and faxing to the Secretariat when the information is available. Additional 
written information and / or copies of test results can be provided separately. The patient should only be 
identified by the assay number, date of birth and initials. The patient's name should not be used in any 
communication. 
3. MFAR SL will notify to the local ethics committee for clinical research (CEIC) and the Autonomous 
Community about the event (according to local standard clinical trial procedures) to the AEMPS.  
 
10. STATISTICAL CONSIDERATIONS  
10.1. Study variables 
10.1.1. Primary efficacy endpoint 
The primary study variable to determine the differences between the two treatment groups will be 


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progression-free survival at 6 months. Said variable will be assessed in patients with glioblastoma who 
have already received 6 cycles of temozolomide (adjuvant) without progressing and who are randomized 
to continue with 6 additional cycles of temozolomide or stop treatment, from the date of randomization 
to the date of defined progression according to RANO criteria. Patients who are alive and without 
evidence of progression at this time will be considered as successes and those who have progressed or 
died at this time will be considered as treatment failures. The diagnosis of progression should be based 
on the RANO criteria. 
Disease progression is defined as:  
1. Radiological worsening (increased contrast uptake area or appearance of new lesions), that is, RANO 
criterion for progression.  
2. significant worsening in FLAIR together with irreversible neurological deterioration 3. 
irreversible neurological deterioration even in the absence of radiological deterioration. 4. 
continuous increase in corticosteroids (> 2 weeks) to prevent neurological deterioration. 
10.1.2. Secondary variables 
It will be determined and compared in both treatment arms:  
Clinical, biological and demographic data: (sex, age, type of surgery, initial MMS, initial IBartel, 
presence of neurological symptoms (mild, moderate, significant), bevacizumab or not in the first-line 
treatment, characteristics of the treatment, second-line treatments.  
Safety / toxicity profile:Type, incidence, severity, frequency, severity and relationship with the 
treatment of the adverse events included in the CRD of the participating patients. It will be studied 
using descriptive statistics techniques, such as frequency tables and contingency tables. 
Tumor Activity: Using RANO criteria, Progression Free Survival, progression-free survival rate at 6 
months, and response rates in patients with measurable disease.  
Overall Survival: Median overall survival. Time from the start of the trial treatment to the date of 
death from any cause. In those patients who are alive at the last follow-up, the OS will be censored as 
of the date of the last follow-up in which the patient was alive. The median OS will be estimated using 
Kaplan Meier curves. 
Changes in the use of corticosteroids: Percentage of patients who have increased / decreased the 
dose of corticosteroids.  
Changes in neurological status: Percentage of patients free of neurological deterioration in both 
arms (MMS / Barthel score).  
MGMT gene methylation: Effects of MGMT gene methylation on study results. Correlation of 
laboratory information with clinical information, response to treatment, toxicity, and overall survival. 
This correlation will be studied using descriptive statistics techniques, such as frequency tables and 
contingency tables. 
10.2. Efficacy evaluation 
All randomized patients will be included in the main response analysis: Progression Free Survival at 6 
months, which will be evaluated according to the RANO criteria.  
Those patients who discontinue treatment for any reason other than disease progression (toxicity or 
patient desire) should undergo MRI every 12 weeks until disease progression as a common healthcare 
practice. Imaging tests should be done using the same method each time (MRI). 
Progression Free Survival (PFS): Time from the start of the trial treatment to the date of the first 
progression according to the RANO criteria (Annex IV of this protocol), or death from any cause. In 
those patients who are alive and have not progressed in the last follow-up, the date of progression will 
be censored to the date of the last follow-up. 
Disease progression is defined as:  
 
1. Radiological worsening (increased contrast uptake area or appearance of new lesions), 
that is, RANO criterion of progression.  
2. Significant worsening in FLAIR together with irreversible neurological 
deterioration 3. Irreversible neurological deterioration even in the absence of 
radiological deterioration.  
4. Continued increase in dexamethasone (> 2 weeks) to prevent neurological deterioration. 
Progression-free survival (PFS) at 6 months as the percentage of patients with / without disease 


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progression 6 months after starting study treatment  
10.3. Safety assessment 
Any patient included in the trial and who has been randomized, having ruled out previous progression, 
or who has received at least a single dose of study medication, will be evaluable for toxicity analysis.  
The safety and tolerability of the study medication will be determined by evaluating the type, incidence, 
severity, frequency, severity, and relationship with the treatment of reported adverse events, physical 
examinations, and laboratory tests. Toxicity will be classified and tabulated using NCI-CTCAE v 4.0. 
Any sign and / or symptom related to the tumor existing at the baseline visit that worsens (in severity 
or frequency) during the trial should be recorded as an adverse event.  
At each visit to this study, all adverse events should be recorded according to NCI-CTCAE version 4.0.  
10.4. Study populations 
Safety population: All patients who have received at least one dose of the study drug will be included.  
Intention-to-treat population (efficacy analysis population): Subject group consisting of all the 
subjects who have been randomized in the trial.  
Population per protocol: All patients who have received 1 dose of study treatment, meet the inclusion 
/ exclusion criteria, have not progressed when randomized and have not incurred major protocol 
deviations during the study will be included.  
10.5. Sample size and statistical analysis 
10.5.1. Sample size 
The sample size is calculated based on the hypothesis defined in the main objective of non-superiority in 
progression-free survival at 6 months in the continuation of 6 to 12 months of treatment between 
methylated and unmethylated patients. 
Based on the data from the EORTC-NCIC study (Stupp et al, 2005, 2009 ref 3), a probability of PFS of 
0.593 in methylated, of 0.285 in non-methylated is expected. 95% confidence is assumed, 80% 
statistical power and it is calculated based on a ratio of 1: 1.5 when having more methylated patients, 
expecting a maximum of 10% losses, the sample must be 32 unmethylated patients and 48 methylated 
(total n = 80) in the TMZ treatment group. In order to respond to the objective of comparison between 
values at 6 and 12 months, the same sample is established in a group of patients without additional 
treatment for 6 cycles with temozolomide. This would lead us to have a sample of 64 unmethylated and 
96 methylated patients for a total of 160 included patients. (n = 160). 
Taking into account that patients with residual disease can potentially benefit from those who do not, 
this variable is established as a randomization factor, and taking into account that 60% residual disease 
is expected in methylated patients and 40% % in non-methylated, 4 randomization groups are 
established so that of the 32 non-methylated 13 are with residual disease and of the 48 methylated 23 
are with residual disease. 
10.5.2. Statistic analysis 
The main objective is to demonstrate that prolonging treatment to 12 cycles does not improve 
progression-free survival at 6 months in the patients included in this study, randomized according to 
MGMT methylation status and whether or not residual disease, to receive 6  additional cycles of 
temozolomide. Therefore the main date is of the progression that can be presented in the form of a 
progression.  
Progression-free survival, response rate, and overall survival will be evaluated to determine the efficacy 
of the treatment. 
To estimate the total survival (and progression-free survival), the non-parametic Kaplan and Meyer 
method will be used. For the univariate comparison of the survival curves according to the different 
variables with potential prognostic effect, the Mantel-Cox test (log rank test) will be used. Finally, in 
order to perform a multivariate survival analysis and to have estimators of the relative risk adjusted for 
potential confounding variables, the Cox regression models will be used. 
Based on the data from the PIVOTAL study, a 6-month probability of PFS is expected of 0.593 in 
methylated and 0.285 in unmethylated. We assume 95% confidence, 80% power, and using the 
comparison formula in the Kaplan-Maier method and log-rank. 


