**2. Incidence and overview**

Regional incidences vary with generally higher number in developed countries and are estimated between 2.2 per million people for low-grade lesions (i.e. WHO grade II

Diagnostic Evaluation of Diffuse Gliomas 201

junctional zone. Tissue from this area instead of the deeper white matter might be not diagnostic or carries the risk of undergrading the tumor. The fixated tissue appears yellowish to gray and may be of varying texture, either softer or firmer than the surrounding normal appearing brain. Larger cysts are uncommon but when present are usually filled with a clear fluid. In cases with extensive microcystic formations of the tumor, a gelatinous appearance is present. Calcifications within the tumor is not the role. In spinal

Astrocytomas might display a wide range of cytologic and histologic features so that some autors even state that "astrocytomas are best defined as infiltrating gliomas that cannot be classified as oligodendrogliomas" (Burger & Scheithauer 2007). One important diagnostic marker is the hypercellularity of the CNS tissue. The number of tumor cells is usually slightly increased with a cellularity of two to five times than normal and the distribution of cells is irregular. Neoplastic astrocytes usually exhibit irregular elongated, hyperchromatic nuclei lacking a perinuclear halo with often minimal fibrillar cytoplasm ("naked nuclei"). The tumor cells lie between myelinated axons which can be visualized with luxol fast blue stains. In some cases there is a prominent pink cytoplasm with short stout processes and eccentrically placed nuclei, a so called "gemistocytic appearance". These tumors are prone to perivascular lymphocytic cuffs which are also seen in glioneuronal tumors (Takeuchi et al., 1976). Nuclei in gemistocytic variants are more rounded and less irregular and might show micronucleoli. Since almost all astrocytomas exhibit some gemistocytic tumor cells, a cut off of more than 20% gemistocytes has been proposed for the gemistocytic variant of astrocytoma (Tihan et al., 2006). Tumor margins in astrocytomas are rarely discernible. The neoplastic astrocytes rest on a fibrillary background which often shows some microcystic changes and increased density of cellular processes. These microcavities are usually absent in reactive gliosis. Cases with extensive mucoid degeneration and rarity of glial processes are designated as protoplasmatic astrocytomas. All three morphologies fibrillar, gemistocytic and protoplasmatic are considered histological variants of diffuse astrocytomas. Since a different clinical outcome for these is not firmly established some authors rather consider these as divergent patterns of differentiation (Louis et al., 2007b). Compared to diffuse astrocytomas, anaplastic astrocytomas exhibit increased cellularity, distinct nuclear atypia and mitotic activity but lack the micovascular proliferation and necrosis of glioblastomas. Multinucleated tumor cells are often diagnostic for a grade III lesion but not required for their diagnosis. One should also be aware of possible previous radiation therapy of the tumor leading to an increase of cell pleomorphism together with a

The original St. Anne-Mayo grade system did not allow mitoses in a low-grade lesion. Current criteria suggest that presence of zero or one mitosis do not alter survival and thus is still compatible with a WHO grade II neoplasm (Giannini et al., 1999). Unfortunately the WHO classification allows for a broad range of interobserver variability in borderline cases of low-grade gliomas, as presence of mitotic activity has to be interpreted in regard to the total sample size (Louis et al., 2007). In small specimens such as stereotactic biopsies a single mitosis suggests at least a grade III lesion but in larger specimens the presence of a single mitosis is not sufficient (Giannini et al., 1999). Cases with low cellularity of astrocytic tumor cells but exhibiting several mitoses should be considered as grade III or IV lesions. Diffuse

cord, cystic lesions may extend from the tumor poles.

decrease of mitotic activity (Gerstner 1977).

**3.2 Histology** 

neoplasms) and up to 4.6 per 100.000 people for glioblastoma (Ohgaki et al., 2005a). There is a strong correlation between age of presentation and histological tumor grade. The mean age of diagnosis for diffuse astrocytoma grade II WHO is 39 years, for anaplastic astrocytomas grade III WHO is 45 years and 61 years for glioblastoma grade IV WHO. The mean age for oligodendroglioma is 43 years, for anaplastic oligodendrogliomas grade III it is 47 years. Mean age for oligoastrocytomas is 40 years, for anaplastic oligoastrocytomas is 44 years (Louis et al., 2007a). A similar age distribution has been observed in our institution. Less than 10% of astrocytic and less than 2% of oligodendroglial tumors develop in the pediatric age group. Thus, the pathologist should always take patients age into mind when considering possible differential diagnoses. There is a slight predominance of males (Ohgaki et al., 2005b) but in contrast to meningiomas or germ cell tumors this is not of diagnostic relevance. Higher socioeconomic status is also a risk factor**.**

