Deciphering the Labyrinthine System of the Immune Microenvironment in Recurrent Glioblastoma: Recent Original Advances and Lessons from Clinical Immunotherapeutic Approaches.

The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.

Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.

BACKGROUND: The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). METHODS: In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. RESULTS: Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. CONCLUSIONS: TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

A novel NDRG1 mutation in a non-Romani patient with CMT4D/HMSN-Lom.

Charcot-Marie-Tooth disease type 4D (CMT4D), also known as hereditary motor and sensory neuropathy Lom type (HMSNL), is an autosomal recessive, early onset, severe demyelinating neuropathy with hearing loss, caused by N-Myc downstream-regulated gene 1 (NDRG1) mutations. CMT4D is rare with only three known mutations, one of which (p.Arg148Ter) is found in patients of Romani ancestry and accounts for the vast majority of cases. We report a 38-year-old Italian female with motor development delay, progressive neuropathy, and sensorineural deafness. Magnetic resonance imaging showed slight atrophy of cerebellum, medulla oblongata, and upper cervical spinal cord. She had a novel homozygous NDRG1 frameshift mutation (c.739delC; p.His247ThrfsTer74). The identification of this NDRG1 mutation confirms that CMT4D is not a private Romani disease and should be considered in the differential diagnosis of recessive demyelinating CMT.

Mutational mechanisms in MFN2-related neuropathy: compound heterozygosity for recessive and semidominant mutations.

Mitofusin-2 (MFN2) mutations are the most common cause of autosomal dominant axonal Charcot-Marie-Tooth disease (CMT, type 2A), sometimes complicated by additional features such as optic atrophy (CMT6) and upper motor neuron involvement (CMT5). Several pathogenic mutations are reported, mainly acting in a dominant fashion, although few sequence variants behaved as recessive or semidominant in rare homozygous or compound heterozygous patients. We describe a 49-year-old woman with CMT5 associated with compound heterozygosity for two MFN2 variants, one already reported missense mutation (c.748C>T, p.R250W) and a novel nonsense sequence change (c.1426C>T, p.R476*). Her mother, carrying the p.R250W variant, had very late-onset minimal axonal neuropathy, whilst the father harboring the nonsense sequence change had neither clinical nor electrophysiological neuropathy. The missense mutation is likely pathogenic according to in silico analyses and a previous report, while the nonsense variant is predicted to behave as a null allele. The p.R250W variant behaves as semidominant by causing only a mild, almost subclinical, neuropathy when heterozygous; the nonsense mutation in the father was phenotypically silent, suggesting that haploinsufficiency for MFN2 is not disease causative, but was deleterious in the daughter who had only one active mutated MFN2 allele.

X-linked Charcot-Marie-Tooth type 1: stroke-like presentation of a novel GJB1 mutation.

X-linked Charcot-Marie-Tooth type 1 (CMTX1) is the second most common type of CMT and is caused by mutations in the Gap-Junction Beta-1 gene (GJB1), encoding connexin 32 which is expressed in Schwann cells as well as in oligodendrocytes. More than 400 GJB1 mutations have been described to date. Many mutation-carrier males have subclinical central nervous system (CNS) involvement, a few show mild CNS clinical signs, whereas only rarely overt though transient CNS dysfunction occurs. We report a 29-year-old man with CMTX1 who, at 16 years, showed short-lived CNS symptoms with transitory white matter abnormalities on cerebral magnetic resonance imaging (MRI) as first clinical presentation of a novel GJB1 mutation (p.Gln99_His100insGln). He had three consecutive episodes of right hemiparesis, together with sensory loss in the paretic limbs and expressive aphasia, all lasting a few hours, over a 2-day period, with concurrent white matter hyperintensity on MRI. These "stroke-like" episodes occurred just after arriving at sea level, after travelling from home at 700 m of altitude. Only a few years later did symptoms of peripheral neuropathy appear. In conclusion, CMTX1 should be included in the differential diagnosis of diseases characterized by transient CNS symptoms and white matter abnormalities on MRI.

Myelin protein zero Arg36Gly mutation with very late onset and rapidly progressive painful neuropathy.