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First, a descriptive study of the main characteristics of the global series of patients will be carried out. 
For continuous variables, the mean and standard deviation will be calculated and for qualitative 
variables the percentages of the corresponding categories will be given without taking into account the 
missing values. To perform the analysis of survival and progression-free survival, the Kaplan-Meier 
method was used and, in case of group comparison, the log-rank test with a 95% CI was used. SPSS 
v15.0 software will be used for this type of analysis. 
Progression Free Survival (PFS): Time from the start of the trial treatment to the date of the first progression according to the 
RANO criteria (Annex IV of this protocol), or death from any cause. For those patients who are alive and have not progressed in the 
last follow-up, the date of progression will be censored to the date of the last follow-up. 
Disease progression is defined as:  
1. Radiological worsening (increased contrast uptake area or appearance of new lesions), 
that is, RANO criterion of progression.  
2. Significant worsening in FLAIR together with irreversible neurological 
deterioration 3. Irreversible neurological deterioration even in the absence of 
radiological deterioration.  
4. Continued increase in dexamethasone (> 2 weeks) to prevent neurological deterioration.  
Progression Free Survival will be determined by the non-parametric Kaplan-Meier method. For the 
univariate comparison of the survival curves according to the different variables with potential 
prognostic effect, the Mantel-Cox test (log rank test) will be used. Finally, in order to perform a 
multivariate survival analysis and to have estimators of the relative risk adjusted for potential 
confounding variables, the Cox regression models will be used. 
Overall survival (TS) is defined as the time from randomization to death from any cause. The TS of 
patients alive at the time of analysis will be censored on the last follow-up date. The median overall 
survival will be estimated, with their respective 95% confidence intervals. Values of p <0.05 will be 
considered statistically significant. 
Toxicities: The safety and tolerability of the study medication will be determined by evaluating the 
type, incidence, severity, frequency, severity and relationship with the treatment of the adverse events 
collected in the CH of the participating patients. It will be studied using descriptive statistics techniques, 
such as frequency tables and contingency tables. 
The variables will be represented by frequency and percentage, the continuous variables will be 
represented as medians and ranges.  
Biomarkers: The frequency of genetic rearrangement will be determined with a 95% confidence 
interval. The correlation with the survival data will be determined using the Kaplan-Meier method. 
11. ETHICAL AND LEGAL CONSIDERATIONS  
11.1. Ethics Committee 
The study will be carried out in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki adopted by the 18th General Assembly of the World Medical Association, 
Helsinki, Finland.  
In accordance with directives 95/46 of the European Parliament and 2001/20 / EC which establish the 
requirements for the development of clinical trials, the information obtained during the clinical trial may 
only be used by the Promoter of the clinical trial to evaluate the results in accordance with the 
mentioned directive.  
In Spain also applies:  
 
The Oviedo Convention, of April 4, 1997, on human rights and biomedicine, ratified in the BOE in 
October 1999.  
In the rules for the adequate protection of personal data, according to the provisions of Organic Law 
15/1999 on the protection of Personal Data.  
The rights and obligations regarding information and clinical documentation, according to the provisions 
of Law 41/2002, of November 14, basic regulating the autonomy of the patient. 
The General Assembly Somerset West, South Africa, October 1996 and the General Assembly 
Edinburgh, Scotland, October 2000.  
Law 14/2007, of July 3, on Biomedical Research.  
11.2. Authorities 


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The study protocol and / or documents inherent to it will be sent to you before starting the trial, as 
established by the national authorities.  
11.3. Informed consent 
The patient must sign 2 informed consents:  
For the clinical trial and for the use of biological samples for centralized pathology review. For patient 
participation in the associated translational substudy. 
The doctor will have to explain the nature, purposes and possible consequences of the clinical trial, in a 
way that the patient can understand. 
The patient must give their consent before being admitted to the clinical study and biological samples 
are taken.  
The subject of the study will give their consent, signing the corresponding model in duplicate. For this 
purpose, each model must bear the signature of the investigator and the patient. The investigator will 
file and keep a copy of the original of each informed consent signed by each patient. 
The investigator will not initiate any investigation for the trial until he has obtained the patient's 
consent.  
The informed consent form used in this study, and the changes made during the course of the study, 
must be prospectively approved by the Ethics Committee.  
11.4. Confidentiality 
In order to guarantee the confidentiality of the test data according to the provisions of the Directive of 
the European Parliament 2001/20 / EC, only the Test Promoter or personnel designated by him will have 
access to them for monitoring / auditing tasks, the researcher and his team of collaborators, the Clinical 
Research Ethics Committee of the corresponding center or the one supervising the trial and the relevant 
health authorities. 
In the aforementioned case of Monitoring / Audits, the researcher must provide direct access to the 
source documents and data. 
The content of the data collection notebooks (CRD) as well as the documents generated during the 
study will be protected from unauthorized uses by people outside the research and, therefore, will be 
considered strictly confidential and will not be disclosed to third parties except to the specified in the 
previous section. 
In Spain, this test will also be carried out in accordance with the provisions of Organic Law 15/1999 on 
the protection of Personal Data  
11.5. insurance 
An insurance policy will be contracted according to the regulatory requirements of each country where 
the trial is carried out.  
In Spain, all patients in this clinical study will be insured through the HDI Hannover Internacional 
Insurance Company (Spain) Seguros y Reaseguros, SA with a policy that will meet the conditions 
stipulated by RD 223/2004. 
11.6. End of trial 
The trial will be considered normatively closed after the data on the primary and secondary variables 
are sufficiently prepared for initial publication.  
 
11.7. Early termination of trial 
This study may be interrupted prematurely if in the opinion of the sponsor there is a sufficient 
reasonable cause. The investigator will receive a written notice that the terminating party documents 
the reason for suspending the study. Circumstances that justify suspension of the study include, but are 
not limited to: 
● Identification of unforeseen, significant or unacceptable risks for patients ● Impossibility of including 
an acceptable number of patients  
● Insufficient compliance with the protocol requirements  
● Plans to modify, suspend or discontinue the development of the study drug.  


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● In case of premature termination of the study, all material (CRD totally or partially completed and 
blank, study drug, etc.) must be returned to GEIS.  
12. PRACTICAL CONSIDERATIONS  
12.1. Diagnostic criteria for the disease under study 
Patients included in this trial must have histological confirmation of the diagnosis of glioblastoma as well 
as the result of the methylation of the MGMT gene.  
Paraffin blocks / tumor slides will be collected from all patients for a central pathology review. 
12.2. Investigator Responsibilities according to Good Clinical PracticesThe 
responsibilities of the principal investigator in each participating Center will be: 1. 
Sign the trial project.  
2. Know in depth the properties of medicines.  
3. Obtain the informed consent of the subjects prior to their inclusion in the trial.  
4. Ensure that patients receive appropriate medical care in the event of adverse events, including 
significant laboratory values, related to the assay.  
5. Collect, record and report the data correctly.  
6. Report serious or unexpected adverse events immediately to the sponsor.  
7. Ensure that all persons involved respect the confidentiality of any information about the trial subjects.  
8. Report regularly to the Ethics Committee for Clinical Research on the progress of the trial.  
9. Take responsibility for the preparation of the final test report, agreeing to it with your signature.  
12.3. Instructions for completing the electronic CRD 
Data will be recorded in accordance with GCP through the electronic documentation system at the 
center.  
The application is designed to function entirely over the Internet. All stages of the processing, except for 
the actual input and display of the data, are carried out centrally on a web / database server. In 
particular, data storage will be done only centrally. 
 
