**5. Oligodendroglioma**

#### **5.1 Macroscopy**

Like all other diffuse growing tumors, oligodendroglioms show diffuse borders. The tumors are usually soft and have a grey to pink color. They may appear hemorrhagic and / or calcified but this is not a specific feature for oligodendrogliomas Superficial growth can expand the cortical grey matter. The anaplastic forms lack a central necrosis typically for glioblastoma but may show focally smaller necroses. Rare disseminating cases may grow as superficial gelatinous mass extending along the spinal cord (Mittelbronn et al., 2005).

#### **5.2 Histology**

In contrast to astrocytomas, oligodendrogliomas are dominated by histologic monotony of the round to oval shaped tumor cells which are best seen in smears. Nuclei have a bland chromatin and prominent nucleoli. The very characteristic perinuclear halo – a fixation artefact resulting from autolytic water absorption – is absent in frozen sections or specimens that have been quickly processed resulting from a short fixation time. Delicate branching capillaries and tumor calcifications that also may affect tumor vessels are more frequent in oligodendrogliomas than in other CNS tumors. Overun cortical areas show a perineuronal satellitosis of the tumor cells and tumor cells may concentrate along subpial structures. In addition cortical structures show often smaller microcystic changes.

Anaplastic oligodendrogliomas show increased nuclear pleomorphism that is mostly restricted to focal areas and increased mitotic activity compared to grade II lesions. Some authors prefer a mitotic cutoff of 6 mitoses per 10 high power fields to discriminate between grade II and grade III lesions (Giannini et al., 2001). Focal elevated cellular areas as nodules do not warrant tumor designation as a grade III lesion in absence of other anaplastic features. In contrast to astrocytomas where endothelial proliferations lead to the diagnosis of glioblastoma, vascular proliferations or extended vascular hyperplasia are typical for anaplastic oligodendroglioma grade III. In addition smaller areas of necrosis may be

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

Like all other diffuse growing tumors, oligodendroglioms show diffuse borders. The tumors are usually soft and have a grey to pink color. They may appear hemorrhagic and / or calcified but this is not a specific feature for oligodendrogliomas Superficial growth can expand the cortical grey matter. The anaplastic forms lack a central necrosis typically for glioblastoma but may show focally smaller necroses. Rare disseminating cases may grow as superficial gelatinous mass extending along the spinal cord (Mittelbronn et al., 2005).

In contrast to astrocytomas, oligodendrogliomas are dominated by histologic monotony of the round to oval shaped tumor cells which are best seen in smears. Nuclei have a bland chromatin and prominent nucleoli. The very characteristic perinuclear halo – a fixation artefact resulting from autolytic water absorption – is absent in frozen sections or specimens that have been quickly processed resulting from a short fixation time. Delicate branching capillaries and tumor calcifications that also may affect tumor vessels are more frequent in oligodendrogliomas than in other CNS tumors. Overun cortical areas show a perineuronal satellitosis of the tumor cells and tumor cells may concentrate along subpial structures. In

Anaplastic oligodendrogliomas show increased nuclear pleomorphism that is mostly restricted to focal areas and increased mitotic activity compared to grade II lesions. Some authors prefer a mitotic cutoff of 6 mitoses per 10 high power fields to discriminate between grade II and grade III lesions (Giannini et al., 2001). Focal elevated cellular areas as nodules do not warrant tumor designation as a grade III lesion in absence of other anaplastic features. In contrast to astrocytomas where endothelial proliferations lead to the diagnosis of glioblastoma, vascular proliferations or extended vascular hyperplasia are typical for anaplastic oligodendroglioma grade III. In addition smaller areas of necrosis may be

glioblastomas as in grade II or III gliomas (Simmons et al. 2001).

addition cortical structures show often smaller microcystic changes.

**5. Oligodendroglioma** 

**5.1 Macroscopy** 

**5.2 Histology** 

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.

Diagnostic Evaluation of Diffuse Gliomas 211

The short tumor cell processes contain microtubuli and ocassionally pericellular spiral laminations but usually lack the abundant intermediate filament of astrocytic tumor cells (Min et al., 1994). Electron microscopy is not used in regular routine practice as combined data from histology, immunistochemistry and molecular pathology is usually sufficient

