This is a prospective multicenter study of hypofractionated radiotherapy for the radiation treatment (RT) of solid tumors and in particular for Glioblastoma (in Aim 2). It is based on the results of ongoing studies at our Institute to validate the efficacy of extremely hypofractionated RT in neoadjuvant settings, which observed immunostimulatory effects of RT and the synergy with immune components. The collaboration between San Raffaele Hospital (Milan), the IRCCS Istituto Nazionale dei Tumori Fondazione G. Pascale (Naples) and the San Giuseppe Moscati Hospital of National Relief and High Specialty (Avellino) will ensure that patient recruitment, treatment and monitoring can be translated into facilities of the National Health System using common procedures. The various departments involved will treat patients with the same methods synergistically exploring the immuno/biological factors related to efficacy (and/or toxicity), based on new radioimmunotherapeutic approaches. Clinical and research activity will be developed jointly, drawing on the expertise in radiotherapy, radiomics, oncology, imaging and immunotherapy skills already available.

This is a prospective multicenter pilot study. Functional and spectroscopic neuroradiological imaging will be adopted for treatment planning. Specialized software will be used to perform radiomic feature extraction and analysis of pre-trained neural networks from the advanced MRI (magnetic resonance imaging) and CT (computed tomography) used for simulation, to identify distribution patterns of aggressive and radioresistant disease areas, with higher probability of disease recurrence, and to intensify the dose on the areas identified as more aggressive, in order to counteract intrinsic radioresistance. The hypofractionated radiotherapy paradigm claims the benefit of reduced treatment times, improved quality of life, better access to specialized treatment centers and potentially improved tumor outcomes with greater disease control and less tumor repopulation. The rationale for neoadjuvant RT is based on the idea of counteracting the tumor's aggressive mechanisms with radiotherapy before the disease is surgically removed, in order to maximize the immunostimulatory potential of RT, and therefore reduce recurrences. Neoadjuvant treatment offers numerous advantages, first of all the ability to adjust the dose to the pathological volume identified by MRI and limit the volume of irradiated healthy brain tissue. Furthermore, the use of imaging derived from functional neuroradiological and spectroscopic techniques for treatment planning would allow us to increase the dose on the areas identified as more aggressive, in order act against intrinsic radioresistance. One of the major risks could be the possibility of developing radionecrosis, but this would not be a cause for concern in the neoadjuvant setting, as all irradiated tissue will then be surgically removed. Patients who agree to participate in the study and who would be candidates for radical surgical treatment, according to the evaluation of the Neurosurgery Department of our Institute, will be treated. These patients will receive neoadjuvant radiation treatment in 5 fractions delivering 30 Gy on PTV and 35-50 Gy on GTV, with a dose-escalation modality that involves increasing the dose to 35-40-42.5-45-47.5-50 Gy in groups of 5 consecutive patients, using standard chemotherapy (TMZ) after surgery.

Current diagnostic brain MRI allows a good definition of the initial disease and its most aggressive areas. Since relapses have always been found to occur in irradiated areas and recent studies have shown that reducing margins does not affect overall survival, smaller margins will be used from GTV to CTV and from CTV to PTV. Therefore, smaller volumes will be generated and treated with hypofractionation. Biological equivalent doses (BED) to the standard prescription will be delivered, with boost to a higher biological equivalent dose, in the most aggressive areas, in order to obtain better local control, maintaining an acceptable level of toxicity and therefore improve the evolution of the disease. CE marked devices (software) will be used according to the approved use, for the definition of the target (CT and MRI) and for the delivery of the treatment (linear accelerators) and the standard drug, which has the authorization for marketing, will be prescribed. Radiomic features related to local response and survival will be identified, to obtain a predictive model. At the same time, we will collect PMBC and patient serum in the biobank to identify presumed immunocorrelated of therapy efficacy and/or predictive biomarkers of response/toxicity to therapy. For comparative purposes, serum from healthy volunteers will also be collected, in numbers equivalent to patients and with sex and age characteristics comparable to the latter.

Inclusion Criteria:

• Diagnosis of Glioblastoma.
• ECOG performance score 0-2 (defined during the first visit)
• Surgically removable lesion (according to the operability criteria established by the Neurosurgery Unit)

For healthy volunteers, people who are as comparable as possible with the patient population in terms of sex and age will be recruited

Exclusion Criteria:

• Previous stroke
• Presence of another primary and/or metastatic tumor For healthy volunteers also, absence of primary and/or metastatic tumor

The data that support the findings of this study (anonymized individual participant data) are available on request from the corresponding author to researchers who provide a methodologically sound proposal. Requests made to the corresponding author will be evaluated by the Lombardy Territorial Ethics Committee

for 2 years after the end of the study

request from the corresponding author approved by the Lombardy Territorial Ethics Committee