For data entry and results printing, the system relies entirely on the so-called "network interface", that 
is, data entry forms and reports are displayed on the client computer as HTML pages (Hyper Text 
Markup Language, the standard language for describing pages on the Internet) through a web browser. 
It is not necessary to install any user-specific software to be able to use the system from the 
investigator's computer. There is the possibility of directly accessing the raw data through an ODBC 
database for further data processing. 
The system checks the correctness of the data by ranges and performs validity and consistency checks. 
Implausible or missing data can be corrected or completed after discussion with the investigator. 
Correction documents are stored (audit trail). 
The system has a system of keys and passwords that restricts access to different areas of the 
application depending on the role assigned by the Promoter. Apart from the researcher, only expressly 
authorized persons who have received specific training for the study may complete the CRD-e. 
All the data collected in the e-CRDs must be able to be documented in measurement records or by 
annotations in the patients' medical records.  
12.4. Final Manuscript and Publications 
Publication of the clinical trial  


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The Investigators Responsible for the study and the researchers who contribute at least 3% of the 
patients will appear as authors.  
The order of authors will strictly depend on the number of patients included by the different 
researchers. The trial coordinators (Dra. Balaña and Dra. Mª Vaz, will occupy the first position, last 
position or 'corresponding author').  
Clinical publication will be carried out by those responsible for the clinical study and clinical researchers.  
It will be the responsibility of the first author, the Principal Investigators of the study and the study 
designer to write the final publications. 
The anonymity of the source subjects of the study samples will be maintained at all times. The results 
or conclusions of the study will be communicated as a priority in scientific publications before being 
disclosed to the non-health public. Procedures of as yet undetermined efficacy will not be released 
prematurely or sensationally. 
Participating investigators should not publish any data, about the patients, that is directly related to the 
objectives of the study, until the trial report is published.  
The trial will be registered in the public access database, www.clinicaltrials.gov 
Publication of the Translational Research 
The results of the translational research will be published in journals of scientific impact, after the 
publication of the main results of the clinical study.  
The first author will be the Coordinator of the Translational Study. All centers that have contributed at 
least 5% of the analyzed material will have the right to a co-authorship in the publication; The centers 
that have contributed a minimum of 15% of the analyzed material will have the right to 2 co-authors. 
The author or authors will be selected at the discretion of each center (pathologists, molecular 
biologists, clinicians ...). All centers that have contributed material for the analysis will be considered in 
the acknowledgments. 
The clinical trial will be registered in "clinicaltrials.gov", and in the database of the National Institute of 
Health of United States of America.  
 
12.5. Monitoring 
The study will be monitored through local visits, telephone calls, and periodic inspection of the CRDs 
frequently enough to verify the following:  
- Patient recruitment rate.  
- Compliance with the approved protocol and amendments, if applicable.  
- That the researcher has received the documents and supplies of the trial necessary to carry out the 
trial properly and in compliance with current legal regulations.  
- That the Investigator and his team are adequately informed about the trial. - Integrity and 
correctness of the data entered in the CRD (according to the monitoring plan). - Informed consent 
(version, date and signature). 
- Eligibility criteria.  
- Selection tests.  
- AAG and notifications from AAG.  
- Main variable of the trial.  
- Collection and storage of biological samples.  
- Completion of CRDs and recording of adverse events.  
- Communicate deviations from the protocol in accordance with good clinical practices and regulatory 
requirements, taking necessary actions to prevent the recurrence of detected deviations. 
The monitoring visits will be made by the studio monitors. It is understood that these monitors will be 
able to access the medical records of the patients after the investigator requests it. The researcher will 
dedicate sufficient time to these visits and will facilitate access to all the documentation to authorized 
persons. 
12.6. Amendments to the Protocol 


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Supplements or changes to the protocol can be made exclusively by the Promoter, who must send them 
to the Ethics Committee and the Regulatory Authorities (in Spain AEMPS) as amendments to the 
protocol. 
12.7. Data processing 
All data (personal, clinical, economic and from studies of biological material) obtained on patients will be 
treated in accordance with the Directive of the European Parliament 95/46 / EC of October 24, 1995 on 
the protection of people with respect to processing of personal data. 
In accordance with the provisions of the aforementioned legislation, patients may exercise their rights 
of access, modification, opposition and cancellation of data, for which they must contact the doctor of 
the clinical trial. 
The content of the CRD as well as the documents generated during the study will be considered strictly 
confidential and will not be disclosed to third parties.  
 