Criteria for mixed astrocytomas / oligodendrogliomas are weakly defined. Not surprisingly interobserver variaibility is great ranging from 9-80% as seen in a study on 155 tumors that were initially classified as oligoastrocytomas (Fuller et al., 2003). Macoscopically these tumors are similar to other diffuse grade II or grade III lesions. Histological diagnosis of oligoastrocytoma requires that both astrocytic and oligodendrogial neoplastic tumor cells are present in the same tumor. These may appear biphasic as two distinct tumor areas or more commonly as intermingled tumor. The minimal amount to which one tumor component has to be present is unfortunately not properly defined. Some authors are satisfied when one single high power field has either astrocytic or oligodendroglial tumor cells, other authors request at least a minimum of 50% neoplastic astrocytes. Separation of astrocytes and oligodendrocytes is not always possible. Every pathologist has seen tumor cells that have features of both lineages. It is important however to distinguish minigemistocytes and gliofibrillar oligodendrocytes in oligodendrogliomas from astrocytes, as they do not warrant the diagnosis of oligoastrocytoma. Single mitoses are compatible with a grade II oligoastrocytoma, however in our institution we have an relaxed approach, when mitoses are increased in a distinct oligodendroglial compartment only. Anaplastic oligoastrocytomas show increased nuclear atypia, elevated cellularity and abundant mitoses. Microvascular proliferations are frequent in grade III oligoastrocytomas. Discrimination of anaplastic oligoastrocytoma from glioblastomas with oligodendroglial differentiation is especially difficult, as WHO criteria allows pseudopalisading necroses to be present in oligoastrocytic tumors. In our institution decision is based on whether necroses are present in astrocytic tumor parts indicating a glioblastoma or is limited to oligodendroglial tumor parts indicating anaplastic oligoastrocytoma. Like histology, immunhistochemistry results are very mixed and represent the immunophenotype of neoplastic astrocytes or oligodendrogytes as discussed in their sections. Grade II tumors

have a MIB-1 proliferation index usually less than 6% (Deckert et al., 1989).

Because of their favourable prognostic value 1p19q codeletion, MGMT promoter methylation and isocitrate dehydrogenase mutations are considered important clinical biomarkers for diffuse gliomas. In addition p53 is useful for diagnostic purposes. These markers are requested with increasing frequency and are discussed in detail below. Even when the diagnosis of a specific glioma type is readily apparent in histological stains, pathologist need to take care, that sufficient tissue is available for future molecular

enough do diagnose an oligodendroglioma or mixed glioma.

**5.4 Electron microscopy** 

**6. Oligoastrocytoma** 

**7. Molecular biology** 

analysis.

present but not typically in the pseudopalisading forms of glioblastoma. The current WHO classification however explicitly allows presence of pseudopalisading necroses in anaplastic oligendrogliomas and thus weakens a sufficient discrimination to glioblastomas with oligodendroglial differentiation. Anaplastic oligodendrogliomas may contain smaller cells with pink cytoplasm and eccentric placed nuclei, so called minigemistocytes and areas with increased fibrillar background and plump process-bearing gliofibrillary oligoendrocytes. These eosionophilic cells are seen more often in grade III than grade II oligodendrogliomas. Finally some oligodendroglioma tumor cells may have sharp delineated borders resembling epitheloid differentiation. In the tumor edges severeal astrocytic cells might be present but unless clearly neoplastic in nature their presence does not warrant the diagnosis of mixed oligoastrocytoma. Focally parallel tumor cell growth may resemble polar spongioblastomas (Louis et al., 2007). Rare cases of oligodendrogliomas may show focally neuopil islands that have to be distinguished from neurocytomas. Tumor cells with signet-ring cell morphology have also been described in oligodendrogliomas (Kros et al., 1997).

#### **5.3 Immunohistochemistry**

There is no distinct single antibody availabe to discriminate reliably between oligodendroglial and astrocytic neoplasms. It is adviseable to use a panel of different antibodies for which expression patterns in these neoplasms has been extensively studied. In our institution we stain routinely gliomas for MIB1, GFAP, MAP2, WT1 and IDH1 R132H. Expression of GFAP is usually absent in tumor cytoplasm of oligodendroglia, however in our daily practice sometime there is ample overlapping of GFAP-positive fibrillary neuropil background. In addition minigemistocytes and gliofibrillar oligodendrocytes are usually positive for GFAP. MAP2 is constantly expressed in oligodendrogliomas, but also found in 92% of astrocytomas and glioblastomas (Blümcke et al., 2004). A perinuclear "capped" expression pattern is more typical for oligodendrogliomas, while in astrocytomas the elongated cell processes are also immunoreactive for MAP2. WT1 in oligodendrogliomas is usually restricted to single WT1 positive tumour cells or completely absent while WT1 is strongly expressed in 83-92% of high grade astrocytic lesions (Schittenhelm et al., 2009). Therefore, in our experience, expression of WT1 in more than 50% of tumor cells indicates either astrocytoma or oligoastrocytoma rather than oligodendroglioma. Nogo-A is found in 71% oligodendrogliomas and 24% glioblastomas but is absent in astrocytomas (Kuhlmann et al., 2008). While Olig2 immunoreactivity is slightly stronger in oligodendrogliomas is also constantly seen in other glial neoplasms (Ligon et al., 2004). Alpha internexin is found in 45- 59% of oligodendrogliomas ans seems to be associated with an 1p19q codeletion (Ducray et al., 2011). Positive IDH1-R132H immunoreactivity is so frequent in oligodendroglial tumors (up to 91% in grade II and 94% in grade III lesions) that this marker is very useful to discriminate oligodendrogliomas from other brain tumors with oligodendroglial morphology (Capper et al., 2011). Diffuse immunoreactivity of p53 is uncommon in oligodendroglial tumors but when present indicates an intact chromosomal 1p arm (Hirose et al., 2010). Oligodendrogliomas with neurocytic differentiation may show synaptophysinpositive neuropil islands and rosettes but usually lack the NeuN nuclear immunoreactivity of neurocytomas. In addition presence of IDH1 or IDH2 mutation strongly favors diagnosis of oligodendroglioma over neurocytoma (Capper et al., 2011).