Mutations in myelin protein zero (MPZ) protein result in a wide spectrum of peripheral neuropathies, from congenital hypomyelinating to late onset sensory and motor axonal forms. In some patients, neuropathic pain can be a prominent symptom, making the diagnosis challenging mainly in those with other risk factors for neuropathy. We describe a 77-year-old woman with impaired glucose tolerance presenting with rapidly progressive axonal neuropathy leading to excruciating pain and severe weakness of lower limbs within 2 years from the onset. Her son abruptly complained of similar painful symptoms at the age of 47 years. Molecular analysis revealed a novel heterozygous missense mutation (c.106A>G) in MPZ exon 2, causing the substitution of arginine-36 with glycine in the extracellular domain. Our observation suggests that MPZ-related neuropathy should be considered in the diagnostic work up of patients with painful axonal neuropathy even presenting with rapid progression and at a very late age of onset.

Co-occurrence of amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease type 2A in a patient with a novel mutation in the mitofusin-2 gene.

Mitofusin-2 gene (MFN2) mutations cause Charcot-Marie-Tooth type 2A (CMT2A), sometimes complicated by additional features such as optic atrophy, hearing loss, upper motor neuron signs and cerebral white-matter abnormalities. Here we report, for the first time, the occurrence of motor neuron disease, consistent with amyotrophic lateral sclerosis (ALS), in a 62-year-old woman affected by early-onset slowly progressive CMT2A, due to a novel MFN2 mutation. After age 60, rate of disease progression changed and she rapidly developed generalised muscle wasting, weakness, and fasciculations, together with dysarthria and dysphagia. Clinical features, EMG findings, and fast progression were consistent with ALS superimposed on CMT.

Novel mutations in the GDAP1 gene in patients affected with early-onset axonal Charcot-Marie-Tooth type 4A.

We report a detailed study of eight patients from four Italian families presenting with autosomal recessive axonal Charcot-Marie-Tooth disease (AR-CMT2), characterized by early-onset and progressive severe weakness of all limbs. Vocal cord paresis was present in two cases. Sural nerve biopsy performed in three patients showed a severe neuropathy characterized by a predominant axonal involvement. Five novel mutations (p.Gln99stop, p.Gln122Lys, p.Arg125stop, p.Val219Asp, p.Asn297Lys) and one previously reported mutation (p.Leu239Phe) were identified in GDAP1 gene. GDAP1 mutations should be considered both in recessive and sporadic cases of early-onset axonal CMT.

A novel founder mutation in the MFN2 gene associated with variable Charcot-Marie-Tooth type 2 phenotype in two families from Southern Italy.

Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy. CMT falls into two main forms: the demyelinating CMT type 1 with decreased nerve conduction velocities and the axonal CMT type 2. CMT2 is further subtyped by linkage analysis into >10 loci, with eight genes identified.Recently, mutations in the mitochondrial fusion protein 2 (MFN2) gene were reported in families with CMT2A1 and additional mutations have been detected in other studies, bringing to 42 the total number of different MFN2 mutations described thus far.2(-)4In the current study, we report a novel MFN2 mutation shared by two apparently unrelated CMT2 families originating from the same area in Southern Italy.

Rapid progression of late onset axonal Charcot-Marie-Tooth disease associated with a novel MPZ mutation in the extracellular domain.

Myelin protein zero (MPZ) is a major component of compact myelin in peripheral nerves where it plays an essential role in myelin formation and adhesion. MPZ gene mutations are usually responsible for demyelinating neuropathies, namely Charcot-Marie-Tooth (CMT) type 1B, Déjèrine-Sottas neuropathy and congenital hypomyelinating neuropathy. Less frequently, axonal CMT (CMT2) associated with MPZ mutations has been described. We report six patients (one sporadic case and five subjects from two apparently unrelated families) with a late onset, but rapidly progressive, axonal peripheral neuropathy. In all patients, molecular analysis demonstrated a novel heterozygous missense mutation (208C>T) in MPZ exon 2, causing the Pro70Ser substitution in the extracellular domain. The diagnosis of CMT2 associated with MPZ mutations should be considered in both sporadic and familial cases of late onset, progressive polyneuropathy. The mechanism whereby compact myelin protein mutations cause axonal neuropathy remains to be elucidated.