In Spain, this study will also be carried out in accordance with Organic Law 15/1999, of December 13, 
on the protection of personal data.  
12.8. Documentation 
The investigator / institution shall maintain the trial documents in accordance with ICH Topic E6 Section 
8, and in accordance with the relevant regulatory requirements.  
Essential documents must be archived in accordance with BPC guidelines or for a longer period of time, 
if required by relevant regulations.  
Original study patient data (medical history) should be stored according to the applicable archiving 
period at the study centers, but not for a period of less than 15 years.  
13. TRANSLATIONAL STUDY  
See annex V to the protocol  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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14. BIBLIOGRAPHY  
1. Crocetti E, Trama A, Stiller C, Caldarella A, Soffietti R, Jaal J et al. Epidemiology of glial and non-glial 
brain tumors in Europe. Eur J Cancer 2012; 48: 1532-1542. 
2. Fuentes-Raspall R, Vilardell L, Perez-Bueno F, Joly C, Garcia-Gil M, Garcia-Velasco A et al. Population-
based incidence and survival of central nervous system (CNS) malignancies in Girona (Spain) 1994-
2005. J Neurooncol 2011; 101: 117-123. 
3. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC et al. Effects of radiotherapy 
with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a 
randomized phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10: 459-466. 
4. Malkoun N, Chargari C, Forest F, Fotso MJ, Cartier L, Auberdiac P et al. Prolonged temozolomide for 
treatment of glioblastoma: preliminary clinical results and prognostic value of p53 overexpression. J 
Neurooncol 2012; 106: 127-133. 
5. Roldan Urgoiti GB, Singh AD, Easaw JC. Extended adjuvant temozolomide for treatment of newly 
diagnosed glioblastoma multiforme. J Neurooncol 2012; 108: 173-177. 
6. Groves MD, Jr., Plummer AB. Helping patients make the best decision regarding duration of 
temozolomide chemotherapy treatment. Continuum (Minneap Minn) 2012; 18: 421-425. 
7. Brandsma D, Stalpers L, Taal W, Sminia P, van den Bent MJ. Clinical features, mechanisms, and 
management of pseudoprogression in malignant gliomas. Lancet Oncol 2008; 9: 453-461. 
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; 352: 997-1003. 
9. Brandes AA, Tosoni A, Spagnolli F, Frezza G, Leonardi M, Calbucci F et al. Disease progression or 
pseudoprogression after concomitant radiochemotherapy treatment: pitfalls in neurooncology. Neuro 
Oncol 2008; 10: 361-367. 
10. Stupp R, Tonn JC, Brada M, Pentheroudakis G. High-grade malignant glioma: ESMO Clinical Practice 
Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010; 21 Suppl 5: v190-193. 
11. Mason WP, Maestro RD, Eisenstat D, Forsyth P, Fulton D, Laperriere N et al. Canadian 
recommendations for the treatment of glioblastoma multiforme. Curr Oncol 2007; 14: 110-117. 
12. Malkoun N, Fotso MJ, Cartier L, Forest F, Auberdiac P, Chargari C et al. [Benefit of a prolonged 
adjuvant treatment with temozolomide for the management of patients with glioblastoma]. Cancer 
Radiother 2011; 15: 202-207. 
13. American Society of Clinical Oncology editor. Gilbert MR, Wang M, Aldape KD, Stupp R, Hegi M, 
Jaeckle KA et al. RTOG 0525: A randomized phase III trial comparing standard adjuvant temozolomide 
(TMZ) with a dose-dense (dd) schedule in newly diagnosed glioblastoma (GBM). Proceedings of the 
2011 ASCO Meeting. June 3-7. Chicago USES. 
14. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W et al. 
MGMTsponsormethylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 2010; 
6: 39-51. 
15. Holdhoff M, Grossman SA. Controversies in the adjuvant therapy of high-grade gliomas. Oncologist 
2011; 16: 351-358. 
16. Franceschi E, Tosoni A, Brandes A. Adjuvant temozolomide: how long and how much? Expert Rev 
Anticancer Ther 2008; 8: 663-665 
17. Balaña C, Vaz MA, Lopez D et al. Should we continue temozolomide beyond 6 cycles in the adjuvant 
treatment of glioblastoma without and evidence of clinical benefit? A cost analysis based on prescribing 
patterns in Spain. Clin Transl Oncology; 2013,(DOI) 10.1007 / s12094-013-1068-3  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Annex I. KARNOFSKY INDEX  
CLINICAL-FUNCTIONAL SITUATION  
PUNCTUATION 
Normal  
100 
Able to perform normal activities  
90 
Minor signs or symptoms  
80 
He takes care of himself; incapable of total normal activity or active 
work 
70 
Requires occasional assistance but capable of caring for most needs 
60 
Requires frequent medical care and assistance  
fifty 
Affected; requires special care and assistance 
40 
Severely affected; indication for hospitalization; death not imminent 
30 
Dying  
10 
Death  
0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Annex II. BARTHEL INDEX 


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Annex III. MINIMENTAL TEST. MMS 
 
 
 
 


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Annex IV. RANO RESPONSE CRITERIA 
 
RC  
RP  
EE  
PROGRESSION 
T1-GD  
0  
≥50% ¯ 
>50% ¯ 
<25% - 
³ 25% - * 
T2 / FLAIR 
=, ¯  
=, ¯ 
=, ¯  
=, - * 
NEW INJURIES  
0  
0  
0  
0. + * 
CORTICOSTERIOIDS  
0  
=, ¯  
=, ¯  
=, - * 
CLINIC  
=, -  
=, -  
=, -  
=, ¯ * 
 
all  
all  
all  
Anyone * 
 
 
COMPLETE ANSWER:  
● Disappearance of the signal in NMR T1Gd  
● Stability or reduction in Flair / T2 images  
● No new radiological lesions  
● Stability or clinical improvement  
● Absence of corticosteroids or stable minimum dose  
PARTIAL ANSWER:  
● Image reduction in NMR by 50% in T1Gd  
● Stability or reduction in Flair / T2 images  
● No new radiological lesions  
● Stability or clinical improvement  
● With reduced or stable steroids  
PROGRESSION: Any of the following  
● 25% tumor growth on T1Gd MRI  
● Significant increase in Flair / T2 images *  
● Appearance of new lesions  
● Irreversible clinical neurological deterioration, even in the absence of radiological deterioration. ● Need to 
increase dexamethasone (> 2 weeks) to prevent progressive neurological deterioration. 
NOTE: Only progression criteria 1,2 AND 3 are applicable for the purposes of including the patient in 
the clinical trial.  
STABLE DISEASE:  
● Image in T1Gd with an increase of less than 25% or a decrease of less than 50%  
● Flair / T2 image stability  
● No new radiological lesions  
● Clinically stable  
● Stable cortisone doses (not to be increased to maintain neurological impairment)  
* not attributable to radiation therapy, demyelination, ischemia, infection, seizures, postoperative 
changes or other effects of treatment  
 
 
 


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Annex V. ASSOCIATED MOLECULAR SUB-STUDY 
The drugs most used in neuro-oncology (nitrosoureas, temozolomide, procarbazine) owe their 
effectiveness to the chloro-ethylating or methylating lesion they produce on DNA. The methylguanine-
O6-methyltransferase (MGMT) repair enzyme repairs DNA through the direct removal of an alkyl group 
from the O6 atom of guanine in the DNA of cells exposed to alkylating agents31-33. In the gene that 
codes for said enzyme (MGMT) there are CpG dinucleotide islets that act as promoters of the gene 
coding.34 If these islets present methylation, the gene is silenced and no repair protein is produced, 
which means that the lesion produced by chemotherapy in the tumor cell it becomes irreversible and the 
cell enters apoptosis.  
TMZ, in a second generation derivative of imidazoltetrazinone, is spontaneously hydrolyzed under 
physiological conditions in the active metabolite and acts as a DNA methylating agent.  
Due to its mechanism of action, it is known that cells that have the ability to repair methylated DNA to 
unmethylated DNA through, among other enzymes, their richness in the enzyme O-6-methyl-guanine-
DNA-methyl-transferase ( MGMT), can overcome DNA damage and therefore not enter apoptosis. The 
enzyme is encoded by the MGMT gene. MGMT may be methylated on GpG islets and not correctly 
transcribed, causing enzyme inactivity. 
The importance of the enzyme and its relationship with the response to alkylating or methylating agents 
in various tumors was known.31, 32 In the EORTC phase III study that provided the indication for 
temozolomide in the first line of treatment, it was determined, in the cases in which there was a tumor 
sample, the MGMT methylation status and it was established that the methylation status was a 
significant predictive factor of response to TMZ. 35 However, it is not useful yet to discriminate between 
patients and decide on a different treatment. During the DNA repair process, the enzyme is consumed 
and has to be synthesized again. Prolonged administration (in continuous regimens or heavy doses) 
could improve the antitumor activity of the drug. 12  
By international consensus, any study performed using temozolomide must be accompanied by a study 
of the MGMT status in tumor cells.37  
Other TMZ resistance enzymes (MSH1 / MSH6) have been described that will be studied a posteriori in 
the Tissue Microarray.  
The histological material in the form of a paraffin block from the material obtained in the initial surgery 
will be sent to the HUGTIP Anatomy Service, (Dr. Carrato) where the histology was confirmed and a 
baseline molecular study of MGMT was carried out using the MSP (methylation -specific polymeration 
chain reaction) technique. (Dr. Carolina Sanz). The MGMT result performed at the originating center will 
be accepted. The results will be reported in the CRO for centralized and stratified randomization and 
subsequent statistical exploitation and will also be sent to the patient's center of origin. Tissue will be 
reserved to prepare a tissue matrix for subsequent immunohistochemical study of IDH1 and TMZ 


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resistance or sensitivity protein (MSH6, MSH2), and future studies that may arise as a result of the test 
results. 
In case of excess material, it will be returned to the center of origin.  
 
