**4.1 Macroscopy**

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 (Stavrinou et al., 2010).

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 mimicking a carcinoma metastasis.

#### **4.2 Histology**

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

Diagnostic Evaluation of Diffuse Gliomas 207

Morphologic variants include granular cell astrocytoma which is characterized by large, PAS-positive cells with a degenerative granular lysosomal content. These look similar to the benign granular cell tumor of the pituitary stalk (Schittenhelm et al., 2010). Another variant is the often subcortical located giant cell glioblastoma showing multinucleated giant cells in more than 50% of tumor cells that can be associated with reticulin deposits (Palma et al., 1989). These tumors need to be distinguished from the more benign subependymal giant cell astrocytomas or pleomorphic xanthoastrocytoma. Another variant contains a biphasic pattern of alternating reticulin-free glial and reticulin-containing mesenchymal deposits and are aptly named gliosarcomas (Louis et al., 2007a). These tumors account for 2% of all glioblastomas (Meis 1991). Metaplastic transformation can be so extensive that chondroid and osseous formations in gliomas are possible (Schittenhelm et al., 2007). Furthermore gliomas can show focal areas of epithelial differentiation that ranges from positive immunreactivity of epithelial antigens to adenoid or squamous formations leading to misdiagnosis of carcinoma (Rodriguez et al., 2008). Rare cases may show a melanotic

In average 3 pseuopalisading necroses are present in a glioblastoma specimen (Brat et al., 2004). Pseudopalisading cells are usually less proliferative and exhibit higher rates of apoptosis due to hypoxic conditions but are usually without a prominent inflammatory infiltrate. More than half of the palisades show a central vascular lumen, in about twenty percent intravascular thrombosis is also seen (Brat et al., 2004). Vascular proliferations may be present throughout the tumor but there is a tendency for these structures to accumulate in the peripheral region of high cellularity corresponding to the contrast-enhancing ring seen in radiological images (Louis et al., 2007a). Tumor vessels in glioblastoma have an increased density and show hyperplasia (Brat et al., 2001). Tumor vessel arrangement in a garland-like fashion is not uncommon. Vascular proliferation in form of glomeruloid bodies in glioblastomas is more frequently than in tumors from any other organ system (Plate et al.,

Infiltrative growth is mostly characterized by small undifferentiated cells growing along axonal structures in the white matter or along the brain surface and blood vessels. These are designated as 'secondary structures of Scherer' (Scherer, 1938). In the spinal cord, tumor cells might extend into the subarachnoid space (Burger & Scheithauer, 2007). Apoptosis of tumor cells is not a major feature but most prominent in areas of pseudopalisading necrosis.

The immunoprofile of glioblastomas is in many ways similar to astrocytomas. The vast majority of glioblastomas express the glial markers GFAP and EAAT1 (Waidelich et al., 2010) but these antigens may occasionally lacking (especially in small glioblastomas). S-100 immunostaining is then helpful to indicate a glial origin of the neoplastic cells. Strong MAP2 immunoreactivity is seen in 90% of glioblastomas (Blümcke et al., 2004). Vimentin immunoreactivity is very unspecific. Diffuse growth of gliomas can be supported by identifying axons with neurofilament stains within the tumor, but extensive neurofilament immunoreactivity of the tumor should prompt the diagnosis of an (anaplastic) ganglioglioma. In gliosarcomas, GFAP is lacking in sarcomatous areas. A complementary reticulin staining pattern in these tumors is diagnostic. The proliferation varies greatly, usually 15-25% of the nuclei are MIB-1 positive, but tumors with small cell morphology can show up to 90% proliferating cells. Tumors with previous radiation or gemistocytic

differentiation (Jaiswal et al., 2010).

**4.3 Immunohistochemistry** 

1999).

Fig. 3. **Glioblastomas** are defined through microvascular proliferations (A) and pesudopalisading necroses (B). The tumor is mitotically active (C) and may show a high degree of anaplasia (D). Glioblastoma cell composition can be so heterogenous with adenoid epithelial metaplasia (E), small cell component (F), focal oligodendroglial differentiation (G) or granular cells (H) in some cases.

Fig. 3. **Glioblastomas** are defined through microvascular proliferations (A) and

or granular cells (H) in some cases.

pesudopalisading necroses (B). The tumor is mitotically active (C) and may show a high degree of anaplasia (D). Glioblastoma cell composition can be so heterogenous with adenoid epithelial metaplasia (E), small cell component (F), focal oligodendroglial differentiation (G)

Morphologic variants include granular cell astrocytoma which is characterized by large, PAS-positive cells with a degenerative granular lysosomal content. These look similar to the benign granular cell tumor of the pituitary stalk (Schittenhelm et al., 2010). Another variant is the often subcortical located giant cell glioblastoma showing multinucleated giant cells in more than 50% of tumor cells that can be associated with reticulin deposits (Palma et al., 1989). These tumors need to be distinguished from the more benign subependymal giant cell astrocytomas or pleomorphic xanthoastrocytoma. Another variant contains a biphasic pattern of alternating reticulin-free glial and reticulin-containing mesenchymal deposits and are aptly named gliosarcomas (Louis et al., 2007a). These tumors account for 2% of all glioblastomas (Meis 1991). Metaplastic transformation can be so extensive that chondroid and osseous formations in gliomas are possible (Schittenhelm et al., 2007). Furthermore gliomas can show focal areas of epithelial differentiation that ranges from positive immunreactivity of epithelial antigens to adenoid or squamous formations leading to misdiagnosis of carcinoma (Rodriguez et al., 2008). Rare cases may show a melanotic differentiation (Jaiswal et al., 2010).

