Vacuolar ATPase and Drug Resistance of High Grade Gliomas: a Study to Investigate Possible Therapeutic Roles for Proton Pump Inhibitors
Conditions: Glioblastoma Multiforme
Sponsor: University of Milano Bicocca

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
1 
 
Title 
Vacuolar ATPase and drug resistance of high grade 
gliomas: a study to investigate possible therapeutic 
roles for proton pump inhibitors. 
Acronim 
GLIODRUG-V 
Protocol Version 
V. 1.0 
Protocol Date 
07-July-2019 
Kind of Study 
Prospective observational study on biological material 
Document Status 
Final 
Promotor 
University of Milano-Bicocca, 
Dipartiment of Medicine and Surgery 
Principal Investigator 
Prof. Carlo Giorgio Giussani, UO Neurochirurgia; ASST Monza, 
Monza, Italia, carlo.giussani@unimib.it 
 
 
Confidentiality statement: 
This document contains confidential information belonging to University of Milano-Bicocca. Except 
as may be otherwise agreed to in writing, by accepting or reviewing these materials, you agree to 
hold such information in confidence and not to disclose it to others (except where required by 
applicable law), nor to use it for unauthorized purposes. In the event of actual or suspected breach 
of this obligation, Milano Bicocca University should be promptly notified. 
 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
2 
 
 
Principal Investigator 
 
Carlo Giorgio Giussani 
 
Associate Professor 
 
Department: University of Milano-Bicocca, Department of Medicine and Surgery 
 
Institution: ASST Monza - Ospedale San Gerardo 
 
City, Nation: Monza, Italia 
 
Firma 
 
Data 
 
 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
3 
 
 
 
 
 
Vacuolar ATPase and drug resistance of high grade gliomas: a study to investigate 
possible therapeutic roles for proton pump inhibitors. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
4 
 
TABLE OF CONTENTS 
1 Introduction  
 
 
 
 
 
 
 
 
 
pag. 4 
2 Experimental Design  
 
 
 
 
 
 
 
 
pag. 5 
2.1 Study Rationale  
 
 
 
 
 
 
 
 
pag. 6 
2.2 Objectives  
 
 
 
 
 
 
 
 
 
pag. 6 
2.3 Type of Study 
 
 
 
 
 
 
 
 
 
pag. 6  
2.4 Study population 
 
 
 
 
 
 
 
 
pag. 6 
2.5 Inclusion criteria  
 
 
 
 
 
 
 
 
pag. 7 
2.6 Exclusion criteria  
 
 
 
 
 
 
 
 
pag. 7 
2.7 Experimental Design of the Study 
 
 
 
 
 
 
pag. 7 
2.8 Withdrawal criteria 
 
 
 
 
 
 
 
 
pag. 7 
2.9 Participating Units 
 
 
 
 
 
 
 
 
pag. 8 
3. Materials and Methods 
 
 
 
 
 
 
 
 
pag. 8 
3.1 Clinical Variables collected 
 
 
 
 
 
 
 
pag. 8 
3.1.1. Anagraphic Data  
 
 
 
 
 
 
 
 
pag. 8 
3.1.2. Anamnestic Data 
 
 
 
 
 
 
 
 
pag. 8 
3.1.5 Pre-operative and post-operative radiological data 
 
 
 
 
pag. 8 
3.1.6 Surgical Data  
 
 
 
 
 
 
 
 
pag. 8 
3.1.7 Histopathological findings 
 
 
 
 
 
 
 
pag. 8 
3.1.8 Follow-up 
 
 
 
 
 
 
 
 
 
pag. 8 
3.2 Study supplies and products 
 
 
 
 
 
 
 
pag. 8 
3.3 Data handling 
 
 
 
 
 
 
 
 
 
pag. 9 
3.4 Surgical methodology 
 
 
 
 
 
 
 
 
pag. 9 
3.5 Tissue and blood samples preservation, storage and management 
 
 
pag. 9 
3.5.1. Sampling and analysis  
 
 
 
 
 
 
 
pag. 9 
3.5.2 Storage and transport  
 
 
 
 
 
 
 
pag. 11 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
5 
 
3.6 Analysis of recurrence  
 
 
 