 
 
 
Annex VI. HELSINKI DECLARATION 
The new update of the Declaration of Helsinki can be found at: 
http://www.wma.net/en/30publications/10policies/b3/  
 
 
 


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Annex VII. NCI COMMON TERMINOLOGY CRITERIA V.4.0 FOR ADVERSE EVENTS 
Version 4.0 of the Common Terminology Criteria for Adverse Events (CTCAE) can be found at the 
following link:  
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReferen 
ce_5x7.pdf  
 
 


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Annex VIII. TEST SCHEME 
 

SAP_000.pdf: 
1 
 
 
TITLE: CLINICAL TRIAL PHASE IIB RANDOMIZED, 
MULTICENTER, OF CONTINUATION OR NON- 
CONTINUATION WITH 6 CYCLES OF TEMOZOLOMIDE 
AFTER THE FIRST 6 CYCLES OF STANDARD FIRST- 
LINE TREATMENT IN PATIENTS WITH 
GLIOBLASTOMA. 
 
 
 
Statistic Plan MAY 2018 
 
 
 
STATISTICAL CONSIDERATIONS 
Sample size 
 
The sample size is calculated based on the hypothesis defined in the main objective of not exceeding 
Progression-free survival at 6 months in the continuation of 6 to 12 months of treatment, between 
methylated and unmethylated patients. 
 
Based on data from the EORTC-NCIC study (Stupp et al, 2005, 2009 ref 3), a probable PFS quality of 
0.593 in methylated, 0.285 in unmethylated. 95% confidence is assumed, 80% of statistical power and 
is calculated based on a ratio of 1: 1.5 to have more methylated patients. 
With a maximum loss of 10%, the sample must be 32 non-methylated patients and 48 methylated 
patients (total n = 80) in the TMZ treatment group. In order to respond to the comparison objective, 
the same sample is established in a group of untreated patients. 
This would lead us to have a sample of 64 patients unmethylated and 96 methylated for a total of 160 
patients included. (n = 160). 
Considering that patients with residual disease can potentially benefit compared to those who do not 
have it, this variable is established as a randomization factor, and taking into account. Reports that 
60% of residual disease is expected in methylated patients and 40% in non-methylated patients, 4 
randomization groups are established so that of the 32 non-methylated 13 are with residual disease and 
of the 48 methylated 23 are with residual disease. 
 
Study variables 
Primary efficacy variable 
 
The main study variable to determine the differences between the two treatment groups will be 
progression-free survival at 6 months. This variable will be evaluated in patients with glioblastoma 
that they have already received 6 cycles of temozolomide (adjuvant) without progressing and are 
randomized to continue with 6 additional cycles of temozolomide or stop treatment, from the date of 
randomization up to date of the progression defined according to the RANO criteria. Patients who are 
living and without evidence of progression at this time will be considered as successes and those who 
have progressed or died at this time will be considered as treatment failures. The diagnosis of 
progression must be based on the RANO criteria. 
 
Disease progression is defined as: 
1. radiological worsening (increase in contrast enhancement area or appearance of lesions new), that 
is, RANO progression criteria. 
2. significant worsening in FLAIR along with irreversible neurological impairment. 


2 
 
3. irreversible neurological deterioration even in the absence of radiological deterioration. 
4. continued increase in corticosteroids (> 2 weeks) to prevent neurological deterioration. 
 
Secondary variables 
The two treatment arms will be determined and compared: 
 
Clinical, biological and demographic data: (sex, age, type of surgery, initial MMS, initial Bartel index, 
presence of neurological symptoms (mild, moderate, important), bevacizumab or not in the treatment 
of 
first line, treatment characteristics, second line treatments. 
 
Safety / Toxicity Profile: Type, incidence, severity, frequency, severity and relationship with treatment 
of adverse events recorded in the CRD of the participating patients. I know will study using 
descriptive statistical techniques, such as frequency tables and contingencies. 
 
Tumor Activity: Using RANO criteria, Progression Free Survival, survival rate progression-free at 6 
months and response rates in patients with measurable disease. 
 
Global Survival (OS): Median of global survival. Time from start of treatment to trial to date of exitus 
for any cause. In those patients who are alive in the last follow-up, the OS will be censored at the date 
of the last follow-up in which the patient was alive. The median OS will be estimated using Kaplan 
Meier curves. 
 
Changes in the use of corticosteroids: Percentage of patients who have increased / decreased the dose 
corticosteroids. 
 
Changes in neurological status: Percentage of patients free of neurological impairment in both arms 
(MMS / Barthel score). 
 
MGMT gene methylation: Effects of MGMT gene methylation on study results. Correlation of 
laboratory information with clinical information, response to treatment, overall toxicity and survival. 
This correlation will be studied using statistical techniques descriptive, such as frequency tables and 
contingency tables. 
 
Statistic analysis 
The main objective is to demonstrate that prolonging the treatment until 12 cycles does not improve 
the progression-free survival at 6 months in the patients included in this study, randomized according 
to the methylation status of the MGMT and by residual disease or not, to receive 6 additional cycles of 
temozolomide.  
 
To determine the efficacy of the treatment, progression-free survival, the rate of response and overall 
survival. To estimate total survival (and progression-free survival) the Kaplan and Meyer 
nonparametic method. For the univariate comparison of the survival according to the different 
variables of potential prognostic effect, the test of Mantel-Cox (log rank test). Finally, to carry out a 
mutivariate survival analysis and have relative risk estimators adjusted for potential confounding 
variables models will be used of Cox regression. 
 
Based on data from the PIVOTAL study, a 6-month PFS probability of 0.593 is expected in 
methylated, of 0.285 in non-methylated. We assume 95% confidence, 80% power, and using the 
comparison formula in the Kaplan-Maier method and log-rank. 
 
First, a descriptive study of the main characteristics of the global series will be carried out. For the 
continuous variables the mean and standard deviation will be calculated and for the qualitative 
variables percentages of the corresponding categories will be given.  


3 
 
To carry out the analysis of survival and progression-free survival, we will use the Kaplan-Meier 
method and, in case of group comparison, the log-rank test with 95% CI. For this type of analysis, the 
SPSS v15.0 software will be used. 
Progression Free Survival (SLP): Time from start of trial treatment to the date of the first progression 
according to the RANO criteria (Annex IV of this protocol), or death for any reason. Those patients 
who are alive and have not progressed in the last 
follow-up, progression date will be censored to the date of the last follow-up. 
Finally to make a mutivariate survival analysis and having relative risk of confusion, estimators 
adjusted to variables 
and Cox regression models will be used. 
 