## **5.4 Electron microscopy**

210 Advances in the Biology, Imaging and Therapies for Glioblastoma

present but not typically in the pseudopalisading forms of glioblastoma. The current WHO classification however explicitly allows presence of pseudopalisading necroses in anaplastic oligendrogliomas and thus weakens a sufficient discrimination to glioblastomas with oligodendroglial differentiation. Anaplastic oligodendrogliomas may contain smaller cells with pink cytoplasm and eccentric placed nuclei, so called minigemistocytes and areas with increased fibrillar background and plump process-bearing gliofibrillary oligoendrocytes. These eosionophilic cells are seen more often in grade III than grade II oligodendrogliomas. Finally some oligodendroglioma tumor cells may have sharp delineated borders resembling epitheloid differentiation. In the tumor edges severeal astrocytic cells might be present but unless clearly neoplastic in nature their presence does not warrant the diagnosis of mixed oligoastrocytoma. Focally parallel tumor cell growth may resemble polar spongioblastomas (Louis et al., 2007). Rare cases of oligodendrogliomas may show focally neuopil islands that have to be distinguished from neurocytomas. Tumor cells with signet-ring cell morphology

There is no distinct single antibody availabe to discriminate reliably between oligodendroglial and astrocytic neoplasms. It is adviseable to use a panel of different antibodies for which expression patterns in these neoplasms has been extensively studied. In our institution we stain routinely gliomas for MIB1, GFAP, MAP2, WT1 and IDH1 R132H. Expression of GFAP is usually absent in tumor cytoplasm of oligodendroglia, however in our daily practice sometime there is ample overlapping of GFAP-positive fibrillary neuropil background. In addition minigemistocytes and gliofibrillar oligodendrocytes are usually positive for GFAP. MAP2 is constantly expressed in oligodendrogliomas, but also found in 92% of astrocytomas and glioblastomas (Blümcke et al., 2004). A perinuclear "capped" expression pattern is more typical for oligodendrogliomas, while in astrocytomas the elongated cell processes are also immunoreactive for MAP2. WT1 in oligodendrogliomas is usually restricted to single WT1 positive tumour cells or completely absent while WT1 is strongly expressed in 83-92% of high grade astrocytic lesions (Schittenhelm et al., 2009). Therefore, in our experience, expression of WT1 in more than 50% of tumor cells indicates either astrocytoma or oligoastrocytoma rather than oligodendroglioma. Nogo-A is found in 71% oligodendrogliomas and 24% glioblastomas but is absent in astrocytomas (Kuhlmann et al., 2008). While Olig2 immunoreactivity is slightly stronger in oligodendrogliomas is also constantly seen in other glial neoplasms (Ligon et al., 2004). Alpha internexin is found in 45- 59% of oligodendrogliomas ans seems to be associated with an 1p19q codeletion (Ducray et al., 2011). Positive IDH1-R132H immunoreactivity is so frequent in oligodendroglial tumors (up to 91% in grade II and 94% in grade III lesions) that this marker is very useful to discriminate oligodendrogliomas from other brain tumors with oligodendroglial morphology (Capper et al., 2011). Diffuse immunoreactivity of p53 is uncommon in oligodendroglial tumors but when present indicates an intact chromosomal 1p arm (Hirose et al., 2010). Oligodendrogliomas with neurocytic differentiation may show synaptophysinpositive neuropil islands and rosettes but usually lack the NeuN nuclear immunoreactivity of neurocytomas. In addition presence of IDH1 or IDH2 mutation strongly favors diagnosis

have also been described in oligodendrogliomas (Kros et al., 1997).

of oligodendroglioma over neurocytoma (Capper et al., 2011).

**5.3 Immunohistochemistry** 

The short tumor cell processes contain microtubuli and ocassionally pericellular spiral laminations but usually lack the abundant intermediate filament of astrocytic tumor cells (Min et al., 1994). Electron microscopy is not used in regular routine practice as combined data from histology, immunistochemistry and molecular pathology is usually sufficient enough do diagnose an oligodendroglioma or mixed glioma.