 
 
 
 
 
pag. 11 
3.7 Neuro-Oncological Follow-up 
 
 
 
 
 
 
 
pag. 11 
3.8 Statistical analysis 
 
 
 
 
 
 
 
 
pag. 11 
3.9 Data management 
 
 
 
 
 
 
 
 
pag. 12 
4. CRF  
 
 
 
 
 
 
 
 
 
 
pag. 13 
      4.1 Rules for completing CRFs 
 
 
 
 
 
 
 
pag. 13 
      4.2 Corrections to CRFs 
 
 
 
 
 
 
 
 
pag. 13 
5. Ethics 
 
 
 
 
 
 
 
 
 
 
pag. 13 
     5.1 Critical documents  
 
 
 
 
 
 
 
 
pag. 13 
6. Significance and Innovation   
 
 
 
 
 
 
 
pag. 13 
7. Responsibilities  
 
 
 
 
 
 
 
 
 
pag. 14 
8. Reports and publications 
 
 
 
 
 
 
 
 
pag. 14 
9. Retention of clinical documentation  
 
 
 
 
 
 
pag. 14 
10. References  
 
 
 
 
 
 
 
 
 
pag. 15 
 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
6 
 
1. Introduction 
Gliomas are primary brain tumors originating from glial cells. They are classified according to the 2016 
WHO classification into three grades from II to IV1. 
Grade III and IV gliomas are considered aggressive primary brain tumors with a poor prognosis which 
depends on several factors. In particular, the most aggressive and malignant of the primary brain tumors 
are the so called Glioblastoma Multiforme (GBM), that are classified as WHO grade IV gliomas1. GBMs 
accounts for 69% of all gliomas and for 12-15% of all primary brain tumors2. Patients with GBM have a poor 
median overall survival (OS) of 14-16 months with gross total surgical resection and adjuvant chemo-
radiation therapy. Nowadays, there are no curative treatments and tumor recurrence is expected. The use 
of alkylating agents has improved the prognosis of HGGs but after the introduction of Temozolomide (TMZ) 
in 2005 as standard first line treatment, no other improvement has been made3. Moreover, so far, there is 
not a standardized second line treatment after tumor recurrence and several drugs or treatment strategies 
are available4. 
 
GBMs have been found to have a small population of cancer stem cells (CSC) that contribute to tumor 
spreading, invasion, proliferation and maintenance. In particular, CSCs seem to be responsible for cancer 
resistance to adjuvant treatments and such resistance is responsible for tumor recurrence during the 
neuro-oncological follow-up5,6.  
Features of drug resistance can also be transmitted cell to cell by the cross-linking network sustained by 
microvescicles (MV) like exosomes and large oncosomes (LO) that transport a varied population of proteins, 
lipids, DNA, and RNA species7 that enhance and influence mechanisms involved in drug resistance8–10. 
Among the molecules that can be transported by this MV network there is H+ Vacuolar ATPase (V-ATPase), 
a multisubunits proton pump that is mainly involved in the acidification of endosomes and lysosomes in 
eukaryotic cells. It is made of a membrane-embedded V0 sector, which regulates protons permeability, and 
an enzymatic V1 ATPase sector11–13. 
 
In case of tumors, the role of V-ATPase seems to be crucial in order to maintain a normal intracellular pH in 
a metabolic setting based on anaerobic glycolytic reactions13. In fact, due to loss of functions of 
mitochondria during tumorigenesis, metabolism of neoplastic cells is based on anaerobic glycolysis which 
can easily afford ATP through the consumption of glucose but that, on the other hand, leads to production 
of lactates. The excess of lactates would determine a reduction of the intracellular pH and, consequently, 
activation of apoptotic reactions leading to the death of neoplastic cells14. In this setting, the excess of 
protons is counterbalanced by the extrusion of H+ from the intracellular to the extracellular environment 
through the activation and overexpression of V-ATPase. For this reason, many tumors show a V-ATPase 
overexpression,whose inhibition is related to a dysregulation of cytosolic pH that ultimately leads to the 
activation of pro-apoptotic proteins, alteration of metabolic chain reactions and of endosomal vesicles14,15. 
Other than providing those functions, V-ATPase plays a crucial role in chemoresistance of solid tumors. In 
fact, excess of protons in the extracellular space determines a reduction of pH that can reduce the 
penetration and the efficacy of chemotherapeutic agents15,16,16. Moreover, in few studies on breast cancer, 
B cell leukemia and melanoma, inhibition of V-ATPase have proved to increase the drug efficacy in vitro15. 
In addition, it is supposed that V-ATPase can act as a pump for extrusion of chemotherapy drugs from the 
intra-cellular to the extra-cellular compartment15,16. 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
7 
 