Total survival (OS) is defined as the time elapsed from randomization to death from any cause. The 
ST of the living patients at the time of the analysis will be censored in the last follow-up date. The 
global survival median will be estimated, with their respective 95% confidence intervals. Values of p 
<0.05 will be considered statistically significant. 
 
Toxicities: The safety and tolerability of the study medication will be determined by evaluating the 
type, incidence, severity, frequency, severity and relationship with the treatment of events. Adverse 
events collected in the clinical records of the participating patients. It will be studied using techniques 
of descriptive statistics, such as frequency tables and contingency tables.  
 
Qualitative variables will be represented by frequency and percentage, continuous variables will be 
represented such as medians and ranges. 
 
Biomarkers: The frequency of genetic rearrangement will be determined with an interval of 95% 
confidence. Correlation with survival data will be determined using the method from Kaplan-Meier. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


4 
 
 
 
INDEX 
 
 
 
INDEX .......................................................................................................................................... 1 
TITLE: Clinical Trial Phase IIB randomized, multicenter, of continuation or non- 
continuation with 6 cycles of temozolomide after the first 6 cycles of standard first-line 
treatment in patients with glioblastoma. .................................................................................................... 3 
1. 
PATIENTS INCLUDED by CENTER .................................................................................................. 3 
1.1 METHYLATION STUDY ...................................................................................................................... 5 
1.2 RESIDUAL DISEASE DESCRIPTION .............................................................................................. 5 
1.3 Distribution of patients according to the two stratification factors ................................ 6 
1.4 FLOWCHART OF PATIENTS ............................................................................................................ 7 
2. 
PATIENT CHARACTERISTICS ............................................................................................................ 8 
2.1 PATIENT STATUS at the time of the report ............................................................................ 10 
3. TOXICITY ....................................................................................................................................................... 11 
3.1 By number of controls/visits ........................................................................................ 11 
3.2 By patient .............................................................................................................................................. 12 
3.4 Relevant differences in toxicity by arm .................................................................................... 13 
4. 
SECOND-LINE THERAPIES ........................................................................................ 15 
5. 
OUTCOME ................................................................................................................................................ 16 
5.1 PRIMARY ENDPOINT: DIFFERENCES IN PROGRESSION-FREE SURVIVAL 
AT 6 MONTHS. .................................................................................................................. 16 
5.2 PROGRESSION FREE SURVIVAL ............................................................................ 16 
5.3 OVERALL SURVIVAL ......................................................................................................................... 19 
5.4 MULTIVARIATE COS ANALYSIS OF PROGRESSION FREE SURVIVAL AND OVERALL 
SURVIVAL DEPENDING ON STRATIFICATION FACTORS AND TREATMENT ARM ....... 24 
5.5. HAZARD RATIO WITH FOREST PLOT FOR A PROGRESSION FREE SURIVAL AND B 
OVERALL SURVIVAL ................................................................................................................................ 25 
6 .TRASLATIONAL STUDY .......................................................................................................................... 26 
7 .ABSTRACTS AND PUBLICATIONS OF THIS STUDY ..................................................................... 28 


5 
 
 
 
TITLE: CLINICAL TRIAL PHASE IIB RANDOMIZED, 
MULTICENTER, OF CONTINUATION OR NON-CONTINUATION WITH 
6 CYCLES OF TEMOZOLOMIDE AFTER THE FIRST 6 
CYCLES OF STANDARD FIRST-LINE TREATMENT IN 
PATIENTS WITH GLIOBLASTOMA. 
 
 
 
SPONSOR PROTOCOL NO.: GEINO 14-01 
 EUDRACT: 2014-000838-39 VERSION: 1.2, 
dated 8 September 2014 
SPONSOR: Spanish Group of Research in Neuro-Oncology - GEINO COORDINATING 
INVESTIGATORS:  
 
1. PATIENTS INCLUDED BY CENTER 
 
 
subject_hospital 
 
Frequency 
Percentage 
% 
Valid 
% 
Cummulat
ive % 
 
 
 
 
 
 
Hospital name 
 
 
 
 
 
 
 
Hospital name 
 
 
 
 
 
 
 
Hospital name 
 
 
 
 
 
 
Total 
 
 
 
 
Number patients included: 
Number patients that started treatment: 
 Reasons not to start: 
 
 


6 
 
Cause of lack of substudy 
 
Frequency 
Percentage 
% 
Valid 
% 
Cummulat
ive % 
 
Reason 
 
 
 
 
Reason 
 
 
 
 
Total 
 
 
 
 


7 
 
 
1.1 METHYLATION STUDY 
 
 
Assessed locally: 
Assessed centrally: 
 
 
 
Number of patients: 
Methylated: 
Unmethylated: 
% Methylated: 
% Unmethylated: 
 
 
 
 
1.2 RESIDUAL DISEASE DESCRIPTION 
 
 
Residual disease was considered and enhancement with a diameter largest than 10mm. It was a 
factor of stratification. There was no central review of images. 
 
 
Frequency 
% 
 
No 
Residual 
disease 
 
 
Residual 
disease 
 
 
Total 
 
 


8 
 
 
 
1.3 DISTRIBUTION OF PATIENTS ACCORDING TO THE TWO STRATIFICATION 
FACTORS 
 
 
 
 
ARM 
MGMT 
status 
Pts 
needed 
Pts 
included 
Residual 
Disease 
Pts 
needed 
Pts 
included 
TMZ 
Met 
 
 
 
 
 
 
 
 
UnMet 
 
 
 
 
 
 
 
 
CONTROL 
Met 
 
 
 
 
 
 
 
 
UnMet 
 
 
 
 
 
 
 
 


9 
 
 
 
 
1.4 FLOWCHART OF PATIENTS 
 
 
 
 
 
 
 


10 
 
 
2. PATIENT CHARACTERISTICS 
 
 
 
[ALL] N= 
CONTRO 
N= 
EXPERI 
N= 
p.over 
all 
TREATMENT 
ARM 
CONTROL 
 
 
 
TMZ 
 
 
 
Sex 
 
Female 
 
 
 
Male 
 
 
 
Age 
 
 
 
 
Residual 
disease>10mm 
 
No 
 
 
 
Yes 
 
 
 
KPS 
 
<70% 
 
 
 
>=70% 
 
 
 
Residual 
Neurological 
symptom 
 
No 
 
 
 
Yes 
 
 
 
DXM dose at 
inclusion 
 
0mg 
 
 
 
0.5-2mg 
 
 
 
>2 
 
 
 
Barthel index 
 
0 
 
 
 
1 
 
 
 
MMSE 
 
<27 
 
 
 
≥27 
 
 
 
NP/ND 
 
 
 


11 
 
Anticonvulsant 
therapy 
 
No 
 
 
 
Yes 
 
 
 
TREATMENT AT DIAGNOSIS PREVIOUS TO ENROLLMENT. 
Initial surgery- 
TREATMENT AT 
DIAGNOSIS. 
 
