**3.3 Immunohistochemistry**

202 Advances in the Biology, Imaging and Therapies for Glioblastoma

astrocytomas (WHO grade II) have normal appearing vessels and a vessel density that is only slightly greater than in normal human brain. Compared to grade II lesions the vessel

Fig. 1. Histology of **diffuse astrocytoma**: HE stains of (A) fibrillary astrocytoma, (B) protoplasmatic astrocytoma and (C) gemistocytic astrocytoma. The proliferation in these tumors, as determined by MIB-1 nuclear immunoreactivity (D) is low (1-2%). Strong, consistent GFAP expression in neoplastic astrocytes (E). IDH1 mutations are found in up to

Differential diagnosis also includes reactive changes of the CNS. In astrocytic neoplasm, tumor cells are morphologically similar but less evenly distributed than reactive cells which are in different stages of activation. In addition reactive astrocytes show longer stellate processes. It is also important to recognize entrapped neurons and differentiate these from the more pleomorphic or even multinucleated neuronal tumor cells of a ganglioglioma.

70% of these tumors, the most common R132H mutation can be detected

immunohistochemically (F).

density increases further in grade III astrocytomas (Brat et al., 2001).

Astrocytomas show an expression of glial fibrillary acidic protein in tumor cells (Yung et al,. 1985). Especially the gemistocytic tumor cell cytoplasm and rare interdispersed Rosenthal fibers show a strong immunoreactivity for GFAP (Tascos et al., 1982). In addition the fibrillary neuropil displays almost always shows a diffusely positive background. Fibrillary astrocytes often show a small perinucelar rim, while interdispersed small round cells might be GFAP-negative. In independent studies all examined diffuse astrocytomas, were at least focally positive for GFAP (Cosgrove et al., 1986, Waidelich et al., 2010). Astrocytomas also express consistently S-100 and vimentin (Tabuchi et al., 1982, Yung et al., 1985). While S-100 is also present in the nuclei, Vimentin is often absent in distant cell processes. The malignancy-associated expression of WT1 is less intense than in high-grade astrocytic lesions (Hashiba et al., 2007, Rushing et al., 2010) but usually more prominent as in reactive lesions or oligodendrogliomas. WT1 is expressed in 52% of all diffuse astrocytomas, but tumors with more than 75% positive WT1 cells should prompt the diagnosis of a high grade glioma (Schittenhelm et al., 2009). A single study demonstrated that oligodendrogliaassociated marker Nogo-a is absent in grade II and III astrocytomas (Kuhlmann et al., 2008). Caution should be employed when using epithelial antigens, such as cytokeratins and epithelial membrane antigen for differential diagnosis of carcinomas, as variable expression of these markers have been observed in astrocytomas (Franke et al.,1991; Ng et al.,1989). A bcl-2 expression in diffuse astrocytomas is more prominent than in reactive lesions and frequently seen in gemistocytic tumor cells (Hussein et al., 2006).

The microtubuli-associated protein 2 which has once been considered as a neuronal marker is expressed in 92-97% of astrocytomas (Wharton et al., 2002). Cytoplasmic staining is preferentially seen in the larger, more pleomorphic, tumour cells and expression is generally more intense in high-grade lesions than in astrocytomas grade II. Some authors propose, that presence of MAP2-positive ramifying cytoplasmic processes aids in differentiating astrocytomas from oligodendogliomas where MAP2 is expressed in a capped fashion highlighting the rounded cells (Blümcke et al., 2004). MAP2 might also help to distinguish astrocytic tumors from ependymal neoplasms which show usually only solitary MAP2 positive cells in one third of cases examined.

Expression of p53 is seen in 72% of diffuse astrocytomas and more prominent in gemistocytic tumor cells and younger patients (Vital et al., 1998). However we have observed p53 in up to 63% of reactive lesions. The phospho-histone H3 marker might be useful to detect mitoses in tumor specimens with a proposed cutoff of 4/1000 between grade II and grade III astrocytomas (Colman et al., 2006). MIB-1 tumor proliferation is usually between 2-3%, rarely exceeding 4% (Sallinen et al., 1994). In protoplasmatic variants the MIB-1 proliferation index is usually lower than in other tumor variants (Prayson et al., 1996). The MIB-1 proliferative activity of astrocytomas grade III usually ranges between 5-10% and there is an overlap on both sides to grade II and grade IV lesions. Rare astrocytoma cases may show focal islands of small oligodendrocyte-like cells with immunoreactivity for synaptophysin and NeuN between conventional glial tumor cells (Barbashina et al., 2007).