In 2015 Di Cristofori and colleagues demonstrated how V-ATPase is overexpressed in HGGs while it is poorly 
expressed in LGGs. From a clinical point of view, it was demonstrated how expression of V-ATPase is related 
to the patient’s prognosis independently from other clinical risk factors. Moreover, it has been 
demonstrated that blocking the V-ATPAse with specific drugs, in in vitro patient-derived GBM cultures, 
increased the efficacy of TMZ if compared with TMZ alone17. 
Highly tumorigenic glioma stem cells secrete in their microenvironment many micro- and macrovescicles 
that are loaded with factors able to influence the behavior of cells in the tumoral niche and cells of the 
immune system. In particular, GBMs secrete LOs loaded with the homeobox genes POU3F2 and HOXA10 
and with the V-ATPase subunit V1G19. Interestingly, such LOs were found to be able to reprogram both 
neoplastic and non-neoplastic cells toward an oncogenic state through the activation of proliferative and 
motility pathways9. So far, it seems that configuration or expression of specific subunits might influence the 
tumor formation or the tumor aggressiveness, probably via Homeobox genes9. In fact, another study by 
Bertolini and Colleagues, reported that neurospheres with high levels of V-ATPase expressed high levels of 
homeobox genes (HOXA7, HOXA10, SHOX2, and POU3F2) In this setting, V-ATPAse can be considered a key 
player involved in multiple functions that are necessary to afford CSCs survival and tumor progression. 
Virtually, V-ATPase is considered a possible target to sabotage the neoplastic metabolic machinery. In 
addition, inhibition of V-ATPase is considered a possible strategy to increase efficacy of antineoplastic 
drugs. This aspect of V-ATPase has not been studied in gliomas14.  
With these premises, the aim of this study is to understand the possible role for proton pump inhibitors 
(PPI) as adjunctive treatments for high grade gliomas.  
 
2. Experimental Design 
 
2.1 Study Rationale 
 
GBMs are still considered tumors with few available treatment options that are able only to achieve a 
temporary local control of the disease. In case of a GBM, tumor recurrence is generally expected within 12 
months and it is due to the presence of marginal tumoral cells with pro-oncogenic molecular phenotypes 
that are resistant to actual chemotherapies and to radiation therapy5,18,19. In particular, according to the 
study of Persano and Colleagues, in case of GBM it is possible to distinguish three neoplastic layers within 
the tumor that show different molecular patterns: the central core; the intermediate layer and the 
peripheral layer5. Nowadays, surgery still represent the first treatment option in case of suspected GBM 
and it aims to remove the contrast enhancing lesion seen at the pre-operative brain MRI. In particular, the 
peripheral layer is made of low replicating cells and it can be considered normal when tissue sampling is 
made far from the tumor cavity5. In fact, Clavreul et al. in 2015 demonstrated that peripheral GBM layer 
contains glioblastoma-associated stromal cell (GASC) that can show different carcinogenic properties and 
that are probably responsible for tumor recurrence20–22. These findings can be considered in line with 
previous studies that showed some invasive tumor cells, various types of reactive cells, and angiogenesis 
with different immunophenotypes in peritumoral brain edema23. 
Nevertheless, some research teams are trying to understand if surgical removal of peritumoral FLAIR 
hyperintensity is able to reduce the tumor recurrence rate prolonging the OS24.  
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
8 
 