In average 3 pseuopalisading necroses are present in a glioblastoma specimen (Brat et al., 2004). Pseudopalisading cells are usually less proliferative and exhibit higher rates of apoptosis due to hypoxic conditions but are usually without a prominent inflammatory infiltrate. More than half of the palisades show a central vascular lumen, in about twenty percent intravascular thrombosis is also seen (Brat et al., 2004). Vascular proliferations may be present throughout the tumor but there is a tendency for these structures to accumulate in the peripheral region of high cellularity corresponding to the contrast-enhancing ring seen in radiological images (Louis et al., 2007a). Tumor vessels in glioblastoma have an increased density and show hyperplasia (Brat et al., 2001). Tumor vessel arrangement in a garland-like fashion is not uncommon. Vascular proliferation in form of glomeruloid bodies in glioblastomas is more frequently than in tumors from any other organ system (Plate et al., 1999).

Infiltrative growth is mostly characterized by small undifferentiated cells growing along axonal structures in the white matter or along the brain surface and blood vessels. These are designated as 'secondary structures of Scherer' (Scherer, 1938). In the spinal cord, tumor cells might extend into the subarachnoid space (Burger & Scheithauer, 2007). Apoptosis of tumor cells is not a major feature but most prominent in areas of pseudopalisading necrosis.

#### **4.3 Immunohistochemistry**

The immunoprofile of glioblastomas is in many ways similar to astrocytomas. The vast majority of glioblastomas express the glial markers GFAP and EAAT1 (Waidelich et al., 2010) but these antigens may occasionally lacking (especially in small glioblastomas). S-100 immunostaining is then helpful to indicate a glial origin of the neoplastic cells. Strong MAP2 immunoreactivity is seen in 90% of glioblastomas (Blümcke et al., 2004). Vimentin immunoreactivity is very unspecific. Diffuse growth of gliomas can be supported by identifying axons with neurofilament stains within the tumor, but extensive neurofilament immunoreactivity of the tumor should prompt the diagnosis of an (anaplastic) ganglioglioma. In gliosarcomas, GFAP is lacking in sarcomatous areas. A complementary reticulin staining pattern in these tumors is diagnostic. The proliferation varies greatly, usually 15-25% of the nuclei are MIB-1 positive, but tumors with small cell morphology can show up to 90% proliferating cells. Tumors with previous radiation or gemistocytic

Diagnostic Evaluation of Diffuse Gliomas 209

Fig. 4. **Oligodendrogliomas** show a typical honeycomb pattern (A). Tumor borders can be discrete infiltrative (B). Anaplastic oligodendroglioma with endothelial proliferations (C) and increased MIB-1 proliferation index (D). Oligodendroglial tumors typically exhibit a marked perinuclear MAP2 immunoreactivity (E) and show far less WT1 immunopositive cells (F) than astrocytomas. **Mixed oligodendroglioma-astrocytoma** can present either as true biphasic tumors (G) or as strongly intermixed (H) as in this anaplastic oligoastrocytoma

with extensive mitotic activity.

morphology may show little proliferating activity. Because of inconsistent laboratory techniques and varying evaluation methods, MIB-1 immunoreactivity has no established cutoffs between low-grade and high-grade lesions. WT1 expression is consistently expressed in glioblastomas (Schittenhelm et al., 2009). In our experience expression is similar in primary and secondary tumors but expression can be reduced in recurrent tumors. In addition there is evidence that tumors that contain a Tp53 mutation show reduced WT1 levels compared to Tp53 wild type glioblastomas (Clark et al., 2007). IDH1 R132H antibody expression is found in 4% of primary and in 71% of secondary glioblastoma (Capper et al., 2010). Tp53 immunoreactivity is less present than in astrocytomas but can be considerably high in giant cell glioblastomas. Microglial markers such as CD68 are regularly found in glioblastomas and can be very extensive in tumors with granular cell component and need to be distinguished from demyelinating lesions. Cytokeratin expression in glioblastomas (especially in giant cell glioblastomas and glioblastomas with true epithelial metaplasia) is an important diagnostic pitfall (Rodriguez et al., 2008). Dot-like EMA immunoreactivity is less frequently observed in glioblastomas than in ependymomas, where usually more than 5 EMA-positive dots per high-power field are seen (Hasselblatt & Paulus 2003). Immunohistochemistry of EGFR wild type protein is more prominent in primary glioblastomas as in grade II or III gliomas (Simmons et al. 2001).