Diagnostic Evaluation of Diffuse Gliomas 205

Utrastructurally astrocytomas contain abundant 7 to 11nm sized not always parallel aligned intermediate filaments independent of fibrillary, gemistocytic or protoplasmic phenotype of the tumor cells (Duffell 1963). Cells contain dilated cisterns of endoplasmatic reticulum, lysosomes and lipid deposits. Eosinophilic granular bodies display as dense osmiophilic masses between intermediate fibrils. The ultrastructural

The most malignant astrocytic glioma widely known by its acronym "GBM" was orginally designated as "glioblastoma multiforme" because of extensive variability of tumor histologies. However, individual tumors can also appear quite monorpous on histology. For this reason the "multiforme" is no longer used by the current WHO classification (Burger & Scheithauer, 2007). In our institution we prefer to use the term "multicentric" for single tumors with radiologically or macroscopically separate lesions and the term "multifocal" for true multiple lesions for which no histological continuum between the tumor centers exists. Common tumor spreading routes include fornix, corpus callosum, anterior comissure and radiation optica because of the high affinity of tumor cells for myelinated structures (Burger et al., 1983). Tumors that reach the dura show often marked desmoplasia leading to a firm texture resembling gliosarcoma or meningioma

The necrotic center of the tumor is often surrounded by a gray rim and varying yellowishgrayish texture of the surrounding white matter. Black hemorrhagic streaks and thrombosed veins are typically for a grade IV lesion. Symmetric tumors spreading over the corpus callosum are called "butterfly gliomas". Glioblastoma tumor borders are usually diffuse but rare cases (especially giant cell pseudoepithelial glioblastomas) can be very circumscribed

The prominent eosinophilic cytoplasm of pleomorphic tumor cells with small fibrillary zones indicates astrocytic heritage of the glioblastoma but this is not the rule for all tumors. Marked nuclear atypia and elevated mitotic activity is common. Either microvascular proliferations or necrosis or both are required to secure the diagnosis. Tumor appearance can be so heterogenous that diagnosis is often based on tissue patterns rather than individual tumor cell morphology. Occasionally perinuclear halos may resemble oligoendrogliomas, however glioblastoma tumor nuclei lack the monotony roundness of true oligodendrogliomas. Small cells with little cytoplasm can appear so monomorphous that small cell glioblastomas mimic anaplastic oligodendrogliomas. Small undifferentiated tumor cells intermingled with gemistocytes are more likely seen in secondary glioblastomas developing from gemistocytic astrocytomas. Some tumors may show prominent perivascular rosettes resembling anaplastic ependymomas but usually lack the more uniform roundness of ependymal tumor cells. Tumor cells can be elongated and arranged in

**3.4 Electron microscopy** 

**4. Glioblastoma 4.1 Macroscopy** 

(Stavrinou et al., 2010).

**4.2 Histology** 

mimicking a carcinoma metastasis.

fascicles so that at the first view sarcoma comes into mind.

picture of glioblastomas is similar.

Fig. 2. **Anaplastic astrocytomas** are histologically characterized by (A) increased cellularity, (B) nuclear pleomorphism, (C) presence of several mitoses. Gemistocytic tumors (D) are prone to undergo a more rapid tumor progression. Immunohistochemistry shows increased MIB-1 prolifation index (E). Extensive p53 nuclear immunoreactivity (F) is more frequent in astrocytomas than oligodendrogliomas or glioblastomas. GFAP (G) also marks the elongated tumor cell processes. Anaplastic astrocytomas have a considerably higher presence of MAP2 positive tumor cells (H) than grade II astrocytomas.

#### **3.4 Electron microscopy**

204 Advances in the Biology, Imaging and Therapies for Glioblastoma

Fig. 2. **Anaplastic astrocytomas** are histologically characterized by (A) increased cellularity, (B) nuclear pleomorphism, (C) presence of several mitoses. Gemistocytic tumors (D) are prone to undergo a more rapid tumor progression. Immunohistochemistry shows increased MIB-1 prolifation index (E). Extensive p53 nuclear immunoreactivity (F) is more frequent in

astrocytomas than oligodendrogliomas or glioblastomas. GFAP (G) also marks the elongated tumor cell processes. Anaplastic astrocytomas have a considerably higher

presence of MAP2 positive tumor cells (H) than grade II astrocytomas.

Utrastructurally astrocytomas contain abundant 7 to 11nm sized not always parallel aligned intermediate filaments independent of fibrillary, gemistocytic or protoplasmic phenotype of the tumor cells (Duffell 1963). Cells contain dilated cisterns of endoplasmatic reticulum, lysosomes and lipid deposits. Eosinophilic granular bodies display as dense osmiophilic masses between intermediate fibrils. The ultrastructural picture of glioblastomas is similar.