Metabolism of GBMs is mainly anaerobial and it is sustained by glycolysis25. Anaerobial glycolysis is a simple 
metabolic reaction that leads to the transformation of glucose in ATP and lactates. Glucose is delivered to 
the tumor through neoangiogenetic processes26. Production of a significant amount of lactates determines 
a decrease of intracellular pH that is counterbalanced by V-ATPase activity through the extrusion of H+ ions 
from the intracellular to the extracellular environment. In vitro inhibition of V-ATPAse has proved to 
increase CSC apoptosis due to decrease of intracellular pH17.  
Moreover, increased V-ATPase activity determines an extrusion of H+ ions in the extracellular environment 
that can positively affect different pro-tumoral activities. In fact, a decrease of extracellular pH leads to 
activation of proteases able to destroy the extracellular matrix. Such activity enhances tumor spreading. 
Moreover, a low extracellular pH can reduce the efficacy of antineoplastic agents since a low pH might 
affect the structural integrity of drugs and their ability to pass through the plasmatic membrane. Finally, V-
ATPase can act as an active pump able to excrete antineoplastic agents15.  
For this reason, PPIs are considered new anti-cancer drugs able to increase tumoral cell death, reduce 
tumor invasion and increase chemotherapy efficacy14. 
Moreover, GBMs with high V-ATPAse expression has proved to be able to transmit highly malignant 
features through a network of MVs and to activate proliferation of GASC in vitro through the transmission 
of G1 subunit of V-ATPAse9,27.  
In this view, our work is intended to study: 1) the effects of PPIs on CSC and GASCs cultures as in vitro add-
on treatments; 2) the MVs load in terms of miRNAs and DNA (ssDNA, exoDNA28) during the neuro-
oncological follow-up in order to understand how it changes after surgery and adjuvant treatments; 3) the 
possible roles of V-ATPase as a clinical marker to be used to check tumor response to adjuvant treatments. 
 
2.2 Objectives 
● Primary objective: to determine and investigate the role of V-ATPase in drug resistance and the 
role of proton pump inhibitors as add-on treatment for high grade gliomas “in vitro” on CSC 
cultures obtained both from tumor and peritumoral margins.  
● Secondary objective: to determine whether MVs content in human blood is similar to the MVs 
content in CSCs “in vitro” in order to study the mechanisms of tumor recurrence and in to study 
possible translational aspects of our research.  
● Tertiary objective: to determine whether mechanisms of drug resistance can be transmitted via the 
cross-linking network sustained by exosomes and how this network is regulated in relation to the 
expression of V-ATPase. 
2.3 Type of Study 
This is a prospective observational cohort study on biological materials. 
 
2.4 Study Population 
Planned number of subjects undergoing surgical removal of intra-axial brain tumor at the Neurosurgical 
Division of Ospedale San Gerardo: almost 45 per year. Thirty patients are expected to have a glioma of 
which 25 patients are expected to be diagnosed with a GBM and 5 with a LGG. We plan to enroll 25 
patients/year. 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
9 
 
2.5 Inclusion Criteria 
Patients => 18 years old; 
Patients with an intra-axial brain tumor suspect for glioma; 
Patients able to sign a consent form for research purpose; 
Patients with planned craniotomy for brain tumor resection. 
2.6 Exclusion Criteria 
Patients < 18 years old; 
Patients with known brain tumors different than gliomas; 
Patients unable to sign a consent form for research purpose; 
Patients undergoing brain tumor biopsy; 
Patients with poor intra-operative or small surgical sample for histopathological diagnosis; 
Histology diagnostic for tumors different than gliomas. 
2.7 Experimental Design 
Our study will use glioma tissues, after all sampling for diagnosis will be collected. Moreover, we will take 
blood samples from patients undergoing surgery for HGGs and during the oncological follow-up. Tumor 
samples will be harvested in order to obtain parallel tumor cultures to test in vitro tumor sensitivity to 
different chemotherapies, with and without inhibition of V-ATPase. Moreover, we will analyze the 
expression of V-ATPase (especially subunit V1G1) in vescicles released by cancer cells, in order to 
understand how V-ATPase can influence drug resistance and tumor progression. HGG-released vescicles 
will be also isolated in the circulation from patients’ blood in order to analyze tumor responses to 
oncological treatments and the genetic and epigenetic status of the tumor, including amplification of 
oncogenes (i.e. c-Myc), and the content of specific coding and non-coding RNA, DNA and retrotransposon 
elements, through the use of next generation sequencing (NGS)28. 
Duration for enrollment: 24months; 
Duration for follow-up analysis: 12 months for each patient. 
Maximum length of the study: 36 months including patients’ follow-up 
2.8 Withdrawal Criteria 
The subject may withdraw at will at any time. The patient may be withdrawn from the trial at the discretion 
of the investigator for safety concerns. Patients who will meet the exclusion criteria after a prior inclusion 
in this study will be withdrawn. A patient withdrawn analysis will be made to clarify the related rate and 
possible impact on the study results. 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
10 
 