The histologic subtypes are not evenly distributed. Diffuse astrocytomas represent 5-10%, anaplastic astrocytomas approximately 10% and glioblastomas between 75-85 per cent of all astrocytic neoplasms (CBTRUS, 2011). This can be explained by the fact that glioblastomas are a heterogenous group of tumors with distinct genetic features but similar morphology. Diffuse astrocytomas show a tendency to progress to a more malignant phenotype during disease progression within 6-8 years, ending finally as secondary glioblastomas, (10-15% of all glioblastomas). However there is no biomarker that can predict the time to progression in individual patients. The majority of glioblastomas develop without a precursor lesion ("de novo") and are genetically distinct from the secondary glioblastomas (Ohgaki et al, 2007). In primary glioblastomas several activated oncogenic pathways are known, but all share a similiar dismal prognosis. Oligodendroglial tumors account for 5-6% of all gliomas and in this group 70% are diagnosed as grade II oligodendrogliomas and 30% as grade III anaplastic oligodendrogliomas (CBTRUS, 2011). While there is no doubt that oligodendroglioma undergo a similar malignant tumor progression as astrocytic neoplasms, there is still debate about how much of these truly develop into glioblastomas. Because of divergent classification criteria true estimates for mixed gliomas vary between 1 and 10% of all gliomas. According to the more stringent CBTRUS criteria, only 2% of all gliomas meet the criteria for a mixed oligodendroglial – astrocytic neoplasm (CBTRUS, 2011).

#### **2.1 General grading of diffuse gliomas**

In 1979 the World Health Organization issued a publication for classification of tumors of the central nervous system which has been updated lastly in 2007. This included a grading scheme based on malignancy behaviour of the tumors. Grading of diffuse gliomas is performed in a four-tiered score ranging from grade I to grade IV, the latter bearing the worst prognosis. Histological factors that influence grading are nuclear atypia, cellularity, mitosis, necrosis and endothelial proliferations. Among diffuse gliomas grade II is assigned to diffuse astrocytoma, oligodendroglioma and oligoastrocytoma. Grade III neoplasms include anaplastic astrocytoma, anaplastic oligodendroglioma and anaplastic oligoastrocytoma. Grade IV is reserved for glioblastoma. This score is used to separate the histologic continuum of diffuse gliomas.

### **3. Astrocytoma**

### **3.1 Macroscopy**

The invaded CNS tissue is usually enlarged, but main anatomical structures remain relatively intact. The overlying cerebral cortex might be affected with a blurred gray-white junctional zone. Tissue from this area instead of the deeper white matter might be not diagnostic or carries the risk of undergrading the tumor. The fixated tissue appears

yellowish to gray and may be of varying texture, either softer or firmer than the surrounding normal appearing brain. Larger cysts are uncommon but when present are usually filled with a clear fluid. In cases with extensive microcystic formations of the tumor, a gelatinous appearance is present. Calcifications within the tumor is not the role. In spinal cord, cystic lesions may extend from the tumor poles.

#### **3.2 Histology**

200 Advances in the Biology, Imaging and Therapies for Glioblastoma

neoplasms) and up to 4.6 per 100.000 people for glioblastoma (Ohgaki et al., 2005a). There is a strong correlation between age of presentation and histological tumor grade. The mean age of diagnosis for diffuse astrocytoma grade II WHO is 39 years, for anaplastic astrocytomas grade III WHO is 45 years and 61 years for glioblastoma grade IV WHO. The mean age for oligodendroglioma is 43 years, for anaplastic oligodendrogliomas grade III it is 47 years. Mean age for oligoastrocytomas is 40 years, for anaplastic oligoastrocytomas is 44 years (Louis et al., 2007a). A similar age distribution has been observed in our institution. Less than 10% of astrocytic and less than 2% of oligodendroglial tumors develop in the pediatric age group. Thus, the pathologist should always take patients age into mind when considering possible differential diagnoses. There is a slight predominance of males (Ohgaki et al., 2005b) but in contrast to meningiomas or germ cell tumors this is not of diagnostic

The histologic subtypes are not evenly distributed. Diffuse astrocytomas represent 5-10%, anaplastic astrocytomas approximately 10% and glioblastomas between 75-85 per cent of all astrocytic neoplasms (CBTRUS, 2011). This can be explained by the fact that glioblastomas are a heterogenous group of tumors with distinct genetic features but similar morphology. Diffuse astrocytomas show a tendency to progress to a more malignant phenotype during disease progression within 6-8 years, ending finally as secondary glioblastomas, (10-15% of all glioblastomas). However there is no biomarker that can predict the time to progression in individual patients. The majority of glioblastomas develop without a precursor lesion ("de novo") and are genetically distinct from the secondary glioblastomas (Ohgaki et al, 2007). In primary glioblastomas several activated oncogenic pathways are known, but all share a similiar dismal prognosis. Oligodendroglial tumors account for 5-6% of all gliomas and in this group 70% are diagnosed as grade II oligodendrogliomas and 30% as grade III anaplastic oligodendrogliomas (CBTRUS, 2011). While there is no doubt that oligodendroglioma undergo a similar malignant tumor progression as astrocytic neoplasms, there is still debate about how much of these truly develop into glioblastomas. Because of divergent classification criteria true estimates for mixed gliomas vary between 1 and 10% of all gliomas. According to the more stringent CBTRUS criteria, only 2% of all gliomas meet

the criteria for a mixed oligodendroglial – astrocytic neoplasm (CBTRUS, 2011).

