Glioblastoma is the most common malignant primary brain neoplasm, representing about 12-20% of all intracranial tumors and accounting for about 50-60% of all astrocytic neoplasms [1]. The astrocytic neoplasms occur in patients of all ages and arise at all levels of the neuraxis. In adults, most occur in the cerebral hemispheres, whereas in children typically occur in the brain stem [2–5] or thalamus [6–9]. Less commonly affected sites in both children and adults include the spinal cord [10–13] and cerebellum [14–16]. The 2007 World Health Organization (WHO) grading system designed three lesions of diffusely infiltrating astrocytic tumors: diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma (grade IV) [17]. Among diffusely infiltrative astrocytomas of the cerebral hemispheres, a close correlation is observed between histologic grade and clinical variables: patient age, duration of symptoms and neurologic performance status. With occasional exceptions, lesions in older patients are more anaplastic, biologically aggressive, recently symptomatic, and destructive of neurologic function.

Sudden death from an undiagnosed primary intracranial neoplasm is an exceptionally rare event, with reported frequencies in the range of 0.02% to 2.1% in medico-legal autopsy series [18–24] and only 12% of all cases of sudden unexpected death due to primary intracranial tumors are due to glioblastomas [25].

We report three cases of sudden unexpected death due to undiagnosed glioblastoma grade IV according to WHO [17] with different brain localization and expression. Complete histological, immunohistochemical and proteomic examinations are presented, to improve diagnosis.

Glioblastoma is highly malignant astrocytic glioma that appears to arise either de novo or in transition from diffuse astrocytoma and anaplastic astrocytoma. Glioblastomas that arise in transition from an often sizable, better-differentiated astrocytic tumor have been referred to as secondary glioblastomas [26]. Other glioblastomas, primary glioblastomas, are densely cellular and homogeneously anaplastic, and exhibit none of the less cellular and better-differentiated components seen in secondary tumors. It remains a matter of debate whether these “primary” variants are truly malignant de novo or have overrun and obscured a precursor lesion.

In the presented cases, the third one showed no neurological symptoms before the death and in the others cases there was a mild neurological symptomatology few hours before the death.

In recent years the concept of two distinct glioblastoma subtypes has been developed, combining clinical, morphological and genetic data. From this concept has emerged a clinical/molecular distinction of “primary” and “secondary” glioblastomas, although it is unclear how to distinguish this differentiation, or the extent to which it is therapeutically and prognostically relevant [31–33]. On the whole secondary types present in younger patients, more often women who have a longer duration of symptoms, and usually lie in the cerebral hemispheres. These tumors have a high frequency of mutation of p53 tumor suppressor gene on chromosome 17p with accumulation of p53 protein, but infrequent amplification of epidermal growth factor receptor (EGFR) involved in control of cell proliferation [32–35]. Loss of chromosome 19q in the region of a presumed tumor suppressor gene(s) as yet unidentified is more common in secondary tumors [36].

The considerably more frequent primary form of glioblastoma appears more abruptly and generally later in life than the secondary type, and more often in men. Genetically, this variant less often has mutations of p53, but exhibits complex genetic abnormalities including deletions, often homozygous, of p16. Amplification of EGFR occurs in 30 to 40 percent of such lesions, and is commonly associated with homozygous deletions in p16^INK4a[32, 33]. Most of molecular and genetic markers are still lacking confirmation; currently, only two markers, O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation and isocitrate dehydrogenase1 (IDH1) mutations, are commonly accepted genetic biomarkers for patients with glioblastoma [37]. As it has been authoritatively stated, primary and secondary glioblastomas are distinct disease entities and develop through distinct genetic pathways with different mRNA and protein expression profiles. Most of these genetic alterations can be ascribed to a determined set of functional pathways (Figure 9). These differences may affect sensitivity to radio- and chemotherapy and should thus be considered in the prognosis and response to therapy, but the genetic analysis is not so relevant for forensic purposes being fundamental, however, for this aim, the completion of diagnostic morphological investigations [38, 39].

In the cases of our observation, the clinical and morphological data are in agreement to the diagnosis of primary glioblastoma. In all cases, in fact, the tumor was very aggressive and there weren't clinical symptoms before the death. Like astrocytomas of lower grade, glioblastomas may be discovered on evaluation for seizures or headache but unlike lower-grade lesions whose infiltrating and insinuating qualities carry the cells unobtrusively into intact parenchyma with little resultant mass effect, at least initially, glioblastoma are often expansive and edema generating. As a result, they are more likely to produce frank neurological deficits and sign of increased intracranial pressure: a subset present in sudden, stroke-like fashion as a consequence of intratumoral hemorrhage.

The features of intratumoral vascular proliferations, in fact, have a constant correlation with the prognosis. In glioblastoma vascular proliferation assumes two forms. Most common is a well-known variant that forms globular masses resembling the glomerular tufts of the kidney, this proliferation, now referred to as “microvascular proliferation”. The second form of vascular hyperplasia has a more legitimate claim to the term “endothelial proliferation” since it is intraluminal and consists largely of endothelial cells within small to medium-sized vessels. Endothelial proliferation is less common than glomeruloid microvascular proliferation and it appears to have a more constant correlation with high-grade gliomas and a poor prognosis.

When evaluating cases of sudden death due to undiagnosed glioblastoma there were several problems in defining the most probable causes of death. Although modern diagnostic imaging techniques have revolutionized the diagnosis of brain tumors, the autopsy and the careful gross examination and section of the fixed brain (with coronal section) is still the final word in determining exact location, topography, mass effects and histology and secondary damage of brain tumor and contributed the elucidation of the cause of death [40]. Immunohistochemistry and proteomic analysis are mandatory in such cases [41–43].

Written informed consent was obtained from the patient's relatives for publication of this case report and any accompanying images.