2.9 Participating Units 
Clincal data and collection of biological samples 
ASST-Monza Ospedale San Gerardo 
● U.O. di Neurochirurgia: Prof. C. Giussani; Dr. A. Di Cristofori; Dr. A. Trezza 
Stem cell cultures and extraction of MVs 
Università degli Studi di Milano Bicocca – Dipartimento di Medicina e Chirurgia  
Laboratorio di Genetica Medica: Dr.ssa Angela Bentivegna 
 
3 Materials and Methods 
 
This study will evaluate patient data and biological tissues collected prospectively and anonymized prior to 
the analysis. The study protocol follows the ethical guidelines of the 1975 Declaration of Helsinki (as revised 
in Brazil 2013). 
3.1 Clinical Variables collected 
3.1.1. Anagraphic Data  
Patient ID (anonymously assigned by the center in consecutive order); Date of Birth; Gender 
3.1.2. Anamnestic Data 
Karnopfsky Performance Status; ASA score; Previous Surgery 
3.1.5 Pre-operative and post-operative radiological data 
Tumor volume on pre- and post-operative brain MRI; extent of resection 
3.1.6 Surgical Data 
Date of surgery; location of the tumor; post-operative complications (e.g. hematoma, seizures) 
3.1.7 Histopathological findings 
Formal histological diagnosis with tumor grade and molecular classification according WHO 2016 
classification of the tumors of the central nervous system. 
3.1.8 Follow-up 
Complications during adjuvant chemo-radiation therapy; treatment response; chemotherapy administered 
with number of cycles and dosage. Dose of radiations delivered. Time to tumor progression and overall 
survival. 
3.2 Study supplies and products. 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
11 
 
Patients will be treated according to local hospital procedure. No additional costs (materials, salaries, 
other) due to the study will be charged to the hospital. Study products will be those commonly used in the 
hospital according to the local rules. 
3.3 Data handling 
Data collection will be performed using an electronic database system, in accordance with the European 
Statement 679/2016/UE. The submitted data will be checked at San Gerardo Hospital (Monza) by clinicians 
(Neurosurgery Division) and by the the Medical Genetics Laboratory (Dr. Angela Bentivegna), of School of 
Medicine and Surgery, University of Milano-Bicocca. Once examined, the record will be accepted into the 
dataset for analysis. 
3.4 Surgical methodology 
Patients will be operated for intra-axial brain tumors. Part of the tumor specimen, will be sent for cell 
culture; while the majority of the tumor specimen will be used for a formal histological diagnosis. In order 
to achieve a gross total resection and confirm it histologically, a sample of marginal tissue of the surgical 
bed will be sent for cell culture and histology. Marginal tissue will be collected from non eloquent brain 
areas. Intraoperative ultrasound will be used to check presence or absence of high cellularity of the sample.  
Patients with brain tumors other than gliomas at histological diagnosis will be excluded from the study. 
Sample of these tumors will be eliminated. 
If the tissue removed from the patient enrolled in this study will not allow to perform the sample for the 
study without affecting the optimal histological diagnosis, the patient will be excluded from the study and 
no sampling will be executed. 
3.5 Tissue and blood samples preservation, storage and management 
3.5.1. Sampling and analysis 
Part 1 – stem cell cultures sampling and processing 
 