In 1979 the World Health Organization issued a publication for classification of tumors of the central nervous system which has been updated lastly in 2007. This included a grading scheme based on malignancy behaviour of the tumors. Grading of diffuse gliomas is performed in a four-tiered score ranging from grade I to grade IV, the latter bearing the worst prognosis. Histological factors that influence grading are nuclear atypia, cellularity, mitosis, necrosis and endothelial proliferations. Among diffuse gliomas grade II is assigned to diffuse astrocytoma, oligodendroglioma and oligoastrocytoma. Grade III neoplasms include anaplastic astrocytoma, anaplastic oligodendroglioma and anaplastic oligoastrocytoma. Grade IV is reserved for glioblastoma. This score is used to separate the

The invaded CNS tissue is usually enlarged, but main anatomical structures remain relatively intact. The overlying cerebral cortex might be affected with a blurred gray-white

relevance. Higher socioeconomic status is also a risk factor**.**

**2.1 General grading of diffuse gliomas** 

histologic continuum of diffuse gliomas.

**3. Astrocytoma 3.1 Macroscopy** 

Astrocytomas might display a wide range of cytologic and histologic features so that some autors even state that "astrocytomas are best defined as infiltrating gliomas that cannot be classified as oligodendrogliomas" (Burger & Scheithauer 2007). One important diagnostic marker is the hypercellularity of the CNS tissue. The number of tumor cells is usually slightly increased with a cellularity of two to five times than normal and the distribution of cells is irregular. Neoplastic astrocytes usually exhibit irregular elongated, hyperchromatic nuclei lacking a perinuclear halo with often minimal fibrillar cytoplasm ("naked nuclei"). The tumor cells lie between myelinated axons which can be visualized with luxol fast blue stains. In some cases there is a prominent pink cytoplasm with short stout processes and eccentrically placed nuclei, a so called "gemistocytic appearance". These tumors are prone to perivascular lymphocytic cuffs which are also seen in glioneuronal tumors (Takeuchi et al., 1976). Nuclei in gemistocytic variants are more rounded and less irregular and might show micronucleoli. Since almost all astrocytomas exhibit some gemistocytic tumor cells, a cut off of more than 20% gemistocytes has been proposed for the gemistocytic variant of astrocytoma (Tihan et al., 2006). Tumor margins in astrocytomas are rarely discernible. The neoplastic astrocytes rest on a fibrillary background which often shows some microcystic changes and increased density of cellular processes. These microcavities are usually absent in reactive gliosis. Cases with extensive mucoid degeneration and rarity of glial processes are designated as protoplasmatic astrocytomas. All three morphologies fibrillar, gemistocytic and protoplasmatic are considered histological variants of diffuse astrocytomas. Since a different clinical outcome for these is not firmly established some authors rather consider these as divergent patterns of differentiation (Louis et al., 2007b).

Compared to diffuse astrocytomas, anaplastic astrocytomas exhibit increased cellularity, distinct nuclear atypia and mitotic activity but lack the micovascular proliferation and necrosis of glioblastomas. Multinucleated tumor cells are often diagnostic for a grade III lesion but not required for their diagnosis. One should also be aware of possible previous radiation therapy of the tumor leading to an increase of cell pleomorphism together with a decrease of mitotic activity (Gerstner 1977).

The original St. Anne-Mayo grade system did not allow mitoses in a low-grade lesion. Current criteria suggest that presence of zero or one mitosis do not alter survival and thus is still compatible with a WHO grade II neoplasm (Giannini et al., 1999). Unfortunately the WHO classification allows for a broad range of interobserver variability in borderline cases of low-grade gliomas, as presence of mitotic activity has to be interpreted in regard to the total sample size (Louis et al., 2007). In small specimens such as stereotactic biopsies a single mitosis suggests at least a grade III lesion but in larger specimens the presence of a single mitosis is not sufficient (Giannini et al., 1999). Cases with low cellularity of astrocytic tumor cells but exhibiting several mitoses should be considered as grade III or IV lesions. Diffuse

Diagnostic Evaluation of Diffuse Gliomas 203

Ventricular tumors with bizarre giant cells but low mitotic activity are often subependymal

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

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-

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

frequently seen in gemistocytic tumor cells (Hussein et al., 2006).

positive cells in one third of cases examined.

cells (Barbashina et al., 2007).

giant cell astrocytomas (WHO grade I).

**3.3 Immunohistochemistry** 

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

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 70% of these tumors, the most common R132H mutation can be detected immunohistochemically (F).

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. Ventricular tumors with bizarre giant cells but low mitotic activity are often subependymal giant cell astrocytomas (WHO grade I).