Biopsy 
 
 
 
Complete resection 
by post-op MRI) 
 
 
 
Complete resection 
without post-op MRI 
 
 
 
Subtotal resection 
 
 
 
ADJUVANT TMZ 
x 6 cycles 
 
 
 
Rxt dose (media) 
 
 
 
 
MOLECULAR DATA 
Methylation 
 
Methylated 
 
 
 
Unmethylated 
 
 
 
IDH 
IDH1-R132 
MUTATED BY 
IHC 
 
 
 
IDH1-R132 non 
MUTATED by IHC 
 
 
 
 
IDH1-R132 mutated 
by sequencing 
 
 
 
IDH2-R172 mutated 
by sequencing 
 
 
 
TOTAL IDH 
(IHC+SEQ) 
 
 
 
 
<= 55y old with 
incomplete IDH 
dataa 
 
 
 
 


12 
 
 
 
 
2.1 PATIENT STATUS AT THE TIME OF THE REPORT 
 
 
 
 
 
Frequency 
% 
Valid % 
No 
Progression 
 
 
 
Progression 
 
 
 
Total 
 
 
 
 
 
 
 
 
Frequency 
% 
Valid % 
No exitus 
 
 
 
Exitus 
 
 
 
Total 
 
 
 


13 
 
3. 
TOXICITY 
 
 
 
3.1 BY NUMBER OF CONTROLS/VISITS: 
 
 
 
 
Number of controls/visits 
Total: 
Control arm: 
Treatment arm: 
 
 
 
HEMATOLOGICAL TOXICITY 
 
CONTROL TMZ %CONTROL %TMZ 
HEMATOLOGICAL 
Anemia 
G1 
 
 
 
 
G2 
 
 
G3 
 
 
 
G4 
 
 
Leucocytes 
G1 
 
 
 
 
G2 
 
 
 
 
G3 
 
 
G4 
 
 
Neutrophils 
count 
G1 
 
 
 
G2 
 
 
 
 
G3 
 
 
G4 
 
 
Platelets 
G1 
 
 
 
 
G2 
 
 
 
G3 
 
 
G4 
 
 
Lymphocytes 
G1 
 
 
 
 
G2 
 
 
 
 
G3 
 
 
 
G4 
 
 
 
ADVERSE EVENTS 
 
CONTROL 
TMZ 
CONTROL% 
TMZ 
G1 & 2 
G3&4 G1&2 G3 G1&2 G3&4 G1&2 G3&4 
Type 
 
 
 
 
 
 
 
 
 


14 
 
 
 
LABORATORY INVESTIGATIONS 
 
 
CONTROL 
TMZ 
CONTROL % 
TMZ% 
G1 & 2 
G3&4 
G1&2 
G3 
G1&2 
G3&4 G1&2 G3&4 
Creatinine 
 
 
 
 
 
 
FALCALINA 
 
 
 
 
 
 
K 
 
 
 
 
 
 
GGT 
 
 
 
 
 
 
 
GOT 
 
 
 
 
 
 
 
GPT 
 
 
 
 
 
 
BT 
 
 
 
 
 
 
GLUCOSA 
 
 
 
 
 
 
NA 
 
 
 
 
 
 
 
 
 
 
 
3.2 BY PATIENT. 
 
 
 
 
 
CONTROL N= (%) 
TMZ N= (%) 
Any 
G1&2 
G3&4 
Any 
G1&2 
G3&4 
Leukopenia 
 
 
 
 
 
 
Neutropenia 
 
 
 
 
 
 
Anemia 
 
 
 
 
 
 
Lymphopenia 
 
 
 
 
 
 
Thrombocytopenia 
 
 
 
 
 
 
Creatinine 
 
 
 
 
 
 


15 
 
Alkaline 
Phosphatase 
 
 
 
 
 
 
Potasium 
 
 
 
 
 
 
Sodium 
 
 
 
 
 
 
GGT 
 
 
 
 
 
 
GOT 
 
 
 
 
 
 
GPT 
 
 
 
 
 
 
Bilirrubin 
 
 
 
 
 
 
Hyperglycemia 
 
 
 
 
 
 
Neurologic 
 
 
 
 
 
 
Nausea and vomiting 
 
 
 
 
 
 
Asthenia 
 
 
 
 
 
 
Anorexia 
 
 
 
 
 
 
Hearing loss 
 
 
 
 
 
 
Skin 
 
 
 
 
 
 
Anxiety 
 
 
 
 
 
 
Pain 
 
 
 
 
 
 
Constipation 
 
 
 
 
 
 
Respiratory 
 
 
 
 
 
 
Infection 
 
 
 
 
 
 
Thromboemolic 
 
 
 
 
 
 
Bone-events 
 
 
 
 
 
 
Cardiac 
 
 
 
 
Second-neoplasia 
 
 
Hospitalization due 
to AE 
 
 
Patients who needed 
a TMZ dose 
reduction 
 
 
Patients who needed 
a delay in cycles 
 
 
Patients who withold 
TMZ due to AE 
 
 
 
 
 
3.4 RELEVANT DIFFERENCES IN TOXICITY BY ARM 
 


16 
 
CONTROL N= (%) 
TMZ N= (%) 
P for 
any 
Any 
G1&2 
G3&4 
Any 
G1&2 
G3&4 
Lymphopenia 
 
 
 
 
 
 
 
Thrombocytopenia 
 
 
 
 
 
 
 
Nausea and vomiting 
 
 
 
 
 
 
 
Fatigue 
 
 
 
 
 
 
 
Leukopenia 
 
 
 
 
 
 
 
Hospitalization due 
to AE 
 
 
 
Second neoplasia 
 
 
 
 
The table includes adverse events that will be different between arms 


17 
 
4. 
SECOND-LINE THERAPIES 
 
 
 
[ALL] 
N= 
(%) 
CONTROL 
N= (%) 
TMZ 
N= (%) 
p. value 
overall 
Type 
 
 
 
 
Type 
 
 
 
Type 
 
 
 
Type 
 
 
 
Type 
 
 
 


18 
 
5. OUTCOME 
 
 
Median follow-up in months. 
 
5.1 PRIMARY ENDPOINT: DIFFERENCES IN PROGRESSION-FREE SURVIVAL 
AT 6 MONTHS. 
 
 
 
 
Treatment ARM 6m-PFS 
95% CI 
Control 
 
 
Experimental 
 
 
 
 
 
5.2 PROGRESSION FREE SURVIVAL 
 
5.2.1 MEDIAN PFS ALL PATIENTS. 
 
 
 
Median PFS 
Median 
SE 
95 % CI 
Lower limit 
Upper limit 
 
 
 
 
 
 
 
5.2.2.MEDIAN PFS BY TREATMENT ARM 
 
 
 
 
ARM 
Median 
Estimate 
SE 
95 % CI 
Lower 
limit 
Upper 
limit 
CONTROL 
 
 
 
 
EXPER. 
 
 
 
 
Global 
 
 
 
 
The estimate is limited to the longest survival time, if censored. 
Figure Kaplan Meier PFS, total and by treatment arm.