Biological samples from patients undergoing surgery for intra-axial brain tumor will be taken and 
processed. Tumor margins will be collected as well, in order to check tumor resection with 
histopathological findings and in order to investigate the role of CSCs in the brain adjacent to tumor in 
determining the tumor recurrence due to resistance to treatments. Both tumor area and its marginal tissue 
will be taken during the surgery and collected for laboratory procedures and histological analysis.  Blood 
samples will be collected 1-3 days before surgery and 1-3 days after surgery and will be processed for 
serum collection and stored for further analysis on MVs. 
The fresh tissue (tumor and marginal tissues) will be treated in order to obtain single cell suspensions and 
neurospheres. Then, each patient-derived primary cell cultures will be expanded and subsequently divided 
into 2 parts: 1) one destined to CSCs cultures for in vitro tests; 2) one for biological storage, for further 
retrospective analyses. The storage of biological samples will be at the Medical Genetics Laboratory (Dr. 
Angela Bentivegna), of School of Medicine and Surgery, University of Milano-Bicocca. All the materials for 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
12 
 
the study will be provided by the same laboratory, so there are no additional costs for the Neurosurgical 
Division and for Hospital San Gerardo. 
 
Part 2 - in vitro study of drug resistance 
 
Neurospheres from marginal tissues will be obtained by co-culturing of marginal tissue with MVs taken 
from CSCs cultures medium, in order to better define the role of V-ATPase in drug resistance and the role of 
MVs in transferring drug resistant phenotypes from a CSCs culture to another. 
CSCs cultures will be treated with antineoplastic drugs with and without PPI. In this way, it will be possible 
to understand the in vitro response to antineoplastic agents and PPI. 
 
Anti-neoplastic agents taken into account for this study are: Temozolomide, Vincristine, Carboplatin, 
Etoposide, Irinotecan (CPT-11), Bevacizumab, BCNU, CCNU, Procarbazine, Irinotecan + Carboplatin, 
Procarbazine + Vincristine + CCNU (PCV). All these agents have been used for treatment of HGGs as first or 
second line therapy.  
 
PPIs used for this study are: concanamycin, Bafilomicine-A1, Archazolide-A. All the procedures will be 
performed for research use at the Medical Genetics Laboratory (Dr. Angela Bentivegna), of School of 
Medicine and Surgery, University of Milano-Bicocca..  
 
Part 3 – serial analysis of blood samples 
 
During the first year of neuro-oncological follow-up, patients will undergo to collection of blood samples 
pre- and post-operatively, three months, six months, nine months and one year after surgery. Each sample 
will be processed in order to study the MVs content of subunits of V-ATPase (especially subunit V1G1) and 
the genetic and epigenetic content (DNA, mRNA and miRNA). The results will be matched with the brain 
MRIs performed as part of the neuro-oncological follow-up. The blood volume will be of about 5-10 mLs 
and it will be taken during the normal blood sampling performed during the neuro-oncological follow-up 
for checking chemotherapy toxicities. No adjunctive invasive procedures will be performed on patients. 
 
During this part of the study, it will be possible to understand the relations between tumor volume and 
MVs concentrations in the bloodstream in order to understand the role of surgery on the existing vesicular 
network between the brain and the rest of the body. Moreover, it will be possible to study the effects of 
adjuvant treatments on the MVs contents during the time of response to treatment and at tumor 
recurrence. Finally, analysis of DNA, miRNA and mRNA contained in the MVs will allow us to explore the 
presence of new markers able to predict tumor recurrence and to understand in vivo any possible changes 
of the neoplastic cells during the treatment. 
These results will be compared with the results obtained in vitro in order to understand if the experimental 
model can reflect the mechanisms active in the patient.  
This part of the research project will allow us to transfer the project from the bench to the bedside and it 
will be the starting point for future projects. 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
13 
 
MVs isolation will be performed as reported below. 
For all patients 20 mL fasting blood sample will be collected in a sterile vacuum tube. The collection will be 
done in the same time of routine preoperative blood samples or planned follow-up controls, since no 
others invasive procedure should be required from the patients since their enrollment in the study. The 
blood sample will be stored at 4°C and handled within 6 hours. Then, 5 mL will be centrifuged at 3000 rpm 
for 10 minutes at 4°C, the plasma phase will be collected and aliquoted in 1.5 mL sterile tubes and stored at 
-80°C until use or shipment. Then, we will isolate peripheral blood mononucleate cells (PBMC) through 
Ficoll gradient from the remaining 15 mL. After isolation, live PBMC will be frozen in standard media 20% 
Fetal Bovin Serum (FBS) 10% Dymethil Sulfoxide (DMSO) and stored in liquid nitrogen until use. All these 
parts of the experiment will be performed at the Medical Genetics Laboratory (Dr. Angela Bentivegna), of 
School of Medicine and Surgery, University of Milano-Bicocca. 
3.5.2 Storage and transport 
The biological material will be shipped and stored to the Medical Genetics Laboratory (Dr. Angela 
Bentivegna), of School of Medicine and Surgery, University of Milano-Bicocca the same day of sample 
collection. No additional costs are required for biological sampling transport since the laboratory is close to 
the hospital. The biological material will be stored for a maximum period of 10 years. 
3.6 Analysis of tumor recurrence 
 
