Neutrophils are the most abundant white blood cells and are the first recruited to inflammatory sites. Neutrophils are an important component of the tumor stroma and can exert both antitumoral and protumoral activities, depending on their maturation and activation state. In human gliomas, the number of circulating and infiltrating neutrophils correlates with the severity of the disease, indicating a prognostic and possible pro-tumoral role for these leukocytes. In glioma preclinical models, neutrophils promote tumor growth and orchestrate the resistance to anti-angiogenic therapies. Nevertheless, recent data indicate that neutrophils can be activated to directly kill tumor cells or to orchestrate the antitumoral response. Here, we review current knowledge about the role of neutrophils in glioma and their possible involvement in new strategies to improve current cancer therapies.

Purpose: Current glioma diagnostic guidelines call for molecular profiling to stratify patients into prognostic and treatment subgroups. In case the tumor tissue is inaccessible, cerebrospinal fluid (CSF) has been proposed as a reliable tumor DNA source for liquid biopsy. We prospectively investigated the use of CSF for molecular characterization of newly diagnosed gliomas. Experimental Design: We recruited two cohorts of newly diagnosed patients with glioma, one (n = 45) providing CSF collected in proximity of the tumor, the other (n = 39) CSF collected by lumbar puncture (LP). Both cohorts provided tumor tissues by surgery concomitant with CSF sampling. DNA samples retrieved from CSF and matched tumors were systematically characterized and compared by comprehensive (NGS, next-generation sequencing) or targeted (ddPCR, droplet digital PCR) methodologies. Conventional and molecular diagnosis outcomes were compared. Results: We report that tumor DNA is abundant in CSF close to the tumor, but scanty and mostly below NGS sensitivity threshold in CSF from LP. Indeed, tumor DNA is mostly released by cells invading liquoral spaces, generating a gradient that attenuates by departing from the tumor. Nevertheless, in >60% of LP CSF samples, tumor DNA is sufficient to assess a selected panel of genetic alterations (IDH and TERT promoter mutations, EGFR amplification, CDKN2A/B deletion: ITEC protocol) and MGMT methylation that, combined with imaging, enable tissue-agnostic identification of main glioma molecular subtypes. Conclusions: This study shows potentialities and limitations of CSF liquid biopsy in achieving molecular characterization of gliomas at first clinical presentation and proposes a protocol to maximize diagnostic information retrievable from CSF DNA.

Introduction: Pembrolizumab demonstrated promising results in hypermutated tumors of diverse origin. Immunohistochemical loss of mismatch repair (MMR) proteins has been suggested as a surrogate of hypermutation in high-grade gliomas (HGG). We evaluated the efficacy and safety of pembrolizumab in relapsing HGGs with immunohistochemical loss of at least 1 MMR protein. Molecular biomarkers of pembrolizumab activity were also analyzed. Methods: Consecutive patients with recurrent HGG and partial or complete loss of MMR protein expression were prospectively enrolled; they received pembrolizumab 200 mg once every 3 weeks until disease progression. The primary endpoint was disease control rate (DCR). Post hoc exploratory analyses included next-generation sequencing to assess tumor mutational burden (TMB), and immunostaining for CD8+ T-cells and CD68+ macrophages. Results: Among 310 HGG patients screened, 13 cases with MMR loss were enrolled: eight glioblastoma, four anaplastic astrocytoma, and one anaplastic oligodendroglioma. Median age was 43 years. DCR was 31%: four patients had stable disease and no patient had complete or partial response. TMB ranged between 6.8 and 23.4 mutations/megabase. Neither TMB nor gene mutations, nor CD8+ T-cell and CD68+ macrophage content, were associated with pembrolizumab activity. Conclusions: pembrolizumab showed no apparent benefit in these patients. No molecular biomarker was found to be associated with pembrolizumab activity.