19 
 
 
Chi-cuadrado 
gl 
Sig. 
Log Rank (Mantel- 
Cox) 
 
 
 
 
 
 
 
5.2.3 MEDIAN PFS BY METHYLATION STATUS 
 
 
 
 
Methylation 
Median 
95 % CI 
Lower limit 
Upper limit 
Met 
 
 
 
Unmet 
 
 
 
Global 
 
 
 
 
 
 
Global comparison 
Chi-cuadrado 
gl 
Sig. 
Log Rank (Mantel- 
Cox) 
 
 
 


20 
 
 
5.2.4 MEDIAN PFS BY RESIDUAL DISEASE 
 
 
Median and 95% CI for each stratification group. 
 
 
5.2.5 MEDIAN PFS DEPENDING ON ARM AND METHYLATION STATUS 
 
Median and 95% CI for each stratification group. 
Figure of methylated patients according to arm. 
Figure of unmethylated patients according to arm 


21 
 
 
 
 
5.3 OVERALL SURVIVAL 
 
 
Number of deaths.  
Total: 
By treatment arm: 
 
Median follow up: (months) 
 
Estimate 
SE 
95 % CI 
Lower limit 
Upper limit 
 
 
 
 
The estimate is limited to the longest survival time, if censored. 
 
 
 
5.3.1 OVERALL SURVIVAL BY TREATMENT ARM 
 
Median OS (months). 
Total: 
By treatment arm: 
 
 
Figure Kaplan Meier for OS.


22 
 
 
5.3.2 OVERALL SURVIVAL DEPENDING ON METHYLATION STATUS. 
 
 
Patients OS stratified by methylation status. 
Estimate 
95% CI 
Lower limit 
Upper limit 
 
 
 
 
 
 
 
 
 
The estimate is limited to the longest survival time, if censored. 
 
Figure Kaplan Meier OS by stratification groups. 
 
MGMT  
Methylated 
No methylated 
Global 


23 
 
 
 
 
5.3.3 OVERALL SURVIVAL DEPENDING ON RESIDUAL DISEASE 
Median OS of patients stratified by residual disease and treatment arm. 
 
 
Estimate 
95 % CI 
Lower limit 
Upper limit 
 
 
 
 
 
 
 
 
 
The estimate is limited to the longest survival time, if censored. 
 
 
 
 
Global comparison 
Chi-cuadrado 
gl 
Sig. 
Log Rank (Mantel- 
Cox) 
 
 
 
 
 
 
Figure: overall survival for patients depending on residual disease 
Residual disease status 
No 
Yes 
Global 


24 
 
 
 
5.3.4 OVERALL SURVIVAL DEPENDING ON ARM AND METHYLATION STATUS 
 
 
Median OS of patients stratified by methylation status and treatment arm. 
 
Figure: overall survival of methylated patients depending on arm 
 
Figure: overall survival of un methylated patients depending on arm. 
 
 
 
 
 
 
 


25 
 
 
 
5.4 MULTIVARIATE COS ANALYSIS OF PROGRESSION FREE SURVIVAL AND 
OVERALL SURVIVAL DEPENDING ON STRATIFICATION FACTORS AND 
TREATMENT ARM 
 
 
 
Multivariate Analyses 
Progression-free Survival 
Overall Survival 
HR 
95%CI 
P 
HR 
95%CI 
P 
Experimental 
arm 
 
 
 
 
 
 
MGMT 
methylation 
 
 
 
 
 
 
Absence of 
measurable 
disease 
 
 
 
 
 
 
IDH wild-type 
 
 
 
 
 
 
IDH status not 
available 
 
 
 
 
 
 
 
 


26 
 
 
 
5.5. HAZARD RATIO WITH FOREST PLOT FOR A PROGRESSION FREE SURIVAL 
AND B OVERALL SURVIVAL 
 
 
 
 
 
 
Forest plot of Hazard ratios of Methylation and residual disease status over PFS and OS. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


27 
 
 
 
6  .TRASLATIONAL STUDY 
 
 
MGMT methylation analysis 
MGMT methylation status will be assessed at the center of origin and centrally. 
Immunohistochemistry (IHC) analysis of the mismatch repair (MMR) deficiency proteins 
(MLH1, MSH2, MSH6, PMS2) and IDH1-R132H 
All cases will be reviewed to confirm glioblastoma. Four tissue microarrays (TMA) will be 
prepared. In samples where hematoxylin-eosin stained whole sections were determined to be 
non-necrotic, centrally located (non-infiltrating border) areas of the tumor will be selected for 
TMA construction. Three cylindrical cores, each measuring 0.6 mm in diameter, will be 
extracted from every donor block using an MTA-1 TMA workstation (Beecher Instruments, 
Silver Spring, MD) and placed in paraffin receptor blocs. 
We will study the protein expression of the mutated form of IDH1-R132H and the expression of 
the proteins codified by the four genes that regulate the MMR system: mutL homologue 1 
(MLH1), mutS homologue 2 (MSH2), mutS homologue 6 (MSH6) and post-meiotic segregation 
increased 2 (PMS2). For each determination, 4μ-thick sections of the four different TMAs will 
be obtained, placed on slides with electrostatic charge, and baked for 30 min at 60°C for proper 
adhesion of the tissue to the slides. Benchmark ULTRA immunostaining automated system 
(Ventana Medical System Inc., Tucson, AZ) will be used for all IHC determinations. Primary 
antibodies used will be IDH1-R132H (clone H-09; Master Diagnostic, Granada, Spain) and 
MLH1, MSH2, MSH6 and PMS2 (clones M1, G219-1129, SP93 and A16-4, respectively; 
Ventana Medical System Inc, Tucson, AZ).  
For IDH1-R132H the IHC result was considered positive if tumor cytoplasmatic positivity was 
seen. 
IDH sequencing 
Genomic DNA from tissue will be obtained using the QIAamp® DNA micro kit (Qiagen, 
Hilden, Germany) according to the manufacturer’s instructions. IDH1 and IDH2 exon 4 will 
amplified independently with Ecotaq (Ecogen, Barcelona, Spain) using the primers described 
by Horbinski et al. Cycling PCR conditions:  
Sequencing will be carried with  ExoProStarTM 1-step (GE Healthcare UK Ltd, Little Chalfont, 
United Kingdom) and sequenced with the BigDye® Terminator v3.1 Sequencing kit (Applied 
Biosystems, Foster City, CA) following the manufacturer’s instructions with an annealing 
temperature of 58ºC. The sequencing product will be purified with ZR DNA Sequencing clean-
up kitTM (Zymo Research, Irvine, USA) and analyzed in an AbiPrism 3130xl Genetic Analyzer 
(Applied Biosystems, Foster City, CA). 
 
Table: Immunohistochemistry results for mismatch repair proteins in the present study and in 
previously reported studies 
 
 


28 
 
MLH1 
N =  
PMS2 
N =  
MSH2 
N =  
MSH6 
N =  
Not evaluable 
 
 
 
 
Negative 
 
 
 
 
<70% positive cells 
(present study) 
 
 
 
 
<70% positive cells 
(Indraccolo) 
 
 
 
 
<80% positive cells 
(Felsberg) 
 
 
 
 
MGMT methylation 
status of patients 
with ≤80% positive 
cells 
(present study) 
 
 
 
 


29 
 
 
7 .ABSTRACTS AND PUBLICATIONS OF THIS STUDY. 
 
 
List of communications in Congress (oral and poster). 
List of publications in scientific juornals.