Tumor recurrence will be evaluated according the RANO criteria. Patients will undergo routine neuro-
oncological and radiological assessment in order to check the brain and the surgical bed during adjuvant 
treatments. 
 
3.7 Neuro-Oncological Follow-up 
 
All patients will undergo to a neuro-oncological follow-up. Each patient will be treated with a standard first 
line treatment according to the Stupp protocol with concomitant chemo-radiation therapy and subsequent 
adjuvant chemotherapy. Every 3 months, each patient will undergo a brain MRI. 
During every neuro-oncological evaluation, each patient will have a blood sample taken in order to check 
the blood count. During this standard procedure, an additional sample of blood will be taken for this study 
in order to extract circulating exosomes and DNA. 
Brain MRIs will be examined to calculate tumor volumes and residual tumor volumes and to diagnose 
tumor recurrence. 
 
3.8 Statistical analysis. 
 
Data will be expressed as median and interquartile range (IQR), number and relative percentage, or hazard 
ratio (HR) with related 95% confidence intervals (CI). Normal distribution of continuous variables will be 
assessed by Kolmogorov-Smirnov test. To compare baseline characteristics of the groups in the univariate 
analysis, continuous variables will be analysed using the Mann-Whitney test or ANOVA according to the 
type of distribution. Categorical variables will be analyzed by Fisher exact test or Chi-Square Test as 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
14 
 
appropriate. A 1:1 propensity-score-analysis, with a caliper of 0.1SD, may be conducted for significant 
variables (p<0.05) at univariate to reduce the retrospective bias of selection. 
Multivariate analysis will be performed by logistic regression analysis or Cox regression as appropriate. DFS, 
OS and TSS will be evaluated by Kaplan-Meier method. Comparison between groups will be performed with 
the log-rank test. A Receiver Operating Characteristic (ROC) curve analysis will be employed as appropriate. 
The optimal cut-off value will be established based on the Youden index. All statistics will be 2-tailed and 
statistical significance will be accepted when p<0.05. 
 
3.9 Data management. 
 
Data will be stored in an electronic database. Each investigator may have access to the whole dataset. 
The subject will be identified by an alphanumeric code linked to the name of the patient that the enrolling 
investigator can see. Appropriate measures such as encryption or deletion will be enforced to protect the 
identity of human subjects in all presentations and publications as required by local/regional/national 
requirements. During the patients entering in the database, data quality will be ensured. At least one 
report analysis will be performed each year.  
Major protocol deviations will lead to exclusion of data from the analysis, while data will not be excluded 
because of minor protocol deviations. The list of major protocol deviations will be detailed and 
documented in the clean file document prior to database release. 
 
The personal data subject of the study will be processed in compliance with the European Regulation on 
the Protection of Personal Data (GDPR), of Legislative Decree 196/2003 and subsequent amendments and 
additions, and of any other Italian law applicable to the protection of personal data ( hence referred to as 
"applicable data protection law"). 
With regard to personal data made available to the Promoter in a pseudo-anonymized form, the Institute 
and the Promoter are considered joint owners, each for their own area of competence, and both will act in 
compliance with the applicable data protection law. 
Furthermore, the Institute and the Promoter will cooperate to take the necessary measures to comply with 
the applicable data protection law, and will also have to implement appropriate technical and 
organizational measures to guarantee the GDPR compliance requirements. 
The Institute is responsible for providing its Principal Investigators and Research Personnel with all 
necessary information concerning the methods through which the Promoter will eventually collect and 
manage their personal data before such information is provided. 
The Promoter ensures the correctness of the processing of personal data as provided by the GDPR. If the 
user becomes aware of a data breach of personal data, the user himself must promptly notify the other 
users of this event, and in any case no later than 24 hours from the knowledge of the event. In this case, 
the user will have to cooperate fully with the other users to remedy the data breach, fulfilling the 
mandatory notification within the set times and managing any damage caused. 
The data breach of personal data is defined by the art. 33 and 34 of the GDPR. 
The roles and responsibilities related to the project will be explained in a further and separate act between 
the Promoter and the institute. 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
15 
 
4 CRF 
 
4.1 Rules for completing CRFs 
The investigator staff must ensure that all information derived from source documentation is consistent 
with the source information. By uploading the complete dataset, the Investigator confirms that the 
information is complete and correct. 
 
4.2 Corrections to CRFs 
Corrections to the data on the CRFs can only be made by communicating to the Data Manager the 
alphanumeric code linked to the name of the patient and asking to modify the data. 
 
5. Ethics 
 
The register will be conducted in accordance with the Declaration of Helsinki and according to local and 
regional ethical standards. 
 
5.1 Critical documents  
Before the Investigator starts the study (i.e., obtains informed consent from the first subject), the following 
documents must be available: 
• Regulatory approval and/or notification as required 
• Signed and dated agreement on the final protocol 
• Signed and dated agreement on any substantial amendment(s), if applicable 
• Approval/favourable opinion from IEC clearly identifying the documents reviewed: the 
protocol, any substantial amendments, subject information/informed consent form and any other written 
information to be provided to the subject, subject recruitment procedures. 
6. Significance and Innovation  
This work is meant to increase our knowledge on glioma progression and on drug resistance of high grade 
gliomas and the role played by V-ATPase. First, this study is meant to better investigate the role of V-
ATPase on drug resistance and the possible roles that V-ATPase inhibitors could play as add-on treatments 
for HGGs. This would be one of the first dedicated study in literature and it could set the theorical bases for 
an in vivo study. 
Secondarily, this study is meant to investigate the molecular changes that tumors may develop through the 
analysis of MVs and circulating DNA during the normal neuro-oncological follow-up. The aim is to find 
molecular settings that might underpin the resistance to adjuvant treatments or that might be involved in 
developing drug resistance during the neuro-oncological follow-up and that could be related with 
development of tumor recurrence. 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
16 
 
This study might allow in future to understand new strategies to increase the efficacy of adjuvant 
treatments against HGGs. These treatments will aim to dismantle the tumoral cell metabolism blocking the 
mechanisms that regulate the intracellular pH. 
7. Responsibilities  
The Investigator is accountable for the conduct of the study. If any tasks are delegated, the Investigator 
should maintain a list of appropriately qualified persons to whom he/she has delegated specified significant 
study-related duties. 
The medical care given to, and medical decisions made on behalf of, subjects should always be the 
responsibility of a qualified physician. 
The Investigator will take all necessary technical and organizational safety measures to prevent accidental 
or wrongful destruction, loss or deterioration of data. The Investigator will prevent any unauthorized access 
to data or any other processing of data against applicable law. 
8. Reports and publications 
The information obtained during the conduct of this study is considered confidential and belongs to the 
investigator group for the purpose of scientific publications. No confidential information shall be disclosed 
to others. Such information shall not be used except in the performance of this study group. 
All the investigators may decide after a consensus, to share the data with other groups or trials 
occasionally. In this case, the agreement of the whole study group is needed. 
9. Retention of clinical documentation 
Subject notes must be kept for the maximum period permitted by the hospital, institution or private 
practice. 
The Investigator must agree to archive the documentation pertaining to the register in an archive after 
completion or discontinuation of the study if not otherwise notified. 
Clinical study documentation must be retained until at least 2 years. 
 
 

STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
17 
 
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STUDY NAME: Vacuolar ATPase and drug resistance of 
high grade gliomas: a study to investigate possible 
therapeutic roles for proton pump inhibitors. 
STUDY CODE: GLIODRUG-V 
Ver. 1.0 
07/06/2019 
 
 
18